bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase
Authors: Gômez-Puyou M. Tuena de , Martins Orlando B. , Gômez-Puyou A.
Published: December 2009

A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.
Relationships between intracellular pH, protein synthesis, and actin assembly during parthenogenetic activation of sea urchin eggs
Authors: Dufresne Louise , Desroches Michel , Bourgault Claire , Gicquaud Claude , Dubé François
Published: December 2009

The relationship between the rate of protein synthesis and the state of assembly of actin was investigated in parthenogenetically activated and fertilized green sea urchin (Strongylocentrotus droebachiensis) eggs. Artificial agents known to raise the intracellular pH (pHi) without affecting the internal calcium concentration were used. Incubation of the eggs with ammonia, procaine, or 12-O-tetradecanoylphorbol 13-acetate (TPA, a protein kinase C activator) resulted in an increase of the protein synthesis rate. At low amounts of ammonia and procaine, this increase was concentration dependent. Higher concentrations of these amine compounds were inhibitory to protein synthesis. Also, chromosome condensation was optimal only in the eggs treated with low concentrations of ammonia or procaine, and was never observed in the TPA-treated eggs. None of these treatments induced detectable changes of the initial amount of G-actin observed in the resting cell, as measured by the deoxyribonuclease I assay. In parallel, no changes of the F-actin content were detected, as determined by quantitative fluorometric measurements of rhodamine–phalloidin labeled eggs. On the other hand, fertilized eggs that showed a higher rate of protein synthesis than parthenogenetically activated eggs were also characterized by a decrease in the total cellular concentration of G-actin. Thus, whereas protein synthesis and chromosome condensation may proceed in the absence of any polymerization of actin in the parthenogenetically activated eggs, it cannot be excluded that actin assembly could be linked to this metabolic activity in the fertilized egg.
The activation of the cyclic-GMP phosphodiesterase via metarhodopsin I: a new model for vertebrate transduction
Authors: Deshpande S. , Abrahamson E. W.
Published: December 2009

Light-scattering spectrophotometry has been used to study rapid photo-induced molecular–cellular changes in vertebrate rod outer segments. Here, we discuss the temporal profiles of the nucleotide-independent P signal as a function of photobleaching, pH dependence in membrane-permeable and -impermeable buffers, angular and wavelength dependence, and cyclic-GMP phosphodiesterase inhibitors. On the basis of these observations, we suggest that (i) the P signal is coupled with the metarhodopsin I photo-intermediate and (ii) processes involved in the P signal invoke activation of cyclic-GMP phosphodiesterase. Furthermore, temperature-dependence studies indicate that the G protein does not participate in the scheme until the metarhodopsin II stage has been reached. This latter finding suggests that GTP-dependent processes are involved principally in the recovery of the system following light absorption. Our results point to a new model for phototransduction in vertebrate vision.
Inhibition by phenylglyoxal of the sodium-coupled fluxes of glucose and phosphate in renal brush-border membranes
Authors: Béliveau R. , Bernier M. , Giroux S. , Bates D.
Published: December 2009

The coupling of phosphate and glucose transport to sodium in brush-border membrane vesicles from rat kidney cortex was studied after chemical modification of arginine residues by phenylglyoxal. Phosphate (10 mM) and sodium (20 mM) uptakes were linear for 6 s and stimulated in the presence of their cosubstrate. The sodium: phosphate stoichiometry measured by a direct method was 1.74. Sodium-independent phosphate and glucose influx were found to be unaffected by phenylglyoxylation. Phosphate- or glucose-independent sodium influx also remained unaltered by the treatment. However, phosphate influx measured with sodium was inhibited by 69% and sodium influx measured with phosphate was inhibited by 40%. When these values were corrected for uncoupled fluxes, the sodium influx coupled to phosphate and the phosphate influx coupled to sodium were inhibited by 93 and 95%, respectively. Glucose influx measured in the presence of sodium was inhibited by 36% and sodium influx in the presence of glucose was reduced by 39%. When the values were corrected for diffusion, these inhibitions were 95 and 100%, respectively. We conclude that the coupling of phosphate and glucose to sodium fluxes by the renal carriers requires the participation of arginine residue(s) in the translocation process. Modification of this arginine by phenylglyoxal leads to a marked inhibition of coupling. These results suggest the implication of arginine residues in the molecular coupling for both glucose and phosphate sodium symporters.
Autolysis of the millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken skeletal muscle
Authors: Nagainis Peter A. , Wolfe Frederick H. , Sathe Shridhar K. , Goll Darrel E.
Published: December 2009

