bluuebunny/crossref_metadata_2025 huggingface dataset:
Two satisfactory methods for purification of human acrosin
Authors: Leyton Lisette , Ioannes Alfredo De , Croxatto Horacio B. , Graham Elise J. , Elce John S.
Published: December 2009

Acrosin has been purified from human sperm cells by two alternative procedures which give purer products and in higher yields than could be achieved previously. The products were characterized by their molecular weight, catalytic action, sensitivity to inhibitors, and reaction with a polyclonal anti-acrosin antibody. After acid extraction of the cells, one method involves removal of acrosin inhibitors by vacuum dialysis, followed by affinity chromatography on a soybean trypsin inhibitor (SBTI) column, and therefore requires that the acrosin be in an active form capable of binding to the inhibitor. The other method involves affinity chromatography on a column of a monoclonal anti-acrosin antibody (MAb) and can be used to provide either active or proenzyme forms of acrosin, by choice of extraction conditions and inclusion of appropriate inhibitors. The yield of human acrosin from the SBTI method was 104% and from the MAb column was 75%. It is hoped that these procedures will make the very scarce human acrosin more readily available for further study.
Cloning and in vitro expression of the measles virus matrix gene
Authors: Greer Peter A. , Hasel Karl W. , Millward Stewart
Published: December 2009

A cDNA library was prepared from Vero cells infected with the Edmonston strain of measles virus. A number of viral specific cDNA clones were isolated and characterized by Northern blot hybridization analysis. A cDNA clone containing a 1500 base pair insert which hybridizes to a viral specific transcript of approximately 1500 nucleotides was subcloned into pSP64 and used as an in vitro transcription template. The resulting RNA transcript was translated in a cell-free system, giving rise to a polypeptide which comigrates on polyacrylamide gels with the authentic measles virus matrix protein and is immunoprecipitated with antisera specific for the matrix protein.
The activity of human kidney arylsulfatases A and B towards catecholamine sulfates
Authors: Demassieux S. , Romain Y. , Bordeleau L. , Gravel D. , Carrière S.
Published: December 2009

Both arylsulfatases (EC 3.1.6.1, ARS) A and B purified from human kidney displayed Michaelis–Menten kinetics towards catecholamine sulfates as substrates with Km values in the range of 4–25 mM. ARS A hydrolyzed adrenaline 3-sulfate and noradrenaline 3-sulfate with a maximal rate lower than that observed for cerebroside 3-sulfate. In contrast, ARS B hydrolyzed adrenaline 3-sulfate and noradrenaline 3-sulfate with a maximal rate similar to that observed for UDP-N-acetylgalactosamine 4-sulfate.
Digitalis-like biological activity in rat cerebellum
Authors: Lintlop Steven P. , Durante Bill , Sunahara Fred A. , Sen Amar K.
Published: December 2009

The cytosolic fraction of rat cerebellum possesses a factor(s) which is capable of inhibiting synaptosomal Na,K-ATPase activity, competing with [3H]ouabain binding to rat brain synaptosomes, and inducing positive inotropy in guinea pig atrial strips. These results demonstrate the existence of a ouabain-like principle in rat cerebella. The inhibitory activity of the factor was found to be partially thermolabile and diminished by a proteolytic agent, and the activity could be augmented by increasing concentrations of Mg2+, suggesting a regulatory mechanism for the endogenous digitalis-like principle.
Comparison of glucose transport mechanisms at opposing surfaces of the renal proximal tubular cell
Authors: Silverman M.
Published: December 2009

This review contrasts the glucose transport mechanisms at opposing surfaces of the renal proximal convoluted tubule: the Na+-dependent D-glucose transporter localized at the brush border membrane and the Na+-independent transporter localized at the basolateral surface. The two sugar transport mechanisms are discussed from the point of view of their specificity, kinetic, and regulatory behaviors. Recent results focussing on molecular characterization of these different carrier proteins are also described, including some newer information on purification of the Na+-dependent glucose carrier from the brush border membrane.
Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes
Authors: Krupka R. M. , Devés R.
Published: December 2009

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: (a) carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; (b) carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulate gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: (a) if sufficiently bulky, they inhibit transport; (b) they bind outside the substrate site (though adjacent to it), (c) they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel; the choline carrier binds monoalkyl-substituted substrate analogs in the outer gated channel and N-ethylmaleimide and a hydrogen ion in the inner gated channel (the hydrogen ion adding to an ionizing group of pKa 6.8). Inhibitors of this kind may be used to provide evidence on the nature of the complementary peptide surfaces forming the channel walls, as well as evidence on the mechanism of gating.
Origins of the phospholipids in animal mitochondria
Authors: McMurray W. C.
Published: December 2009

