bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of rodent cDNA encoding the poly(ADP-ribose) polymerase catalytic domain and analysis of mRNA levels during the cell cycle
Authors: Thibodeau J. , Gradwohl G. , Dumas C. , Clairoux-Moreau S. , Brunet G. , Penning C. , Poirier G. G. , Moreau P.
Published: December 2009

We have isolated a partial 2.0 kb cDNA (pRATC) encoding the entire 489 amino acids of the NAD binding domain located at the C terminus of the rat poly(ADP-ribose) polymerase. pRATC sequences were analysed and compared with the human mRNA. Our analysis reveals a remarkable homology between the rat and human nucleotide and amino acid sequences. Although a few minor amino acid changes were detected, we have found that the total number of possible phosphorylation sites remained constant in the NAD binding domain of both enzymes. We have also found that a 102 amino acid sequence, containing the putative nucleotide binding site Gly-Lys-Gly (position 378), is perfectly conserved between the rat and human sequences. Strong homology was also detected between pRATC and genomic DNA isolated from various vertebrates. In addition, we have analysed the levels of poly(ADP-ribose) polymerase mRNA throughout the cell cycle. Our results show that the levels of mRNA culminate in the G1 phase. We have also found that the increase in enzymatic activity observed in rats following treatment with phenobarbital did not correspond to an increase in the mRNA levels.Key words: poly(ADP-ribose) polymerase.
Hamster liver cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes
Authors: O Kar-Min , Siow Yaw L. , Choy Patrick C.
Published: December 2009

CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) are microsomal enzymes that catalyze the final steps in the syntheses of phosphatidylcholine and phosphatidylethanolamine via the CDP-choline and CDP-ethanolamine pathways, respectively. Both enzyme activities were cosolubilized from hamster liver microsomes by Triton QS-15. Limited separation of these two activities was achieved by ion-exchange chromatography. The partially purified phosphotransferases displayed a higher sensitivity than microsomal phosphotransferases towards exogenous phospholipids and showed an absolute requirement for divalent cations. Upon purification, cholinephosphotransferase was more stable to heat treatment than ethanolaminephosphotransferase. The two enzymes exhibited distinct pH optima and responded differently to exogenous phospholipids. Our results clearly indicate that cholinephosphotransferase and ethanolaminephosphotransferase are separate enzymes.Key words: cholinephosphotransferase, ethanolaminephosphotransferase, phosphatidylcholine biosynthesis, hamster liver.
Mixtures of semisynthetic species of cerebroside sulfate with dipalmitoyl phosphatidylcholine. Thermotropic phase behavior and permeability
Authors: Boggs J. M. , Mulholland D. , Koshy K. M.
Published: December 2009

The phase behavior of mixtures of dipalmitoyl phosphatidylcholine (DPPC) with semisynthetic species of cerebroside sulfate (CBS) containing palmitic acid (C16:0-CBS) or lignoceric acid (C24:0-CBS) in 0.1 M KCl was studied using differential scanning calorimetry. DPPC and C16:0-CBS were miscible in all proportions in the gel phase above 10 mol% CBS and in the liquid-crystalline phase. However, C24:0-CBS was less miscible with DPPC over a wide concentration range in the gel phase. At high CBS concentrations it was probably also not entirely miscible with DPPC in the liquid-crystalline phase. Small amounts of both species of CBS lowered the transition temperature and enthalpy of DPPC, suggesting that they are more soluble in the liquid-crystalline phase of DPPC than the gel phase. The transition temperature at higher CBS concentrations was also less than expected, especially after cycling through the phase transition in the case of C24:0-CBS, suggesting that mixing with DPPC inhibited the intermolecular hydrogen bonding interactions and dehydration of CBS. In C24:0-CBS–DPPC mixtures several populations were present over a wide compositional range, including two solid-solid solutions of fixed composition. At high C24:0-CBS concentrations some C24:0-CBS also phase separated out of the mixture. Structural considerations suggested that the C24:0-CBS which is mixed with DPPC must be interdigitated into the DPPC bilayer. Other populations that are present may have a different structural organization. A fatty acid spin label in these mixtures was a little less ordered than in either lipid by itself. The permeability of these lipids, as well as the two asymmetric species 1-stearoyl-2-caproyl phosphatidylcholine and 1-stearoyl-2-myristoyl phosphatidylcholine (18:10PC and 18:14PC), to a water-soluble spin label tempocholine chloride was also measured. The studies with 18:10PC and 18:14PC indicated that both triple-chain mixed interdigitated bilayers and double-chain partially interdigitated bilayers can trap water-soluble substances and have low permeability. Both species of CBS could also entrap the spin label and had low permeability at 4 °C. However, they rapidly lost the entrapped compound when they transformed into their stable dehydrated phases or into the liquid-crystalline phase. Mixing with DPPC prevented both of these losses. These studies supported the conclusion that a significant amount of the CBS was mixed with the DPPC and that this mixing prevented the dehydration changes which CBS undergoes by itself. They also suggested that the C24:0-CBS can pack with the DPPC in the liquid-crystalline phase in a regular way which must involve inter-digitation of the long C24:0 chain into the DPPC bilayer.Key words: sulfatide, phosphatidylcholine, phase diagram, differential scanning calorimetry, interdigitated bilayer.
Potentiometric and spectroscopic properties of the cytochrome o complex of Escherichia coli
Authors: Withers H. Keith , Bragg Philip D.
Published: December 2009

