bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid
Authors: Labrecque Jean , Bhat Pangala V. , Lacroix André
Published: December 2009

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.
Vanadium as a modulator of cellular regulatory cascades and oncogene expression
Authors: Stern Arnold , Yin Xuefeng , Tsang Siu-Sing , Davison Allan , Moon Jim
Published: December 2009

Vanadium, a trace metal in the environment and in biological systems, influences the behavior of enzymes, mimics and regulates growth factor activity, is a potential mutagenic and carcinogenic agent, and regulates gene expression. The diverse biological actions of vanadium result from its capacity to function as an oxyanion, oxycation, or prooxidant. Vanadium is found in water, rocks, and soils in low concentration and in relatively high concentrations in coal and oil deposits. Vanadium compounds at much higher concentrations than are typically ingested are being considered in the treatment of diabetes mellitus. The actions of insulin and vanadium on the insulin receptor are similar, but the mechanisms are not identical. Vanadium modulates growth-factor-mediated signal transduction pathways. Vanadium promotes cell transformation and diminishes cell adhesion. Consistent with its mitogenic action and its capacity to mimic mitogenic growth factors, vanadium stimulates expression of protooncogenes. In particular, oxygen-derived active species are involved in the expression of the jun protooncogene in the presence of vanadium. The unique cellular activity of vanadium makes it a tool of unparalleled potential for studying mechanisms of cell growth, differentiation, and metabolism.Key words: vanadium, oncogene expression, signal transduction, mitogenesis, transformation.
Metabolism of alkyl lysophospholipid in epithelial cancer cell lines and inhibition of cell growth
Authors: Lu Xiaoli , Zhou Xi , Kardash Dawn , Arthur Gilbert
Published: December 2009

The mechanism of inhibition of cancer cell growth by alkyllysophospholipids is not known. We have investigated the metabolism of 1-O-octadecyl-2-O-methylglycerophosphocholine (ET-18-OCH3) in MCF7, A427, and A549 cell lines to determine whether there is a correlation between metabolism and sensitivity and whether the growth-inhibitory effects are due to ET-18-OCH3 or its metabolites. After 12 h incubation with ET-18-OCH3, less than 1.5% of ET-18-OCH3 taken up in the cells was converted to 1-O-octadecyl-2-O-methylglycerol (OMG). No correlation was observed between the extent of metabolism and sensitivity to the compound. Incubation of cells with 1 μg OMG/mL (2.8 μM) for 12 h resulted in cellular quantities of OMG in MCF7, A427, and A549 that were, respectively, 8-, 5-, and 25-fold greater than those in cells incubated with 5 μg ET-18-OCH3/mL (9.6 μM). While 12 h incubation with 1 μg OMG/mL did not significantly inhibit the proliferation of MCF7 or A427 cells, incubation with 5 μg ET-18-OCH3/mL inhibited MCF7 and A427 growth by 90 and 15%, respectively. A549 cell growth was inhibited 10% by 1 μg OMG/mL, but not by 5 μg ET-18-OCH3/mL. Incubation of cells for 12 h with 5 μg OMG/mL (13.9 μM) inhibited the growth of all three cell lines. Our results indicate that both ET-18-OCH3 and OMG can inhibit cell growth but the low quantities of OMG produced from ET-18-OCH3 are not responsible for the observed inhibition of MCF7 and A427 cell growth.Key words: alkyllysophospholipid, metabolism, epithelial cancer cells, growth inhibition.
Effect of pituitary adenylate cyclase activating polypeptide on vasopressin-induced proliferation of aortic smooth muscle cells: comparison with vasoactive intestinal polypeptide
Authors: Oiso Yutaka , Kotoyori Jun , Murase Takashi , Ito Yoshiaki , Kozawa Osamu
Published: December 2009

