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Andrenogonadal interactions in nicotine-induced alteration of steroidogenesis in male rat.

Chronic nicotine administration in larger doses inhibits steroidogenesis in testis. This inhibition is possibly secondary to increased adrenomedullary norepinephrine activity, since the anti-steroidogenic effect of nicotine can be prevented by alpha-adrenergic blocker like phentolamine. In vitro incubation of testicular slices with nicotine failed to elicit any appreciable effect on delta 5-3 beta-hydroxysteroid dehydrogenase activity indicating lack of direct action of nicotine on testicular steroidogenesis.

The epidermal helix: a model for the bilayer couple phenomenon.

Narrow strips of isolated epidermis rapidly assume a helical conformation when placed in a dilute hypotonic aqueous solution of histamine as a free base. The same belical formations could be induced by dilute hypotonic solutions of alkalis or acids. This coiling resulted from damage to the malpighian cells, with consequent asymmetric osmotic swelling of the epidermis, thus producing torsional forces on the stratum corneum. In essence, the epidermal helix proved to be a pH dependent, osmotically induced, bilayer couple phenomenon.

Ocular findings in metachromatic leukodystrophy. An electron microscopic and enzyme study in different clinical and genetic variants.

Histopathological studies of the eyes from three patients affected with the infantile form of metachromatic leukodystrophy (MLD) showed the storage of metachromatic complex lipids in the retinal ganglion cells, in the optic nerve and the ciliary nerves, as well as the storage of a mucopolysaccharide-like material in the nonpigmented epithelium of the ciliary body. The lesions were limited to the optic, ciliary, and sensory nerves in a fourth patient with the juvenile form of the disorder. These morphological aspects, which are probably related to differences in sulfatase A activities, may explain the variability of the ocular manifestations in metachromatic leukodystrophy. Seven children affected with infantile MLD or with mucosulfatidosis were examined by conjunctival biopsy. Typical lesions of the sensory nerves were obvious and allowed the diagnosis of the disease. However, it seemed impossible to separate the different forms by histopathological studies only. The tear enzymes were assayed in most of the cases and demonstrated a profound deficiency of arylsulfatase A, or of arylsulfatase A and B, in the classical MLD and in mucosulfatidosis, respectively.

Comparison of I.M. pethidine, diazepam and flunitrazepam as premedicants in children undergoing otolaryngological surgery.

Pethidine 1 mg kg-1, diazepam 0.25 mg kg-1 and flunitrazepam 0.02 mg kg-1 i.m. wer compared as premedicants in a double-blind study in 145 children undergoing otolaryngological surgery. Both flunitrazepam and pethidine had an anxiolytic effect in the children of less than 5 yr whereas diazepam had little effect. All of the drugs were anxiolytic in the children aged 5 yr and older. Sleep following thiopentone was restless more often in the younger than in the older children. Cardiovascular responses to thiopentone and to tracheal intubation were most obvious following benzodiazepines in children of less than 5 yr. After anaesthesia 10--33% of the older children could not recall pictures shown to them before anaesthesia. Forty-five (+/-SD 13) min after injection, the concentration of diazepam in serum was similar in both age groups; after 90 min it decreased in the younger and increased in the older children. All concentrations of flunitrazepam were significantly greater in the older compared with the younger children.

Long-term treatment of essential hypertension using nadolol and hydrochlorothiazide combined.

1 The stepped care approach for the treatment of hypertension was adopted in a study at Ain Shams Hospital using hydrochlorothiazide (HCT) and a new beta-blocker, nadolol. Sixty mild to moderately hypertensive patients were studied for 20 weeks (2 weeks no antihypertendive the therapy, 3 weeks placebo, 3 weeks HCT, 4 weeks nadolol + HCT dose titration and 8 weeks nadolol + HCT maintenance). The dose of HCT was 50 mg once daily throughout the study except for six patients who had their HCT dose increased to 100 mg daily during maintenance. The dose of nadolol ranged from 40-240 mg daily. 2 No patient on HCT monotherapy achieved full control of his supine diastolic blood pressure (SDBP less than 90 mm Hg). On combined therapy, 55 patients (91.7%) showed a full response, whereas the remaining five patients a good or adequate response. 3 Thirty-two of these patients agreed to continue in the study for a further 21 months (totalling 2 yr of therapy). To date, 15 of them have completed a total of 10 months, 7 have completed 11 months and 10 have completed 12 months. The delta percentage decrease in supine BP continued to be 28.0 and 19.5 for systolic and diastolic BPs respectively. 4 No significant changes in funduscopies, chest X-rays, ECGs, or full laboratory investigations were noted. A few side-effects of mild nature occurred. None necessitated discontinuation of therapy. 5 Combined therapy with nadolol and HCT is a safe and effective method of controlling hypertension over extended periods.

Arginyl-tRNA synthetase from Escherichia coli K12. Purification, properties, and sequence of substrate addition.

Arginyl-tRNA synthetase from Escherichia coli K12 has been purified more than 1000-fold with a recovery of 17%. The enzyme consists of a single polypeptide chain of about 60 000 molecular weight and has only one cysteine residue which is essential for enzymatic activity. Transfer ribonucleic acid completely protects the enzyme against inactivation by p-hydroxymercuriben zoate. The enzyme catalyzes the esterification of 5000 nmol of arginine to transfer ribonucleic acid in 1 min/mg of protein at 37 degrees C and pH 7.4. One mole of ATP is consumed for each mole of arginyl-tRNA formed. The sequence of substrate binding has been investigated by using initial velocity experiments and dead-end and product inhibition studies. The kinetic patterns are consistent with a random addition of substrates with all steps in rapid equilibrium except for the interconversion of the cental quaternary complexes. The dissociation constants of the different enzyme-substrate complexes and of the complexes with the dead-end inhibitors homoarginine and 8-azido-ATP have been calculated on this basis. Binding of ATP to the enzyme is influenced by tRNA and vice versa.

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides.

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory. When a salf was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO4, MgCl-2, CaCl2) were effective at concentrations lower than those of monovalent cation salts (NACl, KCl, Na2SO4) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5--5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H+ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (-1.9 +/- 0.5) . 10(-3) elementary charge per A2, and the surface potential of about -100 mV in the presence of about 0.1 mM divalent cation of 5 mM monovalent cation were calculated.

Redox titration of electron acceptor Q and the plastoquinone pool in photosystem II.

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve. The titration of Q, as indicated by the dark level of Fi, occurs in two discrete steps. The high-potential component (Qh) has a midpoint potential of +68 mV (pH 7.2) and accounts for approximately 67% of Q. The pH sensitivity of the midpoint potential is -60 mV, indicating the involvement of 1 H+/e. The low-potential component (Q1) accounts for the remaining 33% of Q and shows a midpoint potential near--300 mV (pH 7.2). The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30--40 mV more oxidizing than that of Qh, i.e., at 106 mV (pH 7.2). The Em shows a pH sensitivity of -60 mV/pH unit, indicating the involvement of 1 H+/e. The observation that all 12--14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property. Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.

Inhibition of creatine kinase by iodoalkanes. Further appraisal of the essential nature of the reactive thiol group.

Creating kinase (ATP: creatine N-phosphotransferase) is completely inhibited by low molecular weight iodoalkanes in a pseudo first order reaction. Analysis of this and other data suggests that covalent modification per se is not a sufficient criterion to establish whether or not an enzyme group is essential for catalysis.

The photosensitizing action of phycobiliproteins in the adsorbed state.

In connection with the elucidation of the possibility of photochemical participation of phycobiliproteins in the primary processes of photosynthesis, the ability of a mixture of phycocyanin + allophycocyanin (PC + APC) for photosensitization of redox reactions in the adsorbed state was investigated. It was shown that adsorbates of PC + APC on Sephadexes G-200 and G-25, diethylaminoethylcellulose, carboxyethylcellulose, Bacto-agar, Lifogel, polyethylene glycol, Dowex 50Wx2, and aluminum oxide are capable of sensitizing the photoreduction of methyl red by ascorbid acid. In this case the effectiveness of the sensitizing action depends on the concentration of the adsorbate, the pigment concentration on the carrier, the pH of the medium, and the nature of the solvent. It was shown that in the case of binding to a carrier, the sensitizing ability of PC + APC increases in comparison with that for pigments in the dissolved state. It is suggested that this is promoted by an increase in the concentration and a mutual approach of the reagents after adsorption, and the possible formation of complexes of some or all the participants of the reaction on the surface of the adsorbent.

Characterization of vascular histamine receptors in the rat.

1 In the rat the decrease in blood pressure caused by histamine involves activation of both H1- and H2-receptors. Since arterial pressure measurements alone do not permit the separation of responses into cardiac and vascular components, the following experiments were undertaken to study vascular histamine receptors. 2 Vascular responses were studied in the autoperfused hindquarters of anaesthetized rats. Intra-arterial histamine caused vasodilatation which was only partially attenuated by treatment with mepyramine, an H1-receptor antagonist. Treatment with metiamide, the H2-receptor antagonist, did not affect vasodilatation caused by histamine but did attenuate vasodilatation which persisted after mepyramine. 3 Intra-arterial 4-methylhistamine, an H2-receptor agonist, caused vasodilatation which was reduced by metiamide. The H1-receptor agonist, 2-(2-pyridyl)ethylamine also caused vasodilatation which was blocked by mepyramine. 4 It is concluded that in the rat, histamine causes vasodilatation mediated by both H1- and H2-receptors.

Dendro axonic neurotransmission. II. Morphological sites for the synthesis, binding and release of neurotransmitters in dopaminergic dendrites in the substantia nigra and cholinergic dendrites in the neostriatum.

Morphological evidence is presented indicating sites of synthesis, storage, and release of neurotransmitters in dendrites of dopaminergic cells of the substantia nigra and cholinergic cells of the neostriatum. Smooth endoplasmic reticulum can be identified in dopaminergic neurons touching the dendritic surface. The false transmitter for dopamine, 5-hydroxydopamine (5-OHDA), is localized to smooth endoplasmic reticulum or large vesicular structures which approach the dendritic surface. The dopamine synthesizing enzyme, tyrosine hydroxylase (TH), is localized to microtubules and smooth endoplasmic reticulum which approach the postsynaptic membrane. In the neostriatum, dopaminergic nerve endings make asymmetrical axospinous contacts. The postsynaptic spines often contain a few 'vesicles' near the postsynaptic thickenings. The surface and subsurface structures stain preferentially for choline acetyltransferase (CAT), the synthesizing enzyme for acetylcholine. It is hypothesized that neurotransmitters are released from dendrites as a general phenomenon in the CNS and that they can act upon axonal endings.

Light and electron microscopic identification of monoaminergic terminals in the central nervous system.

A brief critical survey of methods used for light and electron microscopic examination of amine-containing pathways within the CNS. Light microscopic techniques such as fluorescence histochemistry, immunocytochemistry, autoradiography, silver degeneration techniques, and retrograde tracing technique are suitable for studying the topography of pathways but, due to limits of resolution, they are inadequate for identifying terminals. Electron microscopy which is adequate to visualize terminals does not provide an overall view. This review considers various methods which have been devised to specifically detect aminergic nerve terminals. Electrolytic and chemical induced degenerations are described in noradrenergic, dopaminergic, and serotoninergic terminals. Although the individual degenerative alterations are not specific for aminergic terminals, the degenerative process when considered as a whole can be informative. At present no single technique can provide complete information about the origin, course, connections, and terminals of aminergic systems. Concurrent application of light and electron microscopy, experimental surgery, histochemistry, and microsample biochemistry would provide a complete description.

Renal uptake and nephrotoxicity of gentamicin during urinary alkalinization in rats.

1. Effect of urine pH on accumulation of gentamicin in the renal cortex of rats was studied following constant intravenous infusion, and single or repeated i.v. injections with gentamicin. 2. The cortical uptake of gentamicin was moderately inhibited by urinary alkalinization due to sodium bicarbonate treatment, but was unaffected by acidification with ammonium chloride. The altered urinary pH had no effect on urinary excretion of gentamicin. 3. An alkaline urine induced by acetazolamide injections failed to influence cortical accumulation of gentamicin. This effect may be ascribed to 'acidification' of the proximal tubular fluid after carbonic anhydrase inhibition, even though the final urine was alkaline. 4. Nephrotoxicity resulting from chronic treatment of gentamicin was ameliorated by concomitant sodium bicarbonate administration. 5. In conclusion, the intratubular pH of the proximal tubule is a factor which influences the cortical uptake of gentamicin, probably by means of changing the cationic nature of the molecule and, therefore, reduced binding with the luminal membrane.

Biotransformation of 4'-ethynl-2-fluorobiphenyl in the rat. In vitro and in vivo studies.

