bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphism of phosphatidylethanolamine–phosphatidylserine model systems: influence of cholesterol and Mg2+ on Ca2+-triggered bilayer to hexagonal (HII) transitions
Authors: Bally M. B. , Tilcock C. P. S. , Hope M. J. , Cullis P. R.
Published: December 2009

Previous work has shown that Ca2+ can trigger bilayer to hexagonal (HII) polymorphic phase transitions in (unsaturated) phosphatidylserine (PS) – phosphatidylethanolamine (PE) model systems. In this work we examine the influence of cholesterol and Mg2+ on the phase preferences of PS–PE systems. Subsequently, the influence of cholesterol and Mg2+ on the levels of Ca2+ required to trigger bilayer–HII transitions in these mixed systems is studied. It is shown that at 30 °C the presence of equimolar (with respect to phospholipid) levels of cholesterol engenders formation of the HII phase for PE–PS systems containing 15 and 30 mol% PS, whereas bilayer structure is maintained for PE–PS–cholesterol (1:1:2) dispersions. However, the polymorphic phase preferences of the latter system are much more sensitive to the presence of monovalent and divalent cations. In the absence of cholesterol, Mg2+ and high salt concentrations do not affect the polymorphic phase preferences of PE–PS (1:1) systems. In contrast, 8 mM or higher Mg2+ levels or salt concentrations greater than 1.0 M induce HII-phase formation in PE–PS–cholesterol (1:1:2) systems. Further, lower Mg2+ concentrations (2 mM) act as a powerful adjunct to Ca2+ triggering of HII-phase structure in such systems, reducing the Ca2+ concentration required from 4 to 0.25 mM. These results are discussed in terms of Ca2+ concentrations required for fusion events and the influence of cholesterol on the structural preferences of the inner monolayer lipids of the erythrocyte membrane.
Phosphorus-31 nuclear magnetic resonance pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a
Authors: Vogel Hans. J. , Bridger William A.
Published: December 2009

31P nuclear magnetic resonance (NMR), pH titration studies of the phosphoproteins tropomyosin and glycogen phosphorylase a (in the presence of the inhibitor glucose) show that the resonances for the phosphoserine regulatory sites shift with pH. Analysis of line widths indicates that both residues have considerable mobility. These results are in agreement with studies on similar phosphorylated sites on other proteins, leading us to propose that mobility is a general feature of such regulatory sites. pH titration studies on a series of model compounds have indicated that an empirical correlation exists between the Hill coefficient n (a measure of the cooperativity of the titration curve) and the presence of charged groups in the vicinity of the phosphoryl moiety. Moreover, these studies showed that within one class of similarly substituted phosphorus compounds the chemical shifts, the titration behaviour, and the [Formula: see text] were comparable and allow for easy identification of these compounds. The [Formula: see text] values for phosphoserine residues of proteins are in general slightly higher than those of phosphomonoester-containing small compounds. The titration data prompt our estimation that the maximal amount of energy associated with a salt linkage between a phosphoseryl side chain and a positively charged group is about 5 kcal (1 cal = 4.1868 J).
Expression of histone genes during heat shock and in arsenite-treated Drosophila Kc cells
Authors: Tanguay R. M. , Camato R. , Lettre F. , Vincent M.
Published: December 2009

In Drosophila Kc cultured cell lines, heat shock induces an increased synthesis of one of the core histones, H2B. This is accompanied by a reduction in the rate of synthesis of H1 and the other core histones, suggesting a noncoordinated expression of histones during heat shock. Arsenite which has a heat-shock mimicking effect does not induce an increase in the synthesis of H2B. The increased expression of H2B during heat shock shows a temperature dependency similar to that of the low molecular weight heat-shock proteins being observed at temperatures higher than 33 °C. A full heat-shock response is observed after a short 15-min shock at 37 °C, suggesting a rapid transcriptional response of the H2B gene and possibly a decreased transcription of the other histones and (or) an accelerated decay of their corresponding mRNAs. This increased synthesis of H2B seems under transcriptional control since it can be inhibited, like the other major heat-shock proteins, by the addition of actinomycin D.
Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing
Authors: Lepock James R. , Cheng Kwan-Hon , Al-Qysi Hisham , Kruuv Jack
Published: December 2009

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.
The possible role of the superoxide ion in the induction of heat-shock and specific proteins in aerobic Drosophila cells during return to normoxia after a period of anaerobiosis
Authors: Ropp M. , Courgeon A. M. , Calvayrac R. , Best-Belpomme M.
Published: December 2009

In vitro cultured Drosophila melanogaster cells were shown to be aerobic and several kinetic parameters of their respiration were measured. This allowed us to define experimental conditions for a transient period of anaerobiosis followed by a reexposure to normal oxygenation. This treatment, applied without any change of temperature, induced not only the heat-shock proteins, but also a new specific peptide of 27 000 daltons and a twofold increase of the maximal rate of O2 uptake. This evokes a common molecular mechanism activated either by heat or by O2, which could involve the increase of the products of oxygen reduction such as the superoxide ion.
Studies on primate gonadotropin receptors: comparison of follitropin and lutropin receptors in human testis
Authors: Berman M. I. , Sairam M. R.
Published: December 2009

