bluuebunny/crossref_metadata_2025 huggingface dataset:
H3 histone RNA in eggs and embryos of the sea star Pisaster ochraceus
Authors: Banfield D. K. , Boom J. D. G. , Honda B. M. , Smith M. J.
Published: December 2009

Unlike sea urchins, sea stars have little stored histone RNA in their eggs. In an effort to quantify this difference, we have measured H3 RNA concentration in eggs and embryos of the sea star Pisaster ochraceus. The amount of H3 transcript in P. ochraceus 12-h embryos has been measured by RNA excess hybridization kinetics, using a single-strand 32P-labelled coding sequence probe. There are 1 × 105 H3 transcripts in each 12-h embryo. Putative egg H3 transcript concentration was estimated by reciprocal plots. The number of egg H3 homologous sequences (150/egg) is at least an order of magnitude less than rare complex-class, single-copy nuclear DN A transcripts. Slot blots and Northern blots indicate that sea star embryos do not reach the level of H3 transcript abundance seen in sea urchins until at least 16 h of development.
Inhibition of myoblast fusion by bromoconduritol
Authors: Trudel Geralyn C. , Herscovics Annette , Holland Paul C.
Published: December 2009

It has recently been reported that the glucosidase I inhibitor, N-methyl- 1-deoxynojirimycin (MDJN), inhibits myoblast fusion whereas the mannosidase inhibitor, 1 -deoxymannojirimycin (ManDJN), has no effect on fusion. We now report that bromoconduritol, which is an active-site-directed covalent inhibitor of glucosidase II, also inhibits fusion at concentrations that have no effect on the plating efficiency or growth of rat L6 myoblasts. Significant inhibition of fusion was obtained at concentrations as low as 50 μg of bromoconduritol/mL, whereas inhibition of cell growth did not occur until concentrations of 250 μg/mL were reached. Rat L6 myoblasts were grown in the presence and absence of processing inhibitors and were surface labelled with 125I. Analysis of the iodinated proteins by two-dimensional gel electrophoresis demonstrated that a number of high-molecular-weight proteins (>90 000) detected at the surface of control cells were absent from the surface of cells treated with MDJN or bromoconduritol. It is suggested that MDJN and bromoconduritol prevent the translocation of these proteins to the cell surface. The high-molecular-weight proteins detected at the surface of control cells were also detectable in ManDJN-treated cells, indicating that inhibition of N-linked complex oligosaccharide formation does not affect the translocation of these proteins to the myoblast cell surface.
Correlation between the activity of a 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-insensitive puff and the synthesis of major heat-shock polypeptide, hsp70, in Chironomus thummi
Authors: Barettino D. , Morcillo G. , Díez J. L. , Carretero M. T. , Carmona M. J.
Published: December 2009

The induction of puff III-A3b, a major heat-shock puff in Chironomus thummi salivary cells, was insensitive to the transcription inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), whereas no transcriptional activity could be detected at the other heat-shock puffs in the presence of this drug. In these conditions, a polypeptide with the same Mr and isoform pattern as those of the major heat-shock polypeptide, hsp70, was synthesized. These results suggest that hsp70 is encoded by locus III-A3b. In addition to DRB insensitivity, incorporation of [3H]UTP on puff III-A3b took place in an in vitro transcription assay under low-salt conditions (100 mM NaCl); no labelling could be detected at the other heat-shock puffs under these conditions. Although DRB has been reported as a specific inhibitor of RNA polymerase II-directed transcription, and although the low-salt conditions were not propitious for the activity of this enzyme, RNA polymerase II was detected on puff III-A3b and on the other heat-shock puffs by immunofluorescence with anti-RNA polymerase II antibodies.
Effects of antibodies to intact cytochrome-c oxidase and its subunit V on the enzymatic activity
Authors: Nicholls P. , Cooper C. E. , Freedman J. A. , Leece B. D.
Published: December 2009