Pituitary adenylate cyclase activating polypeptide (PACAP) inhibited dose dependently the DNA synthesis stimulated by arginine vasopressin (AVP) in cultured rat aortic smooth muscle cells (SMC). The inhibition was cell cycle dependent and the maximum inhibition was observed when added at the late G1 phase of the cell cycle. Vasoactive intestinal polypeptide (VIP), which shows a considerable homology with PACAP, also inhibited dose dependently the AVP-induced DNA synthesis in a cell cycle dependent manner. The maximum inhibition was also observed at the late G1 phase. The patterns of both the dose-dependent inhibitions were similar, and the inhibition by a combination of PACAP and VIP was not additive. PACAP stimulated dose dependently cAMP accumulation in aortic SMC. VIP also stimulated cAMP accumulation, and the accumulation by a combination of PACAP and VIP was not additive. Both PACAP and VIP had little effect on phosphoinositide hydrolysis in these cells. The suppression of the AVP-induced DNA synthesis by PACAP or VIP was enhanced by 3-isobutyl-1-methylxanthine, an inhibitor for phosphodiesterases. Dibutyryl cAMP, but not 8-bromo-cGMP, inhibited the AVP-induced DNA synthesis, and a combination of PACAP and dibutyryl cAMP was not additive. [Ac-Tyr1, D-Phe2]growth hormone-releasing factor, an antagonist for VIP receptor, reversed the inhibitory effect of PACAP on the AVP-induced DNA synthesis. These results suggest that PACAP has an antiproliferative effect on aortic SMC at the late G1 phase of the cell cycle through cAMP production, and that PACAP and VIP inhibit the AVP-induced DNA synthesis by a common mechanism.Key words: pituitary adenylate cyclase activating polypeptide, vasoactive intestinal polypeptide, arginine vasopressin, DNA synthesis, aortic smooth muscle cells.
Two-dimensional pulsed-field gel electrophoresis of yeast chromosomes: evidence for triplex-mediated DNA condensation
Authors: Hampel Ken J. , Lee Jeremy S.
Published: December 2009

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 μg/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 μM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.Key words: triplex DNA, pulsed-field gel electrophoresis, chromosome structure, DNA mobility, chromosome condensation.
Complete sequence of rabbit kidney aminopeptidase N and mRNA localization in rabbit kidney by in situ hybridization
Authors: Yang Xiao-Feng , Milhiet Pierre Emmanuel , Gaudoux Florence , Crine Philippe , Boileau Guy
Published: December 2009

We have isolated and sequenced a full-length cDNA for rabbit kidney aminopeptidase N (APN). The 3-kilobase cDNA contains 12 nucleotides of the 5′ noncoding region, a 2898 nucleotide long open reading frame, and 113 nucleotides of the 3′ untranslated region. The open reading frame encodes a type II membrane protein of 966 amino acid residues composed of a 10 residue NH2-terminal cytosolic domain, a 23 residue transmembrane domain, and a large 933 residue ectodomain that contains the active site. Rabbit APN has eight potential N-glycosylation sites and seven cysteine residues, one of which is located in the transmembrane domain. Computer analysis showed that the enzymes from human, rat, and rabbit were highly conserved, except for the stalk region immediately downstream from the transmembrane domain. Using in situ hybridization techniques we showed that in rabbit kidney, APN mRNA is present in proximal tubules but not in glomeruli, which corresponds to the localization of the protein observed by immunohistochemistry. Taken together, our results strongly suggest that the expression of APN in kidney is modulated at mRNA levels and not at translational and (or) posttranslational levels.Key words: aminopeptidase N, rabbit kidney, full-length sequence, in situ hybridization.
Equine ovarian aromatase: evidence for a species specificity
Authors: Amri Hakima , Gaillard Jean-Luc , Al-Timimi Ihsan , Silberzahn Pierre
Published: December 2009

Mare granulosa cells and cyclic corpus luteum microsomes are reported to aromatize 19-norandrogens more efficiently than androgens. However, 16α-hydroxytestosterone and epitestosterone were not aromatized by the equine corpus luteum microsomal estrogen synthetase. These results indicate that the equine aromatase system would be different from the human placental microsomal estrogen synthetase, which aromatizes 16α-hydroxyandrogens and epitestosterone but not 19-norandrogens. Furthermore, our data show that the rates of aromatization of androgens and 19-norandrogens were not additive and that 19-norandrogens competitively inhibited the aromatization of androgens, suggesting that a single enzymic system would be involved in the aromatization of androstenedione, 19-norandrostenedione, testosterone, and 19-nortestosterone. Our findings, which are identical to those previously reported for stallion testis and mare placental estrogen synthetases, provide evidence for a strong species specificity of the equine aromatase system.Key words: equine, cyclic corpus luteum, aromatase, androgens, 19-norandrogens.
In vitro growth-stimulatory property of pigeon milk
Authors: Bharathi L. , Shenoy K. B. , Mojamdar M. , Hegde S. N.
Published: December 2009

