bluuebunny/crossref_metadata_2025 huggingface dataset:
The subunit and polypeptide structure of hexosaminidases from human placenta
Authors: Mahuran D. , Lowden J. A.
Published: December 2009

Previous reports have indicated that the hexosaminidases (HEX) are tetrameric enzymes composed of either two dimers of β chains (HEX B) or a β-chain dimer and an α-chain dimer (HEX A). HEX S contains only α chains. The apparent molecular weight of both α and β chains is 25 000. We isolated and purified HEX A and B more than 6000-fold. From HEX A we prepared HEX B and a more anodically migrating HEX S, which contained only α chains. After either extensive reduction and alkylation or performic acid oxidation, HEX B gave only a single protein band in polyacrylamide gel electrophoresis in sodium dodecyl sulfate; HEX A consistently gave two bands indicating it contained chains of two different molecular weights. Other reports note two bands in HEX A but have dismissed the high molecular weight band as a dimer caused by either hydrophobic interaction or an unbroken disulfide bond. To rule out hydrophobic interaction, we determined molecular weight by gel filtration in 6 M guanidine–HC1. To ensure complete disulfide bond breakage we used a harsher reduction and alkylation procedure than previously employed by others. The results were then confirmed by the use of two performic acid oxidation techniques, the stronger method resulting in extensive peptide bond oxidation. Samples of HEX A under reduction and alkylation or the weaker performic acid oxidation procedure, again chromatographed as two approximately equal peaks (50 000 and 25 000). HEX B, and the HEX B prepared from HEX A, resulted in one 25 000-dalton peak. HEX S chromatographed as a single 50 000 peak. We conclude that either the α chain in HEX has a molecular weight of 50 000 with one α chain per α subunit or that two 25 000-dalton chains are united by a nondisulfide crosslink (e.g., an isopeptide bond).
DNA sequence organization in the starfish Dermasterias imbricata
Authors: Smith Michael J. , Lui A. , Gibson K. K. , Etzkorn J. K.
Published: December 2009

The sequence arrangement in the genomic DNA of the starfish Dermasterias imbricata has been examined. Analyses of kinetics of reassociation in solution at DNA lengths of 400, 3100, and 6200 base pairs (bp) demonstrate the interspersion of repetitive and unique DNA. At a fragment length of 400 bp, 45% of the DNA reacts at a rate appropriate for single-copy sequences in a genome of this size (0.54 pg). Interspersion of repetitive sequences is also demonstrated by the reduced hyperchromicity of 3100- or 6200-bp fragments reacted to Cot 10, where only repetitive sequences have formed duplex. S1 nuclease digestion of 3100-bp fragments reassociated to Cot 10 shows that both short (~ 230 bp) and long (≥ 2600 bp) repetitive sequences are present in this DNA. These data demonstrate a short period interspersion pattern in Dermasterias.
Binding of chemical derivatives of Phaseolus vulgaris phytohemagglutinin to pig spleen lymphocytes
Authors: Dupuis Gilles , Clairoux-Moreau Sylvie
Published: December 2009

The mitogenic activity of Phaseolus vulgaris phytohemagglutinin (PHA) and its acetyl, citraconyl, glycinyl, homoarginyl, N-bromosuccinimide, and nitro derivatives has been assayed using pig spleen lymphocytes. Nitrated and N-bromosuccinimide-treated PHA were not mitogenic. Whereas the mitogenicity of acetyl PHA was impaired, the other derivatives were as active as untreated lectin. Binding studies of [125I]PHA and derivatives to purified pig spleen lymphocytes were performed at 37 °C. Results show that the lymphocytes bound various amounts of lectin derivatives. Maximum amounts were observed in the case of mitogenic lectins. Data were analyzed by Scatchard plots and two kinds of binding sites were found for PHA and the mitogenic derivatives. High affinity and low affinity binding sites have been characterized in terms of (apparent) association constant and (apparent) number of sites. Binding of PHA or its chemical derivatives to a small fraction (4.6–9.0%) of all available lectin binding sites was sufficient for maximal lymphocyte stimulation. Nonmitogenic PHA derivatives (nitro or N-bromosuccinimide) bound, with low affinity, to one kind of binding site. The relevancy of these findings to lymphocyte activation is discussed.
Synthesis of very high molecular weight thyroglobulin in three heterologous systems
Authors: Eggo Margaret C. , Burrow Gerard N.
Published: December 2009