Antibodies have been raised in rabbits against whole beef heart cytochrome-c oxidase and purified subunit V. Antioxidase recognizes nearly all the enzyme subunits but reacts very strongly with subunits II and IV. Antisubunit V is quite specific against subunit V. Inhibition of enzyme activity by antioxidase is typically biphasic in time, indicating populations of both rapidly and slowly reacting molecules. Variation of cytochrome c concentration shows partially competitive kinetics, but the antibody also affects "internal" enzymatic events, including the catalytic turnover induced by N,N,N′,N′-tetramethyl-p-phenylenediamine alone and the spin-state change in cytochrome a3 that follows reduction of cytochrome a. No spectral effects can be seen however. Antioxidase also inhibits proteoliposomal respiration with external cytochrome c, but not that with internally trapped cytochrome c. No functionally significant epitopes are detectable on the N side of the membrane in proteoliposomes, although some small effects can be seen with submitochondrial particles. Antisubunit V inhibits the isolated enzyme by at least 60%. The inhibition at high ionic strength induces a biphasic pattern with respect to cytochrome c concentration. Antisubunit V may thus slow the dissociation of cytochrome c from its complex with the enzyme. Antisubunit V has only small effects on the activities of proteoliposomal and submitochondrial particle oxidase in either orientation. On subunit V, some sites, the binding of which can give rise to inhibition, are thus not accessible to antisubunit V when the enzyme is embedded in a functional membrane system.
Effects of cations on binding of human choriogonadotropin
Authors: McIlroy Patrick J.
Published: December 2009

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.
Purification and partial characterization of a plasminogen activator inhibitor from the human glioblastoma, U138
Authors: Rehemtulla A. , Murphy P. , Dobson M. , Hart D. A.
Published: December 2009

A plasminogen activator inhibitor was purified to apparent homogeneity from conditioned media of U138 cells. The inhibitor is a glycoprotein with a pI of 5.4 and an apparent molecular weight of 45 000. The inhibitor forms sodium dodecyl sulfate-stable complexes with plasminogen activators and trypsin but not with plasmin, thrombin, or pancreatic kallikrein. Some biochemical and immunochemical characteristics of the U138 inhibitor distinguish it from other known plasminogen activator inhibitors. The expression of this inhibitor by U138 cells could be modulated by incubation in phorbol myristate acetate, interleukin-1, tumor necrosis factor, and γ interferon, but not in β interferon. Thus, the expression of the plasminogen activator inhibitor can be influenced by biological response modifiers known to be active in the brain and in the neural response to inflammatory stimuli. Therefore, this inhibitor, along with protease nexin, may be involved in brain development and regulation.
Immunochemistry of highly branched N-glycans. Terminal N-acetyl-D-glucosamine (GlcNAc) cluster antigens contain epitopes composed of terminal GlcNAc residues linked to mannose
Authors: Chechik Boris E. , Brockhausen Inka
Published: December 2009

We have previously demonstrated by the immunoperoxidase method the presence of a chicken heterophile antigenic determinant (CHAD-1) in medullary lymphocytes of the bursa of Fabricius and thymus as well as in some nonlymphoid cells. It has been found that the anti-CHAD-1 antibody could be neutralized by absorption with several glycoproteins or glycopeptides containing highly branched, asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, fetuin, desialo-fetuin, and a series of 27 highly purified oligosaccharides with well-defined structures were used to investigate the chemical composition and fine structure of the CHAD-1 epitope. It was shown that anti-CHAD-1 antibody binds to oligosaccharides with at least three terminal N-acetyl glucosamine residues at the nonreducing end. These residues may be linked (β1—2, β1—4, or β1—6 to one, two, or three different mannose residues. The antibody combining site accommodates at least four carbohydrate residues. Oligosaccharides containing five or six terminal N-acetylglucosamine residues at the nonreducing end demonstrated the highest immunoreactivity with the anti-CHAD-1 antibody. Substitution of terminal N-acetylglucosamine residues with galactose, or with galactose and sialic acid, masks CHAD-1. On the basis of this work, epitopes that react with the anti-CHAD-1 antibody will be renamed terminal N-acetylglucosamine cluster antigens (TGCA). Anti-TGCA antibody has potential use in the monitoring of biosynthetic processing of asparagine-linked oligosaccharides and in studies of their cellular distribution and functions.
Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes
Authors: Deliconstantinos George , Kopeikina Lioudmila , Villiotou Vassiliki
Published: December 2009

