bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of testicular hormones on synthesis of soluble proteins by dog prostate slices
Authors: Dubé Jean Y. , Chapdelaine Pierre , Tremblay Roland R.
Published: December 2009

We have submitted adult mongrel dogs to various endocrine manipulations. Prostate slices from these animals were then incubated in vitro in the presence of [3H]leucine or [35S]methionine. We have analyzed the cytosolic proteins by polyacrylamide gel electrophoresis. In intact adult uncastrated dogs, the radioactive amino acids were incorporated into three major bands having respective molecular weights (MW) of 32 000,16 000, and 15 000 in one-dimensional gels in presence of sodium dodecyl sulfate and mercaptoethanol. Two-dimensional electrophoresis revealed heterogeneity of each of these bands, both in isoelectric focussing (IEF) or nonequilibrium pH gel electrophoresis (NEpHGE) conditions. The 32 000 MW proteins showed five to six major radioactive spots and the 15 000 – 16 000 MW proteins showed six to seven spots by IEF. However, the highest incorporation of radioactivity occurred in a 16 000 MW protein seen only in NEpHGE. The lower MW proteins corresponded to some of the major proteins of dog seminal plasma as observed by immunoprecipitation of prostate proteins with antibodies against whole seminal plasma. By contrast, the 32 000 MW proteins were minor proteins of prostate cytosol and seminal plasma by Coomassie blue staining. Castration for 2 weeks completely abolished the synthesis of all these proteins. When castrated animals were treated with 5α-androstane-3α-17β-diol (10 mg/day for 2 weeks), the pattern of protein synthesis returned to the one observed in intact uncastrated animals. These observations show that testicular androgens control the synthesis of dog prostate major secretory proteins.
Macromolecular constituents of basement membranes: a review of current knowledge on their structure and function
Authors: Scott Paul G.
Published: December 2009

Macromolecules which appear to be integral constituents of basement membranes include type IV collagen, the glycoprotein laminin, and heparan sulphate proteoglycan. Another glycoprotein, fibronectin, may occupy an intermediate position between some lining cells and their basement membranes but is not, however, restricted to this location. An additional form of collagen, genetic type V which differs significantly from type IV collagen in structure, appears to be associated with some basement membranes, possibly linking them to underlying connective tissue. The main structural features of each of these macromolecules, as presently understood, are reviewed here as a background to the experimental papers in this "mini-symposium."
Morphological changes at the basement membrane during some tissue interactions in the integument
Authors: Hardy M. H. , Goldberg E. A.
Published: December 2009

Changes at the basement membrane have been reported at the time of tissue interactions during the development of a number of integumentary organs. During the development of vibrissa follicles of the mouse in vitro, gaps appeared in the lamina densa surrounding the dermal papilla at the time of the second, specific dermal message for follicle differentiation. Direct contacts between epithelial and mesenchymal cells were made through these gaps. This area has now been studied in vivo over the same period of follicle development. In addition, the epithelial part of the follicle has been separated from the dermal papilla by (i) enzyme treatment which destroys the lamina densa and (ii) chelating agents which leave the lamina densa attached to the dermal papilla. The resulting surfaces of epithelium and dermal papilla were examined by scanning and transmission electron microscopy. Small gaps first appeared in the lamina densa at the same substage of follicle development as in vitro and were present at the next substage. Processes from the dermal papilla mesenchyme cells traversed these gaps and made close contacts with the epithelial cells just prior to their differentiation into inner root sheath and hair. Instructive interaction through cell surfaces is suggested.
Survey, purification, and properties of sugar phosphate phosphohydrolase among microorganisms
Authors: Choy Francis Y. M. , Lee Ya-Pin
Published: December 2009

Sugar phosphate phosphohydrolase was purified approximately 500- to 600-fold to apparent homogeneity from Escherichia coli B, Escherichia coli C, Escherichia coli var. communior, Escherichia acidilactici, Enterobacter aerogenes, Neisseria meningitidis, and Saccharomyces cereviseae. The molecular weights of the enzyme as estimated by gel filtration ranged from 97 × 103 to 101 × 103. The enzyme was composed of two subunits with the same molecular weight which ranged from 50 × 103 to 52 × 103, as determined by sodium dodecyl sulfate gel electrophoresis. Homogeneous enzyme preparations hydrolyse all the tested α-D-aldohexose 1-phosphate, D-(keto or aldo)hexose 6-phosphate, and pentose phosphate substrates significantly. When the microorganisms were transferred from growth medium with 1% glucose to that without glucose, there were dramatic increases in both the specific and total enzyme activities. At least three isozymes appeared to be present in S. cereviseae, and two appeared to be present in E. coli B, E. coli var. communior, and N. meningitidis. Rabbit antiserum immunized against sugar phosphate phosphohydrolase purified from E. coli B cross-reacted with both the crude extracts and purified preparations of the enzyme from the other microorganisms. The presence of neither sugar phosphate phosphohydrolase activity nor immunocross-reacting material was detected in the following microorganisms: Aspergillus niger, Azotobacter chroococcum, Bacillus subtilis, Bacillus pumilis, Citrobacter freundii, Clostridium butyricum, Corynebacterium xerosis, Flavobacterium aquatile, Flavobacterium synxanthum, Lactobacillus bulgaricus, Micrococcus coralinus, Neisseria perflava, Neurospora crassa, Penicilium expansum, Penicilium notatum, Proteus mirabilis, Proteus vulgaris, Pseudomonas fluorescens, Saccharomyces fermenti, Sarcina lutea, and Streptomyces antibioticus. At present, no conclusive relationship can be established between the phosphoenolpyruvate phosphotransferase system and the enzyme sugar phosphate phosphohydrolase among microorganisms. The physiological role of sugar phosphate phosphohydrolase as a transferase and regulatory enzyme is discussed.
Specific binding of cholesterol to chromatin prepared from mouse spleen cells
Authors: Regenass-Klotz Mechthild , Heiniger Hans-Jörg
Published: December 2009