Calf thyroid RNA has been translated in the wheat germ and messenger-dependent reticulocyte lysate cell free systems and in Xenopus laevis oocytes. Peptides immunologically related to and comigrating with 330 000 molecular weight thyroglobulin as well as both larger and smaller immunopeptides have been synthesized. The smaller molecular weight peptides were due at least in part to degradation during incubation despite the presence of protease inhibitors. The larger molecular weight peptides could represent degradation products of a single polypeptide chain with a molecular weight of 660 000. The 330 000 molecular weight peptides would then represent cleavage of the protomer into two subunits. To support this possibility, thyroid polysomal RNA large enough to code for a peptide with a molecular weight of 660 000 has been shown to be active in thyroglobulin synthesis and to maintain its integrity under denaturing conditions.
Cytochrome c reduction by cysteine plus copper: a pseudosubstrate system for cytochrome c oxidase
Authors: Hill Bruce C. , Nicholls Peter
Published: December 2009

Cysteine alone reduces horse heart cytochrome c very slowly [Formula: see text] with a rate constant virtually identical in high and low ionic strength buffers. Copper catalyzes this reaction increasing the rate by a factor of 105 in 50 mM phosphate and by a factor of 106 in 10 mM Tris buffers. When ferricytochrome c and cysteine are mixed in an oxygen electrode a "burst" of oxygen uptake is seen, the decline in which parallels the reduction of cytochrome c. When cytochrome c oxidase is added to such a mixture two routes of electron transfer to oxygen exist: enzymatic and ferricytochrome c dependent nonenzymatic. Both processes are sensitive to cyanide, but azide inhibits only the authentic cytochrome c oxidase catalyzed process and BCS the ferricytochrome c stimulated reaction.
Purine synthesis de novo and its regulation in rat hepatocytes
Authors: Rosiers Christine Des , Lalanne Marcel , Willemot Joan
Published: December 2009

Purine synthesis de novo and its regulation were studied in freshly isolated hepatocytes from fed adult male rats. These cells incorporated [14C]formate mainly into purine ribonucleotides. The immediate effect of increasing the concentration of inorganic phosphate in the incubation medium was an increase in 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) availability and a stimulation of purine synthesis de novo. However, prolonged incubation of cells in 25 mM phosphate resulted in a decreased PP-ribose-P availability and purine synthesis de novo. Methylene blue and phenazine methosulfate decreased PP-ribose-P availability and purine synthesis de novo although they stimulated considerably the pentose phosphate pathway. In contrast, epinephrine and glucagon increased significantly PP-ribose-P availability and purine synthesis de novo, but they did not change the activity of the pentose phosphate pathway. These results show a relationship between PP-ribose-P availability and purine synthesis de novo in rat hepatocytes. They emphasize the complexity of the regulation of PP-ribose-P availability.
Isolation of deoxyribonuclease II from bovine intestinal mucosa
Authors: Keys D. Stephen , Zbarsky S. H.
Published: December 2009

Mucosa from bovine small intestine was homogenized in Krebs–Ringer phosphate buffer, pH 7.8, the homogenate centrifuged at 16 300 × g, and the supernatant solution filtered through cheesecloth to remove lipid material. The filtrate was centrifuged at 105 000 × g and the supernatant solution chromatographed on DEAE-cellulose. The major peak of DNase II activity, eluted with 20 mM phosphate – 10 mM EDTA buffer, pH 7.8, was purified further by ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-100. The enzyme was purified 78-fold in 13% yield. Evidence was adduced to indicate that the second minor peak of DNase II activity, eluted from the DEAE-cellulose by a potassium chloride gradient in the 20 mM phosphate – 10 mM EDTA buffer, was an artifact arising from the presence of significant amounts of DNA in the 105 000 × g supernatant. The enzyme degraded DNA endonucleolytically to 3′-PO4, 5′-OH oligonucleotides and is similar in its properties to DNase II from other tissues.
Citrate transport in Salmonella typhimurium: studies with 2-fluoro-L-erythro-citrate as a substrate
Authors: Ashton D. M. , Sweet G. D. , Somers J. M. , Kay W. W.
Published: December 2009

The citrate analogue, 2-fluoro-L-erythro-[3,4,5,6-14C]citrate was synthesized as a probe for the citrate transport system of Salmonella typhimurium. This analogue was actively transported by an inducible energy-dependent transport system with high affinity for fluorocitrate (Km = 3.3 μM), and this transport system was inhibited competitively by citrate and isocitrate. Fluorocitrate was shown to be a competitive inhibitor of the citrate-binding protein (C protein) of this organism (Ki = 4–5 μM). Analogue resistant mutants were simultaneously defective in fluorocitrate transport as well as the C protein and the affected allele, tctC, was located at 59 units on the S. typhimurium chromosome map. These tctC mutants were shown to be specifically defective in K+-dependent fluorocitrate transport but still retained another system capable of transporting fluorocitrate in the presence of both Na+ and K+.
Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12
Authors: Bewick Mary A. , Lo Theodore C. Y.
Published: December 2009

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is released by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radio-iodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA – osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA – osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA – osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.
Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins
Authors: Holland Paul C. , Cates George A. , Wenger Byron S. , Raney Barbara L.
Published: December 2009

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate – polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.
Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds
Authors: Dixon Nicholas E. , Riddles Peter W. , Gazzola Carlo , Blakeley Robert L. , Zerner Burt
Published: December 2009

