bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast plasmids have fewer superhelical turns than predicted by the nucleosome repeat of yeast DNA
Authors: Dobranowska-Fishell Alicja , Pulleyblank David E.
Published: December 2009

The superhelical density of three Saccharomyces cerevisiae plasmids was determined with respect to a defined reference state during vegetative growth and stationary phase. The levels of supercoiling determined were ~20% lower than predicted by comparisons with SV40 DNA and reconstituted minichromosomes using histones from higher eukaryotes. In two different plasmids with the ARS1 origin of replication, the level of supercoiling changed substantially as the host cells entered stationary phase. Supercoiling of the endogenous 2-μm plasmid during vegetative growth was lower than in the ARS1 -containing plasmids but did not change significantly upon entry of the cells into stationary phase.Key words: chromatin structure, nucleosome, yeast, DNA supercoiling.
Partial characterization of bovine elastin gene; comparison with the gene for human elastin
Authors: Manohar Advaitanand , Shi Wen , Anwar Rashid A.
Published: December 2009

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5′ direction from the initiation codon and 978 nucleotides in the 3′ direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5′ to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.Key words: elastin gene, elastin gene promoter, bovine.
Transferrin glycosylation in hypoxia
Authors: Regoeczi Erwin , Kay J. Michael , Chindemi Paul A. , Zaimi Ouahida , Suyama Kaye L.
Published: December 2009

The aim of this study was to examine the effect of reduced O2 tension on the glycosylation of transferrin. Rats were placed in a hypobaric chamber (380 mmHg) that corresponded to an altitude of 5486 m above sea level for 21 days. The animals responded with marked increases in hematocrit (from 44 to 76%) and cardiac weight, and with reductions in the concentration of plasma transferrin averaging 15%. Analyses of their plasma transferrin by serial anion-exchange and lectin affinity chromatography revealed no changes in the extent of glycan branching. However, there was a moderate rise in the proportion of fucosylated transferrin molecules (fucosylation index) and a slight decrease in the transferrin fraction bearing a tetrasialylated biantennary glycan. The fucosylation index correlated positively with plasma transferrin concentrations in the test animals, but not in the controls. In contradistinction to the situation with transferrin, hypoxic rats exhibited a reduced fucosylation index of immunoglobulin G.Key words: fucosylation index, hypoxia, immunoglobulin G, lectin affinity chromatography, transferrin.
Covalent modifications of ribosomal proteins in growing and aggregation-competent Dictyostelium discoideum: phosphorylation and methylation
Authors: Ramagopal S.
Published: December 2009

Phosphorylated and methylated ribosomal proteins were identified in vegetatively growing amoebae and in the starvation-induced, aggregation-competent cells of Dictyostelium discoideum. Of the 15 developmentally regulated cell-specific ribosomal proteins reported earlier, protein A and the acidic proteins A1, A2, and A3 were identified as phosphoproteins, and S5, S6, S10, and D were identified as methylated proteins. Three other ribosomal proteins were phosphorylated and 19 others methylated. S19, L13, A1, A2, and A3 were the predominant phosphoproteins in growing amoebae, whereas S20 and A were the predominant ones in the aggregation-competent cells. Among the methylated proteins, eight (S6, S10, S13, S30, D, L1, L2, and L31) were modified only during growth phase, six (S5, S7, S8, S24, S31, and L36) were altered only during aggregation-competent phase, and nine (S9, S27, S28, S29, S34, L7, L35, L41, and L42) were modified under both phases. Five proteins (S6, S24, L7, L41, and L42) were heavily methylated and of these, the large subunit proteins were present in both growing amoebae and aggregation-competent cells. These findings demonstrate that covalent modification of specific ribosomal proteins is regulated during cell differentiation in D. discoideum.Key words: cellular slime mold, cell differentiation, vegetative amoebae, aggregation competence, phosphorylation, methylation, ribosomal proteins.
Sequences of the variable regions of three monoclonal antibodies specific for histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system
Authors: Steeves Teresa , Barry M. Michele , Duckworth Harry W. , Waygood E. Bruce , Lee Jeremy S.
Published: December 2009

The variable regions of three monoclonal antibodies, Jel 42, Jel 44, and Jel 324, specific for the histidine-containing protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system have been sequenced from their respective mRNAs. The Vh gene families were deduced from the percent homology to the concensus gene sequences and the J gene and D gene usage was also analysed.Key words: monoclonal antibodies, gene sequencing.
Purification and immunological characterization of HPLC-purified pertussis toxin subunits
Authors: Chong Pele , Cockle Stephen , Boux Heather , Klein Michel
Published: December 2009