The interaction of 125I-labeled human follitropin (hFSH), human lutropin (hLH), and human choriogonadotropin (hCG) with a particulate fraction prepared from the testes of adult men was investigated. The hormone specific binding was maximal at pH 7.5 and 34 °C in the presence of 10 mM MgCl2. The FSH receptor was relatively more stable than the LH receptor (t1/2 at 34 °C of 14 and 4 h, respectively). The dissociation constants (Kd) calculated for hLH or hCG were very similar (approximately 1.0 × 10−10 – 1.4 × 10−10 M). The affinity of FSH and LH to their receptors did not appear to change with age, as shown from analysis of testes from 17- to 80-year-old men. The number of FSH receptors was greater than the number of LH receptors in these tissues. The hFSH receptor did not show any preference to binding of the homologous hormone, because FSH from nonprimates could displace 125I-labeled hFSH, but the LH receptor showed a marked preference for hLH or hCG as the ligand.
Systemic and local regulation of oxytocin receptors in the rat uterus, and their functional significance
Authors: Fuchs A.-R. , Periyasamy S. , Soloff M. S.
Published: December 2009

Rats were made unilaterally pregnant by tying the right oviduct on the day after mating, to compare the oxytocin receptor concentrations in a nondistended, nonpregnant uterine horn with those in a distended, pregnant horn. On day 20, they were subjected to bilateral ovariectomy and indwelling balloons were inserted into both uterine horns. Following ovariectomy, the rats were injected im with either oil, estradiol benzoate (5 μg/rat per 24 h), progesterone (5 mg/rat per 24 h), or estradiol and progesterone together. For comparison, intact rats were studied on days 21 and 22, 24 and 48 h after insertion of the indwelling balloons. Spontaneous uterine activity and the response to increasing amounts of oxytocin were recorded 20–24 h and 44–48 h after surgery, following which the uteri were excised and assayed for oxytocin and estrogen receptors. The oxytocin receptor concentrations in the two horns were different on day 20 before the treatments were begun, the distended pregnant horn having a higher concentration per milligram DNA than the nonpregnant horn. The various treatments always changed the oxytocin receptor concentrations in the same direction; estrogen increased and progesterone inhibited the estrogen-induced rise in oxytocin receptor concentrations. In intact rats, the distention-induced increase in oxytocin receptor concentrations present on day 20 disappeared near term, but in the absence of the ovaries distention of the uterus had a significant influence on the myometrial oxytocin receptor concentrations, potentiating the effect of estrogen. Progesterone selectively inhibited the distention-induced increase in oxytocin receptor concentrations without inhibiting the hypertrophic effect of distention in general. A good correlation between oxytocin receptor numbers and tissue responsiveness was observed in all instances. The changes in spontaneous activity induced by the various treatments were distinct from the changes in oxytocin responsiveness. Estrogen exerted a strong inhibitory action on the activity stimulated by hormone withdrawal, while progesterone had no inhibitory effect. The pregnant distended horn always showed more spontaneous activity than the nonpregnant horn. There was an overall significant correlation between nuclear estrogen receptor and oxytocin receptor concentrations per milligram DNA, although the partial correlations were not significant in all groups (oil and progesterone). It is concluded that ovarian hormones exert the major regulatory influence on myometrial oxytocin receptor concentrations, but other factors including distention modulate their effect.
Effects of hormones on the maintenance and mitochondrial functions of rat hepatocytes cultured in serum-free medium
Authors: Wilson E. Jane , McMurray William C.
Published: December 2009

The survival and morphology of rat hepatocytes were examined in primary cell cultures that were maintained in serum-free medium supplemented with different hormones. Insulin and dexamethasone improved survival and maintenance of normal epithelial-shaped cells, although triiodothyronine did not alter cell survival or morphology when added to the medium alone or with other hormones. The level of mitochondrial α-glycerophosphate dehydrogenase and cytochromes a(+ a3), b and c, but not c1, were increased in cultured hepatocytes by triiodothyronine. Although induction of α-glycerophosphate dehydrogenase did not require serum or growth hormone, the triiodothyronine effect was potentiated by insulin plus dexamethasone. This permissive effect of dexamethasone parallels its known glucocorticoid action of increasing the activity of the gluconeogenic enzyme tyrosine aminotransferase in the cultured hepatocytes. Glucagon, which also elevated tyrosine aminotransferase activity, had no effect upon the induction of α-glycerophosphate dehydrogenase by triiodothyronine. Since the culture medium was completely defined and triiodothyronine did not alter survival or morphology of the hepatocytes, the effects upon mitochondrial functions are direct cellular actions of the thyroid hormone.
Regulation of phosphoenolpyruvate carboxykinase during postnatal development in the rat
Authors: Hahn Peter , Smale Freda-Anne
Published: December 2009

Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) (PEPcK) activity is high in the liver, brown fat, and small intestinal mucosa of suckling rats. Starvation results in a rise in hepatic activity in newborn and older animals, but a fall in brown fat from rats younger than 14 days. At that age, activity rises in all three tissues on fasting. Both triiodothyronine (T3) and dexamethazone depress activity in gut and (as previously reported) in brown fat, but elevate it in liver. Cold exposure also has a similar effect. Both α-antagonist and agonist injections result in elevated hepatic activity. T3 given together with these more than doubles the effect of either drug or T3 given alone. In brown fat only antagonists decrease PEPcK activity, whereas in gut both agonists and antagonist have an inhibitory effect. It is concluded that PEPcK activity in the three tissues responds differently to the same stimuli with gut mucosa being more similar to, but not the same as, brown fat than to liver.
The biological activity of glucagon–phospholipid complexes
Authors: Liepnieks J. J. , Stoskopf P. , Carrey E. A. , Prosser C. , Epand R. M.
Published: December 2009

Glucagon can form water-soluble complexes with phospholipids. The incorporation of glucagon into these lipoprotein particles reduces the biological activity of the hormone. The effect is observed only at temperatures below the phase transition temperature of the phospholipid and results in a decreased stimulation of the adenylate cyclase of rat liver plasma membranes by the lipoprotein complex as compared with the hormone in free solution. Two- to five-fold higher concentrations of glucagon are required for half-maximal stimulation of adenylate cyclase when the hormone is complexed with dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or bovine brain sphingomyelin. A possible role of lipoprotein-associated hormones in the development of insulin resistance is discussed.
Regulation by ACTH of steroid hormone biosynthesis in the adrenal cortex
Authors: Simpson Evan R. , Waterman Michael R.
Published: December 2009

In this article, current knowledge about the mechanism of action of ACTH will be reviewed. Emphasis will be placed on events which occur subsequent to binding of ACTH to its receptor, stimulation of adenylate cyclase, and activation of protein kinase. In the first part of the review, the acute action of ACTH will be discussed with emphasis on present hypotheses as to the roles of calcium, phospholipids, sterol carrier proteins, and a "labile protein" activator of cholesterol side-chain cleavage. The presumptive role of these factors to increase the binding of cholesterol to the mitochondrial cholesterol side-chain cleavage enzyme will be discussed in the light of the concept that such binding is the step in steroidogenesis which is activated during the acute response of the adrenal cell to ACTH. In the second part of the article, the long-term action of ACTH to increase the levels of steroidogenic enzymes will be reviewed. Recent evidence is indicative that ACTH causes induction of the synthesis of key steroidogenic enzymes present in both the mitochondria and the microsomes. This appears to result from transcription of genes specific for these various species of cytochrome P-450 and ancillary proteins, resulting in increased synthesis of specific forms of mRNA. Whereas the mitochondrial steroidogenic enzymes are synthesized on cytoplasmic polysomes as precursor forms of higher molecular weight, the microsomal proteins are synthesized as forms of similar molecular weight to the mature forms.
Further studies on the corticosteroid-receptor system of the nasal gland of the domestic duck (Anas platyrhynchos)
Authors: Sandor Thomas , Mehdi Afzal Z. , DiBattista John A.
Published: December 2009

The interaction of tritiated corticosterone with the nasal gland corticosterone receptor was investigated. Kinetic studies have shown that the association of [3H]corticosterone–receptor followed second-order reaction kinetics and the dissociation of the ligand from the receptor became "pseudo" first order in the presence of large excess of radioinert steroids at 0, 15, 25, and 35 °C. Similar data were obtained with an ammonium sulfate precipitate of the cytosol. Dissociation rate constants varied from 10−5 to 10−3 s−1 and the association rate constants varied from 0.5 × 104 to 3.8 × 105 M−1∙s−1, depending on the reaction temperature and the cytoplasmic receptor preparation. Equilibrium dissociation constants were in 10−8–10−9 M range. Equilibrium dissociation constants, calculated from kinetic data (kd/ka), showed a marked temperature dependence, while those obtained by saturation analysis varied much less with the reaction temperature. Data obtained in these experiments were used to calculate some thermodynamic parameters of the binding of corticosterone to the cytoplasmic receptor. The enthalpy of dissociation was 101.5 and 79.4 kJ∙mol−1 and the entropy of dissociation was 200 and 280 J∙mol−1∙degree−1 for the crude cytoplasmic receptor and the ammonium sulfate precipitate, respectively. From the equilibrium dissociation constants, the enthalpy and entropy of the equilibrium binding was calculated. Polynomial fitting of Ka values versus 1/T yielded enthalpy (ΔH) values from −0.9 to −88.8 kJ∙mol−1, depending on the nature of the receptor preparation. Entropy values were negative for kinetically derived equilibrium association constants from the crude cytosol at all temperatures and for 0 and 15 °C for the precipitate. Entropy values were positive for Ka values obtained from kinetic rates at 25 and 35 °C and for Ka's calculated from saturation analysis. Further experiments with the precipitate confirmed our previous contention that the nasal gland cytoplasmic corticosterone receptor metabolized the bound ligand to 11-dehydrocorticosterone, though the receptor preparation was corticosterone specific. The following hydrodynamic parameters were obtained on the binding macromolecule: molecular weight, 316 000; s20,w, 8.0; Stokes radius (rs), 77.3 Å (1 Å = 0.1 nm); total frictional ratio (f/f0), 1.71. The labeled receptor preparation translocated to homologous nuclear binding sites following heat activation and, at the nuclear binding sites, the ligand was almost exclusively in its oxidized form. Measurement of the nuclear steroid–receptor complex by exchange assay with [3H]corticosterone confirmed the presence of nuclear binding sites. From these studies, it was concluded that the nasal gland of the duck contains specific, glucocorticoid-type corticosterone receptors and that the effector steroid is probably 11-dehydrocorticosterone or a critical mixture of these two steroids, with the oxidized form predominating.
Uptake of 17β-estradiol and its conjugates by isolated rabbit liver nuclei
Authors: Tong J. H. , Seper E. I. , Layne D. S. , Williamson D. G.
Published: December 2009