Five cell lines were employed to test the growth-stimulating property of pigeon milk in vitro. All the cell lines except A431 showed good growth response to crude homogenates of pigeon milk. Enhancement of DNA synthesis in quiescent Chinese hamster ovary (CHO) cells by pigeon milk was dose dependent up to a concentration of 1%. In vitro growth stimulation by 1% pigeon milk was approximately equal to that by 2% foetal bovine serum (FBS) when CHO cells were used, growth stimulation of Vero cells by 1% pigeon milk was roughly three times of that by 2% FBS. In contrast, 1% pigeon milk was only half as active as 2% FBS on NIH/3T3 cells and five times less active than 2% FBS on human foetal lung fibroblast cells. After dialysis using a relative mass (Mr) cutoff of 3500, the pigeon milk mitogenic activity was retained in the dialyzed solution, although it decreased by 40–60% when dialyzed with Mr cutoffs of 8000 and 12 000 – 14 000. The growth-stimulating activity of pigeon milk was resistant to heat, acid, alkali, and the action of urea, guanidine hydrochloride, dithiothreitol, and trypsin. We suggest that pigeon milk is a new source of growth factor(s) capable of stimulating in vitro the growth of many mammalian cell types.Key words: pigeon milk, growth stimulation, cultured cells.
Coexistence of two biochemically distinct phospholipase A2 activities in human platelet, monocyte, and neutrophil
Authors: Marshall Lisa A. , Roshak Amy
Published: December 2009

The cell-associated phospholipase A2 (PLA2) activities of the human platelet, neutrophil, and monocyte were simultaneously characterized, utilizing the biochemical differences observed between the 14 kDa (kilodalton), type II PLA2 isolated from inflammatory human synovial joint fluid (HSF) and the arachidonic acid (AA) specific, 85-kDa high molecular mass (HMM) PLA2 isolated from the cytosol of a U937 monocytic cell line. The HSF PLA2 can be distinguished from the HMM PLA2 by its resistance to acid treatment, sensitivity to a sulfhydryl reducing agent, lack of preference for the fatty acid on the sn-2 position of phospholipid substrate, and inhibition by the C-7 phosphonate–phospholipid transition-state PLA2 inhibitor. Evaluation of all three cell types revealed that HMM-like PLA2 activity was found predominantly in the cytosolic fractions, although detection in neutrophil cytosol required more concentrated preparations and the use of high specific activity [3H]AA-labeled Escherichia coli. HSF-PLA2-like activity was measured in microsomal and cytosolic fractions of all three cell types, but was found in neutrophil cytosol only after treatment with acid. Further HMM-PLA2-specific interfering agents in neutrophil cytosol were observed and exemplifies one problem in assigning the existence of this enzyme in crude broken cell preparations using activity measurements alone. The role that these two enzymes play in eicosanoid production of the respective cell types remains to be studied.Key words: phospholipase A2, arachidonic acid, neutrophil, platelet, monocyte, cytosol, microsome.
Purification and characterization of D-β-hydroxybutyrate dehydrogenase expressed in Escherichia coli
Authors: Jones Les , Churchill Sharon , Churchill Perry
Published: December 2009

D-β-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been cloned into pUC18, expressed in Escherichia coli, and purified to homogeneity. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 129 μmol/(min∙mg). The functional properties of the enzyme expressed in E. coli were compared with the enzyme purified from rat liver. The specific activities, kinetic parameters, and phospholipid activation profiles were virtually identical. These results indicate that the expression of the enzyme in E. coli is a viable method for producing active functional BDH and should allow for the production of specifically altered BDH molecules.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression, lipid requiring.
Structure of the lipopolysaccharide O-antigen of Pseudomonas cepacia serotype A
Authors: Beynon Linda M. , Perry Malcolm B.
Published: December 2009