The millimolar Ca2+-requiring form of the Ca2+-dependent proteinase from chicken breast skeletal muscle contains two subunit polypeptides of 80 and 28 kDa, just as the analogous forms of this proteinase from other tissues do. Incubation with Ca2+ at pH 7.5 causes rapid autolysis of the 80-kDa polypeptide to 77 kDa and of the 28-kDa polypeptide to 18 kDa. Autolysis of the 28-kDa polypeptide is slightly faster than autolysis of the 80-kDa polypeptide and is 90–95% complete after 10 s at 0 °C. Autolysis for 15 s at 0 °C converts the proteinase from a form requiring 250–300 μM Ca2+ to one requiring 9–10 μM Ca2+ for half-maximal activity, without changing its specific activity. The autolyzed proteinase has a slightly lower pH optimum (7.7 vs. 8.1) than the unautolyzed proteinase. The autolyzed proteinase is not detected in tissue extracts made immedately after death; therefore, the millimolar Ca2+-requiring proteinase is largely, if not entirely, in the unautolyzed form in situ.
Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes
Authors: Brissette Louise , Noël Simon-Pierre
Published: December 2009

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.
Processing and targeting of proteins in the eucaryote
Authors: Bergeron J. J. M.
Published: December 2009

Cell-free systems have helped to elucidate the molecular constituents regulating the selection and translocation of proteins into and across the endoplasmic reticulum membrane, chloroplasts, mitochondria, and peroxisomes, the transport to and through the Golgi apparatus, and the sorting of proteins to the lysosomes, the plasmalemma, and the extracellular milieu. The use of cell-free systems has also been instrumental in defining the endosomal apparatus and its functional significance in the sorting of incoming ligands and receptors, the selective processing of internalized ligands such as insulin, and transmembrane signalling, especially of the epidermal growth factor and insulin receptor tyrosine kinases. Predicted use of cell-free systems to study interorganelle relationships may help to identify the majority of molecular constituents regulating membrane traffic in the eucaryote.
The glycosphingolipid composition and glycosyltransferase activities of the small intestinal mucosa of testosterone-treated rats
Authors: Dahiya Rajvir , Ahlawat Ravinder S. , Sharma Arun
Published: December 2009

Prior studies have demonstrated that sex hormones can influence the glycosphingolipid composition of different organs, including small intestine. However, to date, the effects of testosterone on glycosphingolipids of rat small intestinal mucosa have not been examined. Experiments were conducted to examine the effect of subcutaneous administration of synthetic testosterone (500 μg/100 g body wt.) on the gangliosides and neutral glycosphingolipids of rat small intestinal mucosa. Their results demonstrated that testosterone administrations: (i) increased the ganglioside content including hematoside (GM3); (ii) increased the total content of neutral glycosphingolipids, which was due to the increases in glucosylceramide and globotriaosylceramide; (iii) increased the activities of cytidine 5′-monophosphate-N-acetylneuraminic acid : lactosylceramide sialyltransferase, and UDPgalactose : lactosylceramide galactosyltransferase; (iv) increased the percentage of the long chain base phytosphingosine in hematoside, glucosyl-, and globotriaosylceramide; and (v) significantly altered the fatty acid composition of each of these glycosphingolipids. These results demonstrate that administration of testosterone induces alterations in glycosphingolipid composition and glycosyltransferases activities in rat small intestinal mucosa.Key words: glycosphyngolipid, glycosyltransferase, small intestine, testosterone, rat.
Regulation of the synthesis of DNA polymerase-α in regenerating liver by calcium and 1,25-dihydroxyvitamin D3
Authors: Sikorska Marianna , Belle Ian de , Whitfield James F. , Walker P. Roy
Published: December 2009