The present study compares the uptake and metabolism of 17β-estradiol, 17β-estradiol 3-glucoside, and 17β-estradiol 3-glucuronide by a highly purified preparation of rabbit liver nuclei. The uptake of the three estrogens was rapid and equilibration was reached within 60 s. The order of uptake was 17β-estradiol (64 fmol∙mg protein−1) > 17β-estradiol 3-glucoside (10 fmol∙mg protein−1) > 17β-estradiol 3-glucuronide (6.5 fmol∙mg protein−1). Thin-layer chromatography of the estrogens taken up by rabbit liver nuclei indicated the presence of a β-glucosidase activity associated with the nuclear preparation. The apparent Km value of this enzyme for 17β-estradiol 3-glucoside (3.5 μM) was about 10-fold higher when compared with the cytosolic enzyme. The uptake of the three estrogens was linearly proportional to the substrate concentration from 1 to 100 nM. No competition for uptake was observed among the steroids and the presence of diethylstilbestrol did not reduce the uptake of the steroids. These findings suggest that 17β-estradiol, 17β-estradiol 3-glucoside, and 17β-estradiol 3-glucuronide are taken up by nuclei by a nonsaturable diffusion process. The effect of cytosol on the uptake of estrogens by purified nuclei was also investigated. It was observed that cytosol reduced the uptake of 17β-estradiol but had little effect on that of its conjugates.
Distribution of elongation factor 2 between particulate and soluble fractions of the brine shrimp Artemia during early development
Authors: Yablonka-Reuveni Z. , Fontaine J. J. , Warner A. H.
Published: December 2009

The ADP-ribosylation of elongation factor 2 (EF-2) in vitro was used to quantitate EF-2 and to determine its subcellular distribution in extracts of Artemia embryos at different stages of development. In extracts from dormant cysts of Artemia 40–45% of EF-2 is complexed to macromolecules smaller than ribosomes, whereas the remainder is soluble or free in the cytosol. During early development the amount of "complexed" EF-2 decreases markedly concomitant with an increase in the pool of soluble EF-2. Complexed EF-2 was found to be associated with macromolecules which sediment at 16S–20S and 40S–50S and not with monoribosomes or polyribosomes as reported for mammalian systems. The data show that the decrease in complexed EF-2 is associated with the resumption of development in Artemia.
Pharmacological antagonism of calmodulin
Authors: Roufogalis Basil D. , Minocherhomjee Arda-E-Viraf M. , Al-Jobore Ameera
Published: December 2009

The widespread role of calmodulin in mediating Ca2+-dependent activities raises the possibility that calmodulin antagonists might be used effectively to alter cellular function. Current progress toward the development of calmodulin antagonists of suitable selectivity and specificity is considered in this review. Phenothiazines and other antipsychotic agents, including trifluoperazine, are among the most potent antagonists of calmodulin actions. Their antagonism of calmodulin function correlates with binding to calmodulin, probably at a hydrophobic region induced by Ca2+. Binding to this region is not stereospecific nor restricted to antipsychotic agents, as a wide range of hydrophobic molecules interact similarly and inhibit calmodulin activity. Trifluoperazine is not a specific antagonist of calmodulin, as it interacts with other Ca2+-binding proteins and also inhibits many lipid-dependent enzyme activities, including a widely distributed Ca2+ and phospholipid-dependent protein kinase. New data is presented showing that trifluoperazine, in addition to its binding to calmodulin, binds to the activated states of calmodulin-sensitive enzymes, in particular erythrocyte Ca2+- and Mg -stimulated ATPase. The implications of the lack of selectivity of presently available "calmodulin antagonists" are assessed.
Sequences complementary to cellular deoxypolypyrimidines are localized in the 3′ end of L-cell mRNA
Authors: Dodd Janice Green , Straus Neil A.
Published: December 2009