The O-antigenic polysaccharides of the lipopolysaccharides produced by three isolates of Pseudomonas cepacia serogroup A were analysed by composition, methylation, periodate oxidation, and two-dimensional nuclear magnetic resonance methods. They were found to be identical linear unbranched polymers of a repeating trisaccharide unit containing 2-acetamido-2-deoxy-D-galactose and L-rhamnose residues (2:1) and have the structure[Formula: see text]Key words: lipopolysaccharide, Pseudomonas cepacia, O-antigen.
Control of glycogenosis and effects of exercise on phosphorylase kinase and cAMP-dependent protein kinase in rainbow trout organs
Authors: Mehrani Hussein , Storey Kenneth B.
Published: December 2009

To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed. Other modes of GPK activation were not apparent, for the enzyme in crude extracts was stimulated only by incubation with cAMP and did not respond to cGMP or Ca2+ + phorbol 12-myristate 13-acetate. The data indicate that the cAMP-activated, PKA- and GPK-mediated cascade is key to the activation of glycogenolysis in both the skeletal muscle and liver during burst swimming exercise by trout.Key words: exercise, glycogen phosphorylase kinase, protein kinase A, cAMP, Oncorhynchus mykiss, control of glycogenolysis.
Influence of exogenous growth factors on the expression of plasminogen activators by explants of normal and healing rabbit ligaments
Authors: Murphy Patricia G. , Loitz Barbara J. , Frank Cyril B. , Hart David A.
Published: December 2009

In this investigation we have demonstrated that specific growth factors are able to modify plasminogen activator (PA) activity in explants from both normal and healing ligaments. Specifically, the addition of insulin-like growth factor 2 (IGF-2; 100 ng/mL) and transforming growth factor β1 (TGF-β1; 2.5 ng/mL) to explants of unoperated and healing ligaments resulted in a decrease in both PA and PA – PA-binding protein complex in conditioned medium from several tissues, and particularly in medial collateral ligament (MCL) scar tissue. This effect was observed in the explants from 3-, 6-, and 12-week healing scar tissue. In addition, IGF-2 and TGF-β1 influenced PA release from MCL tissue derived from the unoperated knee joint of 3-week healing animals. In contrast, basic fibroblast growth factor (b-FGF; 10 ng/mL) and IGF-1 (100 ng/mL) did not cause a detectable alteration of PA and PA – PA-binding protein complex activity in any of the healing or unoperated ligaments analyzed. These results indicate that specific growth factors are able to modify the activity of cells in explants of ligament and healing ligament in a distinct manner.Key words: plasminogen activators, ligaments, growth factors.
Ageing, differentiation, and gene expression in rat epididymal preadipocytes
Authors: Kirkland James L. , Hollenberg Charles H. , Gillon Wanda S.
Published: December 2009

Ageing results in decreased replicative potential of preadipocytes, as well as reduced capacities for the lipid accumulation and increases in lipogenic enzyme activities during differentiation of preadipocytes into fat cells. To determine whether decreased differentiation is associated with decreased levels of mRNA for differentiation-dependent genes and whether early as well as late components of the differentiation programme are affected by ageing, we measured β-actin, α-tubulin, lipoprotein lipase, and glycerol-3-phosphate dehydrogenase mRNA levels in undifferentiated and differentiated epididymal preadipocytes from 3-, 17-, and 24-month-old Fischer 344 rats. During ageing, diminished differentiation-related changes occurred in mRNAs affected early (actin, tubulin), midway through (lipoprotein lipase), and late (glycerol-3-phosphate dehydrogenase) in the preadipocyte differentiation process. Hence, early as well as late phases of the differentiation programme were affected by ageing. The effects involved changes in gene transcription or mRNA processing. Our results were not consistent with the hypothesis that age-related decreases in replication are caused by an increased tendency for cell differentiation.Key words: senescence, adipocyte precursors, β-actin, α-tubulin, lipoprotein lipase, glycerol-3-phosphate dehydrogenase.
Acid secretion in α-toxin-permeabilized gastric glands
Authors: Thibodeau Alain , Yao Xuebiao , Forte John G.
Published: December 2009