Mouse spleen cell suspensions were incubated with tritiated cholesterol for various time intervals. The chromatin of these cells was then isolated, washed with Triton X-100, and fractionated on Sephadex columns. It was found that cholesterol binds specifically to the chromatin. The binding was saturated after 45 min of incubation and also displayed characteristics typical of dose-dependent binding. The number of cholesterol molecules bound per nucleus was estimated to be on the order of 10 000. Oxygenated sterols, such as 25-hydroxycholesterol and 7-ketocholesterol, did not compete for the binding with [3H]cholesterol if added in 20-fold molar excess to the incubation medium of the cells. Chromatographic analyses on Sephadex columns displayed a distinct peak of radioactivity. The protein–sterol complex had an apparent molecular weight of 180 000 ± 27 000. Using extensive digestion with DNase I (EC 3.1.21.1) it could be concluded that DNA, binding to the complex, did not influence the estimate of the molecular weight, whereas digestion with pronase or treatment with sodium dodecyl sulfate destroyed the complex. Additional experiments using sucrose density gradients (5–20%) showed also, that [3H]cholesterol was bound to chromatin by one or several proteins.
The method used in purification of nuclei from three variant cell lines of the Shionogi mouse mammary carcinoma alters their uptake of dihydrotestosterone
Authors: Lefebvre Yvonne A. , Caskey Janice J. , Kline Linda D.
Published: December 2009

Modifications of three isolation methods were used to purify nuclei from an androgen-dependent cell line (AD) and two androgen-independent cell lines (AI1 and AI2) of the Shionogi mouse mammary carcinoma. Yields of nuclei, contamination of the nuclei by whole cells, monitoring of cytoplasmic tags by phase-contrast microscopy, and biochemical analyses were used to compare the methods. Purification with the cationic detergent cetylpyridinium chloride (CPC) resulted in greater yields of nuclei than purification of nuclei using Triton N-101. Purification by glycerol loading followed by hypotonic shock, although resulting in somewhat less whole cell contamination of the nuclei, yielded fewer nuclei per gram wet weight starting tissue. Phase-contrast microscopy showed the relative absence of cytoplasmic tags when nuclei were prepared by either the Triton N-101 or CPC methods. However, the yield of protein per nucleus was less when nuclei were prepared using CPC. Androgen uptake by nuclei of three cell lines was markedly reduced in those nuclei prepared by the CPC method as compared with those prepared by the Triton N-101 method. In the case of the AD tumour cell line nuclei prepared by the CPC method, both the affinity of the nuclei for dihydrotestosterone and the number of uptake sites were reduced when compared with AD tumour cell line nuclei prepared by the modified Triton N-101 method.
The human preproinsulin gene: synthesis, cloning, gene modification, and expression studies
Authors: Narang S. A. , Brousseau R. , Georges F. , Michniewicz J. , Prefontaine G. , Stawinski J. , Sung W.
Published: December 2009

A 355 base pair (bp) DNA sequence coding for human preproinsulin has been assembled by joining a synthetic DNA leader sequence coding for 24 preregion amino acids to the previously synthesized DNA duplex of 277 bp constituting the sequence of BCA chain. It was next cloned in M13 mp8 single-stranded bacteriophage and subjected to site-specific mutagenesis and phase shifting to allow its inducible expression under lac operator control. An affinity leader sequence of 25 bp has been added in an attempt to facilitate purification of the preproinsulin.
Biochemical genetics of the mouse IgM system
Authors: Shulman Marc J. , Hawley Robert G. , Ochi Atsuo , Baczynsky W. O. T. , Collins Catherine , Pennell Nancy , Potash M. J. , Köhler Georges , Hozumi Nobumichi
Published: December 2009

Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in κ chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing μ heavy chain fragments and provide information on the requirements of IgM assembly and μ gene expression. We also describe a transfer system for the μ and κ genes which will be useful in analyzing the structural basis of IgM function.
Chicken gizzard tropomyosin: head-to-tail assembly and interaction with F-actin and troponin
Authors: Sanders Clive , Smillie Lawrence B.
Published: December 2009

Chicken gizzard and rabbit striated muscle tropomyosins have been compared with respect to their abilities to polymerize head-to-tail and to interact with rabbit skeletal F-actin and troponin. By both viscosity and sedimentation equilibrium measurements, the smooth muscle protein was shown to aggregate more extensively than the cardiac (skeletal) tropomyosin. The stoichiometry and cooperativity of binding of gizzard tropomyosin to F-actin were shown to be similar to the striated muscle protein, as were also the effects of MgCl2 and KCl concentration. Although the gizzard protein was found to interact with rabbit striated troponin as indicated by sedimentation velocity measurements, it was eluted at a lower KCl concentration from a column of immobilized troponin than the cardiac (skeletal) protein. The absence of a significant increase in viscosity upon addition of troponin to gizzard tropomyosin is consistent with a minimal influence on its head-to-tail aggregation. Thus while gizzard tropomyosin resembles rabbit cardiac (skeletal) tropomyosin in its propensity for head-to-tail polymerization and in its F-actin binding properties, it is similar to the nonmuscle tropomyosin from platelets in its interactions with skeletal muscle troponin. These observations suggest that the gizzard protein shares structural features with both striated muscle and platelet tropomyosins.
Isoelectric forms of clusterin isolated from ram rete testis fluid and from secretions of primary cultures of ram and rat Sertoli-cell-enriched preparations
Authors: Blaschuk O. W. , Fritz I. B.
Published: December 2009

Clusterin, a cell aggregating factor isolated from ram rete testis fluid (RTF), is shown to contain 14.7% hexoses, 13.6% glucosamine, and 7.9% sialic acid. The isoelectric point (pI) of the predominant electrophoretic form of clusterin from ram RTF is 3.7. After treatment with neuraminidase, the pI values become more basic, with the majority of the material being eluted from a chromatofocusing column at pH values between 4.9 and 5.1. Intact clusterin binds quantitatively to wheat germ agglutinin – Sepharose 6 MB, but after treatment with neuraminidase only 49% specifically binds. Clusterin isolated from proteins secreted by primary cultures of ram Sertoli-cell-enriched preparations was shown to have properties similar to those of intact clusterin isolated from ram RTF. In contrast, clusterin isolated from proteins secreted by primary cultures of rat Sertoli- or granulosa-cell-enriched preparations has isoelectric forms which more closely resemble those of neuraminidase-treated ram clusterin.
Folylpolyglutamate synthetase from beef liver: assay, stabilization, and characterization
Authors: Pristupa Z. B. , Vickers P. J. , Sephton G. B. , Scrimgeour K. G.
Published: December 2009

A reliable assay for folylpolyglutamate synthetase has been devised and tested. Conditions have been established for the complete separation of [3H]glutamate and the tritium-labelled products on columns of DEAE-cellulose. The availability of this assay has aided us in partially purifying and characterizing the synthetase from extracts of beef liver. Suitable conditions have been found for the stabilization of the activity of both crude and partially purified folylpolyglutamate synthetase. The apparent Km values for L-glutamate (0.82 mM), dl-L-tetrahydrofolate (9 μM), ATP (25 μM with 10 mM MgCl2), KCl (3 mM), and 2-mercaptoethanol (5 mM) have been estimated. Several oxidized pteridine substrates have been tested. Of the antifolates tested, aminopterin is the more active substrate. The chain lengths of folate polyglutamates have been measured by chromatography on columns of DEAE-cellulose, with elution by a gradient of sodium acetate. Conjugates as long as hexaglutamates have been detected. The identities of the polyglutamates of tetrahydrofolate have been verified by hydrolysis in the presence of conjugase and by double-labelling experiments.
DNA methylation in differentiating mouse teratocarcinoma and erythroleukemia cells
Authors: Aujame Luc , Craig Jane , McBurney Michael W.
Published: December 2009

We have measured the content of 5-methylcytosine (5MC) in the genomic DNA of differentiated and undifferentiated cultures of murine embryonal carcinoma (EC) and murine erythroleukemia (MEL) cells. A large proportion of deoxycytosine residues were methylated in EC cells (4.6%) and this proportion dropped significantly (4.1%) following differentiation. The alpha-1 globin genes were heavily methylated at HpaII sites in EC cells and in differentiated derivatives of these EC cells. The MEL cells had only 3.3% of their genomic deoxycytidine methylated and no significant change occurred following differentiation. The alpha-1 globin genes of MEL cells were much less methylated than the same genes in EC cells and no change in this methylation pattern accompanied the induction of hemoglobin synthesis. In EC × MEL cell hybrids which express the alpha-1 globin genes from both parents, the EC-derived genes had become demethylated. These results are consistent with the general model that DNA hypomethylation is correlated with expression of that DNA and that gene activation is accompanied by DNA demethylation. We have also measured the 5MC content of DNA isolated from nuclease-treated EC nuclei. Unexpectedly, the DNase I sensitive chromatin contained a large proportion of 5MC. This result, along with the work of others, suggests that nuclease sensitivity may often reflect the transcriptional activity of chromatin in somatic cells, but is not indicative of the active state in pluripotent EC cells.
Brown adipose tissue thermogenesis, energy balance, and obesity
Authors: Himms-Hagen Jean
Published: December 2009