Cytochrome o purified from cell membranes of Escherichia coli shows two potentiometrically distinct species with midpoint oxidation–reduction potentials of + 265 ± 5 and + 140 ± 15 mV. The component with the higher potential reacted with carbon monoxide and so likely is the oxygen-reacting heme of the cytochrome o complex. It appears to be responsible for the absorption maximum at 564 nm in reduced minus oxidized difference spectra measured at 77 K. The midpoint potential of the other component was sensitive to oxidation by ferricyanide. This latter component had an absorption maximum at about 554 nm. The inhibitor 2-heptyl-4-hydroxyquinoline N-oxide inhibited reoxidation of reduced cytochrome o by oxygen and modified the spectroscopic behaviour of the 564 nm component. The ratio of the heights of the maxima in the alpha-band region of the absorption spectrum differed in cytochrome o prepared from cloned material from that found in cytochrome o from noncloned sources, in spite of the similar polypeptide compositions of the two preparations.Key words: cytochrome o, potentiometry, HOQNO (2-heptyl-4-hydroxyquinoline N-oxide), oxidase, respiratory chain.
Phosphatidylglycerol acetal of plasmenylethanolamine as an intermediate in ether lipid formation in Clostridium butyricum
Authors: MacDonald Dorothy L. , Goldfine Howard
Published: December 2009

The formation and turnover of the recently discovered phosphatidylglycerol acetal of plasmenylethanolamine was investigated in Clostridium butyricum. Incorporation of phosphate into the phospholipids was studied by using [32P]orthophosphate in pulse and pulse–chase experiments in growing cells. Among the ethanolamine-containing lipids, the diacyl form of phosphatidylethanolamine was labeled most rapidly, followed by the phosphatidylglycerol acetal of plasmenylethanolamine and plasmenylethanolamine. The glycerol acetal of plasmenylethanolamine was labeled most slowly. There was rapid turnover of approximately one half of the newly labeled phosphatidylethanolamine pool. Since the kinetics of labeling of the small pool of phosphatidylglycerol acetal of plasmenylethanolamine and the larger pool of plasmenylethanolamine were similar during the early time courses of pulse and pulse–chase experiments, the results argue against the derivation of phosphatidylglycerol acetal of plasmenylethanolamine from plasmenylethanolamine. The results are consistent with the derivation of glycerol acetal of plasmenylethanolamine from either phosphatidylglycerol acetal of plasmenylethanolamine or plasmenylethanolamine.Key words: phosphatidylglycerol acetal of plasmenylethanolamine, plasmenylethanolamine, clostridia, ether lipids, metabolism.
Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy
Authors: Henry Gillian D. , Sykes Brian D.
Published: December 2009

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.Key words: protein dynamics, hydrogen-exchange kinetics, membrane protein, 13C- and 15N-NMR, isotope labels.
Endo-exonuclease of Aspergillus nidulans
Authors: Koa Helena , Fraser Murray J. , Käfer Etta
Published: December 2009

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5′-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical ΦX-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5′-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.Key words: endo-exonuclease, Aspergillus nidulans, purification, properties, inactive form.
Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa
Authors: Dey Chinmoy S. , Majumder Gopal C.
Published: December 2009