Rabbit gastric glands were treated with α-toxin to test for permeabilization of basolateral membrane and retention of functional activity of parietal cells. Treatment with up to 400 U α-toxin/mL resulted in a dose-dependent increase in permeabilization, as judged by nuclear uptake of trypan blue (960 daltons), while causing relatively little loss of cytoplasmic macromolecules in the size range of lactate dehydrogenase (134 000 daltons). In the presence of cAMP and ATP, α-toxin-permeabilized resting gastric glands were stimulated to accumulate aminopyrine by ~10-fold over glands incubated without added nucleotides. Aminopyrine accumulation in stimulated permeabilized glands was inhibited by specific H+,K+-ATPase inhibitors, omeprazole and SCH-28080, and by the selective inhibitor of protein kinase A, H-89 (IC50 = 7.17 ± 2.05 μM; n = 4). Aminopyrine accumulation in the α-toxin-treated glands was dependent on both exogenous ATP and cAMP; however, when no exogenous ATP was present, cAMP-activated aminopyrine accumulation reached ~50% of maximum, and at levels of ATP > 0.05 mM, maximal aminopyrine accumulation occurred without exogenous cAMP. In the presence of ATP alone, aminopyrine accumulation in permeabilized glands achieved 61.1 ± 3.2% (n = 10; range, 50–70%) of the values measured on paired samples of intact glands stimulated with histamine plus isobutylmethylxanthine. These results demonstrate the functional responsiveness of α-toxin-permeabilized resting gastric glands. The participation of a protein kinase A dependent pathway during activation of permeabilized parietal cell is proposed.Key words: aminopyrine accumulation, H+,K+-ATPase, H-89, protein kinase A, oxidative phosphorylation, digitonin.
Regulation of adhesion-related protein tyrosine kinases during in vitro differentiation of retinal pigment epithelial cells: translocation of pp60c-src to the nucleus is accompanied by downregulation of pp125FAK
Authors: Moszczynska Anna , Opas Michal
Published: December 2009

In the present report we show that induction of expression of a differentiated phenotype in cultured retinal pigmented epithelium of chick embryo is accompanied by coordinate regulation of expression and distribution of two adhesion-related nonreceptor protein tyrosine kinases, pp60c-src and pp125FAK∙pp60c-src translocates from the cell surface in flat undifferentiated cells to the nucleus in the packed differentiated cells. In contrast, pp125FAK, abundant in focal adhesions of flat undifferentiated cells, is downregulated in cells that have differentiated and packed into an epithelial sheet.Key words: retinal pigment epithelium, retina, tyrosine kinases, adhesion, differentiation.
Glutathione metabolism in heart and liver of the aging rat
Authors: Stio M. , Iantomasi T. , Favilli F. , Marraccini P. , Lunghi B. , Vincenzini M. T. , Treves C.
Published: December 2009

A comprehensive study on glutathione metabolism in rat heart and liver as a function of age was performed. In the heart, reduced glutathione, total glutathione, and the glutathione redox index showed a decrease during aging, while oxidized glutathione levels increased in 5-month-old rats with respect to the young animals and remained quite constant in 14- and 27-month-old rats. In the liver, the highest levels of reduced glutathione were found in the 2-month-old rats, while oxidized glutathione reached a peak at 5 months. Glutathione-associated enzymes showed age-related changes. Glutathione peroxidase, unaffected by aging in the heart, decreased in the liver of the 27-month-old rats. In the heart and the liver, the highest values of glutathione S-transferase were found at 5 months and 27 months, respectively. Glucose-6-phosphate dehydrogenase followed a similar trend in both heart and liver. Glutathione reductase also showed the same behaviour in heart and in liver, increasing in old rats with respect to the other age groups. A decrease in γ-glutamylcysteine synthetase was found in the heart and liver of 27-month-old rats in comparison with the 2-month-old ones. In conclusion, a decreased antioxidant capability has been demonstrated in both heart and liver of old rats.Key words: glutathione metabolism, age, rat heart, rat liver.
A nuclear magnetic resonance study of the DNA-binding affinity of Cro repressor protein stabilized by a disulfide bond
Authors: Baleja James D. , Sykes Brian D.
Published: December 2009