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), > 90% (S3), > 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.Key words: pertussis toxin, high-performance liquid chromatography, purification, pertussis vaccine.
Expression of the preproenkephalin A gene in the heart of cardiomyopathic hamsters
Authors: Ouellette Michel , Dumont Michel , Lemaire Simon , Brakier-Gingras Léa
Published: December 2009

The expression of the preproenkephalin A gene (Enk gene), which codes for the precursor of enkephalins, was investigated in the heart of hamsters with a hereditary cardiomyopathy at four different stages of the disease: the prenecrotic stage (30 days), the necrotic stage (60 days), the hypertrophic stage (120 days), and the final stage (200 days). In control atria and ventricles, the relative abundance of the Enk mRNA, as assessed by Northern blot analysis, did not change upon ageing. In the ventricles of cardiomyopathic hamsters, however, it increased about two- to three-fold at the necrotic stage, but was unaltered at the other time points studied; whereas in the atria, it progressively decreased to reach about half that of control hamsters at the final stage. Enkephalin levels, as measured by radioimmunoassay, decreased at 60 days in both the atria and ventricles of control hamsters, and also in the atria of cardiomyopathic hamsters, and remained stable thereafter, corresponding to one-third to one-half of those at 30 days. However, in the ventricles of cardiomyopathic hamsters, the peptide level decreased only slightly, the consequence being that at 60, 120, and 200 days, it was about two- to three-fold that of control hamsters. The lack of correlation between peptide levels and the relative abundance of the Enk mRNA suggests that translational and (or) post-translational mechanisms are important in the control of the expression of the Enk gene in the heart of hamsters.Key words: enkephalins, preproenkephalin A, cardiomyopathic hamster.
Intramitochondrial fatty acid metabolism: riboflavin deficiency and energy production
Authors: Parsons H. G. , Dias V. C.
Published: December 2009

Inborn errors of fatty acid β-oxidation have contributed significantly to our understanding of intracellular fatty acid metabolism. The first intramitochondrial step in β-oxidation of fatty acyl-CoA of different chain lengths is catalyzed by the three chain length specific acyl-CoA dehydrogenases. Inherited deficiency of these enzymes has been reported. Some are riboflavin responsive. The first step of fatty acid oxidation is reviewed with specific emphasis on β-oxidation in newborn infants, rendered riboflavin deficient by phototherapy. Given that medium chain fatty acids are not stored as triacylglycerols and undergo rapid β-oxidation, they have been proposed as superior substrates compared with long chain triglycerides in times of metabolic stress. This review also examines medium chain triglycerides as an alternate energy source. When medium chain triglycerides were fed as 50% of total energy, glucose sparing was present with little loss of energy as dicarboxylic acids.Key words: β-oxidation, acyl-CoA dehydrogenase, riboflavin, medium chain triglycerides, dicarboxylic acids.
Folylpolyglutamate synthetase from beef liver: purification and some properties
Authors: Pristupa Z. B. , McAuley C. M. , Scrimgeour K. G.
Published: December 2009

The polypeptide chain of folylpolyglutamate synthetase from beef liver has been isolated and partially characterized. This polypeptide has an apparent molecular weight of 73 000. Its amino-terminal residue is blocked. Amino acid analysis agrees with the hydrophobic properties and the pi (6.0) of this cytosolic enzyme. Polyclonal antibodies to the denatured enzyme have been prepared.Key words: folylpolyglutamate synthetase, folates, polyglutamates, electrophoresis, antisera.
Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937
Authors: Suzuki K. , Sakata N. , Hara M. , Kitani A. , Harigai M. , Hirose W. , Kawaguchi Y. , Kawagoe M. , Nakamura H.
Published: December 2009

Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90° light scatter intensity, one of the basic parameters of flow cytometry.Key words: monocyte, free fatty acid, triacylglycerol, flow cytometry.
Cytochrome c oxidase: structure, function, and membrane topology of the polypeptide subunits
Authors: Cooper Chris E. , Nicholls Peter , Freedman Jo A.
Published: December 2009