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were ~ 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80–90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 μM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 μg/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (~ 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.Key words: spermatozoa, ecto-protein kinases, cAMP, plasma membrane, cell intactness.
Electroporation-mediated uptake of proteins into mammalian cells
Authors: Lambert Helene , Pankov Roumen , Gauthier Johanne , Hancock Ronald
Published: December 2009

Proteins of up to 230 kilodaltons are taken up by Chinese hamster ovary fibroblasts exposed to electroporation under conditions generally similar to those used to mediate DNA transfection. The uptake of catalase, ovalbumin, and histone H1 labelled with fluorescein was visualized by fluorescence microscopy. Under the same conditions, the uptake of colloidal gold particles (20 nm diameter) was visualized by electron microscopy. In optimum conditions, about 25% of the cells remained viable and grew normally and about 25% of these retained labelled proteins during two cycles of further growth. About 6 × 104 molecules of catalase were retained per cell. Proteins were taken up when presented to the cells up to 4 h after electroporation, suggesting that mechanisms other than classical electropore formation may operate in these conditions. The proteins were localized in the cytoplasm in a predominantly vesicular pattern and histone H1 entered the nucleus in some cells.Key words: electroporation, protein uptake, gold particle uptake, endocytosis, cell membrane.
Characterization of horse plasma gelsolin
Authors: Silva Beatriz E. Ruiz , Burtnick Leslie D.
Published: December 2009

Gelsolin can be purified from horse blood plasma by treating the plasma sequentially with an anion-exchange medium in the presence and then the absence of free Ca2+. The purified gelsolin migrates as a 90-kilodalton protein on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. It has an absorption coefficient of 1.4 mL/(mg ∙ cm) and is similar in amino acid composition to other plasma gelsolins. Horse plasma gelsolin has an intrinsic sedimentation coefficient of 4.8S and a Stokes' radius of 3.8 nm. Hydrodynamic calculations suggest it to be a rather globular protein of 75 000 relative mass, a value similar to those calculated for human and pig plasma gelsolins from their amino acid sequences. Horse plasma gelsolin is able to nucleate actin polymerization, i.e., to abolish the lag observed between the initiation of polymerization of monomeric actin by the addition of salts and the rapid elongation phase of actin filament growth. This nucleation activity also results in lower final viscosities of F-actin solutions, as the existence of a larger number of filaments in samples that contain gelsolin requires that their average length be shorter.Key words: plasma gelsolin, actin, hydrodynamics, actin filament severing, nucleation of actin filament growth.
Identification of a 51-kilodalton calmodulin binding protein that changes during estrogen-stimulated cell growth
Authors: Laquerre S. , Poulin R. , Labrie F. , Chafouleas J. G.
Published: December 2009

Calmodulin-binding proteins (CaMBPs) were analyzed during estrogen-stimulated growth in the human breast cancer cell line ZR-75-1. A variety of Ca2+-dependent and -independent CaMBPs were observed to be present in these cells. Calmodulin (CaM) binding to a 51-kilodalton protein was shown to be Ca2+-dependent. Moreover, binding to this protein was reduced in the estrogen-treated cells. This effect occurred early during estrogen-stimulated cell growth and was maintained during exponential growth in the presence of estrogen. 125I-labeled CaM overlay procedure of two-dimensional polyacrylamide gels reveals that this 51-kilodalton protein is composed of at least two distinct isoforms with different isoelectric points. Subcellular localization demonstrates that this protein resides exclusively in the microsomal fraction.Key words: calmodulin, calmodulin-binding protein, estrogen-stimulated cell growth.
Characterization of a mammalian ribosomal protein gene promoter
Authors: Zahradka Peter , Larson Dawn E. , Sells Bruce H.
Published: December 2009

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.Key words: ribosomal protein gene, gene promoter, cell-free transcription.
Rapid quantitation of uncoupling protein in brown adipose tissue mitochondria by a dot immunobinding ("dot blot") procedure: application to the measurement of uncoupling protein in Richardson's ground squirrel, rats, and mice
Authors: Milner Rachel E. , Trayhurn Paul
Published: December 2009