The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage λ. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 °C, whereas the wild-type protein has a melting temperature of about 47 °C. Using 1H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 °C), but a much lower melting temperature is seen for V55C Cro (58 °C). This suggests that between 58 and 67 °C the cross-linked protein exists in a molten globule state with the α-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (Kd = 160 μM) about eight times more weakly than the wild-type protein (Kd = 19 μM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.Key words: nuclear magnetic resonance, circular dichroism, Cro repressor, protein engineering, disulfide bond.
Structural comparison of the histidine-containing phosphocarrier protein HPr
Authors: Jia Zongchao , Quail J. Wilson , Delbaere Louis T. J. , Waygood E. Bruce
Published: December 2009

The phosphocarrier protein HPr is a central component of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) that is responsible for carbohydrate uptake in many bacterial species. A number of three-dimensional structures of HPrs from both Gram-positive and Gram-negative bacteria have been determined; the overall folding topology of HPr is an open-faced β-sandwich composed of three α-helices and a β-sheet. A detailed structural comparison of these HPrs has been carried out. Besides the overall main chain folding, many detailed structural features are well conserved in all HPr structures. The three x-ray structures of HPrs from Escherichia coli, Streptococcus faecalis, and Bacillus subtilis show considerable overall similarity with respect to the positions of the Cα atoms. A significant structural difference between HPrs from Gram-positive and Gram-negative bacteria is found in the region of Gly54, owing to the steric effects of Tyr37 in HPrs from the Gram-positive species. The region around Gly54 is involved in the binding of HPr to other PTS proteins and the differences in this region may be responsible for some of the poor functional complementation between HPrs from Gram-positive and Gram-negative species. The active center region, residues 12–18, appears to have significant differences in the comparisons between the overall structures. These differences support the proposal that phosphorylation and dephosphorylation of the active site His 15 is accompanied by conformational changes. However, a local structural comparison of residues 12–18 from the x-ray structures of HPrs from E. coli and B. subtilis, and the two-dimensional nuclear magnetic resonance structure of B. subtilis HPr suggests that there is a conserved active center involving residues His15, Arg 17, and Pro18, which shows little conformational change during the phosphorylation cycle. The results of other experimental approaches, including site-directed mutagenesis and NMR spectroscopy, are in some cases difficult to rationalize with some of the details of the structures, but do appear to favour the conclusion that little conformational change occurs.Key words: phosphoenolpyruvate: sugar phosphotransferase system, histidine-containing protein, active center, phosphorylation, protein structure, structural homology.
Purification and characterization of a protein kinase from goat sperm plasma membrane
Authors: Mitra Sutapa , Nath Debjani , Majumder Gopal C.
Published: December 2009

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP, is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 μg/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40–50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (~ 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe. The data are consistent with the view that the isolated enzyme is an ecto-protein kinase whose catalytic site is oriented primarily to the surface of viable sperm cell to cause phosphorylation of the endogenous outer cell-surface phosphoproteins.Key words: spermatozoa, plasma membrane, protein kinase, phosphoproteins, ectoenzyme.
Carbonic anhydrase in mouse skeletal muscle and its influence on contractility
Authors: Côté Claude H. , Jomphe Nicolas , Odeimat Abdul , Frémont Pierre
Published: December 2009

Carbonic anhydrase III (EC 4.2.1.1) is the most abundant cytosolic protein in type I skeletal muscle fibers. Investigations of its physiological role have mostly been conducted with rat muscles, which sometimes are unsuitable for in vitro studies. The objective of the present study was to characterize the carbonic anhydrase in the mouse soleus muscle to verify if this muscle can be used as a model to further study the enzyme's function. Total carbonic anhydrase specific activity in the mouse soleus was comparable to the value for rat. However, 60% of the total carbonic anhydrase activity in the mouse was of the sulfonamide-sensitive type and, therefore, not related to carbonic anhydrase III. Electrophoretic analysis revealed the presence of a 29-kDa protein in total and cytosolic extracts of the mouse soleus. Immunoblotting with an antibody developed against rat carbonic anhydrase III showed that it was also specific for this 29-kDa peptide, which presumably is the mouse carbonic anhydrase III. Inhibition of the sulfonamide-sensitive activity had no effect on contractile and fatigue characteristics, whereas inhibition of the sulfonamide-resistant carbonic anhydrase III activity led to a significant increase in resistance to fatigue. We conclude that the mouse soleus may represent an excellent model to understand the contribution of different carbonic anhydrase isoforms to muscle physiology.Key words: muscle fatigue, carbonic anhydrase III, sulfonamide, metabolism, enzyme.
Observations with a residualizing label in use to map the catabolic sites of plasma proteins
Authors: Regoeczi Erwin , Chindemi Paul A. , Bolyos Maria , Hu Wei-Li
Published: December 2009