Mitochondrial cytochrome c oxidase and its bacterial homologs catalyze electron transfer and proton translocation reactions across membranes. The eukaryotic enzyme complex consists of a large number of polypeptide subunits. Three of the subunits (I, II, and III) are mitochondrially encoded while the remaining 6 (yeast) to 10 (bovine) are nuclear encoded. Antibody and chemical-labelling experiments suggest that subunits I–III and most (but not all) of the nuclear-encoded subunits span the inner mitochondrial membrane. Subunits I and II are the catalytic core of the enzyme. Subunit I contains haem a, haem a3 and CuB, while subunit II contains CuA and the cytochrome c binding site. Subunit III and most of the nuclear subunits are essential for the assembly of a functional catalytic enzyme. Some nuclear subunits are present as isozymes, although little functional difference has yet been detected between enzyme complexes composed of different isozymes. Therefore, any additional role attributed to the nuclear-encoded subunits beyond that of enzyme assembly must be tentative. We suggest that enough evidence exists to support the idea that modification of the larger nuclear subunits (IV, V, and possibly VI) can affect enzyme turnover in vitro. Whether this is a physiological control mechanism remains to be seen.Key words: cytochrome oxidase, polypeptide subunits, antibodies, membrane protein, orientation.
Nonlinear steady-state kinetics of chloramphenicol acetyltransferase
Authors: Crabbe M. James C. , Goode Derek
Published: December 2009

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.Key words: acetyl coenzyme A, chloramphenicol, antibiotics, enzyme kinetics.
Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines
Authors: Hurta Robert A. R. , Wright Jim A.
Published: December 2009

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity. This is the first demonstration that reversion of hydroxyurea resistance and a decline in ribonucleotide reductase activity are accompanied by decreased ferritin expression, and supports the concept that ferritin is important in establishing resistance to hydroxyurea, and may play a role in DNA synthesis, through the regulation of functional iron-containing M2 protein levels required for ribonucleotide reduction.Key words: ribonucleotide reductase, ferritin, hydroxyurea, drug resistance.
Effect of transfection manipulations on mouse cell cycle progression
Authors: McBroom Carol A. K. , Sheinin Rose
Published: December 2009

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphate-dimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak Sphase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1–S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.Key words: transfection, cell cycle progression.
Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage
Authors: Wu Tai-Wing , Wu Jun , Li Ren-Ke , Mickle Donald , Carey Doug
Published: December 2009

Synthetic delta-bilirubin (i.e., bilirubin that is covalently bonded to albumin) and, to a lesser extent, bilirubin that is physically adsorbed to albumin substantially prolonged the survival of human ventricular myocytes against in situ generated oxyradicals. This cytoprotection, manifested at micromolar concentrations of either form of bilirubin, surpasses those given by unconjugated bilirubin alone, by equimolar levels of albumin (which has some cytoprotective activity), by known antioxidants ascorbate, mannitol, or Trolox, and by oxyradical scavenging enzymes superoxide dismutase (24 200 IU/L) plus catalase (92 000 IU/L) on the same cells. Our human cell-based data provide the first direct evidence that albumin-bound bilirubins, especially delta bilirubin, are potent circulating cytoprotectors. The latter may be important in defending, among others, the myocardium, which is much less equipped in antioxidant defences than for example the liver or intestines.Key words: albumin-bound bilirubins, delta-bilirubin, myocytes, oxyradicals, cytoprotection.
Validation of the morphologic end point of necrosis in rat hepatocytes subjected to oxyradical damage
Authors: Carey Doug , Wu Jun , Sugiyama Hiroshi , Wu Tai-Wing
Published: December 2009

We have used phase-contrast microscopy to determine a necrotic end point of the order of minutes in primary hepatocytes exposed to oxyradicals generated with xanthine oxidase plus hypoxanthine. This study examines whether the morphologic end point thus determined agrees with other criteria of cell necrosis. When 95–100% of the cells were shown to be necrotic by our morphologic assay, transmission electron microscopy confirmed definitive subcellular evidence of cell death, trypan blue exclusion revealed a 92% loss in the ability of cells to exclude the dye, and there was a 47% specific release of 51Cr (versus a 50% theoretical value). In contrast, the appearance of extracellular aspartate aminotransferase activity was relatively slow and did not corroborate the morphologic end point. In summary, we have validated the morphologic end point in our cell-based assay of oxyradical damage.Key words: necrosis, oxyradical damage, hepatocyte, validation.
A 63-kDa protein with androgen-binding activity is not from the androgen receptor
Authors: Wrogemann Klaus , Podolsky Gary , Gu Jin , Rosenmann Eduardo
Published: December 2009

We have found a new protein in the heart of rat and mice that can be selectively and covalently labelled with the synthetic androgen analog mibolerone. Binding is specific as it can be displaced by excess radioinert ligand. The protein is prominently expressed in liver, kidney, and heart, but not in skeletal muscle. It is water soluble and found in the cytosol. Under denaturing conditions it has a molecular weight of 63 000 and appears on two-dimensional gels with an isoelectric point of 6.3. The protein's affinity for androgen is lower than that of the androgen receptor and it is about 100-fold more abundant than the receptor in the heart. Expression of the protein is not induced by androgen. The presence of this protein in testicular feminization (tfm) mice with a genetical defect of the androgen receptor rules out that it is the androgen receptor or a portion thereof. The biological role of this protein is not yet known.Key words: androgen, androgen receptor, heart, photolysis, steroid binding protein.
Simultaneous uptake of galactose and glucose by Azotobacter vinelandii
Authors: Wong T. Y. , Murdock C. A. , Concannon S. P. , Lockey T. D.
Published: December 2009

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 1010 cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 1010 cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.Key words: Azotobacter, carbohydrate metabolism, melibiose, transport, galactose.
Differences in accumulation and phosphorylation of proteins in vegetatively growing and aggregation-competent cells of Dictyostelium discoideum
Authors: Ramagopal S.
Published: December 2009

A comparison of proteins from whole cell lysates of vegetative amoebae and aggregation-competent cells by high-resolution two-dimensional gel electrophoresis coupled with a sensitive silver staining method revealed distinct differences. In aggregation-competent cells, 16 proteins present in the vegetative amoebae disappeared, and 25 new proteins appeared. A few other proteins showed quantitative variation during the transition of vegetative amoebae to aggregation competence. Identification of phosphoproteins by in vivo labeling with [32P]orthophosphate showed that none of the developmentally regulated cellular proteins were modified. Phosphorylation was observed in four proteins. One protein was phosphorylated exclusively in aggregation-competent cells. The phosphorylation level of two other proteins was higher in aggregation-competent cells compared with vegetative amoebae. The data suggest that phosphorylation of cellular and certain ribosomal proteins may be regulated coordinately in Dictyostelium discoideum.Key words: cellular slime mold, cell differentiation, protein phosphorylation.
Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells
Authors: Marks David I. , Fox Richard M.
Published: December 2009

Previous studies we performed showed that 8-methoxypsoralen in combination with ultraviolet A light (photochemotherapy) caused DNA damage and that this caused nucleotide depletion in peripheral blood leukocytes, secondary to an active form of programmed cell death, poly(ADP-ribosyl)ation. Further studies revealed that 24 h after exposure to 10 J/cm2 ultraviolet A light and 8-methoxypsoralen (300 ng/mL), apoptotic cells increased from 3 (control) to 31% (p < 0.001). Ultraviolet A light alone also significantly increased the number of apoptotic cells. These morphological changes were confirmed by parallel findings on DNA electrophoresis. Treatment with 2 to 5 J/cm2 of ultraviolet A light and 8-methoxypsoralen caused an approximately 30% increase in cytosolic free calcium levels in peripheral blood leukocytes 1 h after exposure. Associated with this was a 51% increase in 45Ca2+ uptake over the first 60 min. Similar findings in a different lymphoid cell (CCRF–CEM) confirmed the results obtained with peripheral blood leukocytes. The use of calcium-free medium prevented a rise in cytosolic free calcium and decreased the number of cells undergoing apoptotic cell death. Cycloheximide inhibited ultraviolet A light – 8-methoxypsoralen induced apoptosis in CCRF–CEM cells; it also decreased calcium levels in control CCRF–CEM cells. This study shows that ultraviolet A light – 8-methoxypsoralen caused apoptotic cell death in lymphoid cells; this appeared to be associated with calcium influx, presumably because of the requirement of endogenous endonucleases for calcium.Key words: 8-methoxypsoralen, poly(ADP-ribosyl)ation, DNA damage.
Calcium-dependent mechanisms of regulation of smooth muscle contraction
Authors: Walsh Michael P.
Published: December 2009

The contractile state of smooth muscle is regulated primarily by the sarcoplasmic (cytosolic) free Ca2+ concentration. A variety of stimuli that induce smooth muscle contraction (e.g., membrane depolarization, α-adrenergic and muscarinic agonists) trigger an increase in sarcoplasmic free [Ca2+] from resting levels of 120–270 to 500–700 nM. At the elevated [Ca2+], Ca2+ binds to calmodulin, the ubiquitous and multifunctional Ca2+-binding protein. The interaction of Ca2+ with CaM induces a conformational change in the Ca2+-binding protein with exposure of a site(s) of interaction with target proteins, the most important of which in the context of smooth muscle contraction is the enzyme myosin light chain kinase. The interaction of calmodulin with myosin light chain kinase results in activation of the kinase that catalyzes phosphorylation of myosin at serine-19 of each of the two 20-kDa light chains (native myosin is a hexamer composed of two heavy chains (230 kDa each) and two pairs of light chains (one pair of 20 kDa each and the other pair of 17 kDa each)). This simple phosphorylation reaction triggers cycling of myosin cross-bridges along actin filaments and the development of force. Relaxation of the muscle follows removal of Ca2+ from the sarcoplasm, whereupon calmodulin dissociates from myosin light chain kinase regenerating the inactive kinase; myosin is dephosphorylated by myosin light chain phosphatase(s), whereupon it dissociates and remains detached from the actin filament and the muscle relaxes. A substantial body of evidence has been accumulated in support of this central role of myosin phosphorylation–dephosphorylation in the regulation of smooth muscle contraction. However, a wide range of physiological and biochemical studies supports the existence of additional, secondary Ca2+-dependent mechanisms that can modulate or fine-tune the contractile state of the smooth muscle cell. Three such mechanisms have emerged: (i) the actin-, tropomyosin-, and calmodulin-binding protein, calponin; (ii) the actin-, myosin-, tropomyosin-, and calmodulin-binding protein, caldesmon; and (iii) the Ca2+- and phospholipid-dependent protein kinase (protein kinase C).Key words: smooth muscle, Ca2+, myosin phosphorylation, regulation of contraction.
Increased transglutaminase in the aortas of cholesterol-fed rabbits: occurrence of buffer soluble and insoluble forms and an inhibitor
Authors: Wiebe Rick I. , Tarr Alan H. , Bowness J. Michael
Published: December 2009

Rabbits were fed for 10–12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl – sucrose – dithiothreitol – Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.Key words: aorta, transglutaminase, inhibitor, cholesterol, atherosclerosis.
Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella Virginia O:8
Authors: MacLean Leann L. , Perry Malcolm B.
Published: December 2009

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella Virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure:[Formula: see text]The disaccharide structure α-D-Abep-(1 → 3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of α-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.Key words: Salmonella, endotoxin, lipopolysaccharide, polysaccharide, nuclear magnetic resonance.
Characterization and localization of large sulfated glycoproteins in the extracellular matrix of the developing asteroid Pisaster ochraceus
Authors: Crawford Terry J. , Crawford Bruce J.
Published: December 2009

In this study techniques commonly used to extract and purify proteoglycans of vertebrates were applied to the embryo of the asteroid Pisaster ochraceus at the early bipinnaria larva stage, a stage in which extensive cell migration is occurring within the extracellular matrix of the blastocoel. Several large sulfated glycoproteins were isolated and shown to consist of protein cores covalently bound to sulfated polysaccharide chains. The polysaccharide chains consisted primarily of neutral sugars and were not susceptible to glycosaminoglycan-degrading enzymes, suggesting that these were not glycosaminoglycans. The sulfated glycoproteins could be fractionated by electrophoresis on sodium dodecyl sulfate – agarose – acrylamide composite gels. Two types of monoclonal antibodies prepared against isolated extracellular matrix of these embryos reacted with two subsets of bands on Western blots of the composite gels. Staining of sections of the embryos with the antibodies showed that the epitopes that they recognized were located throughout the extracellular matrices of the embryos. That these high molecular weight glycoproteins were located within the extracellular matrix of the embryos suggests that they may be involved in the control of morphogenesis and cellular movement.Key words: extracellular matrix, sulfated glycoproteins developing asteroid, monoclonal antibodies.
Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study
Authors: Gruia-Gray J. , Desser S. S.
Published: December 2009

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 μM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.Key words: fluorescence microscopy, Rana catesbeiana, tubulin, vimentin, viral assembly sites.
Two different sites of action for platelet activating factor and l-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine on platelets and leukemic cells
Authors: Salari Hassan , Dryden Peter , Howard Sandra , Bittman Robert
Published: December 2009

2-O-Methyl analogs of platelet activating factor (PAF) are potent anticancer agents. The sites of action and mechanisms of cell toxicity of these agents are as yet unknown. To better understand the mode of action of this class of anticancer agents, we examined the ability of 1-O-hexadecyl-2-acetylglycero-3-phosphocholine with the S or R configuration at C2 ((R)-PAF and (S)-PAF) and 1-O-hexadecyl-2-methoxyglycero-3-phosphocholine with the S or R configuration at C2 ((R)-ET-16-OCH3-GPC and (S)-ET-16-OCH3-GPC) to induce rabbit platelet aggregation and to inhibit [3H]thymidine uptake into WEHI-3B cells, HL-60 cells, and normal blood lymphocytes. The four chiral ether-linked lipids caused aggregation of rabbit platelets with the following order of potency: (R)-PAF > (S)-PAF > (R)-ET-16-OCH3-GPC > (S)-ET-16-OCH3-GPC; the EC50 values were 1 pM, 50 nM, 1 μM, and 50 μM, respectively. The cytotoxic effects of these ether lipids in leukemic cells was in reverse order to that observed for aggregation of platelets. The order of potency for inhibition of [3H]thymidine uptake by WEHI-3B and HL-60 cells was (R)-ET-16-OCH3-GPC = (S)-ET-16-OCH3-GPC > (S)-PAF > (R)-PAF; the EC50 values were 2, 2, 15, and > 40 μM, respectively. PAF antagonists (WEB 2086, CV 3988, triazolam, and SRI 63,441) blocked the action of the four ether lipids on platelets, while SRI 63,441 blocked the antineoplastic activity of the ether lipids on WEHI-3B and HL-60 cells. None of the four lipids was able to kill normal lymphocytes significantly. Scatchard analysis of PAF receptor binding revealed that HL-60 and WEHI-3B cells, which are sensitive to the cytotoxic action of ether-linked lipids, do not possess PAF receptors, whereas both normal lymphocytes and platelets do possess a PAF receptor. The present data indicate that the cytotoxic action of antineoplastic ether-linked lipids does not involve the PAF receptor. The protective role of SRI 63,441 in blocking the proaggregatory activity of the ether lipids in rabbit platelets involves PAF receptor, but cytotoxic activity against WEHI-3B and HL-60 cells does not result from its ability to act as a PAF antagonist.Key words: platelet activating factor, ether-linked lipids, leukemia, cytotoxicity, thrombosis, membrane receptor, leukocyte.
Purification of sn-glycerol-3-phosphate dehydrogenase from Trypanosoma brucei brucei
Authors: Kornblatt Jack A. , Nthale Joseph , McOdimba Francis
Published: December 2009

A protein has been purified from the membranes of bloodstream forms of Trypanosoma brucei brucei. The purified material contained a single polypeptide chain of molecular mass 67 kilodaltons as judged by sodium dodecyl sulfate –polyacrylamide gel electrophoresis; under "native" conditions it migrated through a Sephacryl S-300 column with a similar molecular mass. The purified protein catalysed electron transfer from sn-glycerol 3-phosphate to oxygen with the subsequent formation of water. Electron transfer by the purified enzyme to O2 was dependent on the presence of low concentrations of the mediator phenazine methosulfate. This protein is clearly the major membrane-bound sn-glycerol-3-phosphate dehydrogenase, but it also has some characteristics suggestive of the trypanosome alternative oxidase activities.Key words: trypanosomes, glycerophosphate dehydrogenase, trypanosome alternative oxidase.
Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells
Authors: Purushotham Karnam R. , Bologna Joseph , Nakagawa Yoichi , Humphreys-Beher Michael G.
Published: December 2009

A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the β-agonist isoproterenol. A single-step purification was performed on a calmodulin–agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase > histone > phosphodiesterase > phenylalanine hydroxylase > phosphorylase b > bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 μM Ca2+ and 2 μM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 μmol/(min∙mg protein) with a Km of 1.6 μM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.Key words: galactosyltransferase, hypertrophy, cell proliferation, phosphorylation.
Hydroxyl free radical production by the reaction of N-methyl-N′-nitro-N-nitrosoguanidine with hydrogen peroxide without exposure to light
Authors: Mikuni Tomiko , Tatsuta Masaharu , Kamachi Mikiharu
Published: December 2009

We examined hydroxyl free radical (∙OH) production in the mixture of H2O2 and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) without exposure to light using the electron spin resonance spin-trapping technique. When the mixtures were protected from exposure to light, ∙OH was formed at pH 6.5 and above; it was not formed at pH 5.0 and below, consistent with our previous report. The amount of ∙OH trapped depended on the concentrations of MNNG and H2O2 and the pH. Nitrite ion was also detected colorimetrically at pH 6.5 and above, but not detected at pH 5.0 and below in the mixtures without exposure to light. Moreover, its production depended on the concentrations of MNNG and H2O2 and the pH. The formation of N-methyl-N′-nitroguanidine in the mixture at pH 7.8 was confirmed by thin-layer chromatography and melting point. These results suggest that nucleophilic attack by H2O2 on the nitroso nitrogen of MNNG results in the formation of N-methyl-N′-nitroguanidine and peroxynitrous acid, which degrades homolytically to yield ∙OH and nitrogen dioxide, resulting in the production of nitrite ion, at pH 6.5 and above without exposure to light.Key words: N-methyl-N′-nitro-N-nitrosoguanidine, hydrogen peroxide, hydroxyl free radical, peroxynitrous acid.
Readthrough enhancement and promoter occlusion on the ribosomal genes of Xenopus laevis
Authors: Moss Tom , Larose Anne-Marie , Mitchelson Keith , Leblanc Benoît
Published: December 2009

An RNA polymerase I termination site is found just upstream of the ribosomal gene promoter in mammals and amphibia. It has been suggested that this termination site may actively enhance ribosomal transcription in a process known as readthrough enhancement or that it may simply prevent the disruption of initiation complexes or promoter occlusion. There is, however, a consensus of opinion that the terminator is important for efficient ribosomal transcription. Here we have quantitatively investigated the relative importance of readthrough enhancement and promoter occlusion on the transcription of the microinjected Xenopus laevis ribosomal gene. The results show that, in this system, promoter occlusion is limited and terminator mutations predominantly affect readthrough enhancement. The terminator is shown to be unnecessary for the enhancer activity of the rest of the ribosomal spacer. Model calculations suggest that readthrough enhancement could be explained by polymerase recycling and that it may be unnecessary to postulate a specific mechanism of polymerase handover.Key words: Xenopus, ribosomal, promoter, RNA polymerase I, rDNA.
The ability of bovine mammary tissue to synthesize lipids for 96 h when cocultured with liver and adipose tissue
Authors: Keys J. E. , Fekry A. E. , Wood D. L. , Capuco A. V.
Published: December 2009

Explants of bovine mammary, liver, and adipose tissues were cocultured in the same well for 24, 48, 72 or 96 h and their ability to synthesize lipids was measured by the incorporation of [14C]acetate into triglycerides, free fatty acids, and phospholipids. The model was developed to study the effect of bovine somatotropin on lipid synthesis in mammary tissue, because somatotropin will not affect mammary tissue unless liver tissue is present. Somatotropin reduced incorporation by mammary tissue relative to the control. However, the rate of incorporation remained constant through 96 h with somatotropin, but steadily decreased in control mammary tissue. Phospholipid release into the media increased dramatically from 2% of that synthesized during the first 24 h to 218% of that synthesized from 72 to 96 h. The high release of phospholipids after 24 h suggests that cellular breakdown was occurring. A 24-h incubation is recommended to study lipid synthesis with this system.Key words: bovine somatotropin, lipid synthesis, triglycerides, free fatty acids, phospholipids.
Dolichol metabolism in Chinese hamster ovary cells
Authors: Kaiden Adina , Krag Sharon S.
Published: December 2009

The addition of oligosaccharide to asparagine residues of soluble and membrane-associated proteins in eukaryotic cells involves a polyisoprenoid lipid carrier, dolichol. In Chinese hamster ovary cells, the major isomer of this polyisoprenol has 19 isoprenyl units, the terminal one being saturated. Our laboratory has developed a procedure to analyze the levels and nature of the cell's dolichyl derivatives. Chinese hamster ovary cells contain predominately activated, anionic dolichol derivatives, such as oligosaccharyl pyrophosphoryldolichol, monoglycosylated phosphoryldolichols, and dolichyl phosphate. Our studies show that in growing cells there is continual synthesis of total dolichol. Also, preliminary data suggest there is no catabolism or secretion of this lipid. The level of dolichyl phosphate did not change significantly under a variety of conditions where the levels of enzyme activities utilizing dolichyl phosphate did change. These results suggested that these enzymes had access to the same pool of dolichyl phosphate and had similar Km values for this lipid.Key words: dolichol, dolichyl phosphate, metabolism, Chinese hamster ovary cells.
Enzymes that recognize dolichols participate in three glycosylation pathways and are required for protein secretion
Authors: Orlean Peter
Published: December 2009