The absorption, metabolism, and plasma pharmacokinetics of a novel anti-inflammatory agent, 4'-ethynyl-2-fluorobiphenyl, was studied in the rat. 4'-[1-14C]Ethynyl-2-fluorobiphenyl was quantitatively absorbed from the gastrointestinal tract. Excretion of radiocarbon into urine was greater than excretion into bile. Appreciate amounts of radiocarbon remained in the carcass 24 hr after dosing. The only metabolite in plasma was (2-fluoro-4'-biphenylyl)acetic acid, which also possessed anti-inflammatory activity. Unchanged 4'-ethynyl-2-fluorobiphenyl was present after administration of higher doses. Peak plasma concentrations of (5-fluoro-4'-biphenylyl)acetic acid were observed within 1 hr of administration. The apparent plasma half-life of this acidic metabolite was 4 hr. The major eliminated metabolite was (4-hydroxy-2-fluoro-4'-biphenylyl)acetic acid. In vivo and in vitro metabolism studies suggest that the major metabolic pathway involves microsomal hydroxylation of the C-H bond of the ethynyl moiety to yield, after rearrangement, a highly reactive intermediate metabolite, 2-fluoro-4'-biphenylylketene.

The disposition of l-3-[(dimethylamino)-(m-dioxan-5-yl)methyl]pyridine in man.

l-3-[(Dimethylamino)-(m-dioxan-5-yl)methyl]pyridine hydrochloride (LY 108380) is being evaluated in man as a potentially useful, nonaddicting analgesic agent. This substituted dioxane is structurally different from any currently known analgesic. Following im administration of the 14C-labeled compound to healthy volunteers, the drug was absorbed rapidly (t1/2(abs) = 2--20 min). Pharmacokinetic analyses suggested that LY 108380 was widely distributed and extensively bound in tissues. The drug was not bound to plasma proteins in vitro or in vivo. In the blood, radioactivity was distributed in both red cells and plasma; a cell/plasma radioactivity ratio of 0.5 was maintained for about 1 hr. The t1/2 for elimination of LY 108380-14C from plasma was about 1.3 hr, although radioactivity persisted in plasma for over 100 hr. At the time of peak radioactivity, the parent compound was the major constituent in plasma; quaternary N-glucuronide and N-desmethylated metabolites were also detected in plasma. Levels of radioactivity in saliva were 2--5 times higher than those in plasma shortly after drug administration. About 82% of the radioactivity was eliminated in the urine, 6% in expired air (as 14CO2), and 1% in feces. The major metabolite of LY 108380 (55% of the dose) was a quaternary amine formed by glucuronidation at the pyridine nitrogen. Less than 10% of the dose was N-demethylated to secondary and primary amines, and about 2% was excreted unchanged.

Atenolol: a review of its pharmacological properties and therapeutic efficacy in angina pectoris and hypertension.

Atenolol is a beta-selective (cardioselective) adrenoceptor blocking drug without partial agonist or membrane stabilising activity. Its profile of action most closely resembles that of metoprolol which differs only in that it has some membrane stabilising activity. Atenolol has been well studied and is effective in the treatment of hypertension and in the prophylactic management of angina. Its narrow dose response range obviates the need for highly individualised dose titration. In patients with angina its long duration of beta-blocking activity allows once daily dosage, whereas other beta-blockers, unless in sustained release dosage forms, need to be given in divided doses. Other beta-blockers can be given once daily in hypertension, but at presnt the evidence for effective control with a once daily regimen is more convincing with atenolol. Further studies are need to clarify any important differences in blood pressure control between the various beta-blocking drugs, both in conventional or sustained release dosage forms. As with metoprolol, atenolol is preferable to non-selective beta-blockers in patients with asthma or diabetes mellitus. Atenolol has been well tolerated in most patients, its profile of adverse reactions generally resembling that of other beta-blocking drugs, although its low lipid solubility and limited penetration into the brain results in a lower incidence of central nervous system effects than seen with propranolol. Atenolol is eliminated virtually entirely as unchanged drug in the urine and dosage needs to be reduced in patients with moderate to severely impaired renal function (glomerular filtration rate less than 30 ml/min). There is no need for modification of dosage of atenolol in liver disease.

Artifacts imitating aging of glucose-6-phosphate dehydrogenase in human erythrocytes.

Data in the literature based on the technique of graded osomotic hemolysis have been re-evaluated. Differences were previously found in the glucose-6-phosphate dehydrogenase/hemoglobin ratio and in the heat stability of the enzyme in hemolysates of 'old' and 'young' cells. These differences were believed to be due to the aging of the enzyme. As the erythrocyte membrane acts as a molecular sieve under hypotonic conditions [cf. Cseke, E., Váradi, A., Szabolcsi, G., and Biszku, E. (1978) FEBS Lett. 96, 15--18], the hemolysate obtained when a fraction is lysed does not properly represent the content of the lysed cells. As hemoglobin is lost from cells which are not yet lysed, the enzyme/hemoglobin ratio is underestimated in 'old' cells and overestimated in 'young' cells. It is further shown that the observed differences in the heat stability of glucose-6-phosphate dehydrogenase in the fractions obtained by graded hemolysis are due to the presence of different concentrations of endogeneous NADP. Therefore the published data obtained by graded osmotic hemolysis do not prove the assumption that the enzyme is aging during the lifetime of the erythrocyte.

Studies on the absorption, distribution and elimination of 6-o-chlorophenyl-2,4-dihydro-2(N-methyl-piperazin-1-yl)-methylene-8-nitro-1H-imidazo[1,2-a] [1,4]benzodiazepin-1-one methanesulphonate in the male rat and rabbit.

The fate of a novel imidazo-benzodiazepine (I) was studied in male rats and rabbits using 14C and 3H-labelled I. In both species the compound was rapidly and widely absorbed after an oral dose of 5 mg/kg to give peak tissue and plasma levels after 1 hour in the rat and 4 hours in the rabbit. The highest concentrations of radioactivity were present in the liver (rat) and liver, kidney and subcutaneous fat (rabbit). Plasma levels of radioactivity fell to 3% of the maximum value in 24 hours in the rat but 48 hours were required for a similar fall in the rabbit. The main route of elimination of radioactivity was via the bile followed by excretion in the faeces. For the rat the rate of biliary elimination was 16.6% of the administered dose/hour; for the rabbit this rate was 5.6%/hour. Recovery of administered radioactivity during 0-24 hours for urine and faeces respectively was 4.8% and 69% for the rat and 23.2% and 10.9% for the rabbit. Up to 97% of the radioactivity administered to rats could be recovered in the excreta in the 7 days following dosing. Up to 90% of the dose administered to rabbits appeared in the excreta during 10 days. No unchanged (I) could be detected in the urine or bile. The radioactive metabolites were polar products, some of which were in the form of glucuronide conjugates.

Dopamine-sensitive adenylate cyclase in homogenates of rat nucleus accumbens: structure-activity studies and effects of agonists and antagonists.

A study has been made of the structural requirements for activity on the dopamine-sensitive adenylate cyclase present in homogenates of rat nucleus accumbens. The only active phenylethylamine derivatives tested were those containing hydroxy groups at the 3 and 4 positions on the benzene ring, a two carbon side chain and a terminal nitrogen, either unsubstituted or containing a single methyl group. The alpha- and beta-adrenergic agonists, phenylephrine and isoprenaline respectively, were both inactive. Norsalsolinol was a weak agonist producing only a 50% stimulation of adenylate cyclase activity. The typical neuroleptic drugs, fluphenazine and cis-flupenthixol were both potent antagonists of the dopamine response as opposed to the atypical neuroleptics, metoclopramide and sulpiride, and the alpha- and beta-adrenergic blocking agents, phentolamine and propranolol respectively, which were all inactive. Our results indicate that the dopamine receptors associated with adenylate cyclase in the nucleus accumbens are similar to those in the corpus striatum.

National Cooperative Crohn's Disease Study: adverse reactions to study drugs.

Adverse reactions to the drugs employed in the National Cooperative Crohn's Disease Study were sought prospectively at each patient visit and by retrospective review of all patient charts. Prednisone caused evident side effects in over 50% of patients on high-dose suppressive therapy and in approximately one-third of patients on prophylactic dose. Thirty-two percent of patients on high-dose, and 26% on prophylactic-dose prednisone required dose reduction or withdrawal because of side effects. Comparable figures for sulfasalazine were 14% and 12%, and for azathioprine 32% and 20%. The incidence of nausea, vomiting, or anorexia among patients taking sulfasalazine was 46% and 34%, on high and low dose respectively; however, this incidence was no different than that observed among patients taking placebo. These symptoms occasioned withdrawal from the study of only 4% and 3% of patients on high and low doses of sulfasalazine, respectively. Azathioprine produced leukopenia at a dose of 2.5 mg/kg body weight in 15% of patients and the mean white cell count, lymphocyte count, granulocyte count, and hematocrit all fell significantly in patients on this dose. Pancreatitis occurred in 5% of patients taking azathioprine but in no other patients. Sulfasalazine proved to be the safest effective suppressive drug for Crohn's disease. Prednisone toxicity, though substantial, is acceptable in view of its demonstrated suppressive efficacy. Azathioprine was approximately as toxic as prednisone but no more effective than placebo in suppressing active disease. None of the drugs was effective prophylactically, and all showed appreciable long-term toxicity.

Properties of extracellular neuraminidase produced by group A streptococcus.

Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.

On the optimization of local hyperthermy in tumors based on a new radiofrequency procedure. Local hyperthermy of large body areas using the CMT selectotherm method.

A new radiofrequency procedure, i.c., the CMT Selectotherm technique, permits to convey large heat quantities per volume unit also to deep-seated tumor tissues without causing thermal lesions in healthy tissues near or at the body surface. The improved spatial homogeneity of energy supply attainable by this method is demonstrated by measurements at a gelatine phantom and, in particular, by in vivo measurements on pigs. The appliability of local hyperthermy to tumors localized in different parts of the body is substantially improved (a) by the principle of superimposing local hyperthermy on an elevated temperature level of metabolically induced whole-body hyperthermy (CMT-spontaneous hyperthermy at 40 degrees C) and (b) by the principle of selective increasing the thermal sensitivity of tumor tissues by decreasing the pH in these areas (the CMT main step). It is shown that the temperature dose T. deltat necessary for the selective occlusion of the vasculature in tumor tissues can be obtained by the CMT Selectotherm process also in deep-seated tumors. This process is part of the 1977 CMT concept. The fundamentals of optimizing local hyperthermy with consideration of heat dissipation from the tissue by heat conduction and convection via the blood stream are demonstrated. Temperature profiles are calculated for some practice-relevant, typical examples (inner and outer parts of sphero-symmetrically shaped tumors). Finally, in vivo measurements and calculations on the time course of temperature under certain conditions and for different tissue layers are discussed.

Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa.

Soluble fibronectin is found in body fluids and media of adherent cultured cells and binds to fibrin and collagen. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for Factor XIIIa (plasma transglutaminase) and can be cross-linked by Factor XIIIa to itself and the the alpha-chain of fibrin. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate Factor XIIIa-mediated crosslinking of fibronectin to collagen. At O degrees or 37 degrees C, fibronectin could be cross-linked to iodinated cyanogen bromide fragment 7 of the alpha 1(I) chain. At 22 degrees or 37 degrees C, fibronectin could be cross-linked to isolated alpha 1(I) chains of type I collagen. Fibronectin could also be crosslinked to types I and III collagen, but only at 37 degrees C. alpha 1(I)-CB7, alpha 1(I) collagen chains, type I collagen, type III collagen, and fibrin all blocked cross-linking between 125I-alpha 1 (I)-CB7 and fibronectin. alpha 1(I)-CB7 blocked cross-linking between fibronectin and fibrin. These results indicate that the determinants of fibronectin-fibrin and fibronectin-collagen binding and cross-linking are similar. Cross-linking of fibronectin to collagen likely occurs in vivo and may be important for normal wound healing, collagen fibrillogenesis, and embryogenesis.

Blood flow and permeability changes in the immune lymphocyte transfer reaction.

Blood flow (133Xe clearance) and plasma exudation ([133I]HSA) have been measured in the immune lymphocyte transfer (ILT) reaction and skin grafts in rabbits. Injection of sensitised lymphocytes produced a dose-related increase in plasma exudation and blood flow at 48 hr, reached a maximum at day 3 and faded from day 5 to 8. There was an increased blood flow and plasma exudation on day 4 after grafting autografts and homografts, but the increase in plasma exudation was significantly higher in homografts. In the ILT reaction (48 hr) and the homografts (4 days) but not in autografts, prostaglandin synthetase inhibition caused a significant reduction in the increased blood flow, but did not abolish it nor did they affect the increased plasma exudation. It is concluded that the ILT reaction is a suitable model for the study of mediators of the vascular effects of the early phase of the skin graft reaction. The present experiments suggest that the vasodilatation is partly due to prostaglandin formation, but part of the vasodilatation and all the plasma exudation are mediated by substances other than prostaglandins.

Effects of external calcium concentration and pH on charge movement in frog skeletal muscle.