As is the case for the assembly of protein components of the membranes in animal mitochandria, the bilayer phospholipids arise from a complicated interplay of intra- and extra-mitochondrial reactions. Our early studies indicated that the bulk of mitochondrial phospholipids (typified by phosphatidylcholine) had their origin in the endoplasmic reticulum and were transported to the mitochondria as complexes with phospholipid-exchange proteins. The polyglycerophosphatides (typified by diphosphatidylglycerol) were apparently synthesized in situ by intramitochondrial membrane-bound enzymes using CDP-diglycerides as intermediates. The case for the precursors in the latter pathway is less clear, although evidence has been presented for dual localization of enzymes for glycerophosphate acylation and CTP:phosphatidate cytidylyl transfer in both mitochondria and microsomes. Phosphatidylethanolamine also shows evidence for two sites of origin: by translocation from its site of synthesis in the endoplasmic reticulum and by translocation of phosphatidylserine followed by decarboxylation within the mitochondria. In the latter case mitochondrial phosphatidylserine decarboxylase may play an important role in the regulation of phospholipid metabolism throughout the cell.
Translocation of Mg2+-dependent phosphatidate phosphohydrolase between cytosol and endoplasmic reticulum in a permanent cell line from human lung
Authors: Walton Paul A. , Possmayer Fred
Published: December 2009

Incubation of A549 cells with digitonin for 4 min resulted in the release of over 90% of the lactate dehydrogenase activity into the medium. Approximately 80% of the Mg2+-dependent but only 7% of the Mg2+-independent phosphatidate phosphohydrolase activity was released in the presence of digitonin. Pretreatment of the cells with oleate reduced the efflux of the Mg2+-dependent phosphatidate phosphohydrolase activity to approximately 5% of total. Oleate did not affect the release of lactate dehydrogenase or the release of the Mg2+-independent phosphohydrolase activity. Incubation of A549 cells with [3H]oleate for 60 min led to incorporation of the label into phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, diacylglycerol, monoacylglycerol, and triacylglycerol, in ascending order. When the level of exogenous oleate was increased to over 2.0 mM, there was a marked increase in the incorporation into monoacylglycerol and diacylglycerol. Only small amounts of radioactivity were associated with phosphatidic acid. Time course studies revealed that the amount of radioactive phosphatidate remained low throughout the incubation period. These investigations were interpreted to indicate that free fatty acids can promote the translocation of the Mg2+-dependent phosphatidate phosphohydrolase activity from cytosol to membrane fractions. This translocation could, at least theoretically, function to facilitate the metabolism of increased amounts of phosphatidate.
Correlations between the 44D7 antigenic complex and the plasma membrane Na+–Ca2+ exchanger
Authors: Letarte Michelle , Quackenbush Elizabeth J. , Baumal Reuben , Michalak Marek
Published: December 2009

The exchange of Na+ for Ca2+ across the plasma membrane is mediated by a carrier transport system known as the Na+–Ca2+ exchanger. We have recently reported the specific inhibition of Na+–Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7. In this review, we summarize the properties of the 44D7 monoclonal antibody and the antigenic complex reacting with this antibody. The 44D7 antibody was produced against human acute lymphocytic cells and recognizes a molecular complex composed of two subunits of the apparent molecular weights 95 000 and 38 000, linked by disulfide bonds. Two other monoclonal antibodies react with the same complex: 4F2 which binds to the same epitope as 44D7 and specifically inhibits the Na+–Ca2+ exchanger activity, and 44H7 which reacts with a distinct epitope and does not inhibit exchanger activity. The 44D7 antibody reacts with nerve fibers in brain and proximal convoluted tubules of kidney, both known to possess Na+–Ca2+ exchanger activity. Reactivity of 44D7 antibody with tonsil and thymus sections is restricted to certain subpopulations of cells. The reactivity of the antibody is very weak with resting lymphocytes in suspension; however, activated T lymphocytes and leukemic cells show increased binding to 44D7 antibody. Several malignant cell lines express high levels of the 44D7 antigen. The reactivity of a human hepatoma with 44D7 antibody is much greater than that observed with normal hepatocytes. The inhibition by monoclonal antibody 44D7 of the Na+–Ca2+ exchanger activity and the similarity in tissue distribution of the 44D7 antigenic complex and the exchanger system suggests that these two molecules might be related. Demonstration of a direct correlation between these two entities will require the isolation of a molecule expressing both antigenic and exchanger activities.
Chemical and genetic comparison of the glucose and nucleoside transporters
Authors: Klip Amira , Walker Denise , Cohen Amos , Leung Chungyee
Published: December 2009