Many eukaryotic genomes have been shown to contain long pyrimidine tracts. In mouse L-cells, at least some of these pyrimidine tracts are transcribed and form a significant portion of the poly(A)+ RNA sequences. In this study the results from three different experimental methods indicate that the sequences complementary to polypyrimidines are localized to the 3′ end of mRNA molecules. First, polypyrimidines reacted preferentially with the 3′-end fragments of mRNA generated by limited alkaline cleavage. Second, digestion of [3H]mRNA–polypyrimidine hybrids with RNase H released 3′-end fragments of mRNA which averaged only 250 nucleotides (NT) in length. Third, polypyrimidine tracts were isolated from cDNA which averaged only 200–250 NT extending from the 3′ end of the corresponding mRNAs. These data suggest that the sequences transcribed from pyrimidine tracts are quite close to the 3′ end of the cellular messages in which they occur, probably within the terminal untranslated region.
Pathways for desaturation of oleoyl chains in Candida lipolytica
Authors: Ferrante G. , Kates M.
Published: December 2009

Incubation of microsomes from 25 °C- or 10 °C-grown cells of Candida lipolytica with [14C]oleoyl-CoA ([14C]18:1-CoA) in the presence or absence of NADH resulted in rapid acyl transfer of [14C] 18:1 to phospholipids (mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) and to acylglycerols. Incorporation into PC was greatly enhanced when incubation was carried out in presence of lysophosphatidylcholine (lyso-PC). In all experiments, in the presence of NADH and O2, with and without added lyso-PC, the initial rate of formation of [14C]linoleoyl-PC was much greater than that of [14C]linoleoyl-CoA ([14C]18:2-CoA). These results suggest that the actual substrate for the Δ12-desaturase is the oleoyl-PC, although some desaturation of 18:1-CoA cannot be eliminated. It is concluded that the main pathway for 18:2 formation proceeds from stearoyl-CoA (18:0-CoA) → 18:1-CoA → 18:1-phospholipid → 18:2-phospholipid; the pathway 18:0-CoA → 18:1-CoA → 18:2-CoA → 18:2-phospholipid is a minor pathway. Microsomes from cells grown at 10 °C had a higher content of 18:2 and a lower phospholipid desaturase activity at 25 °C than microsomes from cells grown at 25 °C, suggesting an inverse relationship between desaturase activity and membrane lipid fluidity.
Mucin synthesis. III. UDP-GlcNAc:Galβ1-3(GlcNAcβ1-6)GalNAc-R (GlcNAc to Gal) β3-N-acetylglucosaminyltransferase, an enzyme in porcine gastric mucosa involved in the elongation of mucin-type oligosaccharides
Authors: Brockhausen Inka , Williams David , Matta Khushi L. , Orr Jeanne , Schachter Harry
Published: December 2009

Pig gastric mucosa microsomes have been shown to catalyze the following reaction: UDP-GlcNAc + Galβ1-3(GlcNAcβ1-6)-GalNAc-α-R → GlcNAcβ1-3Galβ1-3(GlcNAcβ1-6)GalNAc-α-R + UDP, where R is o-nitrophenyl or benzyl. The enzyme catalyzing this reaction has been named UDP-GlcNAc:Galβ1-3(GlcNAcβ1-6)GalNAc-R (GlcNAc to Gal) β3-N-acetylglucosaminyltransferase. The β3-GlcNAc-transferase does not act on Galβ1-3GalNAc-α-o-nitrophenyl. The β3-GlcNAc-transferase requires Mn2+ and Triton X-100 for optimal activity. The Vmax for the microsomal enzyme is 8.7 nmol/mg protein per hour and the Km values are 1.6, 0.9, and 1.2 mM for UDP-GlcNAc and the α-o-nitrophenyl and α-benzyl derivatives of Galβ1-3(GlcNAcβ1-6)GalNAc, respectively. Pig gastric mucosa microsomes catalyze the transfer of GlcNAc to lactose to form GlcNAcβ1-3Galβ1-4Glc, but fail to transfer GlcNAc to lactosyl ceramide, Galβ1-4GlcNAc, or Galβ1-4GlcNAc-β-benzyl.
Effect of intravenous infusion of Intralipid, cholesterol, and plant sterols on hepatic cholesterogenesis
Authors: Kakis G. , Kuksis A.
Published: December 2009

