bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel
Authors: Lazier Catherine B. , Breckenridge W. Carl
Published: December 2009

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine∙HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki ~ 4–5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.Key words: antiestrogen, estrogen receptor, cholesterol, phospholipid, triglyceride.
Inhibition of enzymatic degradation of angiotensin II in membrane binding assays: utility of 1,10-phenanthroline
Authors: Koziarz P. , Moore G. J.
Published: December 2009

Studies of 125I-labelled angiotensin II binding to membranes from bovine uterus and aorta are rendered impossible because enzymatic degradation of 90% of the added ligand occurs within 30 min at 23 °C. High pressure liquid chromatographic analysis has demonstrated that a single iodinated product, which is more hydrophilic than intact angiotensin II, is produced by enzymatic cleavage. Investigation of a series of enzyme inhibitors has shown that the carboxypeptidase inhibitor 1,10-phenanthroline completely inhibits degradation of angiotensin II without disrupting ligand binding.Key words: angiotensin II, binding, degradation, bovine uterine membrane, bovine aortic membrane, 1,10-phenanthroline inhibitor.
Thyroxine 5′-deiodinase in brown adipose tissue of the cynomolgus monkey Macaca fascicularis
Authors: Kates Anna-Lisa , Park Ian R. A. , Himms-Hagen Jean , Mueller Rudolf W.
Published: December 2009

Brown adipose tissue was identified in axillary, interscapular, subscapular, and cervical fat deposits of male and female cynomolgus monkeys (Macaca fascicularis) by histological and immunological techniques. Histology included staining of mitochondria with a Novelli stain and identification of mitochondria-rich multilocular cells. Immunological detection involved separation of homogenate proteins by sodium dodecyl sulphate – polyacrylamide gel chromatography, blotting on to nitrocellulose membranes, and identification of the specific uncoupling protein, unique to brown adipose tissue, with an antiserum to purified hamster uncoupling protein followed by detection with 125I-labelled protein A. The activity of thyroxine 5′-deiodinase in monkey brown adipose tissue homogenates was much higher than that seen previously in brown adipose tissue of rats, mice, and hamsters. This is the first demonstration of the presence of this enzyme in brown adipose tissue of a primate species.Key words: uncoupling protein, primate, 3,5,3′-triiodothyronine, thermogenesis; white adipose tissue.
Differential scanning calorimetry of dispersions of products of oxidation of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine
Authors: Keough K. M. W. , Parsons C. S.
Published: December 2009

Ultraviolet light was used to promote the autoxidation of 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC). The extent of oxidation was monitored by ultraviolet spectroscopy, reaction with thiobarbituric acid, fatty acid analysis, and thin-layer chromatography. Fatty acid analysis and thin-layer chromatography appeared to provide the most consistent estimates of oxidation, especially when extensive oxidation had taken place. The oxidized samples were separated by flash chromatography into fractions enriched in different oxidation products. Differential scanning calorimetry of aqueous dispersions of these fractions indicated that oxidation products had higher transition temperatures than the original SLPC.Key words: lipid peroxidation, ultraviolet light, phase behaviour, transition temperatures.
Triacylglycerols of oil palm (Elaeis guineensis var. dura) mesocarp during fruit maturation
Authors: Osagie Anthony U. , Bafor Maureen E.
Published: December 2009

Changes in triacylglycerol pattern in the mesocarp of developing oil palm fruit were studied. The triacylglycerols were isolated by thin-layer chromatography of the neutral lipid fraction on silica gel plates and further resolved by argentation chromatography. The component fatty acids of individual triacylglycerols were analysed and particular distributions and changes were noted. The triacylglycerol content increased with mesocarp age, accounting for over 90% of the mature mesocarp neutral lipids. The immature fruit mesocarp triacylglycerols contained predominantly saturated fatty acids, whereas the major fatty acid component of the mature fruit mesocarp triacylglycerols was monounsaturated. Only one species of triacylglycerol was present in the mesocarp during the first 14 weeks after anthesis. Three triacylglycerol species were present at 16 weeks after anthesis, whereas six triacylglycerol species were identified from 17 weeks on.Key words: Elaeis guineensis, oil palm mesocarp, triacylglycerols, fatty acids, maturation.
Preparation of a fluorescent (anthracene labeled) cardiolipin on a large scale using the metabolic machinery of the bacterium Micrococcus luteus
Authors: Pérochon E. , Bony J. de , Tocanne J. F.
Published: December 2009