Glucose and nucleoside uptake into human red cells occurs through protein(s) which copurify in a complex, known as band 4.5 of relative mass (Mr) 66 000 to 50 000. The specific inhibitor of glucose transport, [3H]cytochalasin B, and the specific inhibitor of nucleoside transport, [3H]nitrobenzylthioribofuranosylpurine ([3H]NBMPR), incorporate covalently into component(s) of band 4.5 upon irradiation with ultraviolet light. Both photolabelled components are shown to be glycoproteins, since their migration in sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) is increased after treatment of photolabelled band 4.5 with endoglycosidase F. Peptide maps of the photolabelled components were compared. Red cell membranes were photolabelled with either [3H]cytochalasin B or [3H]NBMPR and subjected to SDS–PAGE. The region containing band 4.5 was cut and transferred to a second SDS–PAGE system and exposed to either papain or Staphylococcus aureus V8 protease. Papain (5 μg) completely cleaved band 4.5 and produced fragments of Mr 33 000, 26 000, 21 000, 15 000, and 12 500. Of these, the 21 000 fragment was the most conspicuous and it retained the label of [3H]cytochalasin B; the 33 000 fragment retained the label of [3H]NBMPR. The V8 protease (0.75 μg) completely cleaved band 4.5 and produced fragments of Mr 35 000, 28 000,22 000, 16 000,13 500, and 9000. The 28 000 fragment retained the label of [3H]cytochalasin B. The label of [3H]NBMPR was distributed along the gel in several regions comprising the 35 000, 28 000, and 16 000 fragments. Longer treatment with the V8 protease did not alter the position of the 28 000 [3H]cytochalasin B labelled peak, but completely abolished the [3H]NBMPR labelled peaks. Genetic segregation of the glucose and nucleoside transporters was determined in a lymphoma cell line. A mutant (14T−g) of S49 cells was selected which had lost the capacity to transport thymidine or to bind NBMPR. Uptake of either 2-deoxyglucose or 3-O-methylglucose, inhibitable by cytochalasin B, was not impaired in this mutant. It is concluded that the nucleoside and glucose transporters are glycoprotein components of band 4.5, which are differentiated by peptide map analysis. Further, a lymphoblast mutant was isolated which had lost the nucleoside transport function but retained the glucose transport function.
Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+stores
Authors: Klip Amira , Britt Beverly A. , Elliott M. Esther , Walker Denise , Ramlal Toolsie , Pegg William
Published: December 2009

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels. Further, it is suggested that the halothane-mediated increase in [Ca2+]i could be used as the basis for a noninvasive test of malignant hyperthermia.
Monoclonal antibodies to bovine and human acrosin
Authors: Elce John S. , Graham Elise J. , Zboril Gizela , Leyton Lisette , Perez Elcira , Croxatto Horatio B. , Ioannes Alfredo de
Published: December 2009

Monoclonal antibodies to human acrosin were required for studies of immunological interference with fertilization. Since human acrosin was not available in adequate amounts, monoclonal antibodies have been raised in mice against purified bovine acrosin and screened for cross-reaction with human sperm cells. Two of these antibodies are described, B4F6 and C2E5. Data from enzyme-linked immunosorbent assays, immunoblots, immunoprecipitation, and indirect immunofluorescence on sperm cells indicate that B4F6 binds only to bovine acrosin, and that C2E5 binds both to bovine and to human acrosin at a conformationally determined epitope. The antibodies do not inhibit the hydrolysis of benzoylarginine ethyl ester by acrosin, but C2E5 did inhibit the dissolution of the hamster zona pellucida by purified human acrosin. The antibodies have also been used for affinity purification of acrosin and proacrosin.
The immediate induction of extensive cell fusion by Ca2+ addition in Dictyostelium discoideum
Authors: McConachie David R. , O'Day Danton H.
Published: December 2009

In mated cultures (NC4 × V12) of Dictyostelium discoideum containing 1.0 mM CaCl2, cell fusion generates large numbers of binucleate cells which develop into zygote giant cells. In the absence of Ca2+, binucleate formation does not occur. When 1.0 mM CaCl2 is added to Ca2+-deficient cultures at 18 h, 50% of the cells fuse within 45 min producing large multinucleate syncytia. Small, presumptive gametes appear in Ca2+-deficient cultures and reach a peak of about 20% of the cell population by 10 h, but they maintain this plateau and do not fuse. Upon the addition of CaCl2, the presumptive gametes immediately fuse, producing binucleate cells which develop rapidly into morphologically distinct giant cells. Cell fusion continues, resulting in the formation of extremely large (40–80 μm diameter) multinucleate syncytia by 45 min. The induction of this extensive, synchronous cell fusion does not occur in the presence of other chloride salts and EGTA inhibits it, revealing that Ca+ is the regulatory ion.
Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells
Authors: Lund Karen , Ziola Barry
Published: December 2009

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1–2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3–4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5–6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1–3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5–6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1–2.5 h PI and then recovered to control levels by 5–6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3–fold at 1–5 h PI, but dropped to 14% below control levels during 4–8 h PI. Total phospholipid synthesis in infected cells was up 12% and down 11%, respectively, for the same two time periods. These results show that synthesis of each class of mitochondrial macromolecules is uniquely altered in cells infected by HSV 1.
Sea urchin sperm peroxidase is competitively inhibited by benzohydroxamic acid and phenylhydrazine
Authors: Schuel Herbert , Schuel Regina
Published: December 2009

Sea urchin sperm contain a phenylhydrazine-sensitive peroxidase that is believed to use hydrogen peroxide produced by the fertilized egg to reduce sperm fertility and thereby assist in the prevention of polyspermy. Strongylocentrotus purpuratus sperm were treated initially with hypotonic phosphate buffer (pH 7.0) to remove catalase and then extracted with 0.5% Triton X-100 in 0.5 M acetate buffer (pH 5.0). Peroxidase activity in this detergent extract was assayed using 3,3′,5,5′-tetramethyl benzidine (TMB) as oxidizable substrate. Kinetic studies showed that the Km for TMB is 250 μM. Benzohydroxamic acid and phenylhydrazine are known to be competitive inhibitors of a variety of plant and animal peroxidases. These substances were found to competitively inhibit the sea urchin sperm peroxidase: for benzohydroxamic acid, Ki = 51.2 μM, mean inhibitory dose (ID50) = 146.7 μM; for phenylhydrazine, Ki = 201 nM, ID50 = 303 nM. These findings (i) indicate that the biochemical properties of the sea urchin sperm peroxidase resembles those of peroxidases found in somatic tissues where oxygen radicals are produced by phagocytes to kill bacteria and (ii) support our hypothesis that the sperm peroxidase has a functional role in the prevention of polyspermy during fertilization.
Structural requirement for the recognition of luteinizing hormone releasing hormone (LHRH) by monoclonal and conventional anti-LHRH antibodies
Authors: Singh Vinod
Published: December 2009

Immunochemical studies on monoclonal and conventional anti-LHRH antibodies have been reported. The association constants (Ka) were in the order of 109–1010 L/mol when calculated from Scatchard and Steward–Petty plots. Heterogeneity indices (a) calculated from Sips plot were nearly 1.0, indicating the homogeneous nature of monoclonals. Most of the conventional anti-LHRH produced by monkey, baboon, rabbit, and dog, by immunization using LHRH linked to tetanus toxoid by the carbodiimide condensation method, showed a single slope in Scatchard analysis (except two dog antisera) and a values were nearly 1.0. Monoclonals and conventionals reacted most strongly with native LHRH(NH2). Monoclonals showed poor reactivity with LHRH free acid and LHRH fragments containing free acid. The C-terminus tetrapeptide 7–10 showed 10 times more reactivity than tripeptide 4–6. The heptapeptide 4–10 showed 100 and 1000 times more reactivity than the tetra- and tri-peptide, respectively. Introduction of the tripeptide pGlu-His-Trp-OH to heptapeptide 4–10 caused five times more inhibition in reactivity than the heptapeptide. Conventional anti-LHRH antibodies manifested specificity to the C-terminus of LHRH. These sera did not react with LHRH free acid and LHRH fragments of sequence 4–6, 7–10, and 4–10. The complete loss of reactivity of conventional antibodies and poor reactivity of monoclonal antibodies was partly regained when LHRH free acid was coupled to Lys, Lys-MDP, or (Ala)2-tuftsin, suggesting that for monoclonals and conventionals the antigenic determinant was confined to the conformation involving the C-terminus amide of LHRH.
Structure of the lipopolysaccharide O-chain of Yersinia enterocolitica serotype O:5,27
Authors: Perry Malcolm B. , MacLean Leann L.
Published: December 2009