Rat albumin, immunoglobulin G, transferrin, and aglycotransferrin were prepared for the comparison of their sites of degradation in rats. Iodotyramine-cellobiose was used as the residualizing label and a tyrosine-iodinated portion of the corresponding protein was used as the marker of extracellular undegraded protein. Each protein yielded a distinct distribution (or map) of catabolic activity throughout the body when expressed as percent dose accumulated per gram of tissue. The maps for albumin and transferrin were broadly comparable, whereas those for immunoglobulin G and aglycotransferrin were markedly different. As a whole entity, the liver appeared to top the list of organs/tissues contributing to the degradation of albumin and transferrin. Additional experiments aimed at facilitating the interpretation of results with residualizing labels were carried out with denatured albumin, asialofetuin, and human asialotransferrin type 3. These showed that various types of cells retained the label for markedly different periods of time. We feel, therefore, that the technique is more suited for making comparative measurements than for obtaining degradation rates as absolute values in a given anatomical location.Key words: aglycotransferrin, albumin, immunoglobulin G, protein catabolism, transferrin.
Effect of hypoxia on choline metabolism and phosphatidylcholine biosynthesis in isolated adult rat ventricular myocytes: effects of trifluoperazine
Authors: Rabkin Simon W.
Published: December 2009

The purpose of this study was to test the hypothesis that hypoxia alters choline metabolism in adult rat ventricular myocytes and that this is subject to modification by trifluoperazine. Ventricular myocytes were isolated from adult Wistar rats. Choline uptake and metabolism were examined, using [methyl-3H]choline. Choline metabolism to phosphatidylcholine (PC), an important phospholipid for cellular structure and function, was reduced by mild hypoxia while choline uptake was not altered. Trifluoperazine at 10−5 or 10−6 M reduced PC biosynthesis and did not reduce or prevent the effects of hypoxia. Interruption of myocardial metabolism with the combination of carbonyl cyanide m-chlorophenylhydrozone and amobarbital also reduced PC synthesis, although it also reduced choline uptake by the cell. These data indicate that in isolated cardiomyocyte from the adult rat heart that (i) hypoxia impairs PC biosynthesis, (ii) this may be operative in part through inhibition of mitochondrial function, and (iii) trifluoperazine does not act to oppose these effects of hypoxia, but it impairs PC biosynthesis. Thus a component of the membrane phospholipid changes observed with hypoxia may be due to impairment of PC biosynthesis in the cardiomyocyte.Key words: choline uptake, choline metabolism, hypoxia, carbonyl cyanide m-chlorophenylhydrozone, amobarbital, adult rat cardiomyocyte, trifluoperazine.
A comparison of the lytic properties of two ether-linked lipids, one with and one without antineoplastic activity
Authors: Jackson John K. , Burt Helen , Mok Wilson , Salari Hassan , Kumar Erukulla Ravi , Byun Hoe-Sup , Bittman Robert
Published: December 2009