We have explored the structure, function, and membrane topography of enzymes that recognize dolichols and participate in glycosylation pathways in the endoplasmic reticulum. Enzymes that interact with dolichols, including dolichyl phosphate mannose (Dol-P-Man) synthase and UDP-GlcNAc:Dol-P GlcNAc-1-P-transferase, revealed a conserved amino acid sequence in membrane-spanning regions. The consensus is Phe-Ile/Val-Xaa-Phe/Tyr-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe/Tyr, and we propose it is involved in dolichol recognition. We have used yeast mutants to demonstrate the role of dolichols in three glycosylation pathways. At its nonpermissive temperature, a Dol-P-Man synthase mutant (dpm1) was blocked in N-glycosylation, O-mannosylation, and glycosyl phosphoinositol membrane anchoring of protein, most likely because Dol-P-Man serves as mannosyl donor in all three pathways. The secretion mutant sec59 has a similar phenotype to dpm1, and the presence of a dolichol recognition sequence in the SEC59 protein gave a clue to its defect, which is in dolichol kinase. Comparison of yeast glycosylation mutants suggests that the ability to carry out N-glycosylation alone is sufficient to allow yeast to secrete glycoproteins and that an N-linked saccharide of a minimum size must be attached to proteins for cells to be able to secrete them and maintain a functional secretory pathway.Key words: glycosylation, secretion, glycosyl phosphatidylinositol, yeast mutants.
Changes of dolichol and dolichyl fatty acyl esters during the germination and development of soybeans
Authors: Ishinaga Masataka , Yamauchi Toshimi , Egusa Keiko , Kuroda Keiko
Published: December 2009

The amounts of dolichol and dolichyl fatty acyl esters and their composition in various parts of soybean seedlings were determined during germination and development. The dolichol content of cotyledons decreased during germination. Dolichyl fatty acyl esters were identified in cotyledons and the amount was estimated by high performance liquid chromatography. The relative amounts of short-chain dolichols of 15, 16, and 17 isoprene units increased during development of the seedlings. The homologue distribution of free dolichol was different from that of dolichyl fatty acyl esters. The relative amounts of dolichols with 16, 17, and 18 isoprene units were greater in free dolichol than in dolichyl fatty acyl esters. The percentages of long-chain saturated fatty acids in dolichyl fatty acyl esters, specifically 21:0, 22:0, 23:0, 24:0, and 25:0, increased during development. These fatty acids represented more than 40% of the fatty acids in dolichyl fatty acyl esters in stems. These results suggest that dolichyl fatty acyl esters are not a storage form of dolichol. The large accumulation of dolichol and dolichyl fatty acyl esters in the leaves, where photosynthesis takes place, suggests some other function.Key words: dolichol, dolichyl fatty acy ester, soybean, behenic acid, lignoceric acid.
Uptake of dolichol by Vero cells
Authors: Dessel Guido Van , Meirvenne Hermine Van , Lagrou Albert , Hilderson Herwig , Dierick Wilfried
Published: December 2009

Characterization and kinetics of dolichol uptake by a Vero cell line are reported. Vero cells incorporate dolichol in a time- and dose-dependent manner. Optimal uptake is found at 37 °C and at a pH of 7.4. In contrast to cholesterol, an inhibitory effect on the dolichol incorporation is found for farnesol, geraniol, and retinol. Long chain polyprenols were slightly stimulatory. The translocation seems not to be highly energy dependent. The lack of substantial inhibition by chloroquine does not plead for a receptor-mediated endocytosis. Incorporated dolichol was distributed over both membranes and supernatant fractions, paralleling the distribution of the lysosomal marker β-N-acetylhexosaminidase. The incorporated dolichol is subject to a fast efflux process, which is potentiated by the presence of lipid acceptors in the extracellular medium.Key words: dolichol, efflux, subcellular fractionation, uptake, Vero cells.