1. The effects of both external Ca2+ (1.8, 25, 50 and 100 mM) and external pH (pH 5.5, 7.15, and 9.0) on the voltage-dependence of charge movement in frog skeletal muscle were examined using the three intracellular micro-electrode voltage-clamp technique. 2. The two-state model of Schneider & Chandler (1973) was used to describe the voltage distribution of membrane charge. The parameters of this model are: Qmax, the maximum quantity of charge; V, the potential of equal distribution of charge; and k, a constant relating to the steepness of the charge vs. voltage relationship. 3. In 1.8 mM external Ca2+, alterations, in external pH shifted the transition potential, V, from a mean +/- S.E. of mean of -36.5 +/- 0.9 mV at pH 7.15 to -25.8 +/- 1.3 mV at pH 5.5 and to -42.5 +/- 1.8 mV at pH 9.0. These shifts are consistent with surface charge theory. No significant changes in Qmax or k were observed over the range of pH 5.5--9.0. 4. A reasonable fit of surface charge theory to the shifts in V over the range pH 5.5--9.0 could be obtained with surface charge densities and binding constants: sigma 1 = -1 e/165 A2, pK1 = 3.9 and sigma 2 = -1 e/400 A2, pK2 = 8. 5. However, at pH 7.15, both V and k changed with increasing external Ca2+ concentration. V shifted from -34.9 +/- 3.7 mV in 1.8 mM-Ca2+ to -13.8 +/- 5.1 mV, -19.3 +/- 3.6 mV and 3.3 +/- 9.3 mV in 25, 50 and 100 mM-Ca2+ respectively. k increased from 8.3 +/- 0.6 mV in 1.8 mM-Ca2+ to 15.3 +/- 1.4 mV, 14.6 +/- 1.6 mV and 20.0 +/- 2.9 mV in 25, 50 and 100 mM-Ca2+. Changes in k reflect decreases in the apparent charged particle valence from approximately 3 in 1.8 mM-Ca2+ to approximately 1.2 in 100 mM-Ca2+. As the external Ca2+ concentration was raised, Qmax was at least as large as that measured in 1.8 mM-Ca2+. The 43% decrease in the apparent valence of the charged groups cannot be explained by simple surface charge theory and may reflect a specific interaction between external Ca2+ and the charged groups. 6. Shifts in V with alterations in external pH and Ca2+ concentration are consistent with the effects of these agents on the contraction threshold of muscle fibres. This observation lends further support to the hypothesis that the charge movement is involved in gating muscle contraction and that the charged particles respond to changes in the electric field across the muscle cell membrane. 7. No difference was observed in the charge movement parameters of fibres from both room-temperature and cold-adapted frog tested at 2--5 degrees C in 1.8 mM-Ca2+ at pH 7.15.

Familial polyposis coli: heterogeneous polyp expression in 2 kindreds.

We describe 2 extended kindreds supposedly manifesting familial multiple adenomatous polyposis coli (FPC), but which show marked heterogeneity in the phenotypic expression of colorectal adenomatous polyps. In one family, 2 individuals had diffuse polyposis at very early ages (7 and 10 years), while 6 others (aged 23 to 72 years) had solitary polyps only. Of the patients with solitary polyps, 2 had associated colonic malignancies (ages 26 and 35), while another had a prophylactic colectomy performed at age 46. In the second family, 5 of the 11 patients with evidence of polyps showed the classical presentation of FPC, while the remainder showed marked phenotypic variation. The marked variability in frequency and location of colon polyps points to the need to reassess our traditional criteria for diagnosis of FPC. The high risk of early onset colon cancer in patients from these families who have the most minimal manifestation, namely isolated polyps, recommends more careful scrutiny of supposedly unaffected members of all FPC kindreds.

[Principle growth indices of a chemostat Candida utilis culture resistant to acid pH values].

The kinetics of growth of the Candida utilis chemostat culture 1668-3-37 was studied in a synthetic medium with ethanol at different values of pH and temperature. Chemostat curves were obtained for the pH of the medium of 4.5 and 3.0 and the temperature of 32 degrees C. The following growth characteristics were determined: the maximal growth rate (mumax), the economical coefficient (Y), the substrate (saturation) constant (Ks), the rate of ethanol uptake (q), maintenance energy (m). The minimal amount of ethanol inhibiting the yeast growth was assayed in short-term experiments under periodic conditions with shaking. The value of mumax was 0.35 hr-1 when the yeast was cultivated at 32 degrees C and the pH 4.5, and 0.32 hr-1 at the pH 3.0. The value of Ks varied by an order of magnitude at different pH values when the chemostat culture was grown at D=0.2 hr-1: 36.0 mg/litre at the pH of 3.0 and 3.75 mg/litre at the pH of 4.5. The value of m was close to 0 at the pH of 4.5 and equaled 5--7 mg of ethanol per gram of dry biomass per hour at the pH of 3.0; it was still higher when the temperature of cultivation was increased to 38 degrees C. The minimal substrate (ethanol) concentration inhibiting the yeast growth was constant at different cultivation conditions (pH 3.0 or 4.5 and temperature 32 or 38 degrees C), being equal to 0.45% (v/v) of ethanol.

Selective stimulation of central alpha-autoreceptors following treatment with alpha-methyldopa and FLA 136.

1. The accumulation of normetanephrine in the rat brain induced by the monoamine oxidase inhibitor nialamide was inhibited following alpha-methyldopa and 4-amino-3-(2,6-dichlorobenzylidenehydrazino)-1,2,4-triazole (FLA 136). It was not changed following alpha-methylmetatyrosine despite a greater disappearance of noradrenaline than after alpha-methyldopa. The alpha-adrenoreceptor blocking agent yohimbine increased the nialamide-induced accumulation of normetanephrine and completely antagonized the actions of alpha-methyldopa and FLA 136, indicating that the effects of the two drugs are due to stimulation of alpha-adrenoreceptors. 2. The flexor reflex activity of spinalized rats was not influenced by alpha-methyldopa and alpha-methylmetatyrosine at the doses used in the biochemical experiments, as previously found for FLA 136, indicating no stimulation of classical, postsynaptic, central alpha-adrenoreceptors. 3. The biochemical effects of alpha-methyldopa and FLA 136 might be caused by stimulation of alpha-autoreceptors on the cell bodies and the nerve terminals of noradrenaline neurons. A similar mechanism might be involved in the hypotension reported by other investigators following these drugs.

Complexing of reduced technetium and tin(II) by chelating phosphate compounds. II. In vitro stability of pyrophosphate and ethane-1, hydroxy-1, diphosphonate (EHDP) complexes.

The in vitro stability of 99mTc- and 113Sn-pyrophosphate and ethane-1, hydroxy-1, diphosphonate (EHDP) complexes was studied by varying the mode of preparation. The 1-hr distribution in the rat was used as an indicator for complex formation or destruction. A maximum of bone uptake and urinary excretion and a minimum of soft tissue concentration was obtained if there was an excess of phosphate in relation to tin(II) in the equilibrium. Formation of tin(II) colloid was favoured in the presence of an excess of tin(II) in the equilibrium, 99mTc colloid occurred with some delay. After dilution in neutral normal saline the chelates were more or less destroyed, as shown by a 113Sn(II) colloid formation whereas the 99mTc-phosphate complexes were transformed into a 99mTc kidney agent. At pH 11 the 113Sn(II)-phosphate complexes proved to be stable, the 99mTc-phosphate complexes were also transformed into the 99mTc kidney agent. Oxidation of all tin(II) in the equilibrium by hydrogen peroxide did not change the distribution patterns of 113Sn, 99mTc was oxidized to pertechnetate. In general complexes between tin(II) and chelating phosphate compounds proved to be more stable than those with reduced technetium. EHDP was found to form stronger complexes with tin(II) and reduced technetium than pyrophosphate.

Fucntional specialization and binocular interaction in the visual areas of rhesus monkey prestriate cortex.

If is is believed that neural mechanisms mediating stereoscopic vision may be localized in specific areas of the visual cortex, then it becomes necessary to be able to define these areas adequately. This is no easy matter in the rhesus monkey, an animal close to man, where the cytoarchitecturally uniform prestriate cortex is folded into deep sulci with secondary gyri. One way around this awkward problem is to use the callosal connections of the prestriate cortex as the anatomical landmarks. Callosal connections are restricted to regions at which the vertical meridian is represented. Since the visual fields, including the vertical meridian, are separately represented in each area, each has its own callosal connections. These are of great help in defining some of the boundaries of these areas, since the boundaries often coincide with the representation of the vertical meridian. With the visual areas thus defined anatomically, it becomes relatively easy to assign recordings to particular areas. Studies of binocular interactions in these areas reveal that most cells in all prestriate areas are binocularly driven. Hence, theoretically, all of the prestriate areas are candidates for stereoscopic mechanisms. The degree of binocular interaction varies from cell to cell. At the two extremes are cells which either respond to monocular stimulation only and are inhibited by binocular stimulation or ones which respond to binocular stimulation only. Changing, as opposed to fixed, disparity is signalled by two types of cells. In one category are cells activated in opposite directions for the two eyes. Such cells are always binocularly driven. In the other category are cells, some of which are monocularly activated, that are capable of responding to changing image size. In the monkey, both these categories of cells have so far been found in the motion area of the superior temporal sulcus only.

Stereoscopic subsystems for position in depth and for motion in depth.

We describe psychophysical evidence that the human visual system contains information-processing channels for motion in depth in addition to those for position in depth. These motion-in-depth channels include some that are selectively sensitive to the relative velocities of the left and right retinal images. We propose that the visual pathway contains stereoscopic (cyclopean) motion filters that respond to only a narrow range of the directions of motion in depth. Turning to the single-neuron level we report that, in addition to neurons turned to position to depth, cat visual cortex contains neurons that emphasize information about the direction of motion at the expense of positional information. We describe psychophysical evidence for the existence of channels that are sensitive to change size, and are separate from the channels both for motion and for flicker. These changing-size channels respond independently of whether the stimulus is a bright square on a dark ground or a dark square on a bright ground. At the physiological level we report single neurons in cat visual cortex that respond selectively to increasing or to decreasing size independently of the sign of stimulus contrast. Adaptation to a changing-size stimulus produces two separable after-effects: an illusion of changing size, and an illusion of motion in depth. These after-effects have different decay time constants. We propose a psychophysical model in which changing-size filters feed a motion-in-depth stage, and suppose that the motion-in-depth after-effect is due to activity at the motion-in-depth stage, while the changing-size after-effect is due to to activity at the changing-size and more peripheral stages. The motion-in-depth after-effect can be cancelled either by a changing-size test stimulus or by relative motion of the left and right retinal images. Opposition of these two cues can also cancel the impression of motion in depth produced by the adapting stimulus. These findings link the stereoscopic (cyclopean) motion filters and the changing-size filters: both feed the same motion-in-depth stage.

[Preconcentration of trace chalcophile elements by a zincon--loaded resin and its application to neutron activation analysis (author's transl)].

A chelating agent-loaded resin consisting of an anion exchange resin and zincon which has widely been employed as a specific reagent for zinc(II) and copper(II) in spectrophotometry was prepared. The adsorption behavior of some chalcophile elements was studied in detail, with respect to pH, flow rate and exchange capacity. From the results, it was confirmed that the zincon-loaded resin reacts selectively with copper(II), zinc(II), mercury(II) and lead(II) at lower pH region, and the above reaction is stoichiometric as in the case of the reaction of zincon with metal ions in aqueous solution. Furthermore, the zincon-loaded resin was applied to the selective concentration of trace amounts of chalcophile elements in natural water samples prior to neutron activation analysis. Water samples taken from the Watarase River were filtered and the pH of each filtrate was adjusted to ca. 5.5. After preconcentration was made by the column method (zincon-loaded resin: 2 x 10-4 mol/g resin, 1.0 g, 7 mm phi x 35 mm), the resin in the column was washed and dried in a desiccator. The standard material was also prepared according to the above mentioned scheme. The sample and the standard materials packed in polyethylene vials were irradiated for 40 min by a neutron flux of 5 x 10(13 n.cm-2.sec-1 in the JRR-4 of the Japan Atomic Energy Research Institute. After cooling the materials, activity measurements were made. The results were 53 ppb for copper, 0.25 ppb for mercury.

Physical studies on isolated human prothrombin fragment-2. Comparisons with human prothrombin fragment-1.

Variation of pH strongly affects the fluorescence intensity of human prothrombin fragment-1 in a manner suggesting contributions from a number of protropic equilibria including groups with apparent pKa values near 3.0. These results suggest a structural role for pK1a of gamma-carboxyglutamic acid noieties. Added calcium ions (9 mM calcium chloride) quench the fluorescence titration curve uniformly above pH 4. Below pH 4, however, the titration curve in the presence of calcium ions suggests that calcium-ion-dependent processes leading to fluorescence quenching are pH-dependent. Upon back titration of human fragment-1, from pH 9, hysteresis is observed. Human prothrombin fragment-2 fluorescence titration curves are relatively broad at low pH suggesting the titration of normal carboxyl groups. The titration curves of fragment-2 are not affected by the presence of calcium ions, and hysteresis occurs upon back titration from low pH values. Circular dichroism (CD) Cotton effects appear at 232 nm and 280 nm and a trough appears at 203 nm in the CD spectrum of human prothrombin fragment-2. The Cotton effects in the region from 230 nm to 300 nm are sensitive to pH, ellipticity values at 232 nm increasing from approximately 300 at pH 2.5 to 1300 (degree-cm/decimole) at neutral pH and finally become negative at high pH values. In contrast to fragment-1, at neutral pH the fragment-2 Cotton effect at 232 nm is insensitive to the presence of 8 mM calcium chloride.

[Immobilization of aspergillus oryzae aminopeptidase on organic and inorganic carriers].

The process of Asp. oryzae aminopeptidase immobilization on organic (AE-cellulose, sepharose 4B, Sephadex G-200) and inorganic (SCh-2, SCh-3 sylochromes and KCK N 1 silicagel) carriers was studied. Aminopeptidase immobilized on Sephadex G-200 contains the largest amount of protein (80 mg per 1 g of carrier) and is the most active of all other preparations. The immobilized preparations retain the temperature optimum, like the soluble form, at 60 degrees C, except the preparation immobilized on AE-cellulose (50 degrees C). At temperatures of 70, 80 degrees C the obtained preparations are more thermostable than the initial enzyme. The pH-stability zone for the preparations immobilized by means of sylochrome-3 and Sephadex G-200 is wider to some extent as compared to the soluble enzyme. When studying the substrate specificity it is established that leucyl-glycyl-glycine is the best substrate for both the soluble form and the immobilized preparations. Multiple application of the immobilized aminopeptidase is shown to be possible.

[Relationship between interhemispheric irradiation of excitation and the individual characteristics of the animal].

The changes in irradiation coefficient (the ratio between the value of secretion resulting from interhemispheric irradiation of secretion and the main one secreted on the side of excitation evaluated in per cent) were studied in norm and under different functional loads in dogs with exposed symmetrical parts of the tongue following the method of K. S. Abuladse. It is shown that dogs with low and mean level of irradiation of excitation have good mobility of nervous processes and rapid excitation in homonomous structures whereas dogs with high coefficient of irradiation demonstrated inert nervous processes and were not able to show a clear one-way reaction during interhemispheric interaction. A group of dogs with sharply pronounced asymmetry in coefficient of irradiation to the right and to the left is described. This description permitted an assumption about functional heterogeneity of the hemispheres. The obtained data showed that the character of interhemispheric irradiation is determined by individual characteristics of animals.

Acute effects of temazepam and nitrazepam on psychomotor skills and memory.

Twelve pretrained students ingested temazepam, nitrazepam, and placebo, each double blind at one-week intervals in randomized order. Reactive and co-ordinative skills and critical flicker fusion were measured before each drug intake and 1, 2, 3, 6 and 8 hours after it. Short-term memory and paired association learning were measured at 1, 3 and 8 hours. The psychomotor responses to drugs were modified by a sequence effect (not at zero tests) which effect varied depending on the drug and parameter. In multivariance analysis it was included to reveal drug effects. Nitrazepam 10 mg increased reaction and co-ordination errors and also impaired learning and memory. Temazepam 10 mg impaired co-ordinative skills; on a whole it differed from nitrazepam but hardly from placebo. Temazepam 20 mg impaired co-ordination, and learning and memory. Both temazepam 20 mg and nitrazepam were experienced sedative. All drug effects were clearest during the first 3 hours, nitrazepam also impaired learning at 8 hours. Temazepam 20 mg seems suitable as a hypnotic.

Adrenergic innervation of the human uterus. Disappearance of the transmitter and transmitter-forming enzymes during pregnancy.

The uterine adrenergic transmitter is in many animal species dramatically reduced during pregnancy, probably leading to a functional denervation near term. In order to clarify whether similar changes also occur in the human uterus, the adrenergic innervation of the isthmic myometrium during nonpregnant and pregnant conditions was analyzed by fluorescence histochemistry for demonstration of adrenergic nerves, and by quantitative measurements of norepinephrine and its synthesizing enzymes, tyrosine hydroxylase and dopa decarboxylase. At term pregnancy all fluorescent adrenergic nerves in the myometrium had disappeared, and the norepinephrine concentration had been reduced to almost zero. Parallel to this the activities of tyrosine hydroxylase and dopa decarboxylase were markedly reduced. By contrast, the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, was unchanged, indicating that the adrenergic system was selectively affected. The results confirm that the adrenergic nerves in the human uterus, like those in uterine horns of laboratory animals, undergo fundamental changes in the course of pregnancy. This probably reflects entirely different conditions for a sympathetic influence on the myometrium during the last two trimesters of pregnancy compared to the non-pregnant situation.

Status of psychotropic drug blood level assays and other biochemical measurements in clinical practice.

Assays of drug levels in blood and of other biochemical characteristics of psychiatric patients are being proposed for clinical application, although their utility in practice remains uncertain. Exceptions are the assay of blood levels of anticonvulsants and of lithium ion. Assays of antidepressant drugs may be especially helpful in the evaluation of unexpected responses or in the avoidance of unwanted toxic effects and promise to permit more efficient predictions of individual requirements. Assays of platelet MAO activity or urinary MHPG excretion remain clinically less useful. Attempts to correlate blood levels of antipsychotic agents with clinical effects have been disappointing, although newer assay methods may prove more useful.

[Cutaneous reactions to propranolol (author's transl)].

A 17-year-old male patient with eczematous and psoriasiform eruption that developed during long-term therapy with Propanolol (Avlocardyl) has been studied. This eruption disappeared after removal of the drug; oral challenge was soon followed by a vesiculous and bullous eruption of face and extremities; five months later, sun exposure was followed by a severe eczematous eruption in these areas; nails changes were then observed. Most of side-effects of beta-adrenergic blocking drugs have been reported with Practolol: lichenoid, exanthematous, eczematous, psoriasiform rashes; exfoliative dermatitis; oculo-muco-cutaneous reactions; fibrosing polyseritis and drug induced systemic lupus erythematosus manifestations. Adverse effects of other beta-adrenergic blocking agents are less frequent. The pathogenetic mechanism responsible for these adverse reactions is still obscur: these changes might be caused by blockade of the epidermal cells (and T-lymphocytes) beta-receptors, more than by a direct immunologic, allergic or toxic mechanism.

[Echocardiographic diagnosis of obstructive myocardiopathies: study of the systolic anterior motion of the mitral valve and septal hypertrophy as compared with the hemodynamic and mechanographic findings. Evolution under medical treatment].

Characteristic echocardiographic features of hypertrophic obstructive cardiomyopathy were recorded in 24 patients, all of whom had asymmetric septal hypertrophy and systolic anterior motion of the mitral valve (SAM) at rest or after pharmacodynamic stimulation. The relationship between outflow tract obstruction and SAM was assessed by comparison with data obtained at cardiac catheterisation and external mechanography: SAM seems to be a non-specific phenomenon and may be recorded in cases of hypertrophic cardiomyopathy without obstruction during pharmacodynamic stimulation. In forms with obstruction, SAM and the severity of obstruction increase with the degree of spetal hypertrophy. The increased contractility of the left ventricular posterior wall appears to be an important factor in the mechanism of SAM which can be prevented by betablockade in moderate or labile forms. When SAM is permanent, whatever the gradient recorded, it is a sign of anatomical deformation of the left ventricle and may be an additional indication for cardiac surgery.

Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.

Self-association of unconjugated bilirubin-IX alpha in aqueous solution at pH 10.0 and physical-chemical interactions with bile salt monomers and micelles.

Spectrophotometric measurements of bilirubin-IX alpha in water and in aqueous/organic solvent mixtures at pH 10.0 as a function of bilirubin-IX alpha concentration (approx. 0.6--400 microM) are consistent with the formation of dimers (KD - 1.5 microM) in dilute (less than 10 microM) aqueous solution and further self-aggregation to multimers at higher concentrations. Added urea (to 10M) and increases in temperature (to 62 degrees C) obliterate the dimer-multimer transition at 10 microM, but added NaCl (to 0.30 M) promotes strong aggregation of dimers over a narrow concentration range, suggesting a 'micellization' phenomenon. Concentrations of dioxan or ethanol greater than 60% (v/v) in water were required to obtain the absorption spectrum of bilirubin-IX alpha monomers, suggesting that both hydrophobic and electrostatic (pi-orbital) interactions are involved in stabilizing the dimeric state in water. Micellar concentrations of sodium dodecyl sulphate induced spectrophotometric shifts in the dimer absorption spectrum of bilirubin-IX alpha consistent with progressive partitioning of bilirubin-IX alpha monomers into a relatively non-polar region of the micelles and allowed a deduction of the apparent critical micellar concentration that closely approximated the literature values. The pattern of bilirubin IX alpha association with bile salts is complex, since the absorption spectrum shifts hypsochromically below and bathochromically above the critical micellar concentration of the bile salts. Consistent with these observations, bilirubin IX alpha appears to bind to the polar face of bile salt monomers and to the polar perimeter of small bile salt micelles. At higher bile salt concentrations some-bilirubin-IX alpha monomers partition into the hydrophobic interior of the bile salt micelles. Our results suggest that under physiological conditions the natural conjugates of bilirubin-IX alpha may exhibit similar physical chemical properties in bile, in that dimers, highly aggregated multimers and bile salt-associated monomers may co-exist.

The low ionic strength reaction of human blood: relationship between the binding of serum immunoglobulin and complement of red blood cells and surface charge of the cells.

Using the sucrose haemolysis reaction of Hartmann & Jenkins (1966) as a basic model, the low ionic strength reaction (LISR) of human blood was studied to determine: (1) serum Ig uptake by RBC with saline elution and 125I-IgG uptake, and (2) complement fixation (CF) to RBC with lysis of PNH cells and C3H/C4 antiglobulin haemagglutination (AH) of normal cells. The saline eluates were found to contain IgG and IgM with traces of IgA; their pH optima for the uptake by RBC were 6.0 +/- 0.5, 5.5 +/- 0.5 and c 5.0 respectively. The ratio of bound IgG to IgM was linearly related to the uptake pH. Both C4 AH and lysis were found to be optimum at pH 6.0--7.5, whereas the maximum C3 AH was at pH 6.0 +/- 0.5. The LISR performed at a constant pH (6.1 +/- 0.1) showed that an increasing concentration of neuraminidase (VCN) used in pretreatment of RBC was associated with a decrease in both IgG uptake and CF activity. A maximum VCN effect reduced the Ig uptake to c 20% of normal and abolished almost all the CF activity. An impaired LISR to various degrees was also observed with RBC pretreated with ficin, papain, bromelin, trypsin or protamine, and RBC from two individuals of En(a-) type. Preincubation of serum at LIS with and without RBC resulted in respectively a 'complete' and partial consumption of C in the fluid phase. The latter was not enhanced or inhibited by the addition of VCN-treated RBC for preincubation. A hypothesis is proposed suggesting that in the LSR the Ig uptake by RBC is an electrostatic interaction of the oppositely charged RBC and Ig and the CF to RBC results from C activation by the cell-bound IgG and IgM. In addition, a pH-dependent inactivation of the cell-bound C3 in the LISR is demonstrated.

Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters.

The p-nitrophenyl esters of straight-chain fatty acids were used as substrates of the enzyme subtilisin Novo (EC 3.4.4.16) and its chemically produced artificial enzyme thiolsubtilisin. Subtilisin and thiolsubtilisin pH--activity profiles were determined, and kinetic effects of the active site O-S substitution were observed. Among the substrates tested, both enzymes show highest specificity with p-nitrophenyl butyrate. It was also found that subtilisin is more sensitive to changes in substrate chain length than is thiolsubtilisin. Second-order acylation rate constants (k2/Ks) are remarkably similar for both enzymes. However, thiolsubtilisin deacylation rate constants and Km values are lower than analogous subtilisin constants. While thiolsubtilisin deacylation rate constants give a pH profile identical with that of subtilisin, the pH profile of thiolsubtilisin acylation rate constants shows an active site pK value lowered from the subtilisin pK of 7.15 and exhibits an inflection point at pH 8.45, which is absent in subtilisin.

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes.

Ouabain-sensitive Na+ and K+ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl2 and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. It was found that an increased cellular pH reduced the rates of active transport of Na+ and K+ without significantly altering the ratio of pumped Na+/K+. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between -10 and +60 mV at constant internal pH did not affect the rates of active transport of Na+ or K+.

Characterization of cerebroside (monoglycosylceramide) from the sea anemone, Metridium senile. Identification of the major long-chain base as an unusual dienic base with a methyl branch at a double bond.

1. Cerebroside of the sea anemone, Metridium senile, has been isolated (0.6 mg/g dry tissue weight) and structurally characterized. 2. The structure was shown by mass spectrometry, NMR spectroscopy and degradative studies as beta-glucopyranosylceramide. The major fatty acids were 16 : 0 and 20 : 0 D-2-hydroxy fatty acids. The major base was a novel base, D-erythro-1,3-dihydroxy-2-amino-9-methyl-trans-4, trans-8-octadecadiene. 3. Some unusual fatty acids of marine origin are suggested to originate in this long-chain base by metabolic conversion. 4. The implication of the methyl branch position of the base on our current view of sphingolipid function in the plasma membrane is discussed.

Identification of MSH release-inhibiting elements in the neurointermediate lobe of the rat.

Neurointermediate lobes of rats comprise elements which, when excited in vitro, bring about an inhibition of the release of melanocyte stimulating hormone (MSH). Superfusion of neurointermediate lobes of intact donor rats with medium containing 45 mM K+ induced a stimulation of the release of oxytocin, arginine-vasopressin and dopamine (DA) and inhibited the release of MSH. Fluorescence histochemical observations and the results of release studies indicate that electrothermic lesions in the mediobasal hypothalamus induced a more rapid degeneration of dopaminergic than of peptidergic terminals in the neurointermediate lobe. Dopaminergic nerve terminals and the stimulated release of DA had vanished completely on the second day after these lesions, which coincided with the disappearance of K+-induced inhibition of MSH release. Frontal hypothalamic deafferentations resulted in disappearance of peptidergic nerve terminals as evidenced by the development of diabetes insipidus and the strong decline of depolarization-induced release of oxytocin and vasopressin from neurointermediate lobes in vitro. In contrast, the dopaminergic plexus was left intact, as was the K+-induced stimulation of DA release and inhibition of MSH release. We conclude that the K+-induced inhibition of MSH release is mediated by DA rather than by neuropeptides from terminals in the neurointermediate lobe. The results are in agreement with the proposed MSH release-inhibiting role of the dopaminergic tuberoinfundibular neurones.

Utilization of seawater-urea as a culture medium for Spirulina maxima.

The possibilities of utilization of seawater enriched with ureas as the culture medium for a blue-green alga, Spirulina maxima, were investigated. Pretreatment by precipitation with NaHCO3 and (or) Na2CO3 was found essential to remove the excess amounts of Ca2+ and Mg2+ present in seawater prior to cultivation. A culture medium as good as the synthetic medium reported in the literature for the growth of S. maxima was obtained after treating seawater with NaHCO3 (19.2 g/L) at pH 9.2 and 35 degrees C for 2 h, filtering to remove precipitates, and enriching with K2HPO4 (0.5 g/L), NaNO3 (3.0 g/L), and FeSO4 (0.01 g/L). The same results were obtained by substituting a small amount (0.2 g/L or less) of either crystalline or polymerized urea for the NaNO3 in the above medium. Growth of S. maxima was inhibited at higher concentration of urea in the culture medium. The inhibition effect was due to the partial decomposition of urea into ammonia in alkali medium. Tests conducted on the 130-L cultivation open pond also confirmed that the seawater-urea medium supports growth of S. maxima as well as the best known synthetic medium.

Stability and preliminary pharmacokinetic studies of 1-(2-chloroethyl)-3-[1-(5'-paranitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea (RFCNU), a nonimmunosuppressive nitrosourea.

Using high-performance liquid chromatography, the stability of RFCNU was monitored as a function of pH in aqueous buffers at 37 degrees C and as a function of temperature in plasma. The kinetics of degradation of RFCNU are apparently first-order. The log kappa-pH profile demonstrated the hydroxyl ion-catalyzed solvolysis and a maximum stability around pH 3.0. This analytic assay was reliable for quantitating intact RFCNU in biologic fluids. After administration of 400 mg of RFCNU orally to a female patient, no intact drug was excreted in the urine and plasma levels were very low.

An analysis of electrophoretic and microcolumn methods for the separation of hemoglobins A and A2.

Parameters of three techniques for quantitating hemoglobin A2 were studied in order to identify problems affecting repeatability and then to compare intertechnique results under selected conditions. Satisfactory repeatability with cellulose acetate electrophoresis and scanning densitometry required an applicator that delivers a constant volume of sample. For cellulose acetate electrophoresis/elution and chromatographic assays a sophisticated absorption spectrophotometer and pH meter are necessary. Even with the most carefully chosen conditions significant intertechnique variation occurs. Although the colums are the most repeatable, trailing (a problem usually associated with hemoglobin electrophoresis) has also been demonstrated with column chromatography. Isoelectric focusing demonstrated the copresence of hemoglobin A and hemoglobin A2 in all trail fractions between the two major peaks and in some fractions of each peak. Standards of low protein concentration could be prepared from column fractions identified by isoelectric focusing as containing only hemoglobin A or hemoglobin A2. Such standards would be useful for assessing the accuracy of hemoglobin A2 quantitation.

L-asparaginyl-tRNA synthetase and L-asparagine synthetase activities of L-asparaginase-sensitive and -resistant forms of the mouse Gardner lymphoma 6C3HED.

1. The mouse Gardner lymphoma 6C3HED was grown in ascites fluid in a form sensitive to the action of L-asparaginase (line 1), in another form which was resistant to L-asparaginase (line 2) and in a third form with partial sensitivity to L-asparaginase (line 3). 2. The L-asparaginyl-tRNA synthetase activities of extracts of the tumour cells, cultured both in the mouse and in vitro, were determined. Two of the lines, 1 and 3, in early passage numbers, showed a derepression mechanism involving L-asparagine. Mutation occurred with these lines resulting in the L-asparaginyl-tRNA synthetase activity of all the tumour cell lines being the same. 3. Cells of line 1 had low L-asparagine synthetase activity, which was unchanged by altering the supply of L-asparagine in vitro. Cells of lines 2 and 3 exhibited L-asparagine synthetase activities, which changed with the supply of L-asparagine. 4. It is not certain that L-asparagine synthetase activity of L-asparaginase-sensitive cells is controlled by L-asparaginyl-tRNA acting as a corepressor.

Activities of NAD-specific and NADP-specific isocitrate dehydrogenases in rat-liver mitochondria. Studies with D-threo-alpha-methylisocitrate.

The contributions of NAD-specific and NADP-specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase. Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20--40% by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite alpha-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of alpha-methylisocitrate (400 microM) sufficient for 99.99% inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 +/- 6% and 19 +/- 11% (mean +/- SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3--4-fold, decreased NADPH levels by 30--40% and 2-oxoglutarate accumulation by about 40%. The further addition of 600 microM alpha-methylisocitrate decreased the NH4Cl-stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and alpha-methylisocitrate as in their absence. Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase, 85--100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400--900 microM alpha-methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite alpha-methylisocitrate concentration was from 20--40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite alpha-methylisocitrate concentration was about 60% of the uninhibited rate.

Immunocytochemical localization of neuronal antigens: tyrosine hydroxylase, substance P, [Met5]-enkephalin.

Neuronal antigens can be demonstrated histologically by numerous direct and indirect immunocytochemical techniques in which a specific antibody is identified by a marker compound such as fluorescein isothiocyanate, ferritin, or horseradish peroxidase. One of the more sensitive methods for the light and electron microscopic localizations of antigens in sections of tissue is the peroxidase-antiperoxidase (PAP) technique. The experimental procedures and the results obtained using this technique for the localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase, are described. The cellular and ultrastructural localization of the enzyme is demonstrated in perikarya, processes, and terminals of catecholaminergic neurons in rat brain. The immunocytochemical localization of tyrosine hydroxylase is compared to the localization of two peptides, substance P and [Met5]-enkephalin, in the A2 region of the medulla. These studies suggest that a synaptic interaction exists between the catecholaminergic neurons and neurons showing positive immunoreactivity for the peptides. The limitations of the PAP immunocytochemical technique are also discussed in relation to the immunocytochemical localization of tyrosine hydroxylase and other antigens.

Lactoperoxidase binding to streptococci.

There have been conflicting reports regarding the binding of lactoperoxidase to bacterial cell surfaces. We describe here the effects of cell-bound lactoperoxidase on acid production by suspensions of Streptococcus mutans (NCTC 10449) in the presence of hydrogen peroxide and thiocyanate. Saline suspensions of log-phase bacteria were treated with 0.1 mg of lactoperoxidase per ml and were then washed thoroughly. The addition of hydrogen peroxide and thiocyanate markedly reduced the acid production of these lactoperoxidase-treated bacteria but had no effect on the acid production of untreated controls. After a 3-h incubation in saline, the lactoperoxidase-treated bacteria produced acid in the presence of hydrogen peroxide and thiocyanate at the same rate as untreated bacteria. These observations suggest that lactoperoxidase is initially bound to the cell surface in an enzymatically active form at a concentration sufficient to inhibit acid production. The lactoperoxidase is slowly degraded or desorbed as the bacteria stand in saline suspension.

Fish species identification by thin layer isoelectric focusing.

Conventional electrophoretic techniques generally lack the resolution and reproducibility needed for the reliable identification of fish species. Variations in stabilizing media composition, sample application technique, separation time, applied voltage or current, and the analyst's skill all affect the protein pattern. Thin layer polyacrylamide gel isoelectric focusing (TLIEF), a high resolution protein separation technique, has been applied to the identification of fish species. Sarcoplasmic proteins are separated according to their isoelectric points in a stable, reproducible pH gradient. Protein patterns for 12 species of fish are compared in 4.0% polyacrylamide gels with pH 4.0--6.0 and pH 3.5--10 gradients. Similar patterns are shown in commercially prepared 5.0% polyacrylamide gels with pH 4.0--6.5 and pH 3.5--9.5 gradients (LKB PAG plates). The protein patterns are reproducible in each pH gradient and also correlate well between user-prepared and commercially prepared gels. The inherent high resolution and excellent reproducibility of TLIEF should allow the positive identification of fish species without the costly procedure of using known species as standards.

Kinetics of hydrolysis of phenylthiazolones of arginine, homoarginine, norarginine, and canaavanine by trypsin.

Phenylthiazolones (PTAs) of arginine and its homologs and analogs, homoarginine, norarginine (alpha-amino-gamma-guanidinobutyric acid), canavanine, and gamma-hydroxyarginine, were prepared. A steady-state kinetic analysis of the trypsin [EC 3.4.21.4]-catalyzed hydrolysis reactions was carried out and the kinetic parameters for these internal thioesters were compared with those for normal linear ester substrates. PTA-gamma-hydroxyarginine was so labile that hydrolysis by the enzyme could not be followed. PTA-arginine has a specificity constant (Kcat/Km) comparable to that for the Nalpha-unblocked arginine ester substrate, though the value is about 0.1% of that for a specific ester substrate, Nalpha-tosylarginine methyl ester. PTA derivatives of canavanine and homoarginine were hydrolyzed with Kcat/Km walues of the same order of magnitude as that for PTA-arginine. However, PTA-noraginine was much less susceptible to tryptic hydrolysis that PTA-homoarginine, while the linear esters of norarginine are known to be more susceptible than those of homoarginine.

The oxidation-reduction potentials of compound I/compound II and compound II/ferric couples of horseradish peroxidases A2 and C.

The reversibility of the stepwise reduction of Compound I to the ferric state via Compound II was confirmed in horseradish peroxidases A2 and C. The values of E'o (compound I/Compound II) and E'O (Compound II/ferric) were measured from equilibrium data coupled with the K2IrCl6-K3IrCl6 system in a narrow region of pH near 6.3. The ferric enzymes were also oxidized by ferricyanide to Compound II at alkaline pH and the values of E'O (Compound II/ferric) were measured from the equilibrium data. The pH dependence of E'O (Compound II/ferric) was in accord with the equation: E'O = EO + 0.058 log (Kr[H+] + [H+]2)/(KO + [H+]), where Kr and KO are proton dissociation constants in the ferric enzyme and Compound II, respectively. The pH-E'O (Compound I/Compound II) curves were likewise obtained from the equation, E'O = EO + 0.058 log (Kr + [H+]), where Kr is the proton dissociation constant in Compound II. The forward and backward rate constants were measured in each of one-electron transfer reactions of the peroxidases with the K2IrCl6-K3IrCl6 system at various pH values. The E'O values calculated on the assumption that the ratio of the rate constants equals the equilibrium constant were compared with those obtained from the equilibrium data.

Development of a chemically defined liquid medium for growth of Legionella pneumophila.

A chemically defined liquid medium has been developed for the study of the physiology and antigen production of the Legionnaires disease bacterium. The medium contains basal salts, vitamins, alpha-ketoglutaric acid, pyruvate, 0.05% l-cysteine, 0.05% glutathione, and a mixture of 20 additional amino acids, each of 0.01% final concentration, except serine, which was at 0.1%. The medium in shake culture at 37 degrees C with increased CO2 at pH 6.5, supports the maximum rate of growth, the highest cell yields, and the maximum cell surface antigen as distinguished by specific fluorescein isothiocyanate-conjugated antibody. Studies during the development of this medium showed that CO2, pyruvate, and alpha-ketoglutarate strongly stimulated growth; that cysteine and methionine were required for growth; and that serine, threonine, histidine, tyrosine, and tryptophane were energy sources. Glutathione substituted for cysteine, but cystine did not. The organisms did not use glucose and polysaccharides, as judged by cell yields when these carbohydrates were present or absent. The chelators malate, citrate, and ethylenediaminetetraacetic acid totally inhibited growth. Beta-mercaptoethanol, thioglycolate, dithiothreitol, and Tween 80 (0.05%) inhibited growth strongly or completely. Catalase activity was extremely weak or absent. Morphology varied, depending upon conditions and phases of growth. In general, filamentous forms became chains of cigar-shaped bacilli fragmenting to pairs and becoming coccoidal in the late stationary pha-e of growth. The organism grew at 25, 30, and 37 degrees C. Although they varied in their growth characteristics, 10 isolates were passed for five transfers in the chemically defined broth, giving maximum rates of growth, cell yields, and antigen production.

Adoptive immunotherapy of leukemia in the rat, without graft-VS-host complications.

PVG rats bearing a transplantable T cell leukemia were treated with large inocula of lymphoid cells from AUG rats sensitized either against the leukemia or against PVG lymphocytes. AUG and PVG bear identical Ag-B antigens but differ at minor loci, including the Pta loci, which code for differentiation antigens expressed only on peripheral T lymphocytes. Treatment with AUG cells immune to either the PVG leukemia or normal PVG cells resulted in prolonged survival of leukemic rats, a profound but ephemeral leukopenia and prolonged disappearance of leukemic cells from lymphoid tissue. All treated animals, however, eventually died with large, discrete deposits of leukemic cells in both hard and soft tissues. Despite the deliberate mismatching of host and donor cells for minor transplanation antigens, no evidence of GVH symptoms was observed in treated rats. This was interpreted as a result of directing the adoptive immune response to antigens of restricted distribution, i.e., on leukocytes and not on somatic cells.

The fate of fetal and adult B-cell progenitors grafted into immunodeficient CBA/N mice.

The relative ability of various precursors to generate functional B cells in vivo was assessed by transferring normal, chromosomally-marked CBA/H-T6T6 cells to irradiated or unirradiated immunodeficient CBA/N mice. Emergence of donor-derived B cells was monitored by means of a B-cell cloning assay (in which CBA/N cells are inactive), and by karyotpic analysis of lymphoid, myeloid, and stem cell metaphases. Grafts of lymph node, spleen, anti-mu surface immunoglobin suppressed bone marrow, sIg+ cell-depleted marrow, normal marrow, fetal liver, and yolk sac suggest: (a) there is little self-renewal of sIg+ B cells in these models; (b) pre-committed cells have extensive proliferative/differentiative potential and at least initially contribute most of the newly-formed B cells; (c) populations or pre-B cells obtained from various sources differ in their regenerative ability; (d) CBA/N mice are deficient in a category of pre-B cells which are found in fetal liver; and (e) selective B-cell chimerism results from grafting of unirradiated CBA/N mice.

Neutral lipid accumulation in yeast due to inositol deficiency: kinetic studies on the reciprocal regulation by fructose bisphosphate and citrate of yeast acetyl CoA carboxylase.

Neutral lipids, especially triacylglycerols, accumulated due to myo-inositol deficiency both in the cells of Saccharomyces carlsbergensis (Hayashi et al. (1976) J. Biol. Chem., 251, 5759--5769) and in the liver of the rat (Hayashi et al. (1974) Biochim. Biophys. Acta, 360, 134--155). The accumulation of triacylglycerols in the deficient yeast resulted, at least partly, from an enhancement of acetyl CoA carboxylase activity. The activation of the enzyme reflected the fluctuation due to the deficiency in the levels of fructose bisphosphate and citrate (Hayashi et al. (1978) Biochim. Biophys. Acta, 540, 231--237). Thus, the kinetics of the regulation of acetyl CoA carboxylase by these intermediates was studied. In physiological concentrations fructose bisphosphate sigmoidally activated acetyl CoA carboxylase from yeast with the Hill coefficient of 3, while citrate counteracted the fructose bisphosphate activation in a sigmoidal manner with the Hill coefficient of 2. Fructose bisphosphate markedly increased the apparent Vmax value of acetyl CoA carboxylase for the substrate, ATP and slightly decreased the apparent Km value. Citrate greatly decreased the apparent Vmax value increased by fructose bisphosphate.

The distribution of amylobarbitone, butobarbitone, pentobarbitone and quinalbarbitone and the hydroxylated metabolites in man.

Fluid and tissue specimens collected from 30 subjects at autopsy have been assayed for their content of common sedative barbiturates and the corresponding hydroxylated metabolites by g.l.c. Where one barbiturate had been ingested an inverse relationship between lipid solubility of the drug and the distribution in fluids and tissues was observed. In most cases the liver, and in the remainder the spleen, contained the highest concentrations of barbiturate. Bile concentrations were often in excess of those in the corresponding liver. The metabolites of the four sedative barbiturates were usually present in lower amounts than the parent drugs in the fluids and tissues of most subjects but urine often contained much higher concentrations of metabolites--sometimes exceeding that of the parent drug in the liver. Administration of two or more barbiturates together did not appear to affect the distribution and metabolism of the individual drugs.

Comparison of the effects of the isomers of amphetamine, methylphenidate and deoxypipradrol on the uptake of l-[3H]norepinephrine and [3H]dopamine by synaptic vesicles from rat whole brain, striatum and hypothalamus.

The ATP-Mg++-dependent uptake of [3H]dopamine and l-[3H]norepinephrine into purified synaptic vesicles of whole rat brain, rat striatum and rat hypothalamus was inhibited 10-fold more effectively by S-(+)-amphetamine as compared to its corresponding (R-(-)-enantiomer. In contrast, S-(+)-deoxypipradrol and its R-(-)-enantiomer were approximately equipotent inhibitors of 3H-amine uptake into these synaptic vesicular preparations. The 1R:2R-methylphenidate was twice as potent as its 1R:2S-enantiomer as an inhibitor of 3H-catecholamine uptake. These data suggest that the receptor sites on the amine pumps present in the membranes of all three vesicular preparations are similar in so far as they are all sensitive to the stereochemical configuration around the alpha-carbon of amphetamine but are not sensitive to the stereochemical configuration around the analogous carbon of deoxypipradrol and methylphenidate. These observations are the reverse of those previously observed for the phenethylamine pumps present in peripheral and central neuronal membranes.

Chloride, sodium, potassium and hydrogen ion transport in isolated canine gastric mucosa.

1. The fluxes of isotopically labelled Na+, Cl- and K+ in each direction and H+ secretion across isolated dog gastric mucosa were measured under short-circuit conditions. 2. In the non-stimulated state, the net flux of Na+ was 6.61 micronequiv/cm2.hr from mucosal (luminal, secretory) to serosal (nutrient, blood) side, whereas the net flux of Cl- was only 0.79 micronequiv/cm2.hr, and the direction was from serosal to mucosal side. 3. There was a positive correlation between the net flux of Cl- and acid secretion, however, net flux of Na+ was not correlated with acid secretion initiated by secretagogue treatment. 4. With ion substitution studies, only replacement of mucosal Na+ with choline produced a highly significant decrease in potential difference (p.d.). This indicates that active transport of Na+ from the mucosal to the serosal side is the most important source for the generation of the gastric p.d. in dog gastric mucosa. 5. From ion substitution studies, it was also observed that Cl- in either mucosal or serosal solution is necessary for maintaining acid secretion; whereas only serosal Na+ and K+ are essential for acid secretion. Removal of either Na+ or K+ from the mucosal solution had no effect on acid secretion. 6. Substitution of SO2-(4) for Cl- had no effect on active transport of Na+, but choline substitution for Na+ diminished active transport of Cl-.

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane.

The role of phospholipids in the binding of [3H]tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A2 (Crotalus adamanteus and Naja naja) or phospholipase C (Bacillus cereus and Clostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A2, and with phospholipase C the lipid hydrolysis was about 60--70% for a 70--80% reduction in the binding activity. Phospholipase C from B. cereus and Cl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A2 but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A2. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.

Fractional extracellular space and fractional water content of various rat tissues at different extracellular pH values and in uremia.

At different extracellular pH values fractional extracellular space (calculated from the distribution of 3H inulin) and fractional water content showed no significant differences. 72 h after nephrectomy both variables increased significantly. An exception to this was brain, where fractional extracellular space decreased and total intracellular water increased as a sign of brain oedema.

[Formation of higher alcohols by Saccharomyces carlsbergensis from branched-chain amino acids and their keto analogs].

A new method of chemical synthesis of alpha-ketoisocaproic acid (the keto analogue of L-leucine) is described. It has been shown that the resting cells of Saccharomyces carlsbergensis 776, in the stationary state of biomass, produce mainly higher alcohols: isobutanol from L-valine and its keto analogue; optically active amylol only from L-isoleucine and its keto analogue; isoamylol only from L-leucine and its keto analogue. "Nonspecific" formation of n-propanol from L-valine, L-isoleucine and their keto analogues, as well as that of isobutanol from L-isoleucine and its keto analogue, has been also found at pH 7.0. Formation of higher alcohols from alpha-keto acids has an acidic pH optimum while that from L-amino acids has a neutral or a weakly alkaline pH optimum. Formation of isobutanol from L-valine is an exception. The dependence of higher alcohol formation on the pH and the kinetics of their accumulation suggest that higher alcohols are produced from L-amino acids in at least three sequential reactions: transamination, decarboxylation of the keto analogue being formed, and reduction of the aldehyde; formation of higher alcohols from alpha-keto acids involves two reactions: decarboxylation and reduction. Transamination and decarboxylation are limiting steps in the process in the former case, and decarboxylation in the latter.

[Regional differences in the sensitivity of the metabolism of coelenterate larvae to external influences].

The regional differences in the sensitivity of protein synthesis and free radical processes to temperature, trypsin, urea and LiCl were studied in Obelia flexuosa by means of autoradiography. The regional differences were also determined with respect to the rate of incorporation and excretion of a labelled aminoacid under the normal conditions. The metabolic reactions of larvae can be divided in primary (the first 30 min following the effect) and subsequent adaptive ones. The primary reactions are characterized by the greater sensitivity of the anterior larval regions to all factors under study. The subsequent reactions are characterized by synchronous and unidirectional metabolic starts in both the anterior and posterior larval regions, the starts being bigger in the anterior regions. The restoration of the normal ratios of metabolic activities of the opposite larval regions does not always correlate with the restoration of the normal absolute level of metabolism. The adaptive reactions are better expressed for protein synthesis, rather than for free radical processes. The anterior larval region has the greatest metabolic non-stability by a series of indices.

Swimming capacity of mice after prolonged treatment with psychostimulants. III. Effect of fencamfamine on swimming endurance and availability of metabolic substrates.

The effect of long-term treatment with fencamfamine on swimming endurance and availability of metabolic substrates was investigated in mice. Fencamfamine (14 micrograms/g per day orally for 6 weeks) reduced maximum swimming capacity by more than 40%. This effect could not be attributed to motor incoordination or a diminution of pre-swimming levels of metabolic substrates such as liver and muscle glycogen or blood glucose and non-esterfied fatty acids. However, during swimming the hepatic and muscular glycogen stores were depleted more rapidly in the fencamfamine-treated animals. Thus it appears that fencamfamine leads more rapidly to a shortage of combustible substrates in the swimming animals.

Induction by thymic fractions of T cell subsets capable of modulating GVHR intensity.

Two thymic stromal fractions previously shown to have specific enhancing effects on anatomically distinct T cell populations were tested for their capacity to induce functionally distinct T cell subsets. Parental mice were injected with either soluble thymic fraction (STF) or insoluble thymic fraction (ITF), and their lymphoid cells were harvested 11 days later. It was shown that ITF elicited a splenic corticosensitive T cell subset endowed with enhanced graft-versus-host reaction (GVHR)-inducing capacity. On the other hand, STF increased, mainly in lymph nodes, the number of corticoresistant T cells significantly less active in GVHR. Furthermore, lymphocytes from ITF-treated parental donors could become corticoresistant with reduced GVHR activity after a 1-hr in vitro incubation with STF. We have thus shown that two different elements of the thymic microenvironment could modulate the intensity of the GVHR by modifying the equilibrium between two T cell subsets. These are believed to represent two consecutive differentiation stages.

Arterial carbon dioxide tensions during anaesthesia with manual ventilation. A descriptive study of the effects of various non-polluting circuits.

In 660 supine, intubated and anaesthetized, healthy patients scheduled for various elective surgical procedures, the distribution of arterial carbon dioxide tension (PaCO2) was investigated during manual non-monitored ventilation. The study comprised six equal groups: group 1: ventilation with a circle circuit absorber system; group 2: ventilation with the Hafnia A circuit using a total fresh gas flow (FGF) of 100 ml . kg-1 . min-1; groups 3-6: ventilation with a Hafnia D circuit with fresh gas flows of 100, 80, 70 and 60 ml . kg-1 . min-1, respectively. The mean PaCO2's of the first three groups were situated in the lower range of normocapnia (the observations in the first group having the greatest total range), whereas the rebreathing (Hafnia A and D) circuits resulted in a clustering of observed data. Employing the rebreathing circuits, protection against hypocapnia can be achieved by lowering the fresh gas flow. The most satisfying result was obtained with the Hafnia D circuit with a fresh gas flow of 70 ml . kg-1 . min-1 resulting in normocapnia with a modest and limited spread towards hypo- and hypercapnia. FGF in excess of this level must be considered as wasted. The study indicates that corrections of fresh gas flows for age are superfluous. Use of relaxants and type of surgery had no influence on the observations.

Fentanyl concentrations in brain and serum during respiratory acid--base changes in the dog.

It is a clinical impression that less fentanyl is needed for anesthesia during hyperventilation and hypocarbia. If true, it might be due to both increased penetration of fentanyl, a highly lipid-soluble agent, into the brain and increased brain tissue binding. Serum and brain concentrations of fentanyl were determined in dogs anesthetized with halothane during normocarbia, hypocarbia by hyperventilation, and hypercarbia by addition of CO2 to the inspired mixture. Fentanyl, 12.5 micrograms/kg, was injected iv, and serum and brain samples were taken for fentanyl analysis by radioimmunoassay. Brain fentanyl values peaked latest (15--20 min) and were highest during hypocarbia; brain fentanyl values peaked earliest (0--5 min) and were lowest during hypercarbia; values during normocarbia were intermediate in time to peak (10--15 min) and concentration. Thereafter, brain levels declined, but during hypocarbia were significantly higher and during hypercarbia were significantly lower than during normocarbia. Interestingly, serum fentanyl levels were also significantly higher during hypocarbia. The brain--blood fentanyl ratios for each of the three CO2 levels increased for 30 min and thereafter stayed relatively constant. The brain--blood ratios were highest with hypocarbia and lowest with hypercarbia. At 35 min, when clinical analgesia may be considered terminated, hypocarbic brain levels were double those of normocarbia. The authors feel this reflects, to a large extent, higher serum fentanyl concentrations and delayed cerebral wash-out because of decreased blood flow. To a small but unknown extent the higher brain fentanyl levels result from increased brain--blood penetration due to increased lipid solubility, and increased brain tissue binding of fentanyl during respiratory alkalosis.

The role of bacterial interference in the increased prevalence of oropharyngeal gram-negative bacilli among alcoholics and diabetics.

The oral flora of alcoholics, diabetics, and normal control subjects were compared using an agar overlay technique to determine whether the increased prevalence of oropharyngeal gram-negative bacilli among alcoholics and diabetics exists because patients with these diseases have decreased numbers of normal inhibitory bacteria in the oropharynx. Alcoholics generally had slightly lower concentrations of inhibitory bacteria than control subjects, and diabetics had somewhat higher concentrations than control subjects. However, colonized subjects did not differ from noncolonized subjects with respect to concentrations of these inhibitory bacteria in the oropharynx. Characterization of inhibitory bacteria demonstrated a preponderance of nongroupable alpha-hemolytic streptococci in each of the study groups. Stimulated saliva obtained from subjects failed to demonstrate significant differences in pH between study populations or between colonized and noncolonized subjects within each study population. These results suggest that the frequent oropharyngeal colonization of alcoholics and diabetics by gram-negative bacilli involves mechanisms other than that of a deficiency of normal interfering aerobic bacteria in the oropharynx or an altered salivary pH leading to inactivation in vivo of bacteriocins produced by these inhibitory bacteria.

Pasteurization of salted whole egg inoculated with Arizona or Salmonella.

Recently, Arizona bacteria, close relatives of Salmonella, were recovered from salted whole egg that had been pasteurized by the presently recommended process of 63.3 degrees C (146 degrees F) for 3.5 min. Because of this and the fact that the heat resistance of Arizona in salted whole egg had not been determined, the present study was undertaken. Arizona or Salmonella, grown in Trypticase soy broth supplemented with 2% yeast extract in Fernbach flasks covered with aluminum foil over cotton and guaze at 35 degrees C with shaking at 176 rpm for about 96 h, were found to have the greatest degree of heat resistance. As expected, these cells, when inoculated into salted whole egg at 10(7) cells per ml, survived heating at 63.3 degrees C (146 degrees F) for 3.5 min in a two-phase slug flow heat exchanger. To consistently achieve a 7-log kill of typical Salmonella or Arizona, a treatment of 67 degrees C (152.6 degrees F) for 3.5 min was required. However, if a 7-log kill is mandatory, it remains to be determined whether this process affect the functional properties of this product.

Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment.

This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.

Cardiovascular profile of 2-(3,4-diethyoxy-beta-methoxyphenethyl) imino-1-methylpyrrolidine fumarate (McN-2840-46), a preferential atrial anti-arrhythmic agent.

McN-2840-46, 2.5 mg/kg, i.v., protected against atrial tachyarrhythmias induced by three different methods in dogs and monkeys. The compound was inactive against ventricular arrhythmias produced by ouabain and by chloroform-epinephrine interaction at a four-fold higher dose. Significant reversal of ventricular arrhythmias produced by occlusion of the left anterior descending coronary artery in dogs was achieved by infusion of 8.8 +/- 2.4 mg/kg, i.v. of McN-2840-46. Myocardial electrogram studies confirm that the atrium is preferentially affected. McN-2840-46 does not possess beta 1- or beta 2-adrenergic blocking activity when evaluated on isolated rabbit atrial and guinea-pit tracheal chain preparations. McN-2840-46 is vagolytic but not anticholinergic. The vagolytic activity is attributed to its local anesthetic effect. Depression of myocardial function was observed in anesthetized dogs and in the heart-lung preparation. However, the isolated cat papillary muscle was stimulated by McN-2840-46 and doses considerably above the effective anti-arrhythmic dose did not significantly decrease cardiac output in the non-anesthetized dog. The results of these experiments suggest that McN-2840-46 is a potent "preferential" atrial anti-arrhythmic agent.

The partial purification of sodium-plus-potassium ion-dependent adenosine triphosphatase from the gills of Anguilla anguilla and its inhibition by orthovanadate.

1. (Na+ +K+)-dependent ATPase was partially purified from eel gills by a procedure in which the microsomal fraction of crude preparations of chloride cells was selectively extracted with sodium dodecyl sulphate. 2. The microsomal specific activity was increased 2-fold during optimal treatment with detergent. 3. The final preparation (56% pure) had a specific activity of 341 mumol of ATP hydrolysed/h per mg of protein and a turnover number of 3560 min-1. The number of ouabain-binding sties equalled the number of sites phosphorylated by ATP. 4. Both sodium orthovanadate and ouabain inhibited the purified preparation more than the microsomal fraction, vanadate being more effective on an equimolar basis than ouabain. 5. Inhibition by orthovanadate was not enhanced at 28 mM-as compared with 1mM-MgCl2 and was not reversed by beta-adrenergic agonists (cf. Josephson & Cantley (1977) Biochemistry 16, 4572--4578). 6. Of various other metallic oxyanions tested only niobate proved an effective inhibitor of the enzyme although this anion was less effective than orthovanadate. 7. Orthovanadate partially inhibited phosphorylation of the enzyme by ATP in the presence of 28 mM-MgCl2.

The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex.

1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4--5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta.

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.

[Influence of Silybin-dihemisuccinate on fatty acid synthesis in rat liver (author's transl)].

1. The influence of silybin-dihemisuccinate, a derivative of the flavonolignane silybin from silybum marianum L. Gaertn., on fatty acid biosynthesis of rat liver was studied measuring the radioactivity incorporation of [1-14C]-acetate and 3H2O in fatty acids of the postmitochondrial supernatant of liver homogenates and in fatty acids of liver slices as well as the activities of enzymes involved in do novo synthesis of fatty acids. 2. In the postmitochondrial supernatant of liver homogenates or in liver slices, prepared 30 or 60 min after i.v. injection of 150.6 mg/kg silybin-dihemisuccinate, radioactivity incorporation of 14C-acetate or 3H2O in fatty acids was lowered by about 25%. Adding silybin-dihemisuccinate to incubation mixture in vitro in the concentration of 0.45--0.6 mmol/l silybin the radioactivity incorporation was linearly diminished with increased concentration of silybin. 3. After in vitro addition of varying concentrations of silybin to incubation mixtures in the presence of 0.1 mmol/l silybin activities of acetyl-CoA-carboxylase, fatty-acid-synthetase and ATP-citrate-lyase were diminished by about 50%, while activity of NADP-malate-dehydrogenase was lowered by 20% in the presence of 1 mmol/l silybin. 4. Our results suggest that silybin caused an unspecific and, under in vivo conditions, transitory inhibition of fatty acid synthesis in rat liver.

Attachment of Neisseria gonorrhoeae to human sperm. Microscopical study of trypsin and iron.

Pilated Neisseria gonorrhoeae of colony type 1 (T1) and non-pilated bacteria of colony type 4 (T4) were observed by transmission (TEM) and scanning electron microscopy (SEM). No pili were observed on T4 gonogocci, but two types of pili--straight, type a, and bent, type b--were seen on T1 by TEM. When incubated with human sperum and examined by either TEM or SEM, T1 gonococci were seen to attach by individual pili, by several pili wound together as a rope, or by direct contact. Gonococci from T4 colonies attached only by direct contact. Treatment with typsin (1 mg/ml) damaged or removed pili from gonococci. After incubation with trypsin, attachment of pilated gonococci to sperm was decreased significantly, but such treatment did not affect attachment of non-pilated gonococci. Incubation of gonococci from either colony type in 0.1 mmol/l ferric nitrate, followed by incubation with sperm, significantly increased attachment of only T4 bacteria. No pili were seen on T4 gonococci treated with ferric nitrate; thus, it appears that factors other than pili alone are concerned in attachment of these gonococci to sperm.

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.

On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria.

Different approaches have been used to evaluate the stoichiometry of proton translocation linked to cytochrome c oxidase in rat liver mitochondria. A mathematical model was designed that successfully describes the kinetics of redox-linked proton translocation provided that the rate of electron transfer is not too high. With ascorbate as reductant, an essentially pH-independent (in the pH range 6--8.5) proton ejection stoichiometry (H+/e-) is obtained from either initial rates of H+ ejection (0.86 +/- 0.12), or the model (0.87 +/- 0.14). Similar results are obtained with either ferrocyanide, N.N.N',N'-tetramethyl-p-phenylenediamine or externally added cytochrome c mediating between ascorbate and cytochrome c in rotenone- and antimycin-inhibited mitochondria. Oxygen pulse experiments with ferrocytochrome c as substrate show fully uncoupler-sensitive redox-linked proton ejection with a stoichiometry of 0.78 +/- 0.14. With murexide to measure Ca2+ uptake during oxidation of ferrocyanide, we found a stoichiometry of two positive charges taken up/electron transferred, confirming earlier findings. These results provide strong evidence that cytochrome c oxidase functions as a redox-linked proton pump with a stoichiometry of one H+ ejected and two charges translocated/electron transferred. The thermodynamic consequences of the proton pump are discussed and a maximal P/O ratio of 1 1/3 for 'site 3' is predicted in agreement with state 4 redox potentials and phosphate potential.

AMP deaminase from baker's yeast. Purification and some regulatory properties.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) was found in extract of baker's yeast (Saccharomyces cerevisiae), and was purified to electrophoretic homogeneity using phosphocellulose adsorption chromatography and affinity elution by ATP. The enzyme shows cooperative binding of AMP (Hill coefficient, nH, 1.7) with an s0.5 value of 2.6 mM in the absence or presence of alkali metals. ATP acts as a positive effector, lowering nH to 1.0 and s0.5 to 0.02 mM. P1 inhibits the enzyme in an allosteric manner: s0.5 and nH values increase with increase in Pi concentration. In the physiological range of adenylate energy charge in yeast cells (0.5 to 0.9), the AMP deaminase activity increases sharply with decreasing energy charge, and the decrease in the size of adenylate pool causes a marked decrease in the rate of the deaminase reaction. AMP deaminase may act as a part of the system that protects against wide excursions of energy charge and adenylate pool size in yeast cells. These suggestions, based on the properties of the enzyme observed in vitro, are consistent with the results of experiments on baker's yeast in vivo reported by other workers.

Solubilization and stabilization of human liver glycoprotein sialyltransferase.

Triton X-100 is increasingly effective in solubilizing human liver glycoprotein (asialofetuin) sialytransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotien N-acetylneuraminyltransferase, EC 2.4.99.1) activity as its concentration is increased in the homogenizing buffer. At the optimal concentration of 1.5% (v/v), essentially all of the homogenate sialyltransferase activity was solubilized into the supernatant fluid. Higher concentrations of Triton X-100 inhibited sialyltransferase activity. Several kinetic properties of the solubilized asialofetuin-sialyltransferase activity were compared to those of the membrane-bound enzyme(s) (in homogenates made without Triton X-100 or in resuspended pellets). No major difference was apparent, suggesting that solubilization has not significantly altered the properties of sialyltransferase. The solubilized sialyltransferase activity is quite unstable, losing approximately 50% of its activity after one week of storage at 4 degrees C. Various detergents (Zwittergent, sodium taurocholate and sodium deoxycholate) are differentially effective in stabilizing the solubilized activity. Sodium taurocholate (1.5%, w/v) was most effective with no loss in activity after 40 days and minimal loss (14%) after 60 days storage at 4 degrees C. The solubilized sialyltransferase preparation retains full activity after storage in the frozen state (-20 degrees C) for at least 159 days.

Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A.

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the alpha-hemes (g = 6.0) and the beta-hemes (g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A (g = 6.0). The beta-hemes are found to be comparatively insensitive to both effectors while the alpha-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the alpha-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the beta-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The beta-heme spectral changes appear to be pH independent while those arising from the alpha-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the alpha-hemes which undergoes spectral change upon inositol hexaphosphate binding to the beta-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the alpha-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the beta-hemes are in the high spin form.

The reaction of carbonyl cyanide phenylhydrazones with thiols.

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and aminothiols (cysteine, glutathione) to give corresponding N-(substituted phenyl)-N'-(alkylthiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular AdN type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS- ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group). The reactivitiy of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone less than carbonyl cyanide m-chlorophenylhyrazone less than carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pKa constants. On the other hand, the reactivity of thiols increases with their basicity. The reactivity of carbonyl cyanide phenylhydrazone with thiols is comparable with the reactivity of phenyl isothiocyanate and N-ethylmaleimide. It was demonstrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.

The binding of Arg- and Lys-peptides to single stranded polyribonucleotides and its effect on the polymer conformation.

The interactions between basic oligopeptides (Lys2, Lys3, Arg2 and Arg3) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescence followed by coacervation. Arg3 causes coacervation in all polynucleotides used, yet Lys3 only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg3 (150 muM) than for Lys3 (500 muM). 2) Medium concentrations (greater than 10 muM) of Arg3 and Lys3 induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys3, but not by Arg3. 3) The onset of peptide association is observed at very low peptide concentrations (greater than or equal to 1muM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants kR and kD. Using a simple model with independent and "separate" binding sites, K for Arg3 and Lys3 is found to be in the range of 10(6) to 10(7) M-1. In the case of Arg2 and Lys2 K is lower by a factor of about 10. For various polynucleotides KArg3 is only slightly higher than KLys3, except in the case of poly(I), where KArg3/KLys3 approximately 5. Similar data are obtained by application of a "sphere model" (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg3: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interation. Both field jump and stopped flow data demonstrate a high mobility of the peptide ligands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a "sphere" model, which considers a) excluded binding under the condition of fast ligand distribtuion along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 X 10(11) M-1 s-1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.

Immunization of adult rats against 2.5 S NGF: effects on the peripheral sympathetic nervous system.

The biochemical and morphological changes effected by immunization of adult rats with 2.5 S mouse nerve growth factor (NGF) were studied in sympathetic ganglia and in representative target organs. This immunization procedure maintains high levels of circulating anti NGF-antibody for periods of months. Morphological analysis revealed a general reduction in the size of the adrenergic neurons in the superior cervical ganglion (SCG) which was also reflected at the biochemical level by a 30% decrease in total protein content and a 50--60% reduction in the total activities of all norepinephrine-synthesizing enzymes. However, there was no change in total choline acetyltransferase activity. The biochemical and morphological changes observed in the SCG seem to be confined to the neuronal cell body, since at any stage of immunization target organs (the submandibular and the pineal gland) remained unaffected. All sympathetic ganglia investigated--except the superior mesenteric ganglion--responded in a similar way to the immunization against 2.5 S NGF. These changes in the adrenergic cell bodies were largely reversible. The recovery of normal enzyme activities followed closely the decrease of the antibody titer after cessation of immunization boosting. This indicates that cell death is not caused by anti NGF-antibodies in ganglia of adult animals. Thus, in contrast to adrenergic neurons from newborn animals, which depend on NGF or a crossreacting NGF-like material for survival, differentiated adrenergic neurons need this factor for the maintenance of their normal function but not for survival.

The effects of temperature, local anaesthetics, pH, divalent cations, and group-specific reagents on repriming and repolarization-induced contractures in frog skeletal muscle.

Contractures appear during repolarization of frog toe muscles in media containing perchlorate in place of chloride. These contractures were suppressed or delayed by certain procedures which retard the repriming of K contractures, i.e., by sufficient reduction in temperature or by alkaline pH in solutions lacking divalent cations. They also were greatly reduced without interference with repriming after treatment with a reagent which selectively modifies free amino groups. In the presence of appropriate concentrations of procaine, repriming was markedly impaired with only a small reduction in the amplitude of repolarization-induced contractures. Small contractures were produced during repolarization in chloride solutions in the presence of 10 mM procaine at pH 8.0. None of these procedures affected the changes produced by perchlorate solutions in the potential dependence and the time course of K contractures. The results support the view that activation and inactivation of contraction following depolarization are separate potential dependent processes. Tension appears to develop during repolarization when the reversal of inactivation occurs before the reversal of activation is completed, both steps being necessary to recover the reprimed resting state.

Primary amniotic fluid cell, skin fibroblast and liver alpha-L-fucosidase and its relation to cystic fibrosis.

Cultured skin fibroblast and primary amniotic fluid cell alpha-L-fucosidase had a double optimum of pH 5.0 and 6.0. Alpha-L-fucosidase was largely bound as a single peak to DEAE-cellulose at pH 6.6. Sucrose density isoelectric focusing revealed up to seven components with pI values of 4.9, 5.2, 5.4, 5.8, 6.1, 6.5 and 7.1 with their apparent KM values (77--500 mumol/l) being higher than that (57 mumol/l) of the unfocused enzyme. Liver, skin fibroblast and amniotic fluid cell alpha-L-fucosidase was separated into two peaks by gel filtration. Peak one was more active and stable at low pH and more thermostable at 50 degrees C than peak two, while both peaks had an apparent KM of 52 mumol/l. Apart from the different proportions of the peaks separated by gel filtration, the results for the three tissues were similar. The properties of alpha-L-fucosidase studied were similar for control and cystic fibrosis liver or skin fibroblasts.

Peripheral cardiovascular effects in rats after central administration of histamine and antihistamines.

1. Vascularly isolated but nervously intact rat right hind limbs were perfused with blood at a constant flow rate and changes in perfusion pressure (proportional to vascular resistance), heart rate and blood pressure were monitored. 2. Histamine administered into the right lateral cerebral ventricle (i.c.v.) through guide cannulae, induced dose-dependent increases in perfusion pressure, heart rate and blood pressure. 3. Prior i.c.v. or i.v. administration of metiamide (an H2-antagonist) did not prevent the cardiovascular responses to i.c.v. histamine but rather prolonged them. Following i.c.v. or i.v. administration of chlorpheniramine (an H1-antagonist), however, changes in vascular resistance, heart rate and blood pressure were not significant. 4. Metiamide administration appeared to have some agonist activity on its own. Thus the role of H2-receptors in cardiovascular responses to centrally administered histamine remains unclear. 5. The work shows that in rats increases in nervous dishcarge to at least the hind limb vascular bed occur following central administration of histamine and conforms that increases occur in heart rate and blood pressure. These responses appear likely to be mediated through stimulation of central H1-receptors.

Impaired acid neutralization in the duodenum in pancreatic insufficiency.

The influence of severe exocrine pancreatic disease on the acid-neutralizing capacity of the duodenum was studied in five patients with pancreatic insufficiency (PI) and six control subjects using duodenal perfusion-marker technique. Hydrochloric acid (0.1 N containing 1% PEG) was infused at constant rates (1.2, 4.5 and 7.0 ml/min) into the duodenum just distal to the duodenal bulb. Samples were aspirated from the tip of the duodenal perfusion tube located at the ligament of Treitz. All samples were analyzed for volume, pH, titrable acidity, PEG and [14C]PEG (gastric marker) determination. Patients with PI demonstrated significantly diminished ability to neutralize various acid loads as compared to controls who virtually completely neutralized acid loads in the range of maximal gastric acid secretion. Exogenous secretin did not significantly improve percent acid neutralized in PI. These data clearly indicate that patients with PI have significantly impaired ability to neutralize even small loads of acid in the duodenum.

Determination of amitriptyline and its major basic metabolites in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the routine, simultaneous determination of amitriptyline and its basic metabolites in human urine has been developed. 10-Hydroxylated metabolites are analyzed as their 10,11-dehydro analogs, and primary and secondary amines as their N-trifluoroacetyl derivatives. The use of gradient elution enables amitryptyline, nortriptyline trifluoroacetate, desmethylnortriptyline trifluoroacetate, and the corresponding 10, 11-dehydro analogs to be separated from both each other and from the internal standard used. In this way all six compounds may be conveniently measured in a single chromatogram, with good sensitivity and accuracy. Following administration of a single oral dose (25 mg) of amitriptyline hydrochloride to two human subjects, no unchanged drug was found in any of the urine samples analyzed up to 72 hr after dosing, and only small amounts of nortriptyline and desmethylnortriptyline were observed. 10-hydroxynortriptyline was the major biotransformation product (about 40% of the dose) in urine, with 10-hydroxyamitriptyline and 10-hydroxydesmethylnortriptyline present as minor metabolites. During 72 hr after administration, approximately 60% of the dose was recovered as these five metabolites.

Sewage sludge as a source of cadmium in soil-plant-animal systems.

The objective of this presentation is to relate the abundance and mobility of Cd in components of terrestrial ecosystems with implications for land utilization of sewage sludge. The uptake of Cd by crop plants is a function of the quantity of the element in the soil plus other soil factors affecting the Cd ion activity or electrochemical potential at the plant root surface. The natural abundance of Cd in soils has been reported as 0.5 mug/g which is higher than the background level of 0.2 mug/g found in soils studied in Pennsylvania. Experimental results indicate that the plant availability of Cd increases with each soil addition. While the plant availability of Cd is decreased by liming to increase soil pH, it has not been possible to add Cd salts or sewage sludge Cd without significantly increasing plant uptake. Field studies have shown that land application of sewage sludge can be expected to increase the Cd concentration of corn leaves from a range of 0.05-0.1 mug/g to 1-3 mug/g. Two years after the last application of sludge which added up to 10 ppm Cd to the surface soil, corn grain, sorghum grain, wheat grain, and potatoes showed a 10- to 15-fold increase in Cd over background levels. Studies were conducted with chicks, laying hens, and meadow voles (Microtus Pennsylvanias) to assess the impact of this increase in plant Cd upon the food chain. Corn and sorghum plants were grown on soils with either inorganic or sludge fertilizer for the purpose of producing herbage for use in feeding trials with meadow voles. Eight diets and a synthetic control diet were formulated to study the effect of source (plant vs. inorganic) of Cd on tissue accumulation. Significant accumulation of Cd occurred in kidney and liver, but not muscle, of voles fed diets containing sludge fertilized corn (1.09 mug/g) or sludge fertilized sorghum (2.76 mug/g). The source of Cd had little influence on tissue accumulation. In studies with broiler chicks and laying hens, natural diets containing 0.2 ppm Cd were supplemented with 3 ppm of this element. As with the meadow voles, Cd readily accumulated in liver and kidney. Although the results were not statistically significant, 3 ppm dietary Cd doubled muscle Cd content. There was no transfer of Cd to egg in a long term (12 month) experiment with laying hens. Soil management programs have been developed to maintain animal dietary levels of Cd at less than 1.0 mug/g from the use of sewage sludge on land in Pennsylvania. However, it is concluded that this level over time may cause a significant accumulation of Cd in animal tissues. Interpretation of these results in relation to those for human intake of Cd and the long range health effects of Cd is required for the proper monitoring of sewage sludge applications on land used for production of crops which enter the food chain.

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.

Interaction of butene with human hemoglobin A.

The binding of various alkanes by proteins was recognized years ago. We have studied the effect of butene (C4H8), a short-chain aliphatic hydrocarbon, on the functional properties of human adult hemoglobin. Under 1 atm pressure (100 kPa) butene decreased the affinity of hemoglobin (Hb) for oxygen (p50) by 45% without altering the cooperativity of ligand binding. This effect was independent of pH (from 7.0 to 8.0) and of ionic strength. The changes in the affinity of hemoglobin for oxygen were dependent upon the partial pressure of butene and evoked a saturating mechanism of the binding site(s). Mathematical simulation of the curve relating p50 to the concentration of dissolved butene allowed us to calculate the apparent association constants for one single binding site KHb = 10.4 mmol-1 and KHbO2 = 1.53 mmol-1 to Hb and HbO2 respectively. The larger binding of butene by Hb was confirmed by a 25% decrease in K1, the first association constant of oxygen to the tetrameric hemoglobin. It is concluded that butene is an allosteric effector of human Hb which acts most likely through hydrophobic interactions. It is postulated that the oxygen-linked binding site may be located at the alpha 1 beta 2 interface.