Male Wistar rats were infused intravenously with various amounts (2–20 mL/24 h) of Intralipid or Intralipid containing cholesterol or plant sterols (5–100 mg/24 h), and hepatic cholesterogenesis was monitored by measuring the incorporation of [1-14C]acetate into the nonsaponifiable sterols of liver slices. It was observed that the infusion of Intralipid alone resulted in a hypercholesterolemia that varied with the amount of Intralipid administered and that it was accompanied by up to a threefold increase in hepatic cholesterogenesis. Inclusion of cholesterol in the Intralipid at 5 mg/mL prevented the increase in hepatic cholesterol biosynthesis, while an inhibition of up to 95% of control synthesis was achieved when a total of 33 mg of cholesterol in 20 mL Intralipid was infused over a 24-h period. It is concluded that the feedback regulation of cholesterol biosynthesis is operative even when the entry of cholesterol bypasses the intestine and the lipoprotein synthesis taking place there and that Intralipid is a suitable medium for the intravenous introduction of a large mass of metabolically active cholesterol molecules. Similar infusions of mixed plant sterols failed to prevent the activation or inhibition of cholesterol biosynthesis, but the administration of much larger doses of plant sterols (100 mg) brought about a partial inhibition of hepatic cholesterogenesis. It is concluded that the presence of an alkyl group in the side chain prevents the plant sterols from an effective interaction with the critical sites of the feedback regulatory system of cholesterogenesis. The effects of the larger doses of plant sterols were attributed to the displacement of increasing amounts of free cholesterol from the vascular tissues, which resulted in an effective elevation of plasma cholesterol levels. The infusion of either cholesterol or plant sterols over the 24-h period did not appear to have a consistent effect upon the composition or secretion of biliary bile acids.
Comparison of metastable phase behavior of the complexes of dipalmitoyl phosphatidylglycerol with Mg+2 and myelin basic protein
Authors: Boggs Joan M. , Moscarello M. A.
Published: December 2009

Both divalent cations and myelin basic protein (BP) induce metastable phase behavior in phosphatidylglycerol (PG). The stable phase of the divalent cation – PG complex is dehydrated cylinders or sheets of lipid in which adjacent bilayers are probably bridged by the divalent cation. The structure of the stable phase of the BP–PG complex is unknown. To help understand the metastable phase behavior in the BP–PG complex, it was compared with that of the Mg+2–PG complex using fatty-acid spin labels and differential scanning calorimetry. The stable state of the BP–PG complex melts at a similar temperature and with a similar enthalpy as the Na+-salt form of the lipid, while the stable state of the Mg+2–PG complex melts 35° higher than and with an enthalpy 63% greater than the Na+-salt form. BP causes pronounced motional restriction of a fatty acid spin labeled near the terminal methyl group in the metastable and stable states of its complex with PG. Computer resolution of spectra of fatty-acid spin labels in the Mg+2–PG complex revealed that the spectra characteristic of the stable state were exchange broadened, showing that the probes are not soluble in the stable dehydrated state of this complex. The much different behavior of the BP-PG complex suggests that its stable phase is not dehydrated, that the probes are not displaced from the complex, and that their behavior reflects the ability of hydrophobic residues of basic protein to penetrate into the bilayer while its basic residues bind electrostatically to the lipid polar head groups.
The oligosaccharides of human α1-antitrypsin
Authors: Hercz Albert
Published: December 2009

Oligosaccharides were prepared from α1-antitrypsin (PiM) and human serum transferrin by hydrazinolysis and then reacetylated with radiolabelled acetic anhydride. Gel permeation chromatography revealed similarities between the two preparations. The antitrypsin oligosaccharides were resolved into 76% concanavalin A binding and 24% nonbinding sugar chains by lectin affinity chromatography; both fractions were further resolved by preparative paper electrophoresis. Small amounts of neutral oligosaccharides, as well as approximately 30% "monosialo" and 67% "disialo" biantennary oligosaccharides, were identified in the lectin-binding pool. The disialo oligosaccharide fraction was susceptible to sequential degradation with exoglycosidases. Upon incubation of the monosialo biantennary oligosaccharide fraction with CMP – [14C] sialic acid and a rat liver Golgi preparation as a source of transferase, a new doubly labelled peak was formed that eluted from the gel permeation chromatography column in the same volume as the disialo biantennary oligosaccharide fraction. The paper electrophoretogram of the lectin-nonbinding oligosaccharides revealed the presence of limited amounts of neutral oligosaccharides and oligosaccharides carrying two and three negative charges. The lectin-nonbinding oligosaccharide fraction with two charges was tentatively identified as disialo biantennary oligosaccharide containing a bisecting N-acetylglucosamine residue, and the fraction with three negative charges was tentatively identified as trisialo triantennary oligosaccharide.
Studies on the microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: isolation, characterization, and solubilization of the hydroxylase system
Authors: Madyastha K. M. , Jayanthi C. R. , Madyastha P. , Sumathi D.
Published: December 2009

A suitable procedure for the preparation of cell-free extract with high 11α-hydroxylase activity from the induced vegetative cell cultures of Aspergillus ochraceus was developed. The presence of EDTA (10 mM), glycerol (10%), and dithiothreitol (5 mM), in the grinding medium was found to be necessary for optimal hydroxylase activity. Although the pH optimum of the hydroxylase was found to be 7.7, the ideal pH for preparing the cell-free extract from mycelium was 8.3. Microsomes (2.0 mg) isolated from the cell-free extract hydroxylated progresterone in very high yields (85–90% in 30 min) in the presence of NADPH and O2. Cytosolic involvement in the hydroxylation reaction was not noticed. The apparent Km for NADPH and progesterone were 0.052 and 0.625 mM, respectively. The hydroxylase activity was inhibited by metyrapone, carbon monoxide, SKF-525A, and p-chloromercuribenzoate indicating the involvement of cytochrome P-450 system in the reaction. Inhibition of the hydroxylase activity by cytochrome c and the presence of significant levels of NADPH–cytochrome c reductase in the microsomal fraction suggested that the reductase could be one of the components of the hydroxylase system. The membrane-bound hydroxylase was solubilized using sodium cholate. Solubilized membrane fraction contained considerable levels of cytochrome P-450 and NADPH–cytochrome c reductase, besides hydroxylase activity.
Kinetic studies of rat renin and tonin on purified rat angiotensinogen
Authors: Gutkowska Jolanta , Corvol Pierre , Figueiredo Amintas F. S. , Inagami Tadashi , Bouhnik Jacob , Genest Jacques
Published: December 2009