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5–5 μM) follows an exponential curve described by the general formula y = a∙ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 μM cholesterol glucoside, with a Hill coefficient of h = 2.98 ± 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 μM) or its glucoside (up to 50 μM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 μM) incorporation. Cholesterol (5 μM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 ± 1.1 °C in control SPM, which was elevated to 29.5 ± 1.4 °C in SPM treated with cholesterol glucoside (50 μM) and abolished in SPM treated with cholesterol (5 μM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F− and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 μM) produced an increased packing of the bulk lipids, while cholesterol (5 μM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.Key words: cholesterol, cholesterol glucoside, (NA+/K+)ATPase, Ca2+-ATPase, membrane fluidity, fluorescence polarization.
Conformational analysis of thioredoxin using organoarsenical reagents as probes. A time-resolved fluorescence anisotropy and size exclusion chromatography study
Authors: Brown Simone B. , Turner Raymond J. , Roche Rodney S. , Stevenson Kenneth J.
Published: December 2009

Reduced thioredoxin was subjected to chemical modification studies employing organoarsenical reagents specific for "spatially close" thiols. Modification was monitored by the loss in the free thiol content, by the percent incorporation of radiolabelled organoarsenical reagents, and by observing the changes in the amounts of the various thioredoxins by size exclusion chromatography. The rate of modification depends upon the polarity, rigidity, and size of the reagents. Small nonpolar organoarsenical reagents readily modified reduced thioredoxin, whereas polar and large reagents do not. Modifications resulted in the formation of stable 15-membered cyclodithioarsenite ring structures with no apparent changes in the secondary structure of the protein. Modification was reversed by the extrusion of the arsenical moiety by addition of 2,3-dimercaptopropanol. We have further characterized the oxidized, reduced, and modified thioredoxins by size exclusion chromatography and fluorescence anisotropy decay measurements. Both techniques show an increase in the hydrated volume of the protein upon reduction. Upon modification, the hydrodynamic volume of the protein further swells. Fluorescence anisotropy decay reveals that with modification there is loosening of the protein so that a "domain" containing the fluorophores can relax independently of the whole protein structure.Key words: thioredoxin, organoarsenical modification, protein conformation, size exclusion HPLC, fluorescence anisotropy.
Purification and characterization of activated human erythrocyte prolidase
Authors: Richter Andrea M. , Lancaster Gerald L. , Choy Francis Y. M. , Hechtman Peter
Published: December 2009

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 °C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58 000 when measured by SDS–PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58 000.Key words: prolidase purification, human erythrocytes.
Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases
Authors: Kornblatt Mary Judith , Klugerman Abbey
Published: December 2009

Two isozymes of enolase, αα and γγ, have been purified from rabbit brain and characterized. The kinetic properties of αα and γγ (pH optimum, Km for phosphoglycerate and phosphoenolpyruvate, requirement for a divalent cation) are very similar to those of rabbit enolase, form ββ, and to those of enolase isozymes from other species. However, several novel properties were observed. (i) All the enolases studied were inhibited by Na+ and Li+. (ii) The rabbit enolases, but not yeast enolase, were activated by K+, NH4+, Cs+, and Rb+. (iii) Rabbit enolase is more susceptible to inhibition by excess Mg2+ than is the yeast enolase; the increased inhibition by Mg2+ above pH 7.1 accounts, at least in part, for the observed differences between mammalian and yeast enolases in their pH optima for activity.Key words: enolase, isozymes, kinetics.
The concentration of uncoupling protein correlates with Mg2+ activated mitochondrial binding of GDP
Authors: Henningfield Mary F. , Swick Robert W.
Published: December 2009

Rats were housed at 4 °C for periods of up to 26 days. As little as 2 h of cold exposure caused an increase in the binding of [3H]GDP to mitochondria from brown adipose tissue. Incubation of mitochondria in vitro with 10 mM Mg2+ caused a marked increase in the subsequent binding of GDP to mitochondria from rats housed at 28 °C and a smaller increase in that from rats exposed to 4 °C for 2 h. Chronic exposure to cold led to an even greater increase in the amount of GDP bound to mitochondria incubated with Mg2+. The time course for the increase in the concentration of uncoupling protein was compared with that for GDP binding to mitochondria with and without Mg2+ treatment. The concentration of uncoupling protein appears to be correlated with the GDP-binding values for mitochondria treated with Mg2+ (r = 0.70) but not with the GDP binding to untreated mitochondria (r = 0.36). Therefore, the binding of GDP to untreated mitochondria may represent thermogenic activity at the time of death, whereas that after treatment with Mg2+ may more closely reflect total thermogenic capacity of the mitochondrion.Key words: concentration of uncoupling protein, cold adaptation, unmasking of GDP binding sites.
Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II)
Authors: Turner R. J. , Roche R. S. , Mani R. S. , Kay C. M.
Published: December 2009

The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (λem 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.Key words: S-100b, time-resolved fluorescence, tyrosinate fluorescence, time-resolved emission spectra.
Characterization of a monoclonal antibody specific to a flavonol 2′-O-glucosyltransferase
Authors: Latchinian Lilian , Ibrahim Ragai K.
Published: December 2009

A monoclonal antibody to a partially purified preparation of 2′-O-glucosyltransferase was produced by in vitro immunization of spleen cells from BALB/c mice, followed by fusion with mouse myeloma cells. Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay for (i) their ability to produce immunoglobulins and (ii) their immunoreactivity with a partially purified enzyme preparation. The majority of the immunoglobulin-producing hybridomas were IgM secretors. Two highly immunoreactive IgM-secreting clones were chosen for further characterization. The supernatant fraction from a culture of one of these clones displayed 50% inhibition of the 2′-O-glucosyltransferase activity. The native form of the 2′-O-glucosyltransferase was essential for recognition, suggesting that the epitope recognized by the antibody is a conformational discontiguous one.Key words: monoclonal antibody, in vitro immunization, flavonoid, O-glucosyltransferase.
Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase
Authors: Loe D. W. , Glover J. R. , Head S. , Sharom F. J.
Published: December 2009

5′-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5′-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5′-nucleotidase showed "break points" for all detergents in the temperature range 30–37 °C. SDS-PAGE of pure 5′-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200–630 kDa, suggesting that lymphocyte 5′-nucleotidase may be present in high molecular mass aggregates in its native state.Key words: 5′-nucleotidase, plasma membrane, detergents, solubilization, stability, activation energy.
Structure of the lipopolysaccharide antigenic O-chain produced by Salmonella livingstone (O:6,7)
Authors: Di Fabio Jose Luis , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

The lipopolysaccharide produced by Salmonella livingstone (O:6,7) was composed of an antigenic O-polysaccharide which was shown by composition, methylation analysis and high resolution nuclear magnetic resonance studies to be a high molecular weight polymer containing D-glucose, 2-acetamido-2-deoxy-D-glucose and D-mannose residues (1:1:4) composed in a repeating hexasaccharide unit having the structure:[Formula: see text]Key words: Salmonella livingstone, lipopolysaccharide, O-polysaccharide.
Evolution of vacuolar H+-ATPases: immunological relationships of the nucleotide-binding subunits
Authors: Manolson Morris F. , Percy Judith M. , Apps David K. , Xie Xiao-Song , Stone Dennis K. , Harrison Michael , Clarke David J. , Poole Ronald J.
Published: December 2009

The evolution of the endomembrane systems of eukaryotic cells can be examined by exploring the evolutionary origins of the endomembrane H+-ATPases. Recent studies suggest that certain polypeptides are common to all H+ pumps of this type. Tonoplast H+ -ATPase from Beta vulgaris L. was purified and antibodies raised to two of its subunits. Each of these antisera reacted with a polypeptide of the corresponding size in bovine chromaffin granules, bovine clathrincoated vesicles, and yeast vacuolar membranes, suggesting common structural features and a common ancestor for endomembrane H+-ATPases of different organelles and different kingdoms. The antiserum raised against the 57-kDa polypeptide of plant tonoplast H+ -ATPase also reacted with subunit "a" of the H+-ATPase from the obligately anaerobic bacterium Clostridium pasteurianum and to the α subunit of the H+ -ATPase from Escherichia coli. There was no reactivity with chloroplast or mitochondrial ATPases. These results are discussed in relation to recent sequence data which suggest that endomembrane H+-ATPases may be evolutionarily related to the F0F1 ATPases.Key words: H+ -ATPase, evolution, immunology, vacuole, endomembrane.
Thioredoxin and glutaredoxin systems of rat liver cytosol are not influenced by thyroid dysfunction
Authors: Bhat Ganesh B. , Hummel Brian C. W. , Walfish Paul G.
Published: December 2009

The relation of thyroid hormone status to the function of hepatic cytosolic components activating microsomal reverse triiodothyronine (rT3) 5′-monodeiodination was studied in rats. Hyperthyroidism was induced by administration of thyroxine and hypothyroidism, by thyroidectomy. The DTT-stimulated microsomal rT3 5′-monodeiodination rate was increased by 125% in hyperthyroid rats and reduced to about 30% in hypothyroid rats (when compared with euthyroid animals). Thyroid status was unrelated to NADPH-dependent activation of microsomal 5′-deiodinase by cytosol components or to cytosolic concentrations of thioredoxin and glutaredoxin, which stimulate in vitro microsomal deiodination of thyroid hormone.Key words: hyperthyroidism, hypothyroidism, glutaredoxin, thioredoxin, 5′-deiodinase.
The binding of G-protein to rod outer segment phospholipids at the nitrogen–water interface
Authors: Salesse C. , Lamarche F. , Leblanc R. M.
Published: December 2009

In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption–desorption in the monolayer of each main ROS phospholipid. From examination of surface pressure and ellipsometric isotherms at the nitrogen–water interface, we have determined that once incorporated into the monolayer, the G-protein remains associated, independent of surface pressure, thus providing evidence against the hopping model.Key words: rod outer segment, visual process, hopping model, G-protein, phospholipid monolayer, ellipsometry.
Freeze fracturing properties of native and heat-adapted Chinese hamster ovary (CHO) cells
Authors: Dinda Maria , Quirt Carol , Mackillop William , Singer Michael
Published: December 2009

Chinese hamster ovary cells were either maintained at 37 °C (native cells) or heat adapted by exposure to a temperature of 40 °C for 2 h. Thereafter, native and heat-adapted cells were incubated at different temperatures for various times, harvested, fixed with glutaraldehyde and glycerol, and studied by freeze fracture microscopy. We observed that the fracture plane either passed through the cell, exposing cytoplasm, or stayed within the plasma membrane, and that the location of the fracture plane was strongly determined by the previous thermal history of the cell. We believe that these differences reflect changes in membrane lipid packing arrangements.Key words: freeze fracture, heat adaptation, Chinese hamster ovary cells.
Plasmid library for the transcription of RNA probes complementary to the entire genome of the human immunodeficiency virus type 1 (HIV-1)
Authors: Roy Chitra , Knaak Christian , Garvie Pauline , Limmer Brian , Campione-Piccardo José
Published: December 2009

A plasmid (pBH10R3) containing a 9-kb Sst I fragment of HIV-1 (clone BH-10) inserted in pSP64, an in vitro expression vector, has been used for the transcription of anti-sense HIV-1 RNA. With this system, the transcripts obtained in vitro were not usually full length (1 to 2 kb long) and they predominantly span the 3′ end ORF and ENV regions of the viral genome. We have rearranged the HIV-1 genomic sequences with respect to the SP6 promoter in the pSP64 vector and have obtained a series of new constructs allowing the expression in vitro of RNA transcripts complementary to other regions in the HIV-1 genome, including the 5′ end of the ENV region as well as the TAT, POL, and GAG regions. In fact, the combined use of these constructs as templates for in vitro transcription allows the production of RNA probes spanning the entire viral genome. Compared with the 1- to 2-kb probes mentioned above, the combined use of such probes results in a several-fold increase in the sensitivity of molecular hybridization for the detection of HIV-1 nucleic acid sequences. Also, these constructs enable the preparation of RNA probes that have the potential to detect restriction polymorphisms throughout the HIV-1 genome.Key words: HIV-1, RNA probes, in vitro transcription.
The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ
Authors: Rogers Kem A. , Sandig Martin , McKee Nancy H. , Kalnins Vitauts I.
Published: December 2009