We have synthesized two ether lipids: 2′-(trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (compound 1) with antineoplastic activity and a maltosyl derivative (compound 2) without antineoplastic activity. We have compared the antineoplastic activity of these two compounds against WEHI-3B cells with their ability to disrupt the membranes of erythrocytes or neutrophils. Since ether lipids are highly hydrophobic molecules, it is possible that they may exert their cytotoxic action by inducing the nonspecific perturbation of cellular membranes, causing lysis and cell death. Membrane disruption was monitored by the lysis of cells or the change in erythrocyte membrane microviscocity and compared with the effect of detergents (known nonspecific lytic agents). Both compounds 1 and 2 caused the lysis of erythrocytes and neutrophils. The rate of lysis of erythrocytes was comparable to the rate of change of erythrocyte membrane microviscosity caused by both compounds 1 and 2. Both compounds caused the lysis of erythrocytes via a noncolloid osmotic mechanism that displayed features of the lysis caused by detergents at high concentrations.Key words: ether-linked lipids, antineoplastic, lysis, anisotropy.
Radiolabelling of bovine myristoylated alanine-rich protein kinase C substrate (MARCKS) in an ADP-ribosylation reaction
Authors: Chao D. , Severson D. L. , Zwiers H. , Hollenberg M. D.
Published: December 2009

In an ADP-ribosylation reaction, we have observed the radiolabelling of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent molecular mass (80–90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS. To establish the identity of this radiolabelled constituent in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation.Key words: MARCKS, ADP-ribosylation, neuronal proteins, B-50/GAP-43, kinase C, calmodulin.
Elastin-derived peptide binding to a hydrophobic domain on neutrophil elastase
Authors: Tyagi Suresh C. , Simon Sanford R.
Published: December 2009

To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at ~410 nm when excited at 320 nm, indicating the presence of desmosine and (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase–EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor [Formula: see text], but bound with high affinity to neutrophil elastase [Formula: see text], as detected by its ability to quench elastase endogenous fluorescence. The complete emission spectrum of the mixture of elastase and EDP obtained at excitation wavelengths specific for tryptophan and desmosine/isodesmosine suggests that the EDP was in a hydrophobic environment which was close to at least one of the three tryptophan residues in the enzyme. Based on fluorescence energy transfer, we have estimated a distance between the elastase and EDP of ~10 ± 3 Å (1 Å = 0.1 nm) during elastinolysis. This pattern of binding to a hydrophobic site on neutrophil elastase without competitive inhibition of amidolytic activity was consistent with the importance of hydrophobic interactions between neutrophil elastase and elastin within a region of the enzyme removed from the active site.Key words: proteinase, elastase, elastin, extracellular matrix, elastin-derived peptide.
Antiapoptosis potential of bcl-2 oncogene by dephosphorylation
Authors: Haldar Subrata , Jena Nilamani , Croce Carlo M.
Published: December 2009

The antiapoptosis potential of bcl-2 has now been well established. But the biochemical mechanism of bcl-2 action is still poorly understood. Using the phosphatase inhibitor okadaic acid (OA) or chemotherapeutic agents such as Taxol and 5′-fluorouracil, we found that bcl-2 can be phosphorylated. Since OA or Taxol treatment leads to apoptosis, it seems that phosphorylation of bcl-2 leads to its inactivation. Exposure of several lymphoid cell lines expressing differential amounts of bcl-2 protein to OA resulted in apoptosis of the cells and hyperphosphorylation of bcl-2. Interestingly, the lymphoblastoid cell lines that did not phosphorylate bcl-2 following OA exposure did not undergo apoptosis. Moreover, pro-B cells isolated from patients with acute lymphoblastic leukemias exhibited endogenous phosphorylated forms of bcl-2 and a large number of apoptotic cells, even without OA treatment. Treatment with the phosphatase inhibitor or with chemotherapeutic agents (Taxol, 5′-fluorouracil) led to severe apoptosis of these cells, along with hyperphosphorylation of bcl-2. Phosphoamino acid analysis reveals that bcl-2 is phosphorylated at a serine residue. In summary, our investigation indicates that the phosphorylation pathway involving bcl-2 can be the determinant of cell death in lymphocytes.Key words: bcl-2, oncogene, apoptosis, phosphorylation, dephosphorylation.
The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation
Authors: Kolesnick Richard N. , Haimovitz-Friedman Adriana , Fuks Zvi
Published: December 2009

Recent evidence suggests that tumor necrosis factor α, Fas, and ionizing radiation employ the sphingomyelin pathway to trigger apoptosis. The sphingomyelin pathway is initiated by hydrolysis of plasