bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of infrared spectroscopy for assessment of vinyl polymers of bile-pigment gallstones
Authors: Rege R V , Webster C C , Ostrow J D , Carr S H , Ohkubo H
Published: August 2015

The i.r. spectra of bilirubin isomers that differ in number and position of vinyl groups were examined to verify the assignment of the 988 cm−1 peak of bilirubin (991 cm−1 peak in calcium bilirubinate) to its pendant vinyl groups. There were only small changes in this peak with changes in position of vinyl groups (exo-2- and −18-vinyl versus endo-3- and −17-vinyl), but progressive loss of peaks in this region was observed when vinyl groups were reduced to ethyl groups (dihydrobilirubin and mesobilirubin). Methylvinylmaleimide, a monopyrrole derived from the outer (A and D) rings of bilirubin, has a pendant vinyl group and exhibits a prominent peak at 986 cm−1, but haematinic acid methyl ester derived from the inner (B and C) rings has no vinyl group and shows no peak near 988 cm−1. These observations verify the assignment of the 988 cm−1 peak of bilirubin to its pendant vinyl groups. This supports our previous proposal that a decrease in the peak at 985-995 cm−1 in the i.r. spectra of pigment gallstones, as compared with unconjugated bilirubin or calcium bilirubinate, indicates a consumption of vinyl groups in the process of formation of the polymer in the pigment stones.
The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria
Authors: Vitorica J , Satrústegui J
Published: August 2015

The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed.
Negative co-operativity in glutamate dehydrogenase. Involvement of the 2-position in glutamate in the induction of conformational changes
Authors: Bell E T , LiMuti C , Renz C L , Bell J E
Published: August 2015

The 2-position substituent on substrates or substrate analogues for glutamate dehydrogenase is shown to be intimately involved in the induction of conformational changes between subunits in the hexamer by coenzyme. These conformational changes are associated with the negative co-operativity exhibited by this enzyme. 2-Oxoglutarate and L-2-hydroxyglutarate induce indications of co-operativity similar to those induced by the substrate of oxidative deamination, glutamate, in kinetic studies. Glutarate (2-position CH2) does not. A comparison of the effects of L-2-hydroxyglutarate and D-2-hydroxyglutarate or D-glutamate indicates that the 2-position substituent must be in the L-configuration for these conformational changes to be triggered. In addition, glutarate and L-glutamate in ternary enzyme-NAD(P)H-substrate complexes induce very different coenzyme fluorescence properties, showing that glutamate induces a different conformation of the enzyme-coenzyme complex from that induced by glutarate. Although glutamate and glutarate both tighten the binding of reduced coenzyme to the active site, the effect is much greater with glutamate, and the binding is described by two dissociation constants when glutamate is present. The data suggest that the two carboxy groups on the substrate are required to allow synergistic binding of coenzyme and substrate to the active site, but that interactions between the 2-position on the substrate and the enzyme trigger the conformational changes that result in subunit-subunit interactions and in the catalytic co-operativity exhibited by this enzyme.
Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis
Authors: Lowe P N , Hodgson J A , Perham R N
Published: August 2015

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.
Regulation of fructose 2,6-bisphosphate concentration in white adipose tissue
Authors: Rider M H , Hue L
Published: August 2015

Injection of insulin to fed rats diminished the concentration of fructose 2,6-bisphosphate in white adipose tissue. Incubation of epididymal fat-pads or adipocytes with insulin stimulated lactate release and sugar detritiation and also decreased fructose 2,6-bisphosphate concentration. Such a decrease was, however, not observed in fat-pads from starved or alloxan-diabetic rats. Incubation of adipocytes from fed rats with various concentrations of glucose or fructose led to a dose-dependent rise in fructose 2,6-bisphosphate which correlated with lactate output and detritiation of 3-3H-labelled sugar. In adipocytes from fed rats, palmitate stimulated the detritiation of [3-3H]glucose without affecting lactate production and fructose 2,6-bisphosphate concentration. Incubation of epididymal fat-pads from fed rats in the presence of antimycin stimulated lactate output but decreased fructose 2,6-bisphosphate concentration. Changes in lipolytic rates brought about by noradrenaline, insulin, adenosine and corticotropin in adipocytes from fed rats were not related to changes in fructose 2,6-bisphosphate or to rates of lactate output. In fed rats, the activity of 6-phosphofructo-2-kinase was not changed after treatment of adipocytes with insulin, noradrenaline or adenosine. It is suggested that the decrease in fructose 2,6-bisphosphate concentration observed after insulin treatment can be explained by the increase in sn-glycerol 3-phosphate, an inhibitor of 6-phosphofructo-2-kinase.
Tumour-promoting phorbol esters increase basal and inhibit insulin-stimulated lipogenesis in rat adipocytes without decreasing insulin binding
Authors: van de Werve G , Proietto J , Jeanrenaud B
Published: August 2015

In isolated rat adipocytes, tumour-promoting phorbol esters caused (1) dose-dependent stimulation of lipogenesis in the absence of insulin and (2) inhibition of the lipogenic effect of submaximal concentrations of insulin, but without affecting insulin binding. The possible involvement of protein kinase C in insulin action is discussed.
Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase
Authors: Gibbs P N B , Gore M G , Jordan P M
Published: August 2015

The reaction of human 5-aminolaevulinate dehydratase with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.
Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity
Authors: Lakey T , Mac Neil S , Humphries H , Walker S W , Munro D S , Tomlinson S
Published: August 2015

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.
Comparison of different techniques for estimating rates of protein synthesis in vivo in healthy and bacteraemic rats
Authors: Pomposelli J J , Palombo J D , Hamawy K J , Bistrian B R , Blackburn G L , Moldawer L L
Published: August 2015

Previous studies have reported that use of a flooding dose of radiolabelled amino acid is a more precise technique than the constant infusion of tracer quantities for determining rates of protein synthesis in rapidly turning-over tissues in the rat. However, there has been little direct investigation comparing different methods under comparable conditions. Initially, 12 healthy male Sprague-Dawley rats, weighing approx. 100 g, were randomized to receive either a bolus intravenous injection of 100 mumol of L-leucine (containing 30 microCi of [1-14C]leucine)/100 g body wt., or a continuous 2 h tracer infusion of [14C]leucine. In the second phase of the experiment, 12 additional rats were intravenously injected with 1 × 10(8) colony-forming units of Pseudomonas aeruginosa and 16 h later randomized to receive one of two infusions described above. Total protein synthesis as well as fractional synthesis rates were determined in liver, rectus muscle and whole body. Synthesis rates measured in liver, muscle and whole body were significantly higher in bacteraemic rats than in healthy rats. The flooding-dose methodology gave significantly higher estimates of protein synthesis in the liver, skeletal muscle and whole body than did the continuous-infusion method using direct measurement of the acid-soluble fraction from the respective tissue. Indirect estimates of whole-body protein synthesis based on plasma enrichments and stochastic modelling gave the lowest values.
Intracellular DNA damage produced by a series of diacridines
Authors: Roos I A G , Wakelin L P G , Henry S J
Published: August 2015

The intracellular DNA damage produced by a series of diacridines after a 2 h pulse treatment of L1210 cells in culture was investigated by using the alkaline-elution technique. Like other intercalating agents, diacridines produce single-strand breaks and protein-DNA links. There is a large increase in both types of damage as the alkane chain linking the two 9-aminoacridine residues is increased beyond five methylene groups, which is consistent with the previously observed change from monofunctional to bifunctional intercalation [Wakelin, Romanos, Chen, Glaubiger, Canellakis & Waring (1978) Biochemistry 17, 5057-5063]. For linker chains of less than six methylene groups these agents produce less DNA damage than does the parent 9-aminoacridine at the same drug concentration. Unlike the monofunctional intercalators previously investigated [Ross, Glaubiger & Kohn (1979) Biochim. Biophys. Acta 562, 41-50; Zwelling, Michaels, Erickson, Ungerleider, Nichols & Kohn (1981) Biochemistry 20, 6553-6563; Zwelling, Kerrigan & Michaels (1982) Cancer Res. 42, 2687-2691; Zwelling, Michaels, Kerrigan, Pommier & Kohn (1982) Biochem. Pharmacol. 31, 3261-3267], there is no correlation between the number of single-strand breaks and protein-DNA links produced by these diacridines.
Human liver cathepsin L
Authors: Mason R W , Green G D J , Barrett A J
Published: August 2015

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.
Aldehydes or dicarbonyls in non-enzymic glycosylation of proteins
Authors: Beswick H T , Harding J J
Published: August 2015

The non-enzymic post-translational glycosylation of certain proteins has been implicated in the production of diabetic sequelae. In the present paper the possibility that it is not the glucose aldehyde that binds to proteins but a dicarbonyl autoxidation product is investigated. Earlier experiments may not have distinguished between these two possibilities. The rate of binding of 2-deoxyglucose (a non-autoxidizable sugar) to lens alpha-crystallin is compared with that of glucose (an autoxidizable sugar). The stabilized Schiff-base adducts was investigated by using proton n.m.r. and fast-atom-bombardment mass spectroscopy to distinguish whether they are the product of aldehyde or dicarbonyl addition. We conclude that it is the open-chain aldehyde of glucose that binds initially to amino groups and that there is no participation of dicarbonyl autoxidation products in the initial non-enzymic protein glycosylation reaction.
Spectroscopic evidence for a photosensitive oxygenated state of ammonia mono-oxygenase
Authors: Shears J H , Wood P M
Published: August 2015

Photoinactivation of ammonia oxidation by Nitrosomonas europaea cells by near-u.v. light was confirmed and further shown to occur with the same rate constant as loss of bromoethane-oxidation activity. Hydroxylamine oxidation was much less photosensitive. Protection against inactivation was afforded by anaerobiosis, organic substrates of ammonia mono-oxygenase such as bromoethane, or metal-ion-chelating agents such as thiourea. The presence of 10 mM-NH4+ or 1 mM-hydroxylamine made little difference, whereas hydrazine had a potentiating effect. Illumination of cells also caused a bleaching in the absorption spectrum around 380 nm, along with changes in the cytochrome gamma-band region. Similar effects below 400 nm were obtained when organic substrates and inhibitors of the mono-oxygenase were added to cells in the dark. The copper proteins haemocyanin and tyrosinase have a photosensitive oxygenated state with a near-u.v. absorption band of similar half-width. They also have a sensitivity to chelating agents similar to that of ammonia mono-oxygenase. The experimental results are explained in terms of a three-stage catalytic cycle analogous to that for tyrosinase. In resting cells most of the enzyme is believed to be in an oxygenated (Oxy) form, which absorbs maximally at 378 nm and is photosensitive. In the presence of a substrate, one O atom is inserted into the substrate and the other is reduced to water, leaving the enzyme in an oxidized (Met) state. This is followed by a two-electron reduction of the proposed binuclear copper site to give a reduced (Deoxy) state, which can bind O2 to complete the cycle.
Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis
Authors: Verduyn C , Van Kleef R , Frank J , Schreuder H , Van Dijken J P , Scheffers W A
Published: August 2015

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.
Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor
Authors: Davies Jones E , Hashim F A , Kajita Y , Creagh F M , Buckland P R , Petersen V B , Howells R D , Rees Smith B
Published: August 2015

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.
Effects of detergents and cytochrome c binding on scalar and vectorial proton ejection by proteoliposomes containing cytochrome oxidase
Authors: Nicholls P , Shaughnessy S
Published: August 2015