The concept that thermogenesis in brown adipose tissue can play a role as an energy buffer has developed during the last 5 years. The history of this development is reviewed. Control of brown adipose tissue thermogenesis resides in regions of the brain, located primarily but not exclusively in the hypothalamus, that control the activity of the sympathetic nervous system in response to diet and to environmental temperature. Brown adipose tissue mitochondria are uniquely specialized for thermogenesis, possessing a specific proton leakage mechanism that is regulated by the concentration of fatty acids in the cells of the brown adipose tissue. The level of fatty acids is in turn controlled by the lipolytic action of noradrenaline on the tissue. Sympathetic stimulation also exerts a trophic influence on brown adipose tissue. Effective thermogenesis in brown adipose tissue is associated with leanness and decreased metabolic efficiency, as in the rat rendered hyperphagic and hypermetabolic, by either cold acclimation or cafeteria feeding. Conversely, food restriction is associated with suppressed thermogenesis in brown adipose tissue and increased metabolic efficiency. Defective brown adipose tissue thermogenesis is associated with obesity in a number of different types of obese animals. In three of these (the genetically obese fa/fa Zucker rat, the mouse with hypothalamic damage induced by gold thioglucose, the rat with a surgically induced hypothalamic lesion), diet-induced thermogenesis is defective in brown adipose tissue, but cold-induced thermogenesis is normal. In another type of obese animal, the genetically obese (ob/ob) mouse, control of brown adipose tissue is defective. Studies of this control are complicated by the frequency of torpor in the fed state. The unusual cold sensitivity of the ob/ob mouse is probably due to inappropriate entry into torpor and coincident suppression of brown adipose tissue thermogenesis. Whether the knowledge that defective thermogenesis in brown adipose tissue contributes to obesity in laboratory animals can be applied to humans is presently under investigation. Current research and problems in this area are discussed.
Molecular packing in steroid–lecithin monolayers. IV. Mixed films of epicoprostanol with dipalmitoyl phosphatidylcholine
Authors: Cadenhead D. A. , Müller-Landau F.
Published: December 2009

Studies of the two-component monomolecular film system, dipalmitoyl phosphatidylcholine (DPPC) – epicoprostanol have been carried out at 21 °C utilizing a wide compositional range of films. The results obtained indicate that on initial mixing of the two components, epicoprostanol condensed expanded DPPC films to a greatly reduced degree and expanded condensed DPPC films to a significantly greater degree compared with cholesterol. It is thought that, even on an initial compressional procedure, partial microscopic segregation of epicoprostanol takes place. The segregation is postulated to result from an epicoprostanol–epicoprostanol overlap, leading to row packing with an accompanying reduction in the number of adjacent acyl chains. Subsequent decompression and recompression of DPPC–epicoprostanol films leads to macroscopic segregation of pure or nearly pure epicoprostanol; however, partial miscibility below 20 mol% epicoprostanol may persist. The phase diagram of the initially compressed film may be of the eutectic type, but the mixed system is unstable. The inability of epicoprostanol to substitute for cholesterol appears to be due to a combination of two effects: the tilting of the sterol at the air–water interface, to satisfy the immersion requirements of the 3α-OH group, and the bent α-face of the sterol. The α-OH group of epicoprostanol is postulated to play an indirect role in the weak condensation capabilities of this sterol by inducing a tilt in the molecule and reducing epicoprostanol–acyl chain hydrophobic interactions.
Les interactions moléculaires dans les membranes des photorécepteurs visuels. Étude en film monomoléculaire mixte
Authors: Tancrède Pierre
Published: December 2009

We have examined the interactions in a mixed monomolecular film between the 11-cis and all-trans isomers of retinal and different saturated and unsaturated phosphatidylcholines and phosphatidylethanolamines. The phase diagrams of the different binary mixtures are drawn using the surface pressure isotherms measured on 10−3 M phosphate buffer at 21 ± 1 °C. Analysis of the results shows that the unsaturated chain phospholipids (dioleoyl L-phosphatidylcholine and dioleoyl L-phosphatidylethanolamine extracted from bovine retinas) and the retinal isomers are miscible at the interface. In addition, the excess free energies of mixing are identical for these mixtures. On the other hand, the saturated phospholipids (distearoyl L-phosphatidylcholine and distearoyl L-phosphatidylethanolamine) are not miscible with the retinal isomers under the same conditions. The comparison between the results obtained with the saturated lipid and unsaturated lipid mixtures shows the importance of hydrophobic forces in the thermodynamics of the interactions of these systems. In addition, the isotherms of the mixtures containing phosphatidylethanolamine do not vary with the pH of the subphase in the region studied, pH 5.5 to 8.2. These results raise serious doubts about the existence of a Schiff base between phosphatidylethanolamine and the retinals at the interface. The possible implications of this phenomenon for the disc membrane are analyzed, particularly with respect to the stability of the retinals in vivo.[Translated by the journal]
Plasma membranes from Physarum polycephalum Plasmodia: purification, characterization, and comparison with amoebae plasma membranes
Authors: Pallotta Dominick , Barden Anne , Martel Rémi , Kirouac-Brunet Josée , Bernier François , Lord André , Lemieux Gérald
Published: December 2009

Two methods are described for preparing plasma membranes from Physarum polycephalum plasmodia. In the first method Plasmodia were broken in homogenizing medium (250 mM sucrose, 1 mM ZnCl2, 10 mM Tris (pH 8.0)), and the nuclei were removed by low speed centrifugation. A crude membrane fraction was collected and the membranes were purified by centrifugation in a linear 30–65% (w/w) sucrose gradient. The membranes sedimented in a single band at a density of 1.26 g/cm3. In the second method the plasmodia were broken in the same homogenizing medium and a crude membrane fraction was prepared by differential centrifugation. The membranes were purified by the Dextran – polyethylene glycol aqueous two-phase system. Membranes from both preparations were found by enzymatic assay and by electron microscopy to be virtually free of contaminating cell organelles. Analyses by one- and two-dimensional polyacrylamide gel electrophoresis showed a great similarity in the proteins from two-phase and sucrose gradient prepared membranes, indicating that either technique can be used to prepare membranes for protein studies. An analysis by two-dimensional gel electrophoresis of plasma membrane proteins from amoebae and plasmodia showed differences in some of the major proteins, indicating that changes occur in plasma membrane proteins during differentiation.
The effects of detergents on CDP-choline: 1,2-diacylglycerol phosphocholine transferase from hamster heart
Authors: Arthur Gilbert , Tam Stanley W. , Choy Patrick C.
Published: December 2009

The effects of detergents on CDP-choline: 1,2-diacylglycerol phosphocholine transferase from hamster heart were examined. Maximum enhancement of enzyme activity was produced by Tween-20, while Triton X-100 caused a small increase in enzyme activity. No enhancement of enzyme activity was observed with deoxycholate or ethanol. Although enzyme activity was enhanced by different concentrations of Tween-20 (15–120 μg/mL), a substantially higher concentration of diacylglycerol was required when the concentration of Tween-20 in the assay was greater than 30 μg/mL. In the presence of Tween-20, a sigmoidal curve was obtained when enzyme activity was plotted versus diacylglycerol concentrations. The sigmoidal curve became more prominent when a higher amount of Tween-20 was used. It appears that the enhancement of enzyme activity by Tween-20 was not caused by the direct action of the detergent on the enzyme, but probably resulted in a better dispersion of the lipodal substrate which facilitates better utilization by the enzyme. The acyl specificity of the enzyme in the presence of detergent was found to be rather limited and the highest enzyme activity was obtained with diacylglycerols containing a monounsaturated acyl group at the C-2 position.
Fatty acyl chain structure, orientational order, and the lipid phase transition in Acholeplasma laidlawii B membranes. A review of recent 19F nuclear magnetic resonance studies
Authors: Macdonald P. M. , Sykes B. D. , McElhaney R. N.
Published: December 2009

The orientational order parameters of monofluoropalmitic acids biosynthetically incorporated into membranes of Acholeplasma laidlawii B in the presence of a large excess of a variety of structurally diverse fatty acids have been determined via 19F nuclear magnetic resonance (19F NMR) spectroscopy. It is demonstrated that these monofluoropalmitic acids are relatively nonperturbing membrane probes based upon physical (differential scanning calorimetry), biochemical (membrane lipid analysis), and biological (growth studies) criteria. 19F NMR is shown to convey the same qualitative and quantitative picture of membrane lipid order provided by 2H-NMR techniques and to be sensitive to the structural characteristics of the membrane fatty acyl chains, as well as to the lipid phase transition. Representatives of each naturally occurring class of fatty acyl chain structures, including straight-chain saturated, methyl-branched, monounsaturated, and alicyclic-ring-substituted fatty acids, were studied and the 19F-NMR order parameters were correlated with the lipid phase transitions (determined calorimetrically). The lipid phase transition was the prime determinant of overall orientational order regardless of fatty acid structure. Effects upon orientational order attributable to specific structural substituents were discernible, but were secondary to the effects of the lipid phase transition. In the gel state, relative overall order was directly proportional to the temperature of the particular lipid phase transition. Not only the overall order, but also the order profile across the membrane was sensitive to the presence of particular structural substituents. In particular, in the gel state specific fatty acyl structures demonstrated a characteristic disordering effect in the membrane order profile. These various observations can be merged to provide a unified picture of the manner in which fatty acyl chain chemistry modulates the physical state of membrane lipids.
Is intracellular Ca2+ involved in insulin stimulation of sugar transport? Fact and prejudice
Authors: Klip Amira
Published: December 2009