Digestion of nuclei from normal or partially hepatectomised rat livers with endogenous nucleases liberated a pool of cryptic DNA polymerase-α activity which had previously gone unrecognised. Most of this activity is released into the supernatant as free enzyme molecules (11S), but a small fraction of it is released as a complex of 16S (probably with DNA primase). About 40% of the enzyme remains in the pellet, which contains undigested chromatin and components of the residual nuclear matrix and nucleoskeletal structures. Virtually all of this remaining activity is extracted by 2.0 M salt. The activity of DNA polymerase-α increases equally in all nuclear fractions during the period of DNA replication in regenerating rat liver. Lowering of the serum calcium level by thyroparathyroidectomy does not affect basal DNA polymerase-α activity, but prevents induction of the enzyme during the later stages of prereplicative development. However, an injection of 1α,25-dihydroxy vitamin D3 into the rat during the first 6 h after partial hepatectomy restores the ability of the hepatocytes to induce DNA polymerase-α activity and initiate DNA synthesis. These results are discussed in terms of the role of calcium ions in the regulation of the critical stage of prereplicative development which commits the cells to DNA replication.Key words: replication, nuclear, matrix, complex, lamina, reductase.
Removal of apoprotein-B-containing lipoproteins by plasmapheresis using immobilized phosphorylcholine-binding protein affinity adsorbent
Authors: Saxena U. , Francis-Collins J. , Hall J. , Legal Y. , Barrowman J. , Nagpurkar A. , Mookerjea S.
Published: December 2009

Rat serum phosphorylcholine binding protein was earlier shown to bind lipoproteins containing apoproteins B and E from human very low and low density lipoproteins. The present studies were undertaken to show the effectiveness of rat serum phosphorylcholine-binding protein immobilized on Sepharose affinity column to remove apoprotein-B-containing lipoproteins from normal and hypercholesterolemic rabbit plasma, when used in a plasmapheresis system. The maximum in vitro binding of very low and low density lipoproteins from hypercholesterolemic rabbit plasma to the affinity adsorbent was Ca2+ dependent, and the cholesterol bound to the column at the optimum calcium concentration (2.5 mM) was 21% of the total plasma cholesterol applied. The in vivo binding of total cholesterol from normal and hypercholesterolemic rabbit plasma during plasmapheresis ranged from 0.22 to 7.7%. Total mass of cholesterol bound ranged from 3.86 and 27.52 mg at plasma cholesterol concentrations 13.8 and 282 mg/dL, respectively. Most (>95%) of the bound cholesterol was associated with very low and low density lipoproteins. These studies show the ability of immobilized rat serum phosphorylcholine-binding protein to lower the atherogenic apoprotein-B-containing lipoproteins from plasma of hypercholesterolemic rabbits.Key words: plasmapheresis, rabbit plasma, cholesterol, C-reactive protein, rat phosphorylcholine binding protein.
Differential transport and integration into the nuclear lamina for lamins A, B, and C
Authors: Dagenais André , LeMyre Andrée , Bibor-Hardy Viviane
Published: December 2009

Lamins A, B, and C are the major proteins of the mammalian nuclear lamina and have been well studied in BHK-21 cells. Using in vivo labelling, cell fractionation, and immmunoprecipitation, we have found that lamins have different patterns of nuclear transport and solubility. Newly synthesized lamin A is translocated to the nucleus faster than lamin C or B. It is the most tightly bound lamin and cannot be extracted from the lamina by nonionic detergent or high-salt buffers. Lamins B and C migrate more slowly to the nucleus. Partitioning between cytoskeleton and detergent-soluble fractions shows that integration of lamins B and C is not completed before a 1-h chase. For lamin C this process is dependent upon protein synthesis and can be inhibited with cycloheximide. Even though lamins A and C are almost identical, lamin C is never firmly bound to the lamina and can be partially solubilized upon high-salt treatment.Key words: nuclear proteins, lamins, nuclear transport.
Purification of D-β-hydroxybutyrate dehydrogenase from rat brain
Authors: Zhang Wei-Wei , Churchill Perry
Published: December 2009

D-β-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been purified to homogeneity from rat brain using a new improved method. The purified rat brain BDH has a subunit molecular mass of 31 kilodaltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 125 μmol/(min∙mg). This is 625-fold greater than the activity in the mitochondrial fraction. The new purification procedure involves chromatography using a quaternary amine Sepharose resin followed by a sulphonate Sepharose resin, and eliminates the need for glass bead adsorption chromatography.Key words: mitochondria, membrane, phospholipid, dehydrogenase, lipid dependent.
A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues
Authors: Baszczynski Chris L.
Published: December 2009

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.Key words: anti-kanamycin antibodies, immunoprecipitation, neomycin phosphotransferase II assay, transformation.
15-Lipoxygenase products affect protein phosphorylation in Friend erythroleukemia cells
Authors: Beckman B. S. , Jeter Jr. J. R.
Published: December 2009

Endogenous arachidonic acid metabolism and protein phosphorylation have been examined in Friend erythroleukemia cells in response to the induction of differentiation by dimethyl sulfoxide and hexamethylene bisacetamide. 15-Hydroxyeicosatetraenoic acid levels were elevated in cells differentiated with hexamethylene bisacetamide or dimethyl sulfoxide compared with undifferentiated cells. Protein phosphorylation decreased markedly in differentiated cells compared with undifferentiated cells and the addition of 15-hydroperoxyeicosatetraenoic acid specifically decreased the phosphorylation of a 28-kilodalton protein. These findings indicate that products of 15-lipoxygenase may act as intracellular messengers in Friend erythroleukemia cells by affecting protein phosphorylation.Key words: arachidonic acid, protein phosphorylation, proliferation, differentiation, 15-lipoxygenase.
Traces of brain microtubule-associated proteins affect dynamic properties of microtubules
Authors: Keates Robert A. B.
Published: December 2009

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin–colchicine complex in the mixture to block the repolymerization. For a total of 24 μM tubulin in a polymerization mixture, 10 μM of the sample polymerized originally under the conditions used. When 1.05 μM tubulin–colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23–2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.Key words: dynamics, microtubules, tubulin, microtubule-associated proteins, self-assembly.
Comparison of the structures of operator DNA free and in complex with λ repressor
Authors: Baleja James D. , Sykes Brian D.
Published: December 2009

The structures of operator DNA unbound and in complex with λ repressor protein are compared. The conformation of the left 10 base pairs of a λ right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to λ repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of λ repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.Key words: DNA structure, λ repressor, DNA–protein recognition, nuclear magnetic resonance.
Use of mutant RNAs in studies on yeast 5S rRNA structure and function
Authors: Nazar Ross N. , Ryk Don I. Van , Lee Yoon , Guyer C. David
Published: December 2009

The expression of mutant yeast 5S rRNA genes in vivo is reviewed as a basis for further studies on the structure, function, and regulation of the ribosomal 5S rRNA. Specific base substitutions, insertions, or deletions can result in substantial structural changes which can be detected readily by gel electrophoresis, permitting the assay of mutant RNA synthesis and utilization. Furthermore, the use of high and low copy shuttle vectors, as well as alternate growth conditions, permits a wide adjustment of the mutant RNA concentration. Under optimized conditions more than 80% of the cell's RNA can be replaced with mutant molecules. The application of this strategy to studies on the biosynthesis and structure of the 5S rRNA are demonstrated through recently isolated mutations.Key words: site-specific mitogenesis, 5S RNA, ribosomes, yeast transformation.
Deoxyribonucleoside triphosphate pools of herpes simplex virus infected cells: the influence of selective antiherpes agents and the role of the deaminase pathway
Authors: Aduma Philip J. , Gupta Sagar V. , Stuart Allan L. , Tourigny Guy
Published: December 2009

The effect of 5-methoxymethyl-2′-deoxycytidine (MMdCyd), in combination with tetrahydrodeoxyuridine (H4dUrd) and 5-methoxymethyl-2′-deoxyuridine (MMdUrd) on deoxyribonucleoside triphosphate pools was assessed. The dNTP pool content was almost 5 times as high in herpes simplex virus (HSV) infected VERO cells compared with mock-infected cells. Significant differences in dNTP pool sizes were observed with the different treatments. Treatment of HSV-infected cells with MMdCyd and MMdUrd resulted in a massive expansion of the dTTP pool, whereas pools of dCTP and dGTP were not affected substantially. MMdUrd and MMdCyd produced dATP pools that were 4 and 2.5 times that of the controls, respectively. Treatment with H4dUrd resulted in the dCTP pool increasing 12 times and barely detectable levels of dTTP. MMdCyd in combination with H4dUrd resulted in a marked reduction of the total deoxyribonucleoside triphosphate level. These results indicate that during viral replication the bulk of the thymidine nucleotides are derived from the dCyd/dCMP deaminase de novo pathway.Key words: herpes simplex virus, methoxymethyldeoxycytidine, tetrahydrodeoxyuridine, dNTP pools, deaminase pathway.
Impaired binding of low density lipoprotein to hepatic membranes from uremic guinea pigs
Authors: Shapiro R. Jean
Published: December 2009

Chronic renal failure is associated with abnormalities in lipoprotein metabolism that may contribute to premature atherosclerosis and early mortality in patients on dialysis. In previous studies, we found that plasma clearance of radiolabelled low density lipoprotein (LDL) was retarded in nephrectomized guinea pigs left with one-sixth of normal functioning renal mass. To elucidate potential mechanisms of delayed LDL clearance, we compared binding of LDL to hepatic membranes from both normal and uremic guinea pigs. One hundred micrograms of the 8000 – 100 000 × g hepatic microsomal protein was incubated with 125I-labelled normal guinea pig LDL (10–150 μg/mL) for 1 h at 37 °C, and the membrane washed and pelleted by centrifugation in a Beckman Ti 42.2 rotor. Parallel incubations with excess unlabelled LDL were done to determine specific binding. LDL specific binding to uremic hepatic membranes was significantly impaired compared with normal ones. The major abnormality, as determined by Scatchard transformation of the binding data, was a reduction of the apparent maximal binding of LDL to uremic membranes, with an average Bmax of 4.1 μg/mg protein compared with 6.6 μg/mg protein for normal hepatic microsomes. The affinity of LDL for uremic liver membranes was only slightly diminished with a mean apparent Kd of 35.2 μg/mL in comparison with 21.8 μg/mL for normal liver membranes. These results provide a biochemical explanation for the diminished LDL clearance in uremia and may account for the dyslipidemia of renal failure.Key words: hepatic microsomes, low density lipoprotein, uremia.
Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli
Authors: Churchill Sharon , Churchill Perry
Published: December 2009

A rat liver bacteriophage λ expression library was probed using polyclonal antibodies raised to purified rat liver D-β-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression.
Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus
Authors: Gicquaud Claude , Paré Michel
Published: December 2009

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.Key words: phalloidin, virotoxin, motility, actin, membrane.
ATP-stimulated protease activity in brown fat mitochondria: response to a 24-h fast in mice
Authors: Desautels M.
Published: December 2009

Brown fat mitochondria have [3H]casein-hydrolyzing activity at pH 8.0 associated with both membrane and soluble fractions. An ATP-stimulated proteolytic activity inhibited by vanadate and N-ethylmaleimide was found in the soluble fraction. Membrane-associated proteolytic activity was inhibited by phenylmethylsulfonyl fluoride and trypsin inhibitor, suggesting that it is a serine protease. A 24-h fast in mice caused a significant loss of mitochondrial proteins from the tissue, but had no effect on protease activity of isolated mitochondria with or without ATP. The ATP-stimulated release of amino acids or peptides from isolated mitochondria, as measured with fluorescamine, was not influenced by food deprivation. Thus, brown fat mitochondria possess an ATP-stimulated proteolytic pathway that does not appear to be involved in the bulk removal of mitochondrial proteins from brown fat of fasting mice.Key words: protein degradation, energy balance, body weight, nonshivering thermogenesis.
Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins
Authors: Barbeau Dominique , Marcellus Richard C. , Bacchetti Silvia , Bayley Stanley T. , Branton Philip E.
Published: December 2009

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36–69 within conserved region 1 (CR1), and residues 121–138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.Key words: DNA tumour viruses, transformation, oncogenes, cell cycle, protein complexes.
Resistance of ADP-ribosylated histones and HMG proteins to proteases
Authors: Boulikas Teni , Poirier Guy G.
Published: December 2009

Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or α-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid – urea – Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis.Key words: poly(ADP-ribose), proteases, histones, poly(ADP-ribose) polymerase.
Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein
Authors: Mymryk Joe S. , Oakes Janice D. , Muthuswamy Senthil K. , D'Amico Pietro , Bayley Stanley T. , Lee Raymond W. H.
Published: December 2009

Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of β- and γ-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, α-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.Key words: actin, adenovirus 5 E1A, BC3H1 myoblasts, myogenin.
Physiological levels of hydrostatic pressure alter morphology and organization of cytoskeletal and adhesion proteins in MG-63 osteosarcoma cells
Authors: Haskin Christine , Cameron Ivan
Published: December 2009

The response of human MG-63 osteosarcoma cells to physiological levels of hydrostatic pressure was studied. Cell cultures were subjected to a 20-min, 4-MPa hydrostatic pressure pulse. Adhesion was measured at 20 min and 2 h post-hydrostatic pressure. Morphometric measurements of cell shape and immunofluorescent assays of cytoskeletal and adhesion proteins were done pre- and post-hydrostatic pressure. Pressure-treated cells showed increased adhesion (resistance to deadhesion by trypsinization) with increased recovery time. Indirect immunofluorescence demonstrated increased heterotypic adhesion receptor at cell–cell interfaces and increased α3,β1-integrin at cell–substrate interfaces. Indirect immunofluorescence demonstrated depolymerization of α-tubulin, vimentin, and actin during the pressure pulse. Actin reorganization was slower than that of α-tubulin and vimentin, with stress filaments not well organized even after 1 h postpressure. The depolymerization of α-tubulin, vimentin, and actin observed at relatively low levels of hydrostatic pressure suggests disintegration of the integrin–cytoskeletal attachment complex. The increased resistance of the cells to trypsinization and the increase in both heterotypic adhesion receptor and the α3,β1-integrin at cell interfaces suggest that cells compensate for loss of cytoskeletal integrity by increasing attachment to both adjacent cells and the extracellular matrix.Key words: hydrostatic pressure, integrins, tubulin, vimentin.
Are prosome particles formed by the disintegration of proteasomes?
Authors: Blaker Timothy W. , Locke Michael , Atkinson Burr G.
Published: December 2009

Quail reticulocyte 19S "prosome" fractions isolated on sucrose density gradients contain two kinds of particles: cylindrical proteasomes and ferritin. When samples of this fraction are prepared for electron microscopy using the one-step stain protocol described in this paper, most of the particles have a rectangular image resembling the proteasome. However, when samples are prepared for electron microscopy using the two-step stain protocol described here, there are few rectangular images. Their place is taken by round particles that resemble the prosome. Thus it appears that the round, raspberry-shaped particles called prosomes and the ring-like proteasome particles may be artifacts of specimen preparation for electron microscopy. We propose that proteasome particles may disintegrate when prepared for electron microscopy by methods such as the two-step stain protocol and that prosome particles represent the component parts of the proteasome. Furthermore, based on the enhancement of proteasome images obtained using the one-step main protocol we propose that, instead of consisting of a stack of four rings, the proteasome is constructed of three components, i.e., a spherical central particle flanked by two flat hexagonal end caps.Key words: proteasome, prosome, ferritin, reticulocytes.
Rate of alteration of hepatic mixed-function oxidase system in rats fed different dietary fats
Authors: Ammouche A. , Dinh L. , Youyou A. , Clément M. , Bourre J. M.
Published: December 2009

Studies were carried out to evaluate and relate the rate of alteration in mixed-function oxidase system with the changes of the fatty acid composition of rat microsomes induced by different dietary lipids. Male weanling rats were fed from day 21 to 120 with a commercial rat diet or a semisynthetic diet containing no fat or 10% fat consisting of peanut–rapeseed oil, sunflower oil, or salmon oil. In rats fed a fat-free diet, the cytochrome P-450 concentration and aniline hydroxylase, aminopyrine N-demethylase, and NADPH–cytochrome-c reductase activities of liver microsomes at 120 days were, respectively, 26, 16, 10, and 24% lesser than those of rats fed the control diet. However, cytochrome b5 concentration and NADH–cytochrome-b5 reductase activity were, respectively, 33 and 43% higher than those of the control group at the same time. When rats were fed the sunflower oil diet, the cytochrome P-450 concentration and NADH–cytochrome-b5 reductase activity at 120 days were, respectively, 11 and 23% lesser than those of control group. But the cytochrome b5 concentration was 10% higher than that of the control group. In rats fed the fish oil diet, the cytochrome P-450 concentration and NADPH–cytochrome-c reductase, aniline hydroxylase, and aminopyrine N-demethylase activities at 120 days were, respectively, 30, 48, 41, and 31% higher than those of rats fed the control diet. These enzymes were correlated very well (0.84 < r < 0.93), P < 0.05 with dietary Σ polyunsaturated fatty acids (n−3). The fatty acid composition of liver microsomes of rats fed a fat-free diet revealed high levels of 16:1(n−7), 18:1(n−9), and 20:3(n−9) acids. A dramatic decrease in 18:2(n−6) and 20:4(n−6) acids was observed. Animals fed a sunflower oil diet showed high levels of 22:4(n−6) and 22:5(n−6), and low levels in monounsaturated fatty acids. However when rats were fed a fish oil diet, the liver microsomes were highly enriched in 20:5(n−3) and 22:6(n−3) acids and simultaneously there was a decrease in arachidonic acid. From these findings it is concluded that dietary fats induce changes not only in the fatty acid composition of liver microsome lipids, but also in the activities of mixed-function oxidase system. Also, the results suggest that essential fatty acids might play a role in regulating the intrinsic membrane protein activities.Key words: cytochrome P-450, cytochrome b5, NADH–cytochrome-b5 reductase, NADPH–cytochrome-c reductase, aminopyrine N-demethylase, aniline hydroxylase, microsomes, rat liver.
Gene expression after resumption of development of Artemia franciscana cryptobiotic embryos
Authors: Escalante Ricardo , García-Sáez Alberto , Ortega Maria-Asunción , Sastre Leandro
Published: December 2009

The steady-state levels of six different mRNAs have been studied during Artemia franciscana development. Some of these mRNAs are present in the cryptobiotic cyst, like those coding for cytoplasmic actins, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, and the Na+,K+-ATPase α-subunit isoform coded by the clone pArATNa136. The expression of these mRNAs is markedly induced during cyst development. A small increase in mRNA levels can be observed for some genes at very early stages of development (2 h). The main increase is observed between 4 and 16 h of development for all these genes, although the time course of mRNA accumulation is different for each one of the genes studied. Some other genes, like those coding for muscle actin (actin 3) or the Na+,K+-ATPase α-subunit isoform coded by the cDNA clone α2850, are not expressed in the cyst before resumption of development and their expression is induced after 10 or 6 h of development, respectively. These data on the kinetic of mRNA accumulation provide the information required to determine transcriptionally active developmental stages, necessary to study in more detail the mechanisms of transcriptional regulation during activation of cryptobiotic cysts and resumption of embryonic development.Key words: Artemia, gene expression, actin, Na,K-ATPase, Ca2+-ATPase.
Posttranscriptional regulation of ribonucleotide reductase R1 gene expression is linked to a protein kinase C pathway in mammalian cells
Authors: Chen Frank Y. , Amara Francis M. , Wright Jim A.
Published: December 2009

A rate-limiting reaction in DNA synthesis is catalyzed by ribonucleotide reductase, the enzyme responsible for reducing ribonucleotides to provide the deoxyribonucleotide precursors of DNA. In this study, we have tested the hypothesis that posttranscriptional regulation of ribonucleotide reductase R1 gene expression is controlled by a protein kinase C signal pathway. We show that mouse BALB/c 3T3 fibroblasts treated with the potent and highly specific protein kinase C inhibitor bisindolylmaleimide GF 109203X contain significantly reduced steady-state levels of R1 mRNA and protein. Message half-life studies demonstrate that this is due, at least in part, to a marked decrease in R1 message stability in cells treated with the protein kinase C inhibitor. Furthermore, the protein kinase C signal pathway appears to be specifically involved in this regulation since 8-bromo-cAMP, a modulator of the protein kinase A pathway, had no effect on R1 mRNA levels or stability properties. Cross-linking assays revealed that the binding activity of a R1 mRNA 3′-untranslated region binding protein (R1BP), which was previously shown to be involved in the regulation of R1 mRNA stability, was significantly elevated after treatment of the cells with GF 109203X, in a dose-dependent manner. However, treatment with 8-bromo-cAMP at concentrations up to 2.5 mM did not obviously affect the basic level of the R1BP–RNA interaction. These observations provide a better understanding of the biochemical signals that are critical for the cis–trans interaction-mediated posttranscriptional regulation of ribonucleotide reductase R1 gene expression.Key words: protein kinase C, ribonucleotide reductase, mRNA stability, RNA–protein interaction, bisindolylmaleimide GF 109203X, 8-bromo-cAMP.
Effect of the antirheumatic agent Tenidap on CD3, CD4, and CD8 expression and interleukin-1 and leukotriene B4 secretion in human peripheral blood mononuclear cells
Authors: Conti Pio , Reale Marcella , Barbacane Renato C. , Stuard Stephano , Placido Fernanda
Published: December 2009

Thymocytes that express the complete CD3−T-cell receptor (TCR) complex are CD4− and CD8−. The CD4+ T-cell population can be subdivided into at least two quite distinct subsets, TH1 and TH2 cells, based upon cytokine expression. Interleukin-1 (IL-1) appears to be required for optimal proliferation of T cells in response to antigen and it seems that in the absence of IL-1, TH2 clones proliferate less in response to antigen. Tenidap is an antirheumatic agent that has an inhibitory effect on IL-1 production. In these studies, we show that isolated human peripheral blood mononuclear cells (PBMCs) treated in vitro with Tenidap (15 μg/mL) for 48-h incubations significantly (p < 0.05) enhanced the present of CD4+ expression compared with untreated cells (control), as determined by cytofluorimetric analysis. Lipopolysaccharide and Bacillus Calmette-Guérin were used as positive controls. When the cells were tested for CD3 or CD8 receptor expression, no differences were found between the untreated PBMCs and the treated (15 μg/mL Tenidap) cells. No change was found when cells were incubated for 72 h. Moreover, our data show a strong dose-dependent inhibitory effect of Tenidap (15 μg/mL) on IL-1α, IL-1β, and leukotriene B4 secretion in PBMCs treated overnight. The increased CD4+ expression by Tenidap in PBMCs may suggest an important role for this new antirheumatic agent in immunity and may hold future therapeutic promise for diseases involving IL-1 and leukotriene B4 as mediators.Key words: Tenidap, lymphocyte receptors, leukotriene B4, interleukin-1, lipopolysaccharide.
Role of apoptosis in the immune system
Authors: Williams G. T.
Published: December 2009

Apoptosis is becoming recognized as a fundamental component of the immune system, i.e., in its development and its regulation. It is involved in many diverse areas that fall into two broad categories: firstly, the development and shaping of the immune receptor repertoire and, secondly, immune effector mechanisms. We have employed several model systems for analysing the development of the immune system, e.g., haemopoietic progenitor cell development and thymocyte development and selection, as well as the role of apoptosis in CD4+ T-cell-mediated cytotoxicity. These studies have helped illustrate the fundamental role of active cell death in the physiological functioning of the immune system. Failure of such a fundamental process would be expected to have serious consequences and we have been particularly involved in analysing the role of inappropriate suppression of apoptosis by the BCL2 family of genes both in oncogenesis in the immune system and in the development of cancer cell resistance to therapy. It seems likely that this is only one of many mechanisms by which apoptosis may be disrupted with pathological consequences.Key words: apoptosis, bcl-2, clonal deletion.
Membrane permeation and intracellular trafficking of long chain fatty acids: insights fromEscherichia coliand 3T3-L1 adipocytes
Authors: Mangroo Dev , Trigatti Bernardo L. , Gerber Gerhard E.
Published: December 2009

Long chain fatty acids are important substrates for energy production and lipid synthesis in prokaryotes and eukaryotes. Their cellular uptake represents an important first step leading to metabolism. This step is induced in Escherichia coli by growth in medium containing long chain fatty acids and in murine 3T3-L1 cells during differentiation to adipocytes. Consequently, these have been used extensively as model systems to study the cellular uptake of long chain fatty acids. Here, we present an overview of our current understanding of long chain fatty acid uptake in these cells. It consists of several distinct steps, mediated by a combination of biochemical and physico-chemical processes, and is driven by conversion of long chain fatty acids to acyl-CoA by acyl-CoA synthetase. An understanding of long chain fatty acid uptake may provide valuable insights into the roles of fatty acids in the regulation of cell signalling cascades, in the regulation of a variety of metabolic and transport processes, and in a variety of mammalian pathogenic conditions such as obesity and diabetes.Key words: acyl-CoA synthetase, adipocyte, Escherichia coli, fatty acid, transport, uptake.
Processing of eukaryotic pre-rRNA: the role of the transcribed spacers
Authors: Nues Rob W. van , Venema Jaap , Rientjes Jeanette M. J. , Dirks-Mulder Anita , Raué Hendrik A.
Published: December 2009

The 17–18S, 5.8S, and 25–28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase I. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.Key words: ribosome, processing, precursor rRNA, eukaryote, transcribed spacer.