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.Key words: microfilaments, in situ, endothelium, wound healing, aorta.
The CD4/CD8:p56lck complex in T lymphocytes: a potential mechanism to regulate T-cell growth
Authors: Rudd Christopher , Helms Stuart , Barber Elizabeth K. , Schlossman Stuart F.
Published: December 2009

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These receptors have also been found to regulate T cell growth in a manner independent of MHC recognition. In this report, we describe recent work showing that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase, p56lck, from T lymphocytes. The p56lck protein is a member of the src family, which plays a crucial role in the activation and transformation of various mammalian cells. The CD4/CD8:p56ck complex is catalytically active as shown by its ability to phosphorylate at 55–60 kDa. Two-dimensional, nonequilibrium gel electrophoresis demonstrated the similarity of p56lck associated with the CD4 and CD8 antigens. Detergents were found to vary in their ability to solubilize the CD4:p56lck complex in a catalytically active form. We further demonstrated by in vitro phosphorylation that members of the CD3 complex including the γ, δ, and ε chains, as well as a putative ζ subunit can be phosphorylated at tyrosyl residues by the CD4/CD8:p56lck complex. Thus, this interaction may play an important role in the activation of T cells, and may mediate the cooperative interaction between the CD4/CD8 antigens and the Ti(TcR)/CD3 complex. This interaction also represents a possible precedent by which other members of the src family (c-src, c-yes, c-fgr, etc.) may be found to interact with mammalian growth receptors.Key words: CD4, CD8, protein-tyrosine kinase p56lck.
A transfected H-ras oncogene does not inhibit differentiation of cardiac and skeletal muscle from embryonal carcinoma cells
Authors: Rudnicki Michael A. , Reuhl Kenneth R. , McBurney Michael W.
Published: December 2009

P19 embryonal carcinoma (EC) cells can be induced to differentiate in vitro into a variety of cell types, including cardiac and skeletal myocytes. We have isolated P19 cells stably transformed with either the activated human H-ras oncogene or with a chimeric gene in which the H-ras oncogene was controlled by a muscle-specific promoter. These P19 lines exhibited ubiquitous and muscle-specific expression of the activated H-ras protein, respectively. In both lines of P19 cells, normal cardiac and skeletal muscle differentiation was observed. Since the activated H-ras prevents differentiation of myoblast cell lines, our results suggest that the EC-derived muscle progenitor cell differs from continuous myoblast cell lines, perhaps by lacking a complementing oncogene responsible for myoblast immortalization.Key words: embryonal carcinoma, oncogene, ras, differentiation, myogenesis.
Snake venom components and their cross-reactivity: a review
Authors: Berger Bradley J. , Bhatti A. Rashid
Published: December 2009

Snake venoms are complex mixtures of organic and inorganic compounds, many of which display biological activity. It has been demonstrated that antisera raised against whole venom or a single purified venom protein from one species of snake will react with proteins in the venom of other species. This cross-reactivity between species may have applications in determining snake phylogeny, but recent studies on the variation of venom components within a species make these evolutionary conclusions questionable.Key words: snake, venom, cross-reactive, evolution.
Factors influencing the heat shock response of Xenopus laevis embryos
Authors: Nickells Robert W. , Browder Leon W. , Wang Theresa I.
Published: December 2009

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 °C), whereas others are synthesized only at higher temperatures (i.e., above 37 °C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 °C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.Key words: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), heat shock proteins (hsps), heat shock response, thermotolerance, Xenopus laevis.
Acidic ribosomal proteins of Dictyostelium discoideum that show electrophoretic similarity to Escherichia coli proteins L7 and L12
Authors: Ramagopal S.
Published: December 2009

This study documents the presence of three acidic proteins, A1 (pI 4.95), A2 (pI 4.85), and A3 (pI 4.70), in Dictyostelium discoideum ribosomes. All three proteins showed an apparent molecular mass of 13 000 by two-dimensional, sodium dodecyl sulfate gel electrophoresis. They were selectively released by treatment of ribosomes with 50% ethanol – 1 M NH4Cl. The amino acid compositions of A1, A2, and A3 were identical and indicated a predominant amount of alanine. All the above properties are shared by Escherichia coli proteins L7 and L12 and acidic ribosomal proteins in many eukaryotes. Unlike other eukaryotic systems, the acidic proteins of D. discoideum were found associated with the 40S rather than the 60S ribosomal subunit. Acidic proteins analogous in size and electrophoretic mobility to those of D. discoideum were also detected in several other cellular slime mold strains. Not one of the cellular slime mold acidic proteins reacted with antibodies to E. coli proteins L7 and L12 in immunodiffusion tests. In D. discoideum, the distribution of acidic proteins was altered during development. Amoebae contained all three proteins. In spores, A, was absent and the relative amounts of A2 and A3 were lower than in amoebae. In addition, nine other acidic ribosomal proteins exhibited differences between vegetative amoebae and spores.Key words: acidic ribosomal proteins, development, cellular slime mold, L7 and L12 proteins, two-dimensional gel electrophoresis.
Effects of epidermal growth factor (urogastrone) on gluconeogenesis, glucose oxidation, and glycogen synthesis in isolated rat hepatocytes
Authors: Moreno Francesc , Pastor-Anglada Marcal , Hollenberg Morley D. , Soley Maria
Published: December 2009

Using isolated rat hepatocytes, we studied the effect of epidermal growth factor (urogastrone) (EGF-URO) on the incorporation of [3-14C]pyruvate into glucose and glycogen, on the incorporation of [U-14C]glucose into glycogen, and on the oxidation of [U-14C]glucose to 14CO2. The effects of EGF-URO were compared with those of glucagon and insulin. EGF-URO, with an EC50 of 0.2 nM, enhanced by 34% (maximal stimulation) the conversion of [3-14C]pyruvate into glucose; no effect was observed on the oxidation of glucose to CO2 and on the incorporation of either pyruvate or glucose into glycogen. The effect of EGF-URO on pyruvate conversion to glucose was observed only when hepatocytes were preincubated with EGF-URO for 40 min prior to the addition of substrate. Glucagon (10 nM) increased the incorporation of [3-14C]pyruvate into glucose (44% above control); however, unlike EGF-URO, glucagon stimulated gluconeogenesis better without than with a preincubation period. Neither insulin nor EGF-URO (both 10 nM) affected the incorporation of [U-14C]glucose into glycogen during a 20-min incubation period. However, at longer time periods of incubation with the substrate (60 instead 20 min), insulin (but not EGF-URO) increased the incorporation of [14C]glucose into glycogen; EGF-URO counteracted this stimulatory effect of insulin. In contrast with previous data, our work indicates that EGF-URO can, under certain conditions, counteract the effects of insulin and, like glucagon, promote gluconeogenesis in isolated rat hepatocytes.Key words: rat hepatocytes, EGF-URO, gluconeogenesis, glycogen synthesis.
Conformation-dependent inactivation of tryptophan decarboxylase from Catharanthus roseus
Authors: Fernandez J. A. , Kurz W. G. W. , Luca V. De
Published: December 2009

Catharanthus roseus tryptophan decarboxylase (TDC) was irreversibly inactivated in crude extracts and in purified form by incubation of the enzyme at 37 °C. The half-life of inactivation of TDC was approximately 0.75 h, whereas addition of Mn2+ or Mg2+ cations increased this half-life to 9.5 and to over 30 h, respectively. The evidence presented suggests that TDC could exist in vivo in equilibrium between a dimeric active form, which is stabilized by Mg2+ cations, and a monomeric form, which is susceptible to irreversible inactivation. When the equilibrium is shifted to monomer production and inactivation, TDC was quickly degraded by a proteolytic process, which could involve ATP. The results suggest that TDC activity may be regulated by posttranslational controls that shift the equilibrium between conformational states, some of which may be susceptible to irreversible inactivation followed by proteolysis.Key words: tryptophan decarboxylase, Mg2+-dependent conformational changes, posttranslational modifications.
Pyruvate fermentation by Clostridium acetobutylicum
Authors: Janati-Idrissi Rachid , Junelles Anne-Marie , Kanouni Abdellah El , Petitdemange Henri , Gay Robert
Published: December 2009

Clostridium acetobutylicum ATCC 824 using pyruvate as the sole carbon source produced mainly acetate and butyrate as end products of fermentation. Acetate and butyrate kinase activities were higher in cells growing in the presence of pyruvate than glucose, whereas the level of the acetoacetate decarboxylase, an enzyme involved in solvent formation, was lower. Similar activities of glyceraldehyde-3-phosphate dehydrogenase were found in cells grown in pyruvate and glucose mediums. The transfer of C. acetobutylicum from pyruvate to glucose medium suggested that pyruvate represses the "solventogenesis."Key words: Clostridium acetobutylicum, pyruvic acid, kinase, acetoacetate decarboxylase.
Tubulin synthesis, structure, and function: what are the relationships?
Authors: MacRae Thomas H. , Langdon Carrie M.
Published: December 2009

In most eukaryotes, families of tubulin genes give rise to multiple isoforms of tubulin, which may be modified post-translationally. The synthesis of isotubulins is spatially and temporally regulated, leading to the presence of different tubulins within an organism. The cellular localization of tubulin is also nonrandom with discrete isoforms residing in specific regions of some cells. Much work, dependent upon interrelated molecular and immunological technologies, has gone into determining why cells produce multiple isotubulins. One proposal would have us believe that isotubulins are functionally discrete and that the isotubulin composition of a microtubule determines its function. A second idea is that tubulin multigene families arose by gene duplication and subsequent genetic drift. The duplicated genes survive in modified form, as they provide the cell with the ability to effectively regulate tubulin synthesis at the transcriptional level while yielding multifunctional tubulins. Analysis of mutant tubulin genes is revealing functionally important regions of tubulin, prompting a return to the consideration of those properties of the molecule that are fundamental to microtubule formation rather than function per se. A model for autoregulation of tubulin synthesis has been developed, but tittle is yet known of other molecular signals or mechanisms involved in regulation of tubulin production. Further study of flagella formation promises to change this situation. Complementing the genetic studies is the examination of tubulin posttranslational modifications including detyrosination/tyrosination, acetylation/deacetylation, and phosphorylation. Enzymatic mechanisms mediating tubulin posttranslational changes are partially elucidated, as are the influences of the modifications on microtubule properties. Potential functions of the posttranslational changes are the modulation of microtubule-associated protein binding to microtubules, the provision of a mechanism to indicate microtubule age, or as mediators of cellular morphogenesis, a role which could be dependent upon the first two functions. Despite the progress that has been made, the physiological significance of having several isotubulins within a single cell and the molecular details of microtubule function remain obscure.Key words: tubulin genes, multitubulin hypothesis, autoregulation, posttranslational modifications.
Ribosomal protein S1 and initiation factor IF3 do not promote the ribosomal binding of ~ 19-nucleotide-long mDNA and mRNA models
Authors: Laughrea Michael , Tam John
Published: December 2009

A number of model mDNAs and mRNAs, about 19 nucleotides in length, were obtained by automated synthesis and T7 RNA polymerase directed transcription from synthetic DNA templates, respectively. These mDNAs and mRNAs had Shine-Dalgarno sequences that were four to eight nucleotides long and their sequence was similar to the R17 coat protein initiation site. The effect of S1 or initiation factors (IFs) on the rate and extent of ribosomal binding of these mDNAs and mRNAs was investigated by nitrocellulose filtration. No effect was detected, suggesting that the main function of S1 or IFs included neither the direct recognition or binding of short messengers to the 30S subunits, nor their rejection as "false" for possessing the wrong sugars or an insufficient length. A pulse–chase experiment with one of the mRNAs also showed that S1 or IF3 did not influence the exchange rate of mRNA bound to the 30S subunit.Key words: ribosome, initiation, mRNA, ribosomal protein S1, initiation factor IF3.
Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems
Authors: Coorssen Jens R. , Rand R. P.
Published: December 2009

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (Lα) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single Lα structure to a mixture of Lα and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.Key words: diacylglycerol, cholesterol, bilayers, phosphatidylcholine, phosphatidylethanolamine.