Insulin stimulates the rate of glucose transport in muscle and fat tissue by incorporation of transporters from internal membranes into the plasma membrane. It is conceivable that cell Ca2+ ions could play a role in transporter translocation. Indeed Ca2+ has been thought to mediate insulin action, but the evidence remains highly controversial. Experiments to this effect include (i) determinations of a requirement for extracellular Ca2+ in the hormonal response, (ii) stimulation of glucose transport by agents thought to elevate cytosolic Ca2+, [Ca2+]i, and (iii) determinations of Ca2+ efflux. Actual measurements of the effect of insulin on [Ca2+]i were missing until recently. Current methods to measure [Ca2+]i include Ca2+-selective intracellular electrodes, metallochromic dyes, and photoproteins. Main drawbacks of these procedures have been the requirement of microinjection for their incorporation, which restricts their use to large cells, and their interaction with cytoplasmic Mg2+ and H+. Recently, a fluorescent Ca2+ chelator, quin-2, has been devised, which circumvents these difficulties. A permeable, non-chelating precursor of quin-2 penetrates cells and once in the cytosol becomes an impermeant, fluorescent Ca2+ chelator. With this technique it was shown that insulin does not change [Ca2+]i while stimulating glucose transport in L6 muscle cells.
Structural analysis of the specific polysaccharide of Streptococcus pneumoniae type 9L (American type 49)
Authors: Richards James C. , Perry Malcolm B. , Kniskern Peter J.
Published: December 2009

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9L (American type 49) is composed of D-galactose (one part), D-glucose (one part), D-glucuronic acid (one part), 2-acetamido-2-deoxy-D-mannose (one part) and 2-acetamido-2-deoxy-D-glucose (one part). Partial acid hydrolysis, periodate oxidation, nitrous acid deamination, optical rotation, methylation and 13C and 1H nuclear magnetic resonance studies showed that the polysaccharide is an unbranched high molecular weight linear polymer of a repeating pentasaccharide unit having the structure:[Formula: see text]
Complexes of cytochrome caa3 from the thermophilic bacterium PS3 formed with ligands and during catalytic activity
Authors: Sone N. , Nicholls P.
Published: December 2009

PS3 (thermophilic bacterium) cytochrome caa3 reacts slowly with cyanide which forms a low-spin complex with the CuBa3 centre. Partial reduction under catalytic conditions increases the rate of cyanide binding, and the reaction constant is rather similar to that of the mammalian enzyme, but the partially reduced complex dissociates more rapidly than does the corresponding eukaryotic complex. A simple biphasic reaction can account for the results obtained. The azide complex of partially reduced PS3 cytochrome caa3 shows an α-peak blue shift similar to that of the mammalian enzyme. PS3 cytochrome caa3, forms an oxyferri ("oxygenated") species like the mammalian enzyme, but does not undergo high- to low-spin changes during the aerobic steady state with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine as substrates. Interactions seen in cytochrome oxidase – ligand reactions and its spin-state changes are therefore intrinsic to the enzyme's large catalytic subunits and do not require the presence of the small nuclear-encoded subunits found in eukaryotic systems.
Synthesis of an antheridiol-inducible polypeptide during sexual morphogenesis of Achlya ambisexualis E87
Authors: Horton J. Stephen , Horgen Paul A.
Published: December 2009

Changes in in vivo polypeptide synthetic patterns during sexual morphogenesis of Achlya ambisexualis E87 were investigated using polyacrylamide gel electrophoresis. Autoradiography of [35S]methionine-labelled polypeptides revealed the apparent qualitative synthesis of a 64 000 dalton polypeptide, commencing 30–60 min after antheridiol addition and continuing for at least 6 h. Synthesis of this pheromone-inducible polypeptide was detected throughout male sexual morphogenesis in still culture matings of the male strain E87 and the female strain 734. Two other species of Achlya were also found to synthesize the 64 000 dalton polypeptide in response to antheridiol. The inducible polypeptide did not appear to be associated strictly with a branching response, as no detectable synthesis occurred when asexual branching was induced by the exogenous addition of either casein hydrolysate or lactalbumin hydrolysate. Two-dimensional gel electrophoretic analysis revealed quantitative changes in the synthesis of many other polypeptides as a result of antheridiol action. In addition, both equilibrium and nonequilibrium two-dimensional gel electrophoresis showed the inducible polypeptide to be basic, demonstrating an isoelectric point of more than 8. Studies using the RNA synthetic inhibitor actinomycin D suggested a transcriptional control of the inducible polypeptide.
Site-specific cleavage of tobacco mosaic virus RNA: A study of factors influencing the cleavage
Authors: Saleem M. , Pelcher L. E.
Published: December 2009

DNA oligomer directed ribonuclease H (RNase H) methodology is applied to specifically cleave tobacco mosaic virus (TMV) RNA. Using a synthetic DNA oligomer P(dT8)dCdC, complementary to a region from nucleotide 5545 to nucleotide 5554 at the 3′ end of TMV RNA, we have cleaved the RNA at the site of polynucleotides complementary to the DNA oligomer. Factors such as secondary structure of the RNA, concentrations of DNA oligomer, RNase H and magnesium ions in the reaction mixture, and time of incubation were optimized for the RNase H cleavage of TMV RNA – DNA oligomer complex. Denaturation of TMV RNA with 50% dimethyl sulphoxide at 50 °C is essential for the site-specific cleavage.
Mammalian lymphocytes: stress-induced synthesis of heat-shock proteins in vitro and in vivo
Authors: Rodenhiser David , Jung Jack H. , Atkinson Burr G.
Published: December 2009

Mammalian (human, mouse, and rabbit) white blood cells (lymphocytes) maintained in culture respond to a brief incubation at an elevated temperature (at or above 41 °C) by (i) the new and (or) enhanced synthesis of a small number of proteins (the so-called heat-shock proteins; HSPs) having molecular masses of approximately 110 000, 100 000, 90 000, 70 000, 65 000, and 26 000 daltons and (ii) the depressed synthesis of proteins normally made at 37 °C. The HSPs synthesized in culture by human, rabbit, and mouse (peripheral and splenic) lymphocytes are similar in number, molecular mass, and distribution on two-dimensional (isoelectric focusing and sodium dodecyl sulfate – polyacrylamide) electrophoretic gels to those synthesized in vivo by lymphocytes in hyperthermic mice. Since the level of hyperthermia used to induce HSP synthesis in mouse lymphocytes in vitro and in vivo is of a magnitude (41 °C) also used to promote thermotolerance in mice and is similar to temperatures attained during febrile episodes in rabbits and in humans, we suggest that the in vitro and in vivo synthesis of HSPs by mouse lymphocytes, demonstrated in this study, represents a relevant, physiological response which mammalian lymphocytes may normally use to survive periods of thermal stress.
The catabolism of very low density lipoproteins
Authors: Breckenridge W. Carl
Published: December 2009

The lipolysis of very low density lipoproteins (VLDL) in vitro is a useful model for the study of the process of conversion of this triacylglycerol rich lipoprotein into low (LDL) and high (HDL) density lipoproteins. Data is reviewed to show that a portion of surface cholesterol and phospholipid which becomes redundant during lipolysis is lost from the lipoprotein. In the absence of HDL, the material forms lipoprotein-X (LpX) like vesicles which are not readily disrupted by HDL once they are formed. In the presence of HDL during lipolysis, the material is largely incorporated into HDL. The data is used to suggest a mechanism of formation of LpX-like vesicles in conditions where the production of surface remnants exceeds the capacity of HDL to disrupt them. Evidence is also provided to show that apolipoproteins (apo) C-II, C-III, and E are lost from VLDL and that this loss is primarily in association with a neutral core particle of HDL size which can subsequently exchange lipids and apolipoproteins with plasma HDL. Such a mechanism could account for the removal of apo E and excess cholesteryl ester which is necessary for conversion of VLDL to LDL. The role of hepatic lipase in this process remains speculative. Recent evidence is reviewed and used to propose that the enzyme may serve to rearrange the neutral core and surface composition of LDL and HDL subfractions to allow for the packaging of cholesteryl esters and the cycling of apolipoproteins.
The structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 11F (American type 11)
Authors: Richards James C. , Perry Malcolm B. , Kniskern Peter J.
Published: December 2009

The specific polysaccharide of Streptococcus pneumoniae type 11F (American type 11) is composed of 2-acetamido-2-deoxy-D-glucose (one part), D-glucose (one part), D-galactose (two parts), ribitol (one part), phosphate (one part), and O-acetyl (two parts). Hydrolysis, dephosphorylation, periodate oxidation, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies showed that the polysaccharide is an unbranched linear polymer of a ribitol-phosphate substituted repeating tetrasaccharide unit having the structure:[Formula: see text]The specific capsular polysaccharides of S. pneumoniae type 11B and 11C (American types 76 and 53) were found to have the same tetrasaccharide repeating unit as the 11F polysaccharide, but differed from it in their mode of O-acetylation and the replacement of the ribitol phosphate by glycerol phosphate in the 11C specific polysaccharide.
Modifications of membrane lipid structure and their influence on cell growth, passive permeability, and enzymatic and transport activities in Acholeplasma laidlawii B
Authors: McElhaney Ronald N.
Published: December 2009

Acholeplasma laidlawii B is a simple procaryotic microorganism, without a cell wall, whose membrane lipid fatty acid composition and cholesterol content can be dramatically modified. It is thus possible to markedly vary the temperature and cooperativity of the gel to lipid-crystalline phase transition, thereby altering the phase state and "fluidity" of the A. laidlawii membrane lipids. By varying the chain length of the exogenous fatty acids supplied, it is also possible to modify the thickness of the membrane lipid bilayer. Acholeplasma laidlawii B cell growth and most membrane functions are strongly dependent on the phase state of the membrane lipids. When gel-state lipid predominates at physiological temperatures, cell growth declines sharply, nonelectrolyte permeability exhibits a pronounced local maximum, net glucose transport is markedly reduced, and ATPase activity declines moderately. Upon complete conversion of the lipid to the gel state, cell growth ceases, passive permeability is markedly reduced, and net glucose transport is abolished, but appreciable ATPase activity remains. Provided that the membrane lipid is predominantly or exclusively in the liquid-crystalline state, cell growth and ATPase activity are almost independent of membrane lipid fatty acid composition and cholesterol content, except that cell growth is inhibited by the presence of "hyperfluid" lipid. In contrast, passive permeability and active glucose uptake are influenced by alterations in membrane lipid composition, with increases in lipid fluidity resulting in higher nonelectrolyte translocation rates. Alterations in the length of the membrane lipid hydrocarbon chains per se also appear to affect A. laidlawii growth and membrane function. Thus lipids containing fatty acids with effective chain lengths of 13 carbons or less do not support cell growth or normal enzymatic and transport activities, and lipids with effective hydrocarbon chain lengths of 19 carbons or more do not support normal cell growth.
The effects of delipidation on the major antigenic determinant of purified human intestinal mucin
Authors: Mantle M. , Forstner J. F.
Published: December 2009

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidyl-ethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to < 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.
Role of ornithine decarboxylase in proliferation of prolactin-dependent lymphoma cells
Authors: Elsholtz Harry P. , Shiu Robert P. C. , Friesen Henry G.
Published: December 2009

Mitogenic stimulation of Nb2 lymphoma cells by lactogenic hormones (prolactin, human growth hormone) caused a dramatic early increase in ornithine decarboxylase (ODC) activity that achieved a maximal level by 6–8 h. A marked increase in ODC activity was also generated when cells which had reached a growth plateau were transferred to fresh medium that did not stimulate growth. Furthermore, low concentrations of human growth hormone (20 pg/mL) elicited a proliferative response, but did not cause a detectable early increase in ODC activity. The early peak of ODC activity thus appeared not to be directly involved in mediating lactogen-stimulated growth nor was it required to support the mitogenic response. However, prolonged suppression of ODC activity by DL-α-difluoromethylornithine (DFMO) (200 μM) attenuated the growth of Nb2 cells (50–60% inhibition), indicating that normal cell growth was dependent on ODC and polyamine biosynthesis. Under these conditions, putrescine, the enzyme product, or the polyamines spermidine and spermine restored normal cell growth when added at a concentration of 1 μM or greater. Nb2-SP cells, variants which proliferate in the absence of prolactin, were about two times more resistant to the growth suppressive effects of DFMO than prolactin-responsive Nb2 cells.
The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells
Authors: Lim Peter , Cornell Rosemary , Vance Dennis E.
Published: December 2009

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline: 1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 μM for CDP-choline. A similar Km value of 104 μM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 μM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 μM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.
A comparison of two procedures used for complexing Fe(III) with human apotransferrin: I. physicochemical properties of the Fe(III) transferrin products
Authors: Berry Leslie R. , Hatton Mark W. C.
Published: December 2009

Samples of human Fe∙transferrin (Fe∙HTr) were prepared from a single batch of apotransferrin (apo∙HTr) by either the Fe(III)–citrate or the Fe(II)–ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe∙HTr prepared by the citrate method and 55Fe∙HTr prepared by the ceruloplasmin method contained 2.2–2.3 and 2.0 Fe/mol, respectively. For both 55Fe∙HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe∙HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55–60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe∙HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe∙HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo∙HTr were 0.275 and 0.309 for Fe∙HTr (citrate) and Fe∙HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe∙HTr (citrate) and Fe∙HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe∙HTr preparations. Dialysis against H2O caused Fe∙HTr (citrate), but not Fe∙HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe∙HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe∙HTr (citrate). In contrast, these properties of Fe∙HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe∙HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe∙HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe∙HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo∙HTr by these two different procedures. The two Fe∙HTr products may differ, conceivably, in their abilities to transfer Fe to cells.