A dot immunobinding ("dot blot") method for measuring uncoupling protein in brown adipose tissue mitochondria is described. Mitochondrial proteins were solubilized in sodium dodecyl sulfate and applied directly to a nitrocellulose membrane housed in a 96-well microfiltration manifold. Spare binding sites on the nitrocellulose membrane were blocked with bovine serum albumin and then anti-(uncoupling protein) serum was applied. The antigen–antibody complex was detected by the addition of 125I-labelled protein A. Each nitrocellulose "dot" was cut out and its radioactivity was counted. A calibration curve was constructed from purified uncoupling protein standards, taken through the entire procedure. The dot immunobinding method is sensitive (nanogram quantities of uncoupling protein), and in contrast to conventional radioimmunoassay and enzyme-linked immunosorbent assay procedures, it is also rapid and appears to be very robust. The method has been successfully applied to the measurement of uncoupling protein in brown adipose tissue mitochondria of Richardson's ground squirrel, rats, and mice.Key words: brown adipose tissue, uncoupling protein, dot immunobinding, immunoassay, thermogenesis.
Detoxification of secondary products of lipid peroxidation in the cytosol of a mouse fibroblast cell line
Authors: Brophy Peter M. , Barrett John
Published: December 2009

A cytosolic fraction prepared from a mouse fibroblast cell line reduced secondary products of lipid peroxidation, including alkenals, alkanals, alk-2-enals, and alka-2,4-dienals, using NADH or NADPH. An alternative route for detoxification of alk-2-enals and alka-2,4-dienals in the cell line cytosol was via glutathione conjugation. The major glutathione transferase in the cell line cytosol was partially characterized by glutathione-agarose chromatography and chromatofocusing. High glutathione peroxidase activity suggested that the enzyme was an alpha family glutathione transferase. The major glutathione transferase also efficiently catalyzed the conjugation of alk-2-enals and alka-2,4-dienals with glutathione.Key words: lipid peroxidation, glutathione transferase, detoxification.
Induction of plasminogen activator inhibitor type 2 expression during differentiation of human K562 cells towards a macrophage phenotype
Authors: Rehemtulla A. , Arndt A. , Hart D. A.
Published: December 2009

Human K562 cells, a multipotential cell line of hematopoietic origin, were found to differentiate towards a macrophage-like cell when incubated in the presence of phorbol myristate acetate (PMA). Differentiation was accompanied by the expression and secretion of a plasminogen activator inhibitor (PAI). Incubation of the cells in the presence of other agents (dimethyl sulfoxide, retinoic acid, vitamin D3) capable of inducing differentiation towards other phenotypes did not lead to the induction of PAI expression. The inhibitor induced by PMA was partially purified and it exhibited biochemical characteristics similar to PAI-2. The molecule is a glycoprotein with a pI of 5.8. Northern blot analysis of mRNA isolated from control and PMA-treated cells revealed the presence of an approximately 2-kilobase mRNA from treated cells that hybridized to a PAI-2-specific synthetic oligonucleotide. This mRNA did not hybridize with a PAI-I-specific oligonucleotide and was absent from control cells. These observations are consistent with the literature regarding PAI-2 expression by cells of the monocyte–macrophage lineage. Therefore, commitment of this multipotential cell line towards the macrophage lineage results in specific activation of the PAI-2 gene. Further analysis of the regulatory elements of the PAI-2 gene may provide additional insight into the relationship between this gene and the macrophage phenotype.Key words: macrophage differentiation, proteinase inhibitor, cell differentiation.
Triacylglycerol synthesis and diacylglycerol acyltransferase activity during skeletal myogenesis
Authors: Sauro Victor S. , Strickland Kenneth P.
Published: December 2009

The role that diacylglycerol acyltransferase (DAGAT) may play in the switch in lipid metabolism from predominantly triacylglycerol- and phospholipid-synthesizing myoblasts to predominantly phospholipid-synthesizing myotubes has been studied during L6 skeletal myogenesis. Fatty acid induced triacylglycerol (TAG) accumulation in vivo was found to be optimal with long-chain, unsaturated fatty acids. The fatty acid induced TAG accumulation was significantly greater in myoblasts than that in myotubes. DAGAT activity in vitro was found to be associated with the particulate (membrane) fraction only. The inhibition by many thiol-specific reagents (N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5′-dithiobis(2-nitrobenzoic acid)) suggest that a thiol group is at or near the active site. In general, optimal DAGAT activity in vitro was observed when long-chain unsaturated acyl-CoAs and diacylglycerols (DAGs) containing long acyl chains were used as substrates for in vitro TAG synthesis (although 1,2-didecanoin was also very effective). DAGAT activity (expressed relative to DNA) was shown to decline over twofold during skeletal myogenesis when measured in the absence of exogenous DAG. However, in the presence of exogenous (1 mM) DAG, there was no significant change in DAGAT activity, suggesting that the levels of this enzyme are not altered during skeletal myogenesis. These results indicate that endogenous DAG levels are limiting TAG synthesis in L6 myotubes. However, DAG content of myotubes was significantly greater than that of myoblasts, suggesting that there may be an increased competition for DAG (perhaps owing to enhanced phospholipid synthesis) during skeletal myogenesis. The combined effects of decreased synthesis and increased degradation (reported earlier) of TAG may account for the decrease in endogenous TAG contents observed during skeletal myogenesis.Key words: diacylglycerol acyltransferase, TAG synthesis, skeletal myogenesis.
Isozymes of ribonuclease and the changes in their relative levels during development in the cellular slime mould Dictyostelium discoideum
Authors: Uchiyama Saburo , Isobe Kazumasa , Nagai Shin-Ichi
Published: December 2009

The isozymes of ribonuclease were analyzed in cell-free, crude extracts of Dictyostelium discoideum by activity staining of polyacrylamide gels after electrophoresis. The relative levels of three isozymes were then examined during the growth and during the first stages of multicellular development. We observed the replacement of two of these three isozymes by two other isozymes at the pseudoplasmodial stage. These isozymes were different from ribonuclease T1 in terms of their mobility in polyacrylamide gels during electrophoresis. The mobilities of two of the isozymes, DdI and DdII, were 59 and 42% of that of ribonuclease T1. The changes in the relative levels of the isozymes during development are discussed.Key words: ribonuclease, isozyme, cellular slime mould, development, Dictyostelium discoideum.
DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation
Authors: Hunting Darel J. , Gowans Bonnie J. , Dresler Steven L.
Published: December 2009

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.Key words: DNA polymerase, excision repair, ultraviolet radiation.
Hormonal control of macrophage function and glutamine metabolism
Authors: Rosa L. F. B. P. Costa , Cury Y. , Curi R.
Published: December 2009

Murine macrophages have been reported to utilize glutamine at high rates. However, the role of glutamine in macrophage function is still unknown. In the present study, the maximum glutaminase activity of macrophages was investigated under several endocrine dysfunctions that are known to cause alterations in macrophage function. The results obtained suggest that glutamine might play an important role in the onset of phagocytosis in inflammatory macrophages. Moreover, the studies show that insulin, glucocorticoids, and thyroid hormones may be responsible for the regulation of glutamine metabolism and, consequently, of macrophage function.Key words: macrophage, glutamine, insulin, glucocorticoids, thyroid hormones, catecholamines.
Conjugation of N-acylated amino sugars to protein by reductive alkylation using sodium cyanoborohydride: application to an azo derivative of α-amanitin
Authors: Mullersman Jerald E. , Preston III James F.
Published: December 2009

Reductive alkylation mediated by cyanoborohydride is an attractive approach to the conjugation of small molecules, such as drugs, to proteins. This reaction is specific for protein amino groups and can be conducted under mild conditions with little risk of protein polymerization. However, the lability of the aldehyde function that is needed in such reactions presents a difficulty. We have investigated the use of derivatives of D-galactosamine and D-glucosamine in reductive alkylation, since these sugars contain aldehyde groups that are inherently protected and that may be readily linked to other molecules through their amino groups. The amino groups of these sugars were acylated with N-4-nitrobenzoylglycylglycine. Studies of the reductive coupling of the resultant adducts to bovine serum albumin revealed that conjugation to albumin is strongly dependent on cyanoborohydride, is much faster in the presence of borate, and shows a marked increase in rate between pH 7.0 and 9.0. In the presence of borate, the glucosamine derivative coupled much more rapidly than did the galactosamine derivative. The aryl nitro group of the glucosamine adduct was selectively reduced to an amine, diazotized, and reacted with α-amanitin to form an azo compound. This azo derivative was reductively coupled to form conjugates that inhibit calf thymus RNA polymerase II.Key words: α-amanitin, borate, bovine serum albumin, cyanoborohydride, protein modification, reductive alkylation.
Surface denaturation of proteins: the thermal inactivation of β-galactosidase (Escherichia coli) on wall–liquid surfaces
Authors: Edwards Robert A. , Huber Reuben E.
Published: December 2009