Micrococcus luteus bacterium has been used as a tool for the preparation of a fluorescent (anthracene labeled) cardiolipin by a method involving two steps. First, 9-(2-anthryl)nonanoic acid is incorporated into the lipids of the bacterium by usual metabolic pathways; then phosphatidylglycerol, a major lipid in this bacterium, is converted into cardiolipin by endogeneous cardiolipin synthetase. It is shown that cardiolipin synthetase transforms anthracene–phosphatidylglycerol into anthracene–cardiolipin at a high rate. The method can be extended to the synthesis of cardiolipin molecules bearing other hydrophobic marker groups.Key words: cardiolipin, fluorescence, biosynthesis, Micrococcus luteus.
Effects of membrane polyunsaturated fatty acids on adenosine receptor function in intact N1E-115 neuroblastoma cells
Authors: Murphy M. G. , Byczko Z.
Published: December 2009

We have examined the effects of increasing membrane polyunsaturated fatty acids (PUFAs) on adenosine receptor function in intact N1E-115 neuroblastoma cells. Addition of linoleic acid to the culture medium for 48 h resulted in an approximate threefold increase in the amount of ω6 fatty acids esterified to membrane phospholipids. Basal cAMP accumulation was significantly higher in the PUFA-enriched cells than in controls, although the differences could be diminished by approximately 75% by treatment of the cells with adenosine deaminase or 8-phenyltheophylline. Exposure of the cultures to the stable adenosine analogue 5′-N-ethylcarboxyamide adenosine (NECA) resulted in concentration-dependent increases in cAMP accumulation. Data from saturation experiments indicated that the maximum amount of cAMP that could be formed in response to NECA in the PUFA-enriched cells was twice that in control cells. Also, the amount of agonist required to elicit half maximal stimulation in the supplemented cells was significantly less than in the control cells (mean values for EC50, 0.85 and 1.43 μM, respectively). The results of this study demonstrate that membrane PUFA have the ability to modify interactions between adenosine receptors and adenylate cyclase in neural cells, a fact that is of potential importance in considering the central role that adenosine plays as a neuromodulator in the nervous system.Key words: polyunsaturated fatty acids, neuroblastoma cells, adenosine receptors, cAMP.
Properties of kinesin isolated from human prostatic DU 145 tumor cells and bovine brain
Authors: Stearns Mark E. , Piazza Gary A.
Published: December 2009

We have isolated and compared the 116-kilodalton (kDa) kinesin heavy chain from DU 145 human prostatic tumor cells and bovine brain. Comparative sodium dodecyl sulfate – polyacrylamide gel electrophoreses (SDS–PAGE), Western blots, and proteolytic digestion analysis all showed that the 116-kDa polypeptides from both sources were indistinguishable. Polyclonal antibodies raised against sea urchin kinesin cross-reacted with both brain and DU 145 kinesin on Western blots. SDS–PAGE and A-5 m chromatographic studies indicated that kinesin forms a quaternary heteropolymer of ~ 400 kDa. DU 145 cells had three proteins of 116, 72, and 64 kDa forming the heteropolymer, in a 2:1:1 ratio, whereas brain cells appeared to have equimolar amounts of the 116-kDa heavy chain and a 64-kDa light chain.Key words: kinesin, DU 145, prostate tumor cells, bovine brain.
Differential expression of the epidermal K1 and K10 keratin genes during mouse embryo development
Authors: Ouellet Thérèse , Lussier Marc , Babaï Feridoun , Lapointe Line , Royal André
Published: December 2009

Induction of genes coding for the K1 and K10 keratins during mouse development was studied by measuring the accumulation of their respective mRNAs in day 10 to 17 embryos using an RNase protection assay. Although these two keratins are coexpressed in the suprabasal layers of the epidermis, it was found that while K1 mRNA was detectable as soon as day 10, K10 mRNA was not detectable before day 12. The expression of these genes at this stage of development was not expected since they are specifically associated with keratinization, a process that does not begin before day 17 of gestation. Histological examination of the epidermis of day 10 to 17 embryos suggests that both genes are induced in cells committed to epidermal differentiation, after stratification has started but before the onset of keratinization. It was also found that the two mRNAs increased in abundance steadily and significantly until day 16 and that, in spite of the expectation that filaments should contain equivalent amounts of each subunit, K1 mRNA remained more abundant than K10 mRNA at all times including in adult epidermis. These observations indicate that the two genes are regulated independently during development.Key words: intermediate filaments, keratinization, differentiation, RNase protection, histology.
Reconstitution into liposomes of highly purified PO glycoprotein from avian peripheral nerve myelin
Authors: Földvári Marianna , Mezei Michael , Mezei Catherine
Published: December 2009

To explore the adhesive, possibly fusogenic properties of the major protein of peripheral nerve myelin (PO protein), three methods were employed for the reconstitution of this glycoprotein into liposomes of various lipid compositions. The most successful technique for the preparation of proteoliposomes was a detergent solubilization that employed Bio-Beads for the removal of Triton X-100 from the PO protein – lipid – detergent mixture. This method yielded a similar level of PO protein relative to lipids in the liposomes to that found in myelin. The protein to lipid ratio of the reconstituted proteoliposomes was only 1.4-fold lower than the initial protein to lipid ratio employed to prepare the liposomes. In contrast, the solvent evaporation and reverse-phase evaporation methods provided liposomes with 30 times lower protein to lipid ratio when compared with PO protein to lipid ratio initially used for the preparation of proteoliposomes. The incorporation of PO protein was dependent on the lipid composition of the vesicles and on the degree of purification of the protein. Proteolytic enzyme treatment showed that proteoliposomes containing the highly purified form of PO protein were partially or completely sensitive to trypsin treatment, whereas in liposomes containing a partially purified form of PO protein, a 22.4-kilodalton fragment was completely protected from trypsin digestion, indicating a transmembrane orientation of the partially purified polypeptide. Endoglycosidase F treatment of the liposomes established that all of the oligosaccharide moieties were removed from the liposome-associated protein, indicating the oligosaccharide moiety was exposed to the outside surface of the liposomes. Taken together, the results of limited proteolysis and endoglycosidase treatments did not provide strong evidence for the transmembrane orientation of highly purified PO in the liposomes, but were consistent with a surface of partial hairpin configuration, where the extracellular domains are facing the outside milieu. Electron microscopic examinations revealed highly convoluted, elongated shapes arid extensive fusion of PO liposomes containing lipids capable of adopting the hexagonal (H11) phase. Proteoliposomes prepared with the simple lipid composition of dipalmitoyl phosphatidylcholine -cholesterol (10:1 molar ratio) yielded stable, unilamellar liposomes of 80 nm average diameter, that encapsulated water-soluble drugs efficiently into the intravesicular compartment.Key words: liposomes, PO glycoprotein, myelin, peripheral nerve.
Studies on the lipopolysaccharide of a virulent and an avirulent strain of Vibrio vulnificus
Authors: Bahrani K. , Oliver James D.
Published: December 2009

Vibrio vulnificus is a marine bacterium associated with both primary septicemias and wound infections in humans. The lipopolysaccharides of a virulent and an avirulent strain of Vibrio vulnificus were compared with respect to their chemical constituents and electrophoretic characteristics. 2-Keto-3-deoxyoctonic acid, a normal constituent of the lipopolysaccharide of typical Enterobacteriaceae, was not found in the lipopolysaccharide of either strain. Hexadecenoate (C16:1) was the predominant fatty acid of the lipid A moiety of the lipopolysaccharides and of the membrane phospholipids of both strains. Hydroxy fatty acids composed 44% of the total fatty acids of the lipid A of the avirulent and 40% of those in the virulent strain. In addition, odd-numbered fatty acids were detected in both lipopolysaccharides. The electrophoretic profile was similar for both strains, but demonstrated no "ladder-like" pattern characteristic of "smooth" lipopolysaccharides. The result of this study showed no significant differences between the lipopolysaccharides of the virulent and avirulent strains of Vibrio vulnificus. The possible role for lipopolysaccharide in pathogenesis of Vibrio vulnificus infections is discussed.Key words: Vibrio, lipopolysaccharide, pathogenesis.
Relationship of alcohol production to lipid composition of yeast in a continuous flow bioreactor
Authors: Gil G. H. , Jones W. J. , Tornabene T. G.
Published: December 2009

Saccharomyces cerevisiae was cultivated in a controlled aerated, dual-stage (column), continuous flow bioreactor in a hybrid free-cell and immobilized-cell state. The yeast cells maintained an ethanol concentration of 58–64 and 91–98 g/L in stages I and II, respectively. The lipid composition of the cells cultivated under these conditions was correlated to the effects of aeration by interrupting the aeration on days 113 and 266 of continuous operation. Under conditions of aeration or nonaeration, an alternating increase and decrease in the contents of squalene, sterols, and fatty acids of the respiratory-competent and -deficient unattached free cells was observed. The cellular free lipid compositions of the immobilized cells in the aerated and nonaerated conditions were similar and characteristic of respiratory-deficient cells with the exception of the immobilized cells exposed to a higher ethanol concentration (stage II). These cells contained a broader range of sterol components and increased levels of unsaturated fatty acids than immobilized cells at a lower ethanol concentration (stage I). The neutral lipid to phospholipid ratio decreased for respiratory-deficient cells with phosphatidylethanolamine and phosphatidylinositol being the principal phospholipids. The data demonstrated the essentiality of the hybrid bioreactor design for continuous long term performance and the importance of maintaining specific yeast lipid constituents for continuous high alcohol productivity.Key words: yeast immobilization, lipids, fatty acids, sterols, phospholipids, continuous flow, aeration, ethanol.
Regulation of synthesis of oligosaccharyl pyrophosphoryl dolichol during seed germination
Authors: Rip Jack W. , Williams Joyce A. , Crick Dean C. , Carroll Kenneth K.
Published: December 2009

Subsaturating (approximately physiological) concentrations of [1-14C]dolichol were supplied in vitro to soybean embryo microsomes, to enable the formation of [1-14C]dolichyl phosphate ([1-14C]Dol-P). By including saturating amounts of nonradiolabeled UDP-GlcNAc, GDP-Man, and UDP-Glc individually or in combination in the assay, the extent to which newly formed [1-14C]Dol-P is directed into [1-14C]dolichyl pyrophosphate ([1-14C]Dol-PP) – GlcNAc, [1-14C]Dol-PP-(GlcNAc)2, [1-14C]Dol-P-Man, and [1-14C]Dol-P-Glc could be followed. The addition of UDP-GlcNAc did not appreciably decrease the rate of accumulation of free [1-14C]Dol-P, whereas GDP-Man and UDP-Glc reduced the free [1-14C]Dol-P content of the assays by about 20 and 50%, respectively. The appearance of Dol-P-Man and (or) Dol-P-Glc accounted entirely for the decreased amounts of free Dol-P present. Even in the presence of all three nucleoside diphosphate sugars, about 40–50% of the total [1-14C]Dol-P formed remained in the free phosphate form. The early intermediates Dol-PP-GlcNAc and Dol-PP-(GlcNAc)2 were produced only in trace amounts by microsomes isolated prior to germination and between 2 and 20 h of germination. The availability of Dol-P does not appear to be the rate-limiting factor in Dol-PP-oligosaccharide formation and N-linked glycoprotein formation in this system.Key words: dolichol, dolichyl phosphate, N-oligosaccharide, synthesis regulation.
Biochemistry of natural freeze tolerance in animals: molecular adaptations and applications to cryopreservation
Authors: Storey Kenneth B.
Published: December 2009

For a wide variety of animals, winter survival in cold climates includes the ability to tolerate ice formation in extracellular body fluids. Among terrestrially hibernating vertebrates, freeze tolerance has been documented for five amphibian and two reptile species. These species may survive for days or weeks in a frozen state with no breathing and no heart beat, and with up to 65% of total body water as extracellular ice. The biochemical mechanisms involved in natural freeze tolerance include (i) the regulation of extracellular ice formation by proteinaceous ice nucleators in body fluids, (ii) the accumulation of high concentrations of low molecular weight carbohydrates as cryoprotectants to regulate cell volume reduction during freezing and stabilize macromolecular structure, and (iii) a well-developed ischemia tolerance that supports the survival of individual organs while frozen. The present article focuses on recent advances in our understanding of the biochemistry of natural freeze tolerance in lower vertebrates and the application of these studies to the improvement of cryopreservation technology for transplantable mammalian organs.Key words: freeze tolerance, cryopreservation, cold hardiness, cryoprotectants.
Esterase activity of high-Km aldehyde dehydrogenase from rat liver mitochondria
Authors: Senior Dave J. , Tsai C. Stan
Published: December 2009

Aldehyde dehydrogenase possessing an esterolytic activity has been purified to homogeneity from rat liver mitochondria. Steady-state kinetic studies suggest that the esterolytic reaction follows an ordered uni-bi mechanism. The formation of an acyl enzyme intermediate via nucleophilic catalysis during the esterase reaction is established kinetically using a series of substrates with varying acyl carbon chains and substituted phenyl octanoates with varying electronic effects. The enzyme was reconstituted into phospholipid vesicles. A significant increase in binding capacity is observed when the enzyme is encapsulated into liposomes containing 4% diphosphatidylglycerol.Key words: aldehyde dehydrogenase, esterase activity.
Purification and partial characterization of a mitogenic protein released from preadipocytes of massively obese subjects
Authors: Roncari Daniel A. K. , Thompson Coral E.
Published: December 2009

A protein released into the culture medium by omental preadipocytes of massively obese persons, which stimulates the replication of rat perirenal preadipocytes, has been purified to a high degree. By gel filtration chromatography, the molecular mass of the mitogenic protein was ~66 000 daltons (Da), while on sodium dodecyl sulfate – polyacrylamide gel electrophoresis, two subunits were obtained, relative masses (Mr) of ~31 000 and ~35 000. The isoelectric point of the ~66 000 Da entity was 5.6 ± 0.2. By specific radioreceptor assay, the purified protein was related to epidermal growth factor and transforming growth factor alpha. It was not related to insulin-like growth factors I and II by radioimmunoassay and radioreceptor assay. We propose that the ~66 000 Mr protein, and other mitogenic proteins released by preadipocytes from massively obese persons, act through paracrine–autocrine mechanisms and may play a role in the development of the hyperplasia of enlarged fat cells characteristic of massive corpulence.Key words: preadipocyte, massive obesity, mitogenic, paracrine.
Endogenous heparin-binding lectin activity in human placenta: purification and developmental expression
Authors: Ceri Howard , Hwang Wei Sek , Cheung Helen
Published: December 2009

Human placental extracts contain a herapin-inhibitable lectin activity. The lectin, which closely resembles those from chicken and rat tissues, was purified by heparin-affinity chromatography. It shares many properties with the previously reported lectins, including hapten specificity, molecular weight of monomers, and immunological cross-reactivity. Sections from different stages of placental development, stained by immunohistochemistry procedures using lectin-specific antibody, showed that the lectin was initially present only in cytotrophoblasts of early first trimester villi. Later in the first trimester, both cytotrophoblasts and syncytiotrophoblasts were stained positively for lectin. From second trimester to term, the lectin was seen only in syncytiotrophoblasts.Key words: lectin, human placenta, development, heparin, cytotrophoblast, syncytiotrophoblast.
Enzymes that degrade phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate have different developmental profiles in chick brain
Authors: St.C. Palmer Frederick B.
Published: December 2009

The activities and subcellular distributions of the hydrolases that degrade polyphosphoinositides were compared in the developing chick central nervous system. Specific activities increased 2- 3-fold and total activities increased 13-to 16-fold. Phosphatidylinositol 4-phosphate phosphatase is localized in membranes (78%), but is preferentially associated with nonmyelin membranes, since the increase in specific activity preceded myelination and proportions of membrane and soluble activities were constant during accumulation of myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphatase is largely soluble in embryonic (57%) and myelinated brain (50%). Although specific activity increased coincident with myelination, approximately equal increases in soluble and membrane activity indicate no preferential association with myelin membranes. Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity increased only in the early stages of myelination, but showed some preferential association with myelin membranes, since the proportion of soluble diesterase declined from 40 to 25%.Key words: brain, development, myelin, phosphodiesterase, phosphoinositide, phosphomonoesterase, phospholipids.
Cortisol catabolism by lymphocytes of patients with chronic lymphocytic leukemia
Authors: Klein Ami , Lishner Michael , Bruser Barbara , Curtis John E. , Amato Dominick J. , Malkin Aaron
Published: December 2009

A low rate of catabolism of cortisol by lymphocytes correlates with high sensitivity of the cells to the steroid and causes them to die at a greater rate than control samples. Since lymphocytes of patients with chronic lymphocytic leukemia respond to treatment with glucocorticosteroids and are cortisol sensitive, we attempted to see whether their capability to catabolize cortisol differs from that of normal lymphocytes. No difference was found between the two groups of cells with regard to the pattern of cortisol metabolites. However, the lymphocytes of the chronic lymphocytic leukemia groups showed a total cortisol catabolism per cell that was significantly lower than that of the control group. Patients with low lymphocyte count in peripheral blood showed a relatively higher cortisol metabolism by lymphocytes per cell than those with high counts.Key words: lymphocytes, cortisol, catabolism, chronic lymphocytic leukemia, morbidity.
Effect of cellular growth state on indices of n − 6 polyunsaturated fatty acid status in human fibroblasts
Authors: Bettger William J. , Driscoll Eric R. , Karmiol Soverin
Published: December 2009

A 2 × 2 design was employed to examine the effect of cellular growth state and medium serum concentration on potential indices of n − 6 polyunsaturated fatty acid (PUFA) status in human skin fibroblasts. The cells were cultured either as nonmultiplying cell monolayers or as medium-density, log-phase multiplying cells. An interaction of cellular growth state and medium serum concentration influenced the accumulation of 20:3(n − 9), but not 22:3(n − 9), in the cellular phospholipids. The 20:3(n − 9)/20:4(n − 6) ratio was the most sensitive index of n − 6 PUFA status; however, the ratio was significantly affected by cellular growth state. The 22:3(n − 9)/22:4(n − 6) ratio appears to be an index of n − 6 PUFA status in fibroblasts that is not significantly affected by the growth state of cells.Key words: n − 9 polyunsaturated fatty acids, fibroblasts, cell culture.
Cross-resistance and biochemical characteristics of second-step mutants of HeLa cells resistant to cardiac glycosides
Authors: Chopra Arvind , Dudani Anil K. , Gupta Radhey S.
Published: December 2009

From ouabain-resistant (OuaR) mutants of HeLa cells which do not show any cross resistance to the digoxin analog SC4453, stable second-step mutants resistant to either SC4453 or those exhibiting increased resistance to digoxin have been isolated. The mutants obtained exhibited highly specific cross resistance towards different cardiac glycosides (CGs) and, based on their cross-resistance patterns, contained more than one type of genetic lesion. Biochemical studies with these mutants showed that cellular uptake of 86Rb was inhibited by specific CGs to which they showed increased resistance. The mutants showed reduced binding of [3H]ouabain and [3H]digoxin in comparison with the parental OuaR cells and about 50–60% of the Na+,K+-ATPase activity in the mutant cell extract was highly resistant to inhibition by ouabain and digoxin. In contrast to the above changes, these mutants showed no evidence of amplification, enhanced transcription, or gross alterations in the genes for the α or β subunits of Na+,K+-ATPase. These observations indicated that these mutants involved a second-specific alteration in Na+,K+-ATPase. In contrast to these mutants, Chinese hamster ovary cells, which naturally exhibit comparable levels of resistance to CGs, showed no significant binding of either [3H]ouabain or [3H]digoxin and all of their Na+,K+-ATPase activity was resistant to inhibition by CGs.Key words: cardiac glycosides, mammalian cell mutants, mutants resistant to cardiac glycosides, Na+,Ka+-ATPase.
Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol
Authors: Hsuanyu Yuchiong , Dunford H. Brian
Published: December 2009

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 °C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant K2,app, was found to fall within the range of 5.8 × 106 ± 0.4 × 106 M−1∙s−1 < K2,app < 1.8 × 107 ± 0.1 × 107 M−1∙s−1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme–substrate complex formation, with kcat = 22 ± 3 s−1, Km = 67 ± 10 μM, and the second-order rate constant k3,app = 2.0 × 105 ± 0.2 × 105 M−1∙s−1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 ± 0.5 s−1, Km = 49 ± 7 μM, and k3,app = 1.03 × 105 ± 0.07 × 105 M−1∙s−1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 ± 5 and 0.52 ± 0.05 s−1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 ± 5 and 0.33 ± 0.02 s−1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate. It is suggested that kinetic studies on this enzyme can be performed in the presence of diethyldithiocarbamate, if one is cognizant of the rate of reaction of the stabilizing agent with compounds I and II and corrects for it if necessary.Key words: prostaglandin H synthase, diethyldithiocarbamate, cyclooxygenase, peroxidase, stabilizing agents.
Ubiquitin immunoreactivity shows several proteins varying with development and sporulation in the basidiomycete Coprinus cinereus
Authors: Kanda Tosaku , Tanaka Nobuharu , Takemaru Tsuneo
Published: December 2009

Crude extracts of mycelia and basidiocarp primordia in the basidiomycete Coprinus cinereus were resolved on sodium dodecyl sulfate – polyacrylamide gels, and ubiquitin and several proteins were detected by immunoblotting with anti-ubiquitin antibody. The molecular masses of the proteins detected were 30 900, 28 600, 27 800, 26 300, 22 500, and 15 400 daltons, respectively. Relative levels of ubiquitin and the ubiquitin-immunoreactive proteins were measured in different stages of development. The levels of ubiquitin and most of the ubiquitin-immunoreactive proteins in basidiocarp primordium formation increased and in basidiocarp maturation decreased in cap and upper stipe, while in lower stipe became high except for the 27 800 dalton protein and ubiquitin. During sporulation, ubiquitin and all the ubiquitin-immunoreactive proteins tended to decrease in the cap of the young wild-type basidiocarp. The levels of 30 900 and 15 400 dalton proteins increased transiently at 6–10 h after the beginning of the last light period, while ubiquitin decreased markedly. No correlation was observed between changes in levels of the ubiquitin-immunoreactive proteins and the blocked stages in sporulation-deficient mutants.Key words: ubiquitin, development, sporulation, Coprinus.
Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells
Authors: Mello Maria Luiza S. , Russo Jose
Published: December 2009

Feulgen–DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.Key words: cell transformation, c-H-ras oncogene, NIH/3T3 cells, image analysis, Feulgen staining.
Effect of sulfonates on the esterase activity of carboxypeptidase A
Authors: Frye Jeffrey L. , Sebastian John F.
Published: December 2009

The effects of sulfonates on the carboxypeptidase A catalyzed hydrolysis of the ester substrate benzoylglycyl-L-phenyllactate were determined. The modifiers examined were benzenesulfonate, p-toluenesulfonate, 2-phenylethanesulfonate, methanesulfonate, ethanesulfonate, propanesulfonate, butanesulfonate, pentanesulfonate, hexanesulfonate, heptanesulfonate, and 2,2-dimethyl-2-silapentane-5-sulfonate. Sulfonate activators of peptide hydrolysis were inhibitors of esterase activity. Of the sulfonates studied, 2,2-dimethyl-2-silapentane-5-sulfonate was the most effective inhibitor. 2-Phenylethanesulfonate, hexanesulfonate, heptanesulfonate, and 2,2-dimethyl-2-silapentane-5-sulfonate exhibited uncompetitive inhibition. The remaining sulfonates either did not inhibit or the inhibition was too weak to properly characterize.Key words: carboxypeptidase A, sulfonate modifiers, esterase activity.
Age-associated changes in the estrogen receptor-active proteases of female rabbit liver
Authors: Williamson Denis G. , Song Marie M.
Published: December 2009

Female rabbit liver cytosol contains a receptor-modifying activity that converts the 250 000 estrogen receptor of liver and uterine cytosol to a 37 000 form. There is an age-dependent increase in this receptor-active protease and in the general protease activity of rabbit liver cytosol, measured with [14C]casein. Sephacryl S-200 chromatography of liver cytosol shows that in the young animal (5 weeks old) the major receptor-modifying activity elutes near the void volume, while in the older animal (13 weeks old) activities having lower molecular weights are present. The general protease activity elution profile is similar to the receptor-active protease profile for the 5-week-old rabbit but not the 13-week-old rabbit. The liver cytosol of the older animal has a high molecular weight protease active toward [14C]casein but not toward the estrogen receptor. The changes in the estrogen receptor forms and the receptor-modifying activity profiles of liver cytosol that occur during development in the rabbit suggest that receptor-modifying activity may initially be associated with the estrogen receptor to form a high molecular weight complex.Key words: liver, estrogen receptor, proteolysis, age dependent.
Dietary orotic acid accentuates the hepatic response to phenobarbital in rats
Authors: Haines D. S. Milton , Wu Margaret , Tokmakjian Sonya D.
Published: December 2009

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.Key words: phosphatidylcholine, phosphatidylethanolamine, cytochrome P-450, phenobarbital, orotic acid, endoplasmic reticulum.
The cytoprotective effect of Trolox demonstrated with three types of human cells
Authors: Wu Tai-Wing , Hashimoto Noriaki , Wu Jun , Carey Doug , Li Ren-Ke , Mickle Donald A. G. , Weisel Richard D.
Published: December 2009

Trolox, a hydrophilic analogue of α-tocopherol, was reported to scavenge peroxyl radicals better than vitamin E in sodium dodecyl sulfate micelles and in liposomes. However, it was not known if Trolox protects human cells against oxyradical damage or if it acts as an antioxidant there. Here we demonstrate that Trolox prolonged substantially the survival of human ventricular myocytes and hepatocytes against oxyradicals generated with xanthine oxidase plus hypoxanthine, and prevented lysis of red cells exposed to an azo-initiator (2,2′-azo-bis(2-amidinopropane) HCl. Note that Trolox did not inhibit xanthine oxidase. In each cell type, the protection by Trolox was dose dependent and surpassed those given by such water-soluble antioxidants as ascorbic acid, superoxide dismutase, and (or) catalase, each examined at or near its optimal level in the same system. Using hepatocytes as a model, we further observed that Trolox reduced markedly the quantity of phospholipid conjugated dienes (a chemical imprint of oxyradical damage) in cells despite their exposure to oxyradicals. These data suggested that Trolox behaves as an antioxidant in cells as illustrated in hepatocytes.Key words: oxyradicals, cytoprotection, hepatocytes, myocytes, erythrocytes.
Sister chromatid cohesiveness: Vital function, obscure mechanism
Authors: Maguire Marjorie P.
Published: December 2009

Observations of chromosome behavior have suggested that it is sister chromatid cohesiveness which is primarily responsible for maintenance of chiasmate association between pachytene and anaphase of the first meiotic division and also for maintenance of sister centromere association until anaphase II. These associations seem essential for assurance of normal distribution of chromosomes into gametes (except in organisms in which alternative means have evolved, such as the male of Drosophila melanogaster). Sister chromatid cohesiveness is also found in varying degrees at mitosis. Reports of observations that are relevant to the nature of this cohesiveness are reviewed here with particular attention to behavior under a variety of conditions which include ploidy changes, presence of mutation effects, chromosome rearrangements, and experimental treatments. Attention is focused on constraints imposed upon model building by the observations, and also on directions for future study, which seem promising.Key words: sister chromatid cohesiveness, chiasmata, topoisomerase II, synaptonemal complex, meiosis.
The discovery of the vinca alkaloids—chemotherapeutic agents against cancer
Authors: Noble Robert L.
Published: December 2009

In folklore medicine, extracts of the leaves of the subtropical plant Catharanthus roseus (L.) G. Don (sometimes known as Madagascar periwinkle) were reputed to be useful in the treatment of diabetes. This review describes how attempts to verify the antidiabetic properties of the extracts led instead to the discovery and isolation of two complex indole alkaloids, vinblastine and vincristine, which are used in the clinical treatment of a variety of cancers. The two alkaloids, although structurally almost identical, nevertheless differ markedly in the type of tumors that they affect and in their toxic properties. These and related alkaloids have been the subject of many pharmacological and biochemical investigations both in vivo and in vitro in the search for improved cancer treatments. A model system used in these studies, a transplantable lymphoma in Noble strain rats designated Nb2 node, has serendipitously led to the development of a highly sensitive and specific bioassay for lactogenic hormones.Key words: Catharanthus roseus, vinblastine, vincristine, cancer chemotherapy, Nb rat lymphoma, lactogen bioassay.
The electrical and spectroscopic properties of planar asymmetrical membranes incorporating chlorophyll a and plastoquinone-9. Model of incorporation of chlorophyll a and plastoquinone-9
Authors: Robert Sylvain , Tancrède Pierre
Published: December 2009

We have studied the incorporation of chlorophyll a and plastoquinone-9 in Montal–Mueller membranes. In particular, we have been interested by the influence of both the lipid: chlorophyll a ratio and the asymmetry of incorporation of the constituents on the electrical and fluorescence spectroscopic properties of the planar membranes built up from these constituents. The phospholipid matrix was made from phosphatidylethanolamine and phosphatidylserine. The monitoring of the fluorescence spectral properties of chlorophyll a incorporated in various concentrations leads to the conclusion that chlorophyll a is incorporated in the bilayers in monomeric form inside microdomains. It is shown that chlorophyll a is positioned in these microdomains in such a way that the porphyric ring is interacting with the polar head of the lipid molecules where the interface polarity shows a dielectric constant varying between 25 and 35. The phytyl chain is embedded in the bilayer core, serving as an anchor, running parallel to the aliphatic chains of the phospholipids. We have also monitored the position of the plastoquinone-9 molecules within the bilayer. We found that plastoquinone-9 is incorporated in the center plane of the bilayer, increasing the thickness of the bilayer. This result confirms evidence, gathered in the literature from monolayer and differential scanning calorimetry studies, that long chain quinones and especially plastoquinone-9 are embedded deeply within the hydrophobic core of the bilayer. We also show that when chlorophyll a and plastoquinone-9 are present together in the bilayer, the quinolic ring of the plastoquinone-9 molecule positions itself in the free volume created by the bulky porphyric ring of a chlorophyll a molecule.Key words: chlorophyll, plastoquinone, bilayer, conformation, capacitance, fluorescence.
Specific inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of their second subunit
Authors: Cosentino Gregory , Lavallée Pierre , Rakhit Sumanas , Plante Raymond , Gaudette Yvon , Lawetz Carol , Whitehead Paul W. , Duceppe Jean-Simon , Lépine-Frenette Carole , Dansereau Nathalie , Guilbault Claire , Langelier Yves , Gaudreau Pierrette , Thelander Lars , Guindon Yvan
Published: December 2009

Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 μM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 μM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.Key words: ribonucleotide reductase, inhibition, human, bacteria.
Two pathways in the biosynthesis of cadystins (γEC)nG in the cell-free system of the fission yeast
Authors: Hayashi Yukimasa , Nakagawa Chiaki W. , Mutoh Norihiro , Isobe Minoru , Goto Toshio
Published: December 2009

Small metal-binding peptides, cadystins, with the general structure of (γ-Glu-Cys)n-Gly ((γEC)nG), were synthesized in a cell-free system of fission yeast to examine the in vivo synthetic pathway. The crude enzyme for cadystin synthesis was prepared by ammonium sulfate precipitation (75% saturation) from the 120 000 × g supernatant of the cell extract, and the excess salt in the enzyme fraction was removed by Sephadex gel filtration. Using this crude enzyme fraction, it was shown that there were two pathways for cadystin biosynthesis. One pathway is γ-Glu-Cys (γEC) dipeptidyl transfer from both glutathione (γECG) and cadystins to glutathione and cadystins. The other one is γEC polymerization from (γEC)n and glutathione to (γEC)n+i, followed by glycine addition with glutathione synthetase.Key words: cadystin, fission yeast, cell-free biosynthesis, dipeptidyl transferase.
Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase
Authors: Nichol Christine P. , Roufogalis Basil D.
Published: December 2009

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by α-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.Key words: acetylcholinesterase, bovine erythrocytes, phosphatidylinositol-specific phospholipase C, phase separation, affinity chromatography.