The detergent lauryl maltoside abolishes respiratory control and proton ejection by cytochrome c oxidase-containing proteoliposomes over a narrow concentration range. Expression of cryptic activity (inward-facing oxidase) is released over the same concentration range. Catalytic functions (Vmax. and Km) of the enzyme are not changed by the detergent. Lipid micelles containing detergent bind approximately the same amount of cytochrome c as do vesicles containing an equivalent amount of lipid. Uncoupler-insensitive proton release is seen when proteoliposomes are pulsed with ferrocytochrome c at low ionic strength. Such uncoupler-insensitive acidification is not seen at higher ionic strength, nor with oxygen pulses of anaerobic solutions previously incubated with cytochrome c. Vesicles at low ionic strength catalyse cytochrome c autoxidation; this process can mimic proton re-equilibration in systems that have pumped protons from inside to the bulk phase. Proton re-equilibration following a pulse of cytochrome c or oxygen is multiphasic. The slowest phases are attributed to vesicle heterogeneity, some internal alkali being retained within vesicles of low intrinsic proton permeability. This can be overcome by the addition of either very low levels of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or high levels of valinomycin.
The active transport of 2-keto-d-gluconate in vesicles prepared from Pseudomonas purida
Authors: Agbanyo F , Taylor N F
Published: August 2015

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.
Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection
Authors: Wake S A , Mercer J F B
Published: August 2015

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.
Absence of keratan sulphate from skeletal tissues of mouse and rat
Authors: Venn G , Mason R M
Published: August 2015

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.
Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate
Authors: Hunter G K , Allen B L , Grynpas M D , Cheng P T
Published: August 2015

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.
Effect of the chaotropic anions thiocyanate and perchlorate on the entry of ricin into Vero cells
Authors: Sandvig K , Olsnes S
Published: August 2015

The effect of different anions on the sensitivity of Vero cells to ricin was investigated. The cells were equally sensitive to ricin when NaCl was replaced by NaBr, NaI, Na2SO4 or with iso-osmotic concentrations of mannitol. In contrast, NaSCN and NaClO4 strongly protected against ricin at pH 7.2, but not at pH 7.6. The possibility that the protective effect is due to the ability of chaotropic anions to decrease the pH close to the membrane is discussed.
The effect of glucagon administration on protein synthesis in skeletal muscles, heart and liver in vivo
Authors: Preedy V R , Garlick P J
Published: August 2015

Infusion of glucagon (0.5 mg/h per 100 g body wt.) into fed rats for 6 h inhibited protein synthesis in skeletal muscle, but not in heart. The order of sensitivity of three muscles was plantaris greater than gastrocnemius greater than soleus. Treatment with glucagon for periods of 1 h or less had no effect. Liver protein synthesis was inhibited by glucagon treatment for 10 min, but stimulated after 6 h. The effect of glucagon on muscle was not secondary to impaired food absorption or to depletion of amino acids by increased gluconeogenesis, since the inhibition of protein synthesis was observed in postabsorptive and amino acid-infused rats. The failure of glucagon to inhibit muscle protein synthesis after 1 h may have been caused by the increase in plasma insulin that occurred at this time, since an inhibition was detected in insulin-treated diabetic rats. The lowest infusion rate that gave a significant decrease in muscle protein synthesis was 6 micrograms/h per 100 g body wt., despite a small increase in plasma insulin. This gave plasma glucagon concentrations in the high pathophysiological range, suggesting that glucagon may be significant in the pathogenesis of muscle wasting in metabolic stresses such as diabetes and starvation.
The latency of rat liver microsomal protein disulphide-isomerase
Authors: Lambert N , Freedman R B
Published: August 2015

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.
Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the mitochondrial uncoupling agent 2,4-dinitrophenol
Authors: Byrne E , Hayes D J , Shoubridge E A , Morgan-Hughes J A , Clark J B
Published: August 2015

Infusion of dinitrophenol intra-arterially into rat hind limb caused an irreversible failure of isometric twitch tension and the induction of a severe progressive contracture. Metabolite analysis of muscle in which the twitch response had grossly fatigued revealed low levels of ATP and phosphocreatine together with lactate accumulation. Studies using 31P-n.m.r. confirmed the decrease in ATP and creatine phosphate concentrations and indicated a fall in intracellular pH. It is concluded that dinitrophenol-induced myopathy does not represent a good model for the human mitochondrial myopathic condition as has been previously suggested.
Experimentally induced defects of mitochondrial metabolism in rat skeletal muscle. Biological effects of the NADH: coenzyme Q reductase inhibitor diphenyleneiodonium
Authors: Hayes D J , Byrne E , Shoubridge E A , Morgan-Hughes J A , Clark J B
Published: August 2015

An animal model for the human condition of mitochondrial myopathy has been established and characterized physiologically and biochemically. The NADH: coenzyme Q reductase inhibitor diphenyleneiodonium [Bloxham (1979) Biochem. Soc. Trans. 7, 103-106] was either infused acutely in vivo into rat hind limb or injected chronically into rats. Both modes of delivery resulted in a reduced muscle oxidative capacity and increased fatigue. Analysis of muscle metabolites by h.p.l.c. and 31P-n.m.r. indicated that ATP concentrations were similar to control values during periods of stimulation and these were maintained by the phosphocreatine pool. During the recovery period after muscle stimulation in the experimental animals the muscle pH remained depressed and the rate of phosphocreatine synthesis was markedly delayed as compared with controls. Factors thought to be involved in the fatigue response are discussed in relation to this model.
Isolation of active subforms of α2-macroglobulin
Authors: Chlebowski J F , Williams K
Published: August 2015

Anion-exchange chromatography is shown to permit resolution and separation of subforms of the serum glycoprotein alpha 2-macroglobulin. The subforms differ dramatically in their stability as judged by differential scanning calorimetry, undergoing thermally induced unfolding at temperatures of 61 and 69 degrees C respectively. In addition, the proteinase-binding stoichiometry of the subforms differs by a factor of 2, with the more- and less-stable forms binding 2 and 1 mol of proteinase per mol of tetramer respectively. The calorimetric stability of the two forms is differentially affected on treatment with neuraminidase, suggesting that the nature of glycosylation may in part account for the observed differences in physical and functional properties.
Heterogeneity of high-mobility-group protein 2. Enrichment of a rapidly migrating form in testis
Authors: Bucci L R , Brock W A , Meistrich M L
Published: August 2015

A determination of the absolute amounts of high-mobility-group proteins 1 and 2 (HMG1 and HMG2) in rat tissues demonstrated that amounts of HMG2 were low in non-proliferating tissues, somewhat higher in proliferating and lymphoid tissues, but were extremely elevated in the testis. This increase was due to a germ-cell-specific form of HMG2 with increased mobility relative to somatic HMG2 on acid/urea/polyacrylamide-gel electrophoresis. To determine if the findings in the rat were a general feature of spermatogenesis, testis (germinal), spleen (lymphoid), and liver (non-proliferating) tissues from various vertebrate species were examined for their relative amounts of HMG1 and HMG2, and for HMG2 heterogeneity. Bull, chimpanzee, cynomologus monkey, dog, gopher, guinea pig, hamster, mouse, opossum, rabbit, rat, rhesus monkey, squirrel and toad (Xenopus) tissues were analysed. Nearly all species showed relatively high contents of HMG2 in testis tissue, whereas HMG1 contents were similar in all species and tissues. Ten of thirteen species showed a rapidly migrating HMG2 subtype in testis tissue, separable by acid/urea/polyacrylamide-gel electrophoresis. Xenopus, which lacks HMG2 in somatic tissues, showed an HMG2-like protein in testis tissue. Although the rapidly migrating HMG2 subtype in species other than rat was not testis-specific, it was always enriched in the testis. This study indicates that increased amounts of HMG2 and the enrichment of a rapidly migrating HMG2 subtype are general features of spermatogenic cells.
Role of uricase in the triggering of germination of Bacillus fastidiosus spores
Authors: Salas J A , Johnstone K , Ellar D J
Published: August 2015

The likelihood that uric acid was the only compound capable of triggering germination of Bacillus fastidiosus spores was reinforced by the finding that ureidoglycollic acid, urea, NH4Cl, 2,8-dihydroxypurine and a combination of L-alanine and O-carbamoyl-D-serine were ineffective as germinants. Uric acid-triggered germination of B. fastidiosus was prevented by a range of inhibitors that also inhibited uricase activity in dormant spore extracts. O2 uptake during germination started immediately after addition of uric acid, possibly as a consequence of the oxidation of uric acid by the enzyme uricase. Germination showed a dependence on uric acid concentration, with a relatively high Km (4-5 mM). During the first 10 min of germination of heat-activated spores there was no detectable change in the number of spore-cortex reducing groups, indicating that selective cortex hydrolysis is not involved in the trigger mechanism of germination of B. fastidiosus. On the basis of the results, a model is proposed in which re-initiation of uricase activity is the mechanism by which B. fastidiosus spores are triggered to emerge from the dormant state.
Exhaustive laccase-catalysed oxidation of a lignin model compound (vanillyl glycol) produces methanol and polymeric quinoid products
Authors: Lundquist K , Kristersson P
Published: August 2015

Laccase-catalysed oxidation of the lignin-related phenol vanillyl glycol results in the initial formation of dimers and subsequent polymerization. The polymerization is accompanied by a liberation of methanol corresponding to 15-20% demethylation. Visible spectra together with reduction experiments suggest the simultaneous formation of o-quinones. The participation of quinone formation in the polymerization process and the possible role of such intermediates in lignin biodegradation is discussed.
Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration
Authors: Di Virgilio F , Vicentini L M , Treves S , Riz G , Pozzan T
Published: August 2015

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.
Evidence for the presence of an essential arginine residue in photoreactivating enzyme from Streptomyces griseus
Authors: Eker A P M
Published: August 2015

Butane-2,3-dione inhibits the enzymic activity of Streptomyces griseus photoreactivating enzyme (PRE). Some characteristics of the inhibition, notably the enhancement by borate buffer and the reversibility, indicate that arginine residues are modified. From the kinetics of inhibition it can be concluded that a single essential arginine residue is involved. U.v.-irradiated DNA, the substrate for PRE, protects the enzyme against inactivation by butane-2,3-dione. This suggests that the essential arginine residue is situated in or near the u.v.-irradiated-DNA-binding site. Non-irradiated DNA at higher concentrations also protects against inactivation, indicating that PRE can form non-specific complexes. From the ratio of complex constants obtained from protection experiments with non-irradiated and u.v.-irradiated DNA it appears that PRE preferably binds to dimer sites.
Adenosine formation and release from neonatal-rat heart cells in culture
Authors: Meghji P , Holmquist C A , Newby A C
Published: August 2015

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter.
Kinetics of protein-modification reactions. Stoichiometry of modification-produced enzyme inactivation: modification of rhodanese by 2,4,6-trinitrobenzenesulphonic acid
Authors: Rakitzis E T , Malliopoulou T B
Published: August 2015

A mathematical treatment is presented for the dependence of enzyme activity loss on the numbers and reactivities of the groups essential for catalytic function, when enzyme protein modification is carried out by the use of concentrations of protein reactive groups well in excess of that of modifying agent. Experimentally obtained data on the modification of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) by 2,4,6-trinitrobenzenesulphonic acid are presented, and it is shown that, at pH9.00, the fractional concentration of rhodanese groups, or of rhodanese group reactivities, essential for enzyme catalytic function is 0.88; this value is found to decrease with decreasing pH of the reaction medium. The possibility that rhodanese inactivation by 2,4,6-trinitrobenzenesulphonic acid is brought about by modification of groups other than amino groups is ruled out by a comparison of the enzyme-inactivation and protein-modification stoichiometries, for putative reaction models for enzyme and modifying agent.
Pancreatic amylase secretion and cytoplasmic free calcium. Effects of ionomycin, phorbol dibutyrate and diacylglycerols alone and in combination
Authors: Merritt J E , Rubin R P
Published: August 2015

Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.
α-adrenergic stimulation of glutamine metabolism in isolated rat hepatocytes
Authors: Verhoeven A J , Estrela J M , Meijer A J
Published: August 2015

The mechanisms by means of which phenylephrine stimulates glutamine metabolism were studied in isolated rat hepatocytes. In the first 2 min after phenylephrine addition there was a rapid fall in the concentrations of intracellular 2-oxoglutarate and glutamate, presumably owing to activation of 2-oxoglutarate dehydrogenase. This was followed 2-3 min later by activation of glutaminase and by increases in glutamate and 2-oxoglutarate. Activation of glutaminase by phenylephrine was due to direct stimulation of the enzyme rather than to reversal of inhibition by the decrease in 2-oxoglutarate and glutamate. The stimulation of glutaminase by phenylephrine is partly due to an increase in the affinity of the enzyme for ammonia, its essential activator. It is concluded that stimulation of steady-state flux through the pathway from glutamine to glucose and urea can only be achieved by stimulation of glutaminase, the first enzyme in the pathway.
A new negatively regulated acute-phase phosphoprotein synthesized by rat hepatocytes
Authors: Le Cam A , Le Cam G
Published: August 2015

The effect of acute inflammation on the production of the major phosphorylated protein (PP63) excreted by rat hepatocytes was investigated. Both intra- and extracellular forms of the protein labelled with [32P]Pi, [3H]fucose and [35S]methionine were immunoprecipitated with monospecific polyclonal antibodies, and relative rates of PP63 synthesis were measured. The hepatocytes of acutely inflamed rats produced and excreted 85% less 32P- and 3H-labelled PP63 than did control cells. This decreased amount of PP63 did not result from an impairment in the phosphorylation or glycosylation processes or from a blockade in excretion, but rather was found to be due to extensive shut-off in biosynthesis of the protein as measured by [35S]methionine incorporation. Thus PP63 would appear to represent a new negatively regulated acute-phase protein.
The effect of progesterone on prolactin stimulation of fatty acid synthesis, glycerolipid synthesis and lipogenic-enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection
Authors: Martyn P , Falconer I R
Published: August 2015

The activities of lipogenic enzymes, such as acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase, and glycerolipid synthesis increased significantly in mammary explants of 11-day-pseudopregnant rabbits in response to prolactin, in the presence of near-physiological concentrations of insulin and corticosterone in culture. Increasing the concentration of progesterone in culture resulted in suppression of glycerolipid synthesis and activities of acetyl-CoA carboxylase and fatty acid synthetase, but not the pentose phosphate dehydrogenases. However, at near-physiological concentration of progesterone, only acetyl-CoA carboxylase activity was decreased. Injection of prolactin intraductally into 11-day-pseudopregnant rabbits stimulated glycerolipid synthesis, fatty acid synthesis and enzymes involved in fatty acid synthesis, after 3 days. Intraductal injection of progesterone separately or together with prolactin had no significant effect on basal or stimulated lipogenesis in mammary glands. Intramuscular injection of progesterone at 10 mg/day did not suppress fatty acid synthesis stimulated when prolactin was injected intraductally, but a significant inhibition was observed at a higher dose (80 mg/day).