Kinetic studies of highly purified rat renin and rat tonin on completely purified angiotensinogen and angiotensin-tetradecapeptide synthetic renin substrate were performed. The Michaelis–Menten constant (Km) for renin, determined at the optimum pH, was 2.8 ± 0.03 μM for angiotensinogen and 28.8 ± 2.69 μM for angiotensin-tetradecapeptide renin substrate. The Km of purified rat tonin was determined as 0.66 ± 0.18 μM for angiotensinogen and 2.33 ± 0.42 μM for angiotensin tetradecapeptide. In comparison with renin, tonin shows higher affinity with respect to angiotensinogen, but the turnover number of natural substrate molecules observed with renin (0.87 s−1) is more than 3700 times higher than that of tonin (2.3 × 10−4 s−1). Renin shows higher affinity to angiotensin tetradecapeptide than tonin, but similar turnover numbers of 2.8 and 10.0 s−1 are observed for hydrolysis of angiotensin tetradecapeptide by renin and tonin, respectively.
The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30)
Authors: Caroff Martine , Perry Malcolm B.
Published: December 2009

The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30) is composed of D-galactose (three parts), D-glucose (one part), 2-acetamido-2-deoxy-D-glucose (one part), phosphate (one part), and glycerol (one part). Hydrolysis, periodate oxidation, methylation, optical rotation, and nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight linear polymer of a pentasaccharide repeating unit having the structure:[Formula: see text]
Characterization of neonatal cardiac myosin heavy chain mRNA and synthesis of complementary DNA
Authors: Jandreski Mark , Liew C. C.
Published: December 2009

A procedure for the extraction of intact RNA from whole heart tissue using guanidine hydrochloride has been accomplished. Subsequent chromatography on oligo(dT)-cellulose of the total RNA and translation of the poly (A)+ RNA in a cell-free reticulocyte translation system has shown that the myosin heavy chain mRNA from rat heart tissue was present. The myosin heavy chain mRNA was identified by several criteria: (i) sizing by sucrose density gradient centrifugation, (ii) migration of a polypeptide translation product coincided with a myosin heavy chain marker in one-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and (iii) immunoprecipitation of the mRNA translation product with a myosin heavy chain specific antibody. A myosin heavy chain mRNA enriched fraction was used to direct cDNA synthesis in an in vitro system. An optimal condition has been elucidated in which large molecular weight cDNA strands are produced. Evidence is presented here for the production of cDNA strands in excess of 4000 nucleotides which includes the heavy chain myosin in a high potassium – low sodium transcription assay, as revealed by dot blot hybridization with a cDNA clone of the myosin heavy chain.
Modification of actin with 2-(N-methylanilino)naphthalene-6-sulfonyl chloride
Authors: Burtnick L. D. , Isaac C. D.
Published: December 2009

2-(N-Methylanilino)naphthalene-6-sulfonyl chloride (Mns–Cl) was used to label up to eight sites, presumed to be lysine residues, on rabbit skeletal muscle actin. The resultant Mns–actin was highly fluorescent, displaying emission bands near 445 and 480 nm. Incorporation of three Mns groups per actin resulted in impairment of the ability of actin to polymerize. Final relative viscosities attained for salt-induced polymers of actin having no bound Mns groups and three bound Mns groups per actin, respectively, were 1.9 and 1.4. More extensive modification further lowered the ability of actin to polymerize, but the changes were less dramatic. The fluorescence properties of Mns–actin samples that contained up to eight Mns groups per actin were not affected significantly by the addition of salt to the samples. It seems that the modified lysines either lie outside a region of direct intersubunit contact in F-actin or that the modification of a particular lysine residue or residues on a particular actin prevents that actin unit from adding on to a growing F-actin chain. Modification of actin with Mns–Cl does not affect its ability to bind and to inhibit DNase I until more than three Mns groups have been incorporated into each actin. The loss of DNase I inhibitory power only becomes apparent after the ability of actin to polymerize has been reduced substantially.
Glucagon inhibits phosphatidylcholine biosynthesis via the CDP-choline and transmethylation pathways in cultured rat hepatocytes
Authors: Pelech Steven L. , Pritchard P. Haydn , Sommerman Eric F. , Percival-Smith Anthony , Vance Dennis E.
Published: December 2009

The short-term influence of insulin and glucagon on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes was investigated. Under conditions in which insulin (100 nM) stimulated [3H]acetate incorporation into fatty acid almost twofold, synthesis of phosphatidylcholine via CDP-choline and from phosphatidylethanolamine were unaffected. By contrast, glucagon (100 nM), even in the presence of insulin (100 nM), reduced the rate of phosphatidylcholine formation from [Me-3H]phosphocholine by approximately 25% (p < 0.05) within 1 h. Similarly, [3H]phosphatidylethanolamine incorporation into phosphatidylcholine was inhibited in cells exposed to glucagon. Insulin and glucagon had little or no effect on [Me-3H] choline uptake by the hepatocytes. No changes in the activities of the phosphatidylcholine biosynthetic enzymes in cytosol and microsomes from glucagon-treated cells could be detected.
The control of accuracy during protein synthesis in Escherichia coli and perturbations of this control by streptomycin, neomycin, or ribosomal mutations
Authors: Brakier-Gingras Léa , Phoenix Pauline
Published: December 2009

This review surveys the different experimental approaches which describe the binding of tRNA to mRNA-programmed ribosomes and the control of tRNA selection. This selection is best described by the two-step model proposed by Hopfield and demonstrated by Thompson and his collaborators. The model involves a first control at the initial reversible binding of tRNA to the ribosome and a second control, the proofreading control, which promotes rejection of the incorrect tRNA from a high-energy intermediate during the transition from the initial to the final binding state. Streptomycin, neomycin, and ribosomal fidelity mutations appear to affect both control steps. Their effect can be related to the location of the mutated ribosomal proteins and to the conformational changes induced in the ribosome by the misreading agents. An alteration of the first control probably results from a distortion of the codon–anticodon interaction, while an alteration of the second control may be caused by a change in the association between ribosomal subunits.
Purine and pyrimidine analogues irreversibly prevent passage of lymphocytes from the G1 to the S phase of the cell cycle
Authors: Boumah Christine E. , Setterfield George , Kaplan J. Gordin
Published: December 2009

Six-hour pulses of the purine analogue 8-azaguanine (8-AG) and the pyrimidine analogue 5-fluorouracil (5-FU) produced a novel irreversible effect on mouse and human lymphocytes. Cells treated with these analogues early during culture with concanavalin A and then washed in presence of excess natural base could pass normally through the various stages of blast formation (e.g., increased K+ transport, increase in nuclear and cytoplasmic volume, disaggregation of chromatin), but showed a severe inhibition of DNA synthesis when this was measured by [3H]thymidine incorporation at 48 h of culture; this was true irrespective of whether the 6-h pulse with analogue occurred at 0, 12, or 24 h of culture in the presence of mitogen. The analogue 6-mercaptopurine, which strongly inhibited DNA and RNA synthesis while present in the medium, had no irreversible effects, unlike 5-FU and 8-AG. The persistence of the effects of 5-FU in presence of excess thymidine in the medium suggested that inactivation of thymidylate synthetase was not responsible for the inhibition observed here. The effect was expressed in the presence or absence of protein synthesis; therefore, the observed inhibition of proliferation was not due to synthesis of a toxic protein, but to an effect on the formation or function of the DNA synthesizing system and (or) on its template, thus preventing the cells from passing from the G1to the S phase of the cell cycle.
Preparation and examination of the oligosaccharides of serum α1-antitrypsin PiZ
Authors: Hercz Albert
Published: December 2009

Oligosaccharides were prepared from serum α1-antitrypsin, PiM and PiZ, by hydrazinolysis and reacetylation with [3H]acetic anhydride. A comparison by gel permeation chromatography indicated that the two variants contain the same kinds of complex oligosaccharides. Specifically, serum PiZ does not contain mannose-rich oligosaccharides in spite of the presence of mannose-rich oligosaccharides in liver PiZ.
Resolution of alkenylacylglycerol moieties of natural glycerophospholipids by gas–liquid chromatography on polar capillary columns
Authors: Myher J. J. , Kuksis A.
Published: December 2009

A sensitive and practical procedure has been developed for the determination of the sn-1-alk-1-enyl-2-acylglycerol moieties of natural glycerophospholipids using capillary gas–liquid chromatography with flame ionization detection. The method gives excellent resolution and quantitative estimates for molecular species according to carbon number and degree of unsaturation. The diradylglycerols are released from the glycerophospholipids by hydrolysis with phospholipase C and the alk-1-enylacylglycerols are resolved by thin-layer chromatography. Either the trimethylsilyl or the tertiary-butyldimethylsilyl ethers are then resolved at 250 °C by gas–liquid chromatography on a glass capillary column wall-coated with a polar cyanopropylsiloxane polymer using hydrogen as the carrier gas. The method has been applied to the analysis of the alk-1-enylacyl- and diacyl-glycerol moieties of the phosphatidylethanolamines of rat heart and kidney.
Calcium-dependent hydrolyses of polyphosphoinositides in human erythrocyte membranes
Authors: Moore R. Blaine , Appel Stanley H.
Published: December 2009

Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 °C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 μM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca–EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.