Irreversible inactivation of dilute β-galactosidase (Escherichia coli) at relatively low temperatures was found to occur as a result of interactions of β-galactosidase with wall–liquid surfaces. The rate of inactivation was directly proportional to the wall–liquid surface area, but independent of the air–liquid surface area, and the rate was also dependent on the wall composition. A small portion of the β-galactosidase molecules was found to bind strongly to the surfaces of vessels in which the β-galactosidase was stored. Bovine serum albumin eliminated the inactivation and it also eliminated the binding of β-galactosidase to the wall. On the other hand, EDTA eliminated the inactivation, but it did not decrease the amount of β-galactosidase bound. The addition of some transition metals increased the rate of inactivation. Protection of β-galactosidase from surface inactivation by EDTA is not, therefore, a result of decreased binding of the enzyme to the walls of the vessels, but is probably a result of the ability of EDTA to scavenge certain trace metal ions present in solution, which are needed for the inactivation. The content of protein in the solution did not change as a result of the inactivation and, thus, the inactive enzyme does not accumulate at the surface. Since β-galactosidase is often stored for long periods of time and since it is used to decrease the lactose content of milk for lactose intolerant individuals, this study may have practical significance. The presence of metal chelators and extraneous proteins should improve the stability of the enzyme, especially for processes that are carried out at elevated temperatures.Key words: β-galactosidase, surface inactivation, denaturation, protein binding.
Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum
Authors: Patriarca Eduardo J. , Kobayashi George S. , Maresca Bruno
Published: December 2009

Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms. In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 °C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium-to yeast-phase transition. The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature. We have found that by inducing thermotolerance, i.e., an initial incubation at 34 °C, the thermosensitive attenuated Downs strain of H. capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures. Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 °C proceeds at rates comparable to those of the virulent strains.Key words: heat shock, thermotolerance, ATPase, 70-kilodalton heat-shock protein, fungal morphogenesis.
The M1 subunit of rat liver ribonucleotide reductase appears to be modified by ubiquitination
Authors: Sikorska Marianna , Kwast-Welfeld Joanna , Youdale Tony , Richards Robert , Whitfield James F. , Walker P. Roy
Published: December 2009

Using a combination of immunoblotting, double immunoprecipitation, immunoglobulin-affinity chromatography, and isoelectrofocusing, we have been able to identify a group of proteins that display CDP-reductase activity and contain antigenic epitopes recognized by anti-ribonucleotide reductase M1 subunit and anti-ubiquitin antibodies. In the cytoplasm of rat liver cells, we could detect a total of five proteins with molecular masses of 92, 89, 56, 45, and 37 kilodaltons which reacted with the anti-M1 subunit serum. All of them, except the 89-kilodalton protein (the nascent unmodified M1), were also recognized by the anti-ubiquitin antibody. In normal liver cells, all of the apparently ubiquitinated species of the M1 protein were found in the cytoplasm, but not in the nuclear envelope associated pool of the enzyme. However, we did not detect ubiquitinated M1 protein fragments in the cytoplasm of Morris hepatoma 5123tc. The level of the apparently ubiquitinated fragments of the M1 subunit increased in parallel to the DNA-synthetic activity of normal liver cells, suggesting that ubiquitination plays a key role in the regulation of the activity of the enzyme during the cell cycle.Key words: ribonucleotide reductase turnover, proteolysis, ubiquitin, DNA replication.
Compensatory effect of distal promoter sequences on the basal expression of a microinjected 70-kilodalton heat shock protein gene after the midblastula transition of Xenopus laevis embryogenesis
Authors: Ovsenek Nick , Heikkila John J.
Published: December 2009

The promoter sequences involved in the basal expression of a human 70-kilodalton heat shock protein (HSP70) gene during Xenopus embryogenesis were analyzed by microinjection of mutant promoters of a HSP70 – chloramphenicol acetyltransferase fusion gene into fertilized eggs and following their expression during early development. While deletion of the HSP70 gene promoter to −100 base pairs (bp) did not affect basal transcription in postmidblastula stage embryos, linker–scanner mutations in the CCAAT and purine box elements blocked expression. However, extension of the 5′ boundary to −188 bp restored full wild-type expression to these mutants. These results suggest that multiple redundant cis-acting regulatory elements present in the human HSP70 gene promoter can function during Xenopus embryogenesis.Key words: heat shock protein gene, Xenopus embryos, microinjection, linker–scanner mutations, transcription.
A dolichol acyltransferase present in rat and human postheparin plasma
Authors: Sindelar P. , Valtersson C.
Published: December 2009

Incubation of small unilamellar vesicles consisting of dioleoyl phosphatidylcholine – dioleoyl phosphatidylethanolamine (3:1) and 2 mol% [3H]dolichol-19 with postheparin plasma from rat resulted in the formation of dolichyl oleate. Normal plasma or heat-treated postheparin plasma contained no activity and, hence, the results indicate the presence of a cell surface associated dolichol acyltransferase that can be released into the blood by heparin. The reaction is strongly stimulated by phosphatidylethanolamine and Ca2+, whereas no stimulation with triglycerides or acyl-CoA was observed. Together with the fact that the only product formed was dolichyl oleate, these results strongly suggest that a transacylation mechanism from the phospholipids to dolichol is operative in the liposomes. Gel chromatography of postheparin plasma yielded a molecular mass of about 350 kilodaltons for the active enzyme and density gradient centrifugation indicated that this high molecular mass complex consists mainly of proteins. Finally, we conclude that this enzyme is not unique to the rat, but is also present in human postheparin plasma.Key words: phospholipids, dolichol, plasma, heparin, acyltransferase(s).
Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol
Authors: Zeng Ling-Hua , Wu Tai-Wing
Published: December 2009

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.Key words: purpurogallin, kidney cells, oxyradical damage, xanthine oxidase inhibition, antioxidant.
Immediate-early genes, neuronal plasticity, and memory
Authors: Robertson H. A.
Published: December 2009

The demonstration that the immediate-early gene c-fos is rapidly and transiently expressed in brain following a variety of manipulations has led to intense study of these genes to determine what physiological role they play. The very wide range of stimuli which lead to induction of immediate-early genes (IEGs) in the brain has raised concerns for the specificity of their actions and the suggestion that they might merely be involved in housekeeping functions. On the other hand, there is evidence that these genes may play a role in the transmission of information from cell surface receptors to the genetic material in many instances of neuronal plasticity, including development of seizure susceptibility (kindling), long-term potentiation, drug-induced changes, the phase shift in circadian rhythms, and spreading neuronal depression. In addition to being a putative third (or fourth) messenger involved in transduction of signals to the genetic material, activation of IEGs has proven to be a useful tool for the study of transsynaptic activation of certain neuronal pathways in the brain. Thus, studies on the induction of IEGs are proving to be especially useful in understanding some important functions and properties of the mammalian brain.Key words: immediate-early genes, brain, memory, neuronal plasticity, gene expression.
Mechanisms of oncogene-mediated alterations in metastatic ability
Authors: Chambers Ann F.
Published: December 2009

Transfected ras oncogenes have been shown to induce metastatic properties in some cells. This altered behavior is likely due to changes in ras-mediated signal transduction pathways, resulting in altered expression of genes important to metastasis. Clarification of the mechanisms by which ras is able to induce metastatic ability in model systems will improve our understanding of tumor progression, even in those cells in which ras activation has not been implicated. Many of the consequences of ras expression also have been detected in cells that have become metastatic in the absence of altered ras, suggesting that there is a set of common changes that can lead to metastasis, with multiple signals capable of eliciting these changes. We have identified several changes in metastatic, ras-transformed NIH 3T3 cells that may contribute to their increased malignancy, including expression of proteolytic enzymes and their inhibitors, and adhesive and calcium-binding proteins. Not all cells, however, respond in this way to expression of oncogenic ras. We have found that murine LTA cells, which are tumorigenic but nonmetastatic, are ras resistant and remain nonmetastatic when expressing high levels of transfected ras, in contrast to NIH 3T3 cells, which are ras sensitive and become both tumorigenic and metastatic in response to comparable levels of ras. LTA cells differ in their patterns of gene expression in response to ras when compared with NIH 3T3 cells, suggesting that the two cell lines process the ras signal differently. Here we review our results with ras-transfected NIH 3T3 and LTA cells. An understanding of the ways in which these cells differ in their response to an oncogenic signal may provide clues to mechanisms of tumor progression.Key words: ras, signal transduction, proteolytic enzymes, osteopontin, calcyclin.
Expression of a mutant regulatory subunit of cAMP-dependent protein kinase in the Caco-2 human colonic carcinoma cell line
Authors: Mihajlovic Vera , Krolczyk Arkadius J. , Auerbach Wojtek , Buchwald Manuel , Schimmer Bernard P.
Published: December 2009

Caco-2 human colonic carcinoma cells were transfected with an expression vector encoding a mutant form of RI (regulatory subunit of the type 1 cAMP-dependent protein kinase), driven by the metallothionein 1 promoter. A stable transformant was isolated that expressed the mutant RI gene in a Zn2+-inducible manner. The consequences of the RI mutation on cAMP-dependent protein kinase activity, cell division, and regulation of chloride efflux were examined. When grown in the absence of ZnSO4, protein kinase activity in the transformant was stimulated 2.5-fold by cAMP and approached the levels of cAMP-dependent protein kinase activty seen in parental Caco-2 cells; when treated with ZnSO4, cAMP-dependent protein kinase activity in the transformant was inhibited by 60%. In the absence of ZnSO4 the transformant grew with the same doubling time and to the same saturation density as the untransformed parent. In the presence of ZnSO4 the transformant exhibited a cAMP-reversible inhibition of cell division, indicating that a functional cAMP-dependent protein kinase was required for the growth of these cells in culture. Induction of the mutant RI gene also abolished forskolin-stimulated chloride efflux from these cells, suggesting obligatory roles for cAMP and cAMP-dependent protein kinase in forskolin's actions on chloride channel activity. We anticipate that this transformant will be useful for further studies on the roles of cAMP and cAMP-dependent protein kinase in the regulation of intestinal epithelial cells, including regulation of cell proliferation and differentiation, and regulation of chloride channel activity by neurohormones and neurotransmitters.Key words: chloride efflux, cell growth, gene transfer, forskolin.
Mutational analysis of a DNA sequence involved in linking gene expression to the cell cycle
Authors: Andrews Brenda J. , Moore Lynda
Published: December 2009

Entry of budding yeast cells into the mitotic cell cycle requires the activity of a conserved regulatory kinase encoded by the CDC28 gene. The kinase is thought to trigger entry into the cell cycle or START, through association with a number of regulatory subunits known as G1cyclins. A number of genes whose transcription is dependent on CDC28 and thus linked to START are controlled by two transcription factors, SWI4 and SWI6. The genes controlled by SWI4 and SWI6 include two known G1 cyclins (CLN1 and CLN2), a putative new G1 cyclin (HCS26), and the HO gene whose product initiates cell type switching. SWI4 and SWI6 act through a repeated sequence element, SCB (SWI4,6-dependent cell cycle box), found 2–10 times in the upstream regulatory sequences of target genes. We have constructed a library of mutants in the SCB using doped oligonucleotide mutagenesis. All single base pair changes examined compromised the ability of the SCB to activate transcription in vivo. Analysis of the behaviour of the mutant SCBs in an in vitro DNA binding assay shows that the inability to activate transcription can be explained by reduced binding of SWI4 and SWI6 to the mutant SCBs. This analysis, together with a consideration of the SCBs found upstream of known SWI4,6-dependent genes, leads to the proposal of a revised consensus sequence for this important regulatory element.Key words: cell cycle control, gene expression, SWI4,6-dependent cell cycle box, SWI4, SWI6.
Factors affecting cellular mosaicism in the expression of a lacZ transgene in two-cell stage mouse embryos
Authors: Kothary R. K. , Allen N. D. , Barton S. C. , Norris M. L. , Surani M. A. H.
Published: December 2009

In the present study, we have analysed the expression pattern of a lacZ transgene (CMZ12) in preimplantation stage mouse embryos. The transgene is expressed at the two-cell stage, where it shows cellular mosaicism due to variable expressivity. The variable gene expression indicates a partial penetrance of the transgene. The extent of variation in expression is influenced by the genetic background of the oocyte. DBA/2 and CFLP genetic backgrounds promote high expression of the transgene, while Balb/c, C57BL/6, DDK, and F1(C57BL/6 × CBA) genetic backgrounds give none or very little lacZ activity. In vitro culture of one-cell embryos to the two-cell stage induces the expression of lacZ in all strain backgrounds tested. The variation in CMZ12 expression is a transient phenomenon and does not affect later stage activity of the transgene. Nuclear transfer experiments and DNA methylation analysis suggests that a heritable modification of the transgene locus has not occurred.Key words: cellular mosaicism, lacZ transgene, mouse embryos, variable expressivity.