The cellular lipopolysaccharide produced by Yersinia enterocolitica serotype O:5,27 was of the S-type and composed of an antigenic O-chain polysaccharide linked through a core oligosaccharide region, which in turn was linked through 3-deoxy-D-manno-octulonosyl units to a lipid A moiety. The O-chain polysaccharide was composed of equal molar amounts of L-rhamnose and D-xylulose. By partial hydrolysis, periodate oxidation, methylation, specific optical rotation, and 13C and 1H nuclear magnetic resonance studies, the structure of the O-chain was established as being a linear backbone of alternating 1,3-linked α-L-rhamnopyranosyl and β-L-rhamnopyranosyl units, to which 2,2-linked β-D-threo-pent-2-ulofuranoside (D-xylulofuranoside) units were present on every L-rhamnopyranosyl residue, as shown below.[Formula: see text]
Prespore and prestalk differentiation of Dictyostelium discoideum as examined by changes of wheat germ agglutinin binding proteins
Authors: Kumagai Akiko , Mori Hideo , Osuka Shinoi , Okamoto Koji
Published: December 2009

Prespore- and prestalk-specific, wheat germ agglutinin (WGA) binding proteins in Dictyostelium discoideum were identified on two-dimensional gels by the use of a peroxidase–antiperoxidase method. Using these proteins as markers, differentiation of the two presumptive cell types was examined during the development. In normal development, two groups of prespore-specific WGA-binding proteins were found: one was detectable when cells formed discrete aggregates without tips (10.5 h) and reached a maximum level at 12 h, while the other appeared at the time of slug formation (17 h). On the other hand, a prestalk-specific WGA-binding protein, having an unusually high pI value (pI ca. 9.5), began to accumulate just before slug formation (13.5 h). The changes of WGA-binding proteins in a shake culture system were similarly analysed and compared with those in normal development.
The effects of cold-temperature stress on gene expression in maize
Authors: Yacoob Reza K. , Filion W. Gary
Published: December 2009

A rapid decrease from the 28 °C incubation temperature of 5-day-old maize seedlings induced a response recorded as an altered synthesis of several polypeptides. The maximum response occurred at 4 °C and included cold-shock proteins with relative molecular masses of 94, 92, 90, 73, 70, 54, 50, 44, 38, 34, 33, 32, 24, 20, and 14 kilodaltons (kDa). Western bolt analysis (probed with polyclonal antibodies against maize heat-shock proteins) and fluorograms prepared from one-dimensional gel electrophoresis of maize heat-shock proteins revealed differences between the cold-induced polypeptides and the maize heat-shock proteins. The abundance of low molecular weight polypeptides and the absence of a marked depression in normal protein synthesis were the most noted differences from the heat-shock response. The cold-shock response, which was induced by as little as a 3 °C reduction in temperature, showed some transitory proteins, was separate from an acclimation response, and lasted up to 18 h (after returning the seedlings to 28 °C) before the normal protein synthetic pattern returned. Seedlings allowed to recover from a 4 °C shock for 4 h at 28 °C showed synthesis of a 250-kDa polypeptide, which lasted less than 2 h and was completely inhibited by actinomycin D.
Ca2+ currents and acid secretion in the isolated parietal cell involved in response to gastrin, compound 48/80, and ethylenediamine tetraacetic acid
Authors: Tsunoda Yasuhiro
Published: December 2009

In intact guinea pig parietal cells, gastrin or compound 48/80 caused an initial increase in cytosolic Ca2+ concentration and subsequent acid secretion, owing to release of intracellulary stored Ca2+ besides the Ca2+ entry from the extracellular space. However, the maximum gastrin-induced Ca2+ entry into the cell was delayed by 60 min, a time which coincided with sustained acid secretion (by gastrin) that was dependent on medium Ca2+. On the other hand, there are two ATP-dependent Ca2+-removal systems detected in either plasmalemma or smooth surfaced membrane besides that of mitochondria. The plasmalemmal Ca2+-removal system was dependent on calmodulin. Smooth surfaced membrane vesicles caused an ATP-dependent Ca2+ uptake that was almost similar to that taken by saponin-permiabilized cell. In this system (permeable cell), myo-inositol 1,4,5-triphosphate (InsP3) caused the release of ATP-accumulated Ca2+ into the cytosol, suggesting an ATP-dependent and InsP3-sensitive Ca2+ pool(s) is in or near the smooth surfaced membranes. The ATP-dependent Ca2+ uptake by vesicles was markedly enhanced by the stimulation of cells with gastrin, compound 48/80, or EDTA. The increase of this Ca2+ uptake in stimulated cells by plasmalemmal vesicles exceeded that by smooth surfaced ones. The increase of the Ca2+ uptake by plasmalemmal vesicles was abolished by the cease of intracellular Ca2+ release without Ca2+ entry. In addition, gastrin or compound 48/80 evoked an early Ca2+ efflux across the plasma membrane owing to a pump that was independent of medium Ca2+ in intact cells. These results suggest that in the first acid secretion by gastrin or others, the Ca2+ released, which may be derived from an ATP-dependent and InsP3-sensitive Ca2+ pool, is mainly pumped out by the plasmalemmal Ca2+-removal system rather than the intracellular Ca2+-removal system; whereas the sustained acid secretion by gastrin required medium Ca2+ and in this phase, Ca2+ efflux across the plasma membrane became lower, suggesting that an ATP-dependent Ca2+ pool may be replenished by Ca2+ entering from the extracellular space.
Purification and properties of two phospholipid transfer proteins from yeast
Authors: Bozzato Richard P. , Tinker David O.
Published: December 2009

Several intracellular proteins of low and intermediate molecular weights have been isolated from a variety of mammalian and plant tissues that possess an ability to catalyze the transfer or exchange of intact phospholipid molecules between different membrane systems. The soluble cytosolic fraction of the yeast Saccharomyces cerevisiae also contains phospholipid transfer activity that varies with both the state of cellular growth and the type of metabolic carbon source. This activity is protein in nature and very unstable, and requires powerful separation techniques for its purification. Here we report the isolation and characterization of two phospholipid transfer proteins from yeast, one of which we believe represents a partial proteolytic product of the other. The two proteins were purified to near homogeneity through a combination of dye–ligand and high performance ion-exchange chromatographic techniques. Transfer protein I (TP-I) is eluted at a lower ionic strength from an anion-exchange column than transfer protein II (TP-II), which reflects the difference in their isoelectric points; TP-I has a pI of 6.3, while that for TP-II is 6.1. Both species have the same apparent molecular weight of 33 400 and virtually identical substrate specificities. The order of the relative rates of phospholipid transfer are phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol > phosphatidylserine.
Catalytic properties of the yeast phospholipid transfer protein
Authors: Bozzato Richard P. , Woolley Drew , Tinker David O.
Published: December 2009

The properties of the phosphatidylcholine (PC) transfer reaction catalyzed by the yeast phospholipid transfer protein (TP-I) were examined in vitro. Donor and acceptor membranes consisted of unilamellar (ULV) and multilamellar (MLV) vesicles, respectively. The phospholipid composition of the membranes participating in the transfer reaction, and in particular that of the MLV acceptors, have a tremendous effect upon the rate of PC-catalyzed transfer. Phosphatidylethanolamine (PE) is an essential component of the acceptor membrane, but it alone is not sufficient to sustain appreciable transfer rates. If combined in an equimolar ratio with PC, there is only a modest increase in transfer rates. On the other hand, when combined with alternate substrates such as phosphatidylinositol (PI) or phosphatidylserine (PS), very high rates of PC transfer occur. The measurement of transfer rates is not affected by the molecular species of PC used as the radioactive tracer. Evidence is also presented to indicate that the two forms of the transfer protein (TP-I and TP-II) are not identical in terms of their interactions with a membrane surface: differences occur in the levels of transfer of PC, PE, PI, and PS at equilibrium. Finally, by kinetic analysis, the mechanism of the protein-catalyzed transfer of PC is shown to conform to a ping-pong bibi model with excess substrate inhibition, analogous to ordinary two-substrate enzyme-catalyzed reactions. Both the rates of desorption and adsorption of the protein from the surface of the ULV are much greater than those describing the similar interactions of the protein with MLV. In fact the rate-limiting step in this reaction is desorption of the protein from the MLV surface.
Effects of oxytocin and vasopressin on the preadipocyte 3T3-F442A cell line
Authors: Wilson E. Jane , Hollenberg Morley D.
Published: December 2009

The 3T3-F442A mouse fibroblast cell line, triggered by factors present in fetal calf serum (FCS), converts either spontaneously or, in the simultaneous presence of FCS and insulin, at an accelerated rate into cells exhibiting the adipocyte phenotype. The effects of the neurohypophysial hormones in differentiated cells on glucose metabolism (glucose oxidation and lipogenesis) were compared with the stimulatory actions of insulin, which had its most pronounced effects in cells differentiated spontaneously with FCS in the absence of insulin. The differentiated 3T3-F442A cells were sensitive to physiological levels of insulin and exhibited manyfold increases in glucose metabolism in response to it. This result demonstrated that these cultured cells respond to insulin, in a manner analogous to freshly isolated adipocytes. In contrast to its insulin-like effects in isolated epididymal adipocytes, oxytocin was not reproducibly able to stimulate glucose metabolism in differentiated 3T3-F442A cells. Vasopressin was similarly inactive. In contrast, both oxytocin and vasopressin blocked adipocyte conversion triggered by FCS, either in the presence or absence of insulin; vasopressin was more potent than oxytocin, indicating that a vasopressin receptor was responsible for the observed inhibition of differentiation. Our work suggests that vasopressin could potentially play a role in the regulation of the adipocyte differentiation process.
Partial purification and characterization of a neutral proteinase with collagen telopeptidase activity produced by human gingival fibroblasts
Authors: Nakano Takuo , Scott Paul G.
Published: December 2009

A neutral proteinase was purified 1930-fold from medium conditioned by the culture of human gingival fibroblasts that had been stimulated to secrete enzymes by concanavalin A. This enzyme had an apparent molecular weight of 35 000 (gel chromatography) and apparent isoelectric point of 4.3 (chromatofocusing). It was inhibited by chelating agents, serum, and nonactivated conditioned fibroblast medium, but not by phenylmethylsulphonyl fluoride or N-ethylmaleimide. This proteinase removes the C-telopeptide from the α1 chain of type I collagen, an activity which could be important in the degradation of collagen in the extracellular matrix. It was also found to digest fibronectin but had no effect on proteodermatan sulphate under the conditions used. It appears to be unrelated to previously described fibroblast extracellular proteinases and we, therefore, tentatively propose the name fibroblast metalloproteinase IV.
The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells
Authors: Pan Y. T. , Hori Hidetaka , Elbein Alan D.
Published: December 2009

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7–9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 μg/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.
The effect of linoleic acid on the sensitivity of human lymphocytes to Cortisol and their capacity to catabolize the steroid
Authors: Klein A. , Bruser B. , Malkin A.
Published: December 2009

We have shown previously that cortisol-sensitive lymphocytes (thymocytes) have a much lower capacity than cortisol-resistant cells to catabolize cortisol. In the present study, we attempt to demonstrate that inhibition of cortisol catabolism may make cortisol-resistant lymphocytes vulnerable to the steroid. Linoleic acid, which has the capacity to inhibit the catabolism of cortisol by lymphocytes, was used for this purpose. By using various concentrations of linoleic acid (20–60 μg/mL) we showed an inverse linear relationship between linoleic acid concentration and the rate of cortisol catabolism by lymphocytes. During this experiment which took 17 h the viability of cells did not change significantly (minimum viability 95%), even at the highest concentration of linoleic acid. Keeping the metabolism of cortisol at a level of 40% of that obtained by the control, by adding linoleic acid to lymphocyte cultures (50 μg/mL) and measuring the viability of the cells for a period of 3 days in the presence or absence of cortisol, we were able to show a rise in the death rate of the cells which started after 24 h of incubation owing to the presence of the steroid.
Structural studies of the capsular polysaccharide from Haemophilus pleuropneumoniae serotype 2
Authors: Altman Eleonora , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

The capsular polysaccharide of Haemophilus pleuropneumoniae serotype 2 (ATCC 27089) is composed of D-glucose (two parts), D-galactose (one part), glycerol (one part), and phosphate (one part). Hydrolysis, dephosphorylation, methylation, enzymic studies, and 1H and 13C nuclear magnetic resonance experiments showed that the polysaccharide is a high molecular weight polymer of a tetrasaccharide repeating units, linked by monophosphate diester and having the following structure:[Formula: see text]
The formation and decay of the oxyferrous complex of beef adrenocortical cytochrome P-450scc. Rapid-scan and stopped-flow studies
Authors: Kashem Mohammed A. , Dunford H. Brian
Published: December 2009

The formation and spontaneous decay of the oxyferrous complex of purified beef adrenocortical cholesterol-bound (high spin) cytochrome P-450scc have been studied by means of rapid-scan spectrometry in the Soret region at 4 °C. The oxyferrous complex, the formation of which occurs within 40 ms with a Soret absorption peak at 422 nm, is unstable and decays spontaneously to the ferric cholesterol-bound cytochrome P-450scc. The rapid-scan spectra for both processes were recorded. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous complex at 418 nm, between oxyferrous complex and ferric cytochrome P-450scc at 411 nm. The kinetics of oxygen binding and spontaneous decay of the oxyferrous complex have also been studied at 4 °C by means of stopped-flow experiments in the pH range 5.1–8.8. The rate constant for oxygen binding is constant at 5.8 × 105 ± 0.8 × 105 M−1∙s−1 over the pH range of the study. On the other hand, the decay process exhibited pH-dependent monophasic first-order kinetics. The rate constant for the decay appears to be influenced by an acid group with a pKa of 7.1 on the oxyferrous complex of cholesterol-bound cytochrome P-450scc.
Partial characterization of the heteropolysaccharide associated with the 7S domain of type IV collagen from placenta
Authors: Leushner James R. A.
Published: December 2009

A major heteropolysaccharide fraction was isolated from the 7S domain of human placental type IV collagen. Analyses revealed that it was an asparagine-linked oligosaccharide. Characterization using molecular sieve chromatography, exoglycosidase and endoglycosidase digestion, and chemical analysis suggested a bianternnary complex with the following structure:[Formula: see text]A microheterogeneity was noted with respect to the addition of the fucose and sialic acid residues. Analysis of component polypeptides of the 7S fraction following endoglycosidase treatment suggested that the most obvious site of heteropolysaccharide attachment was in a polypeptide of relative mass 40 000. Amino acid analysis of this isolated polypeptide indicated that it was rich in collagenous sequences and also contained half-cystine residues.
The involvement of mRNA secondary structure in protein synthesis
Authors: Pelletier Jerry , Sonenberg Nahum
Published: December 2009

Translation initiation in eukaryotes is a complex process involving many factors. A key step in this process is the binding of mRNA to the 43S preinitiation complex. This is generally the rate-limiting step in translation initiation and consequently a major determinant of mRNA translational efficiency. The primary and secondary structure of the mRNA 5′ noncoding region have been implicated in modulating translational efficiency. Translational efficiency was shown to be inversely proportional to the degree of secondary structure at the mRNA 5′ noncoding region. Furthermore, it was shown that cap-binding proteins that interact with the 5′ cap structure (m7GpppN) of eukaryotic mRNAs are involved in the "unwinding" of the mRNA secondary structure, in an ATP hydrolysis mediated event, to facilitate ribosome binding. Thus, cap-binding proteins can potentially regulate mRNA translation. Here, we discuss the available data supporting the notion that eukaryotic 5′ mRNA secondary structure plays an important role in translation initiation and the possible regulation of this process.
Small cytoplasmic RNAs and their location within the cytoplasm
Authors: Bird R. Curtis , Sells Bruce H.
Published: December 2009

These studies were designed to establish the location of various species of small RNAs within the subcellular cytoplasmic compartments. Four cytoplasmic RNA-containing compartments were examined: (A) cytoskeleton-bound polyribosomal ribonucleoprotein (RNP) complexes, (B) soluble-phase polyribosomal RNP complexes, (C) cytoskeleton-bound free RNP complexes, (D) soluble-phase free RNP complexes. The presence of the small cytoplasmic RNA (scRNA) population and histone H4 and actin mRNAs in each compartment was examined to determine their spatial distribution within the cytoplasm. The 7S signal recognition RNA and the 5S and 5.8S rRNAs were distributed among all four compartments, while 4S tRNAs were localized largely in fraction D. Fraction C contained a group of seven abundant scRNAs, of approximately 105–348 nucleotides in length, which were localized almost entirely within the cytoskeleton-bound free RNP compartment. Actin mRNAs were localized in fraction A, the actively translating cytoskeleton-bound compartment. Following cytochalasin B treatment, actin mRNAs were released into the soluble phase, implicating a dependence on the integrity of actin filaments in its binding. Such treatment also released several of the scRNAs from their cytoskeleton-bound location. In contrast histone H4 mRNAs were much more widely dispersed, being present in all four cytoskeletal compartments. Approximately 60% of the H4 mRNAs, however, were localized within the soluble-phase polyribosomes in fraction B. Cytochalasin B treatment released only the small portion of untranslated histone H4 mRNA associated with the cytoskeleton in fraction C, suggesting that the binding of these H4 mRNAs was dependent in some manner upon the integrity of actin filaments.
On the presence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function
Authors: Gallo-Payet Nicole , Pothier Pierre , Isler Henri
Published: December 2009

Using light and electron microscopy, we have observed the presence of rays containing medullary tissue extending across the cortex of rat adrenal glands. Within these rays chromaffin cells, as well as collagen and nerve fibers, were present. It is suggested that these endocrine cells may have a paracrine function within the cortex, possibly via their secretory product.
Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa
Authors: Barua Madhabi , Majumder Gopal C.
Published: December 2009

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, β-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.