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 °C, kcat has the values 5870, 85, 0.55, and 0.075 s−1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato–enzyme intermediate.
Relationship between membrane fluidity and capping of receptors for concanavalin A
Authors: Mak W. Wai-Nam , Wong J. Tze-Fei
Published: December 2009

The activities of a range of phenylalaninol-related compounds on capping of concanavalin A and induction of rounding of Chinese hamster ovary tsHl cells, as well as on the fluidity of phosphatidylcholine–cholesterol (1:1) liposomes, have been examined. These compounds include phenylalanine, histidinol, leucinol, benzyl alcohol, benzylamine, 2-phenylethanol, 2-phenylamine, 3-phenyl-1-propanol, 3-phenyl-1-propylamine, and 3-phenylpropionic acid. The results indicate a strong correlation between the capacities of these compounds to enhance fluidity and their capacities to inhibit capping of concanavalin A. The specificity of this correlation is suggested by the finding that both types of capacities are poorly correlated with the capacities of the various compounds to induce cell rounding.
Metabolism of NG-monomethyl-L-arginines: formation of NG-methylagmatine by Escherichia coli preparation
Authors: Paik Woon Ki , Kim Sangduk , Hutchins Mary Gail Kinzer , Swern Daniel
Published: December 2009

Commercially obtained Escherichia coli preparation degrades NG-monomethyl-L-arginine derived from methylation of protein-bound arginine residues. Elemental analysis and NMR spectroscopy established the reaction product as NG-methylagmatine, a decarboxylation product of NG-monomethyl-L-arginine.
The specificity of sialyltransferase activity in smooth membrane fractions of embryonic chicken liver
Authors: Bendiak Brad , Zalik Sara E.
Published: December 2009

Smooth membrane preparations of 13-day embryonic chicken livers, characterized by electron microscopy and marker enzyme analyses, have been found to contain sialyltransferase activity which displayed precise acceptor specificity. One sialyltransferase transferred N-acetylneuraminic acid (NANA) to galβ1 → 4glcNAcβ1 → R structures. Evidence based on competition studies suggests that a second enzyme is present transferring this sugar to a galβ1 → 3galNAcα1 → R structure. The enzyme capable of adding NANA to galβ1 → 4glcNAcβ1 → R structures has a pH optimum of 5.5, a temperature optimum of 30 °C, and half-saturating values of 17 μM for CMP-NANA and 180 μM for galactoside termini on desialyzed α1-acid glycoprotein. It is activated about 10-fold by Triton X-100, has no exogenous divalent cation requirement, and is inhibited by CTP, CDP, and CMP. The enzyme requires carbohydrate structures underlying the galβ1 → 4glcNAc terminus for maximal catalytic activity; the necessity of such precise specificities of sialyltransferases is discussed in the light of recent structural evidence for the carbohydrate moieties of several glycoproteins.
Mast cell–lysosomal cationic protein interaction: effects of metabolic inhibitors and decay of activated cells on enhanced fluorescence of anilinonaphthalene sulfonate
Authors: Ranadive Narendranath S. , Ruben Deborah H.
Published: December 2009

It has been shown earlier that the interactions of the isolated rat peritoneal mast cells with cationic protein from rabbit neutrophil lysosomes (band 2 protein) can be studied using anilinonaphthalene sulfonate (ANS) as a fluorescent probe. In the present communication, binding of ANS dye to the mast cells interacting with band 2 protein (B2) under various conditions has been reported. The inhibition of histamine release by metabolic inhibitors was found to have no effect on enhancement of ANS fluorescence. On the other hand, inhibition of histamine release at high concentration of Ca2+ (14.4 mM) was accompanied by the decrease in enhanced fluorescence. In the presence of 7.2 mM of Sr2+, the release of histamine was enhanced with small but significant increase in ANS fluorescence. The cells heated to 42 °C partially lost their capacity to release histamine without the loss of enhanced fluorescence. The mast cells interacting with B2 at 10 °C for various time intervals showed time-dependent loss in histamine releasing capacity with concomitant loss in enhanced fluorescence. These studies suggest that the enhancement of ANS fluorescence is associated with the early events of the cell membrane caused by interaction of B2 with the cells. The extracellular cations significantly influence this early event.
Thermodynamics of lipid–proiein associations: the enthalpy of binding of Apo C-III to synthetic phosphatidylcholines
Authors: Pownall Henry J. , Massey John B. , Hsu Fu-Juan , Gotto Jr. Antonio M.
Published: December 2009

The association of synthetic phosphatidylcholines with apolipoprotein (apo) C-III, an apoprotein from human plasma very low density lipoproteins, has been studied by gel filtration, microcalorimetry, and differential scanning calorimetry. Apo C-III associates with a minimum of 35 molecules of dimyristoyl phosphatidylcholine (DMPC) and can accommodate up to 100 molecules of DMPC per apo C-III molecule. These complexes are readily isolated by chromatography on Sepharose CL-4B. Microcalcrimetry of apo C-III with DMPC or dipalmitoyl phosphatidylcholine (DPPC) vesicles reveals a high enthalpy of association of apo C-III with DMPC (−250 kcal∙mol apo C-III−1) and DPPC (−490 kcal∙mol apo C-III−1); these values translate into 2.8 and 4.65 kcal∙mol lipid−1, respectively. These high enthalpy values are observed only near the transition temperatures (Tc) of the lipids. Above and below Tc the enthalpy of association was practically zero. Differential scanning calorimetry of DMPC –apo C-III complexes shows them to be composed of 35–60 mol lipid∙mol apo C-III−1 in which no more than 45 molecules of DMPC form a boundary around apo C-III. It is shown that the effect of apo C-III on the thermal properties of DMPC is to elevate the Tc of the lipid and that an important component of the enthalpy of association of apo C-III with DMPC is due to the crystallization of the lipid acyl chains. Since the enthalpy of association is a term in the free energy of association, we suggest that the apo C-III induced crystallization of DMPC is important in the thermodynamics of this lipid–protein association. Generally, the sign and magnitude of the enthalpies of association should be important in predicting the distribution of lipids and proteins in more complex systems.
Isolation and characterization of 3-acetamido-3,6-dideoxy-L-glucose from the core oligosaccharides obtained from the aquatic gram-negative bacteria Aeromonas hydrophila and Vibrio anguillarum
Authors: Banoub Joseph H. , Shaw Derek H.
Published: December 2009

The amino sugar 3-acetamido-3,6-dideoxy-L-glucose has been isolated and characterized from the core oligosaccharide obtained from the bacterial lipopolysaccharides of Aeromonas hydrophila and Vibrio anguillarum. This is the first occasion in which a dideoxyamino sugar has been confirmed as a constituent of the core oligosaccharide rather than the O-polysaccharide.
Purification of liver cytosolic proteins that stimulate triacylglycerol synthesis
Authors: Roncari Daniel A. K. , Mack Esther Y. W.
Published: December 2009

Heat-stable rat liver cytosolic proteins that stimulate triacylglycerol synthesis catalyzed by adipose tissue or liver microsomal enzymes have been isolated in a highly purified state. Their molecular weight ranges were found to be 35 000 – 45 000, 20 000 – 28 000, and 8000 – 12 000. The protein with molecular weight 20 000 – 28 000 and pI 7.3–7.7 was purified 4716-fold. The cytosolic proteins were stable at 85 °C for 15 min and were inactivated by proteases. They did not reveal any intrinsic phosphatidate phosphohydrolase or diacylglycerol acyltransferase activity, but led to enhanced conversion of phosphatidate to diacylgiycerol and triacylglycerol.
Chromatin-associated deoxyribonuclease activity in liver nuclei isolated from Rana catesbeiana froglets and premetamorphic and T3-induced tadpoles
Authors: Harvey William , Atkinson Burr G.
Published: December 2009

Nuclei and chromatin isolated in the presence of calcium or magnesium from Rana catesbeiana liver tissue exhibit considerable endogenous deoxyribonuclease activity. This activity is present in liver nuclei isolated from froglets as well as in liver nuclei isolated from untreated and thyroid hormone treated premetamorphic tadpoles. Nuclei and chromatin isolated in the absence of divalent cations and in the presence of spermine exhibit no detectable expression of the endogenous deoxyribonuclease activity. The endogenous deoxyribonuclease present, but not expressed, in spermine-isolated tadpole liver nuclei or chromatin is salt extractable. Once dialyzed, the salt-extracted deoxyribonuclease is activated by calcium or magnesium. This deoxyribonuclease shows maximal enzymic activity in 15 mM calcium at pH 8.0 or in 15 mM magnesium at pH 7.4. After Ca2+ activation, deoxyribonuclease activity is maximally inhibited by amounts of spermine similar to that required to completely inhibit DNase I. Destruction of the salt-extracted deoxyribonuclease activity by treatment with proteinase K or heat suggests that it is of a proteinaceous nature. The localization and nature of this enzymic activity established that it is associated with the salt-soluble proteins affiliated with tadpole and froglet liver chromatin.
Cation and local anesthetic conductance induced by the negatively charged ionophore Br-X537A in lipid bilayers
Authors: Roy G. , Okada Y. , Laprade R.
Published: December 2009

The cation conductance induced by Br-X537A was measured for K+ and Ca2+ at different pH and different Br-X537A concentrations. The K+-concentration dependence was different depending on the pH of the solution. At pH 4.3, the conductance depended linearly on [K+] while at pH 7 it depended on [K+]2. About the same dependence on [Br-XS37A] was found at these pH values. From these results it was concluded that two complexes were transporting K+; at pH ≥ 7 the complex is mostly K2X3− and at pH ≤ 5 the complex is mostly KHX3−. From the conductance dependence on [Ca2+], pH, and [Br-X537A] it was found that the only conducting complex is CaX3−. It was also observed that Br-X537A could transport tetracaine. Taking into account the surface charge induced by tetracaine, the conductance depended linearly on the tetracaine concentration. Correcting for the surface charge induced by Br-X537A, the tetracaine conductance was found to depend on [Br-X537A]2. Consequently, the conducting complex is TX2− at pH ≥ 7. No other conducting complexes are formed as the pH is decreased and at pH 4 tetracaine is not conducted. It was also found that procaine was not conducted between pH 8 and 4.
Potential of intense γ-irradiation of host cells for analysis of virus-specified transcription and replication
Authors: Silver Morris , Dales Samuel
Published: December 2009

Intense γ-irradiation from a cobalt source differentially affects macromolecular synthesis of cultured mammalian cells. Exposure of monkey BSC-1 or murine fibroblastic L2 cells to 40 or 70 krad (1 rad = 1 × 10−2 J/kg) abolishes DNA and RNA synthesis almost entirely but reduces the formation of protein much less. A dose–response analysis of irradiation shows that synthesis of total RNA and the messenger component thereof, measured as the poly(A)-containing fraction, are equally diminished. Host cells in which formation of DNA and RNA are minimal can support normal or nearly normal replication and transcription rates of vesicular stomatitis and vaccinia viruses. Therefore, use of pretreatment with γ-irradiation, as employed here, should prove to be generally useful in examining virus-related transcription under circumstances in which application of drugs affecting gene expression, such as actinomycin D, is deemed undesirable.
Meiotic arrest and synaptonemal complexes in yeast ts spo 10 (Saccharomyces cerevisiae)
Authors: Moens Peter B. , Kundu Subhas C.
Published: December 2009

The yeast strain spo 10 is characterized by meiotic prophase arrest at 34 °C but not at 26 °C. At either temperature normal meiotic S phase is followed by prophase where each nucleus contains a set of duplicated spindle pole bodies, synaptonemal complexes (SCs), and frequently a nuclear-dense body (ndb). In arrested cells the SCs become prominent but fragmented and 50% or more of the ndbs contain polysynaptonemal complexes. It is assumed that spo 10 arrest occurs prior to shutdown of SC production but does not affect SC production itself, thus causing an accumulation of SC material in the ndb.
Effect of heat shock on gene expression in human epidermoid carcinoma cells (strain KB) and in primary cultures of mammalian and avian cells
Authors: Atkinson Burr G. , Pollock Michael
Published: December 2009

The brief incubation of human epidermoid carcinoma (KB) cells, and of primary cultures of quail myoblasts and hamster fibroblasts, at an elevated temperature causes the pattern of gene expression to shift from the production of a broad spectrum of different proteins to the enhanced synthesis of a small number of heat-shock proteins. Comparison of the heat-shock polypeptides synthesized by each of these vertebrate cells demonstrates the similarity of some, as well as the uniqueness of other, heat-inducible gene products synthesized by cells from different vertebrates. A major polypeptide, commonly synthesized in response to heat by each of these vertebrate cells, has an apparent molecular weight of 64 000 and an isoelectric point of 5.8. Triton X-100 completely extracts this polypeptide from quail myoblasts and hamster fibroblasts, and partially extracts it from KB cells. This particular response to heat shock, by cells from different vertebrates, suggests that it may involve the expression of a gene(s) with an analogous and potentially crucial cellular function. This specific heat-shock polypeptide, as well as others, is not detectably synthesized in quail cells prior to heat shock or 6–8 h after recovery from heat shock which suggests that in this cell type it may be a product of a normally quiescent gene(s) and that its expression is subject to thermal regulation.
Identification of gene products from cloned fragments of the left arm of λdapB2
Authors: Mackie George A. , Parsons Gay D.
Published: December 2009

A set of recombinant plasmids encompassing the bacterial substitution in λdapB2 (Mackie, G. A. (1980) J. Biol. Chem. 255, 8928–8935) has been characterized genetically by complementation and biochemically by an analysis of the proteins encoded by individual plasmids in vitro. In addition to a cluster of four genes which includes the dapB locus, a total of seven gene products has been identified. One of these is ribosomal protein S20, whose gene (rpsT) has been localized to a 0.56-kilobase segment of DNA bounded by HindIII and HindII sites. Positive identification of this gene on plasmids pGM9 and pGP2 has been achieved by analysis of the products encoded by these plasmids in vitro and by the ability of pGM9 to complement a strain lacking S20 among its 30S subunit proteins. A second gene is that for isoleucyl tRNA synthetase (ileS). Its presence on pGM21 has been ascertained by the latter's ability to direct the synthesis of a protein in vitro with the size anticipated for this gene product. Extracts of cells harbouring this plasmid also exhibit greater isoleucyl tRNA synthetase activity than parental extracts. A third gene is at least 1.3 kilobases distant from rpsT and encodes a protein of 24 000 molecular weight, of unknown function. This locus is in turn about 6.5 kilobases from λ gene E. The latter gene, and several others, all of which likely derive from λ DNA are carried on a 5.2-kilobase fragment inserted in the piasmid pGM11 which includes the junction between phage and bacterial sequences on the left side of λdapB2. This fragment of DNA is inserted into the vector in a manner which permits the coupled transcription and translation of λ genes D and E in vitro, despite the absence of their natural promoter. The most striking feature of the organization of the bacterial genes on λdapB2 is the clustering of genes for polypeptides in the right half of the bacterial substitution in contrast to the apparent minimal use of the coding potential in the left half of the substitution.
Induction of a novel set of polypeptides by heat shock or sodium arsenite in cultured cells of rainbow trout, Salmo gairdnerii
Authors: Kothary R. K. , Candido E. P. M.
Published: December 2009

The heat-shock response has been characterized in cultured fibroblasts of the rainbow trout, Salmo gairdnerii. The response has been elicited by two different stress situations; cells were either subjected to higher temperatures than normal (27 to 29 °C as opposed to 22 °C) or were incubated in medium containing sodium arsenite (15 to 100 μM final concentration). The response of the cells to these conditions is to synthesize a set of new polypeptides, the "heat-shock polypeptides" (hsps), that are not present or present in extremely low amounts in noninduced cells. Furthermore, during prolonged arsenite induction, the synthesis of normal cellular proteins is repressed. In trout fibroblasts, at least six hsps are detectable. These range from 30 000 to 87 000 in molecular weight and are referred to as hsp30, hsp32, hsp42, hsp62, hsp70, and hsp87. The hsp30 and hsp70 components are the most abundant and can be visualized by Coomassie blue staining of gels after prolonged induction. The heat-shock response is a reversible process in trout cells. Results of in vitro translation of mRNA from induced cells indicate that the control of hsp induction may be at the transcriptional level. Hsp70 from trout comigrates with the major hsp from Drosophila melanogaster on sodium dodecyl sulfate – polyacrylamide gels, suggesting that this protein may be highly conserved in evolution.
DNA binding proteins from Tetrahymena thermophila
Authors: Tsao Nora N. G. , Pearlman Ronald E.
Published: December 2009

We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila. Three major proteins which bind to denatured DNA-cellulose were obtained. The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate – polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources. The protein facilitates denaturation of the synthetic copolymer poly[d(A-T)∙d(A-T)], depressing the melting temperature by nearly 40 °C. It also permits the renaturation of poly[d(A-T)∙d(A-T)] in high salt concentration. Two other binding proteins have molecular weights of 25 000 and 23 000 in sodium dodecyl sulfate – polyacrylamide gel electrophoresis. The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase. These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities. These three proteins are abundant in the cell with approximately 1.0 × 106 to 10.0 × 106 molecules of each protein monomer per cell. One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay. Peptide mapping of the three proteins suggests that they are all distinct. We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.
A calorimetric study of the thermal transitions of Halobacterium cutirubrum
Authors: Cho K. C. , Chow K. C. , Mark K. K.
Published: December 2009

The thermal transitions of Halobacterium cutirubrum have been examined by differential scanning calorimetry. Two distinct peaks corresponding to the denaturation of two major protein components were observed in the heating curves. One of the peaks has been assigned to the denaturation of the envelope glycoprotein. The variations of the denaturation temperatures with the addition of glucose, glycerol, NaNO3, and NaSCN are consistent with the previous proposal that hydrophobic interactions are essential in stabilizing the glycoprotein.
Photolysis of pyridinoline, a cross-linking amino acid of collagen, by ultraviolet light
Authors: Sakura Sachiko , Fujimoto Daisaburo , Sakamoto Kikuo , Mizuno Akio , Motegi Katsutoshi
Published: December 2009

Pyridinoline, a cross-linking amino acid of collagen, was degraded by irradiation of ultraviolet light. The decomposition rate varied with pH of the solution and wavelength of irradiation light. The maximum of the degradation rate at individual pH coincides with the ultraviolet absorption maximum. Namely, it was maximally degraded by irradiation at 295 nm in acidic solution and at 325 nm in neutral and alkaline solution. At the optimum wavelength, the photolysis occurred more rapidly in neutral and alkaline solution than in acidic solution. The quantum yield in neutral solution was approximately 0.11 and independent of wavelength. One of the photolysis products was identified as hydroxylysine on an amino acid analyser, indicating that the cleavage of the pyridinium ring occurred.
A cell-free system from Streptococcus faecium for studies on the biosynthesis of triterpenoid carotenoids
Authors: Taylor Richard F. , Davies Brian H.
Published: December 2009

A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.
Sexual development in a homothallic fission yeast: synthesis of readiness proteins resolved by gel electrophoresis
Authors: Calleja G. B. , Johnson Byron F. , Walker Teena
Published: December 2009

Sexual development of a homothallic strain of Schizosaccharomyces pombe was monitored by radiolabelling and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Of more than 60 bands detected by Coomassie brilliant blue and by autoradiography, about 30 bands synthesized during development were discrete enough for experimental analysis.About a dozen bands are preferentially vegetative, another dozen preferentially developmental. However, vegetative bands as a group are also synthesized during development. Their synthesis is relatively unaffected by low concentrations of cycloheximide or by chloramphenicol and is not temperature sensitive at 37 °C nor catabolite repressible. Only band 40 (ca. 40 000 daltons) seems to be exclusively vegetative.The synthesis of developmental bands 13, 18, 24, 30, and α, all of which first appear during late-log phase, is catabolite repressible. Developmental band 51 is also synthesized throughout the vegetative phase. The synthesis of bands 24, 30, 51, and α is temperature sensitive at 37 °C during the development, but that of band 18 is not. The synthesis of band 13 during development is not temperature sensitive, but its earlier synthesis during late-log phase is. The synthesis of all these six developmental bands is immediately inhibited by cycloheximide, but not by chloramphenicol. Their appearance as a group of radioactive bands is greatly diminished in cultures grown in cycloheximide, in chloramphenicol, or in ethidium bromide.Developmental bands 13, 18, 24, and 30 may be called readiness proteins. They first appear prior to the earliest morphological signs of sexual activity. Their developmental synthesis is inhibited by conditions mat inhibit sexual development. Such inhibitory conditions include anaerobiosis, restrictive temperature, aging in stationary phase, the presence of inhibitors of cytoplasmic protein synthesis and of mitochondrial function, and catabolite repression. Readiness proteins may be regulating the switch from vegetative metabolism.
The effects of experimental inflammation on turnover of fatty acid synthetase and levels of fatty acid synthetase messenger RNA in rat liver
Authors: Langstaff John M. , Burton David N.
Published: December 2009

Suppression, by experimental inflammation, induced by subcutaneous injection of oil of turpentine, of the usual increase in liver fatty acid synthetase (FAS) activity resulting from fat-free feeding following starvation (adaptive synthesis) was shown to result entirely from lowered hepatic content of FAS protein. Comparison of changes in the relative rate of synthesis of FAS, determined radioimmunochemically during adaptive synthesis with and without inflammation, with concomitant changes in FAS activity, revealed that inflammation partially suppressed the increased rate of synthesis characteristic of adaptive synthesis, but insufficiently to account entirely for the suppression of enzyme activity. Inflammation accelerated the relative rate of degradation of FAS, causing a 50% decrease in enzyme half-life and a corresponding increase in kd and turnover index. Levels of translatable FAS mRNA rose only fivefold after 24 h of adaptive synthesis, while the relative rate of FAS synthesis increased 12-fold indicating the operation of both transcriptional and translational control. Inflammation, induced at the start of adaptive synthesis, caused a 65% lowering of die relative rate of FAS synthesis after 24 h and a 60% decrease in mRNA translatable as FAS, but was without effect on the total translational activity of the mRNA although alterations in the size distribution of RNA species in the mRNA fraction were noted.
The separation and properties of phosphoprotein phosphatase modulators from brown adipose tissue of young rats and the changes in their concentration during development
Authors: Knight Brian L. , Skala Josef P.
Published: December 2009

The heat-stable modulators of phosphoprotein phosphatase activity have been partially purified from brown adipose tissue. A nonphosphorylatable inhibitor of phosphorylase phosphatase (inhibitor 2) and an activator of phosphohistone phosphatase were similar to the corresponding modulators from muscle and liver in both their physical properties and in their relative effects upon three different preparations of phosphatase. An inhibitor of phosphorylase phosphatase that was only active when phosphorylated was eluted from DEAE-cellulose by 80 mM NaCl (inhibitor 1′). Only a small amount of inhibitor was eluted at 20 mM NaCl (inhibitor 1), which is the concentration that eluted the bulk of the phosphorylatable inhibitor in muscle and liver. Inhibitor 1 and inhibitor 1′ had similar physical properties but differed in their activities towards the different phosphatases. It is suggested that they are different forms of the same protein and that inhibitor 1′ more closely resembles the native inhibitor. The modulators did not compete with each other and were probably not subunits of a phosphatase complex. However, the direction and timing of the changes in their concentration in brown fat during the developmental period indicate that the inhibitors, at least, perform some useful physiological function in the tissue. The concentration of inhibitor 2 was high before birth and for 10 days after birth, when the tissue was proliferating. The concentration of the phosphorylatable inhibitor was highest immediately after birth and for the next 16 days, which is the period of greatest thermogenic activity of brown fat.
Identification of hyaluronic acid as a component of human teratocarcinoma-derived cells of line PA 1
Authors: Rasilo Maija-Liisa , Wartiovaara Jorma , Renkonen Ossi
Published: December 2009

Ovarial teratocarcinoma-derived cells (PA 1) of human origin, which are known to be capable of differentiating in vitro, incorporate label from radioactive galactose and 2-amino-2-deoxy-D-glucose into hyaluronic acid. This macromolecule, which is prominent in many differentiating embryonic and malignant cells, was separated from sulfated glycosaminoglycans and from large-sized glycopeptides by chromatography on concanavalin A – Sepharose. It was identified by a number of chemical and enzymatic degradations as well as by several chromatographic methods.
Reorganization of unique and repetitive sequences during nuclear development in Tetrahymena thermophila
Authors: Brunk Clifford F. , Tsao Smiley G. S. , Diamond Catherine H. , Ohashi Pamela S. , Tsao Nora N. G. , Pearlman Ronald E.
Published: December 2009

Genomic libraries of macro- and micro-nuclear DNA of the ciliate protozoan Tetrahymena thermophila were constructed in the bacteriophage vector λgtWESλB. Screening of these libraries with a probe for the repeated hexanucleotide sequence C4A2 showed many phage from the micronuclear library but few if any macronuclear sequences having homology to this probe. This is consistent with C4A2-repeating elements being present predominantly if not exclusively at or near the termini of macronuclear DNA. Sequences flanking C4A2-repeating elements were isolated from a number of purified phage and were used as hybridization probes to restriction endonuclease digested macro- and micro-nuclear DNA. These experiments revealed a repeated sequence family as well as unique sequences present only in micronuclear DNA. The repeated sequence element appears to be dispersed throughout the genome. Phage-containing individual members of this micronucleus limited sequence family were purified from the micronuclear library. Some of these phage contained sequences which hybridized to macronuclear DNA. These fragments therefore contain a "transition" region between micronucleus-limited sequences and sequences present in both nuclei.
Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties
Authors: Kwan Chiu-Yin , Davis Robert C.
Published: December 2009

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)–enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenolpyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)–enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.
Studies on the uptake of exogenous phosphoglycerides by Escherichia coli B cells
Authors: Proulx P. , Hellion P. , Mackenzie J.
Published: December 2009

The uptake of exogenous lipids by Escherichia coli B was studied under various conditions. Lysophosphoglycerides were absorbed more readily than diacyl analogues when sonicated dispersions of a single lipid were used. When these same lipids were admixed with coliform lipids and dispersed as vesicles, the uptake of lysophosphoglyceride diminished and was approximately equal to that of diacyl analogues. Neutral lipids such as diglyceride, fatty acid, and cholesterol were also absorbed when admixed and dispersed as vesicles with coliform lipids. The uptake of lysophosphoglyceride was stimulated slightly by all divalent cations tested except Mg2+; monovalent cations were ineffective. Uptake was accompanied by conversion of lysophosphoglyceride to diacyl analogues. In the presence of Ca2+, lysophosphatidylethanolamine also formed a more polar lipid product yet unidentified. The uptake of lipid did not cause release of 3H label into the medium from cells that had been grown in [3H]acetate-containing medium. Also, the [3H]phosphoglyceride content of such labelled cells remained constant. Thus the uptake process did not involve exchange of membrane lipid with the medium or an enhanced hydrolysis of endogenous lipids, but it represented a net gain of lipid by the cell. The uptake did not seem to involve a stable adsorption of lipid at the surface of the cell as could be judged from electron microscopic examination of the cells after incubation; the cell surfaces were devoid of adsorbed vesicle or liposomal types of structures and did not display evidence of expansion. [3H]Cholesterol–[32P]phospholipid mixtures were taken up without a change in isotopic ratio. This result together with the other evidence presented indicate a net uptake of exogenous lipid by a process likely involving fusion.
Convenient biosynthetic preparation of isomeric spin-labelled radioactive phosphatidic acids
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

A convenient method for the enzymatic preparation of sn-3-[2-3H]phosphatidic acids carrying also 5-, 12-, or 16-nitroxide stearic acids, from sn-3-[2-3H]glycerophosphate and isolated guinea pig liver microsomes, is described in detail. The procedure allows a simultaneous preparation of three spin-labelled sn-3-[2-3H] phosphatidic acids of yields 3–3.5 μmol of each compound which is > 99% pure in respect to the radioactivity and which contains 25 mol% of spin-labelled fatty acids. These phosphatidic acids were approximately equally distributed between the primary and the secondary hydroxyl when 12- or 16-nitroxide stearic acids were used or predominantly (75%) associated with the secondary hydroxyl of sn-3-[2-3H]phosphatidic acid when 5-nitroxide stearic acid was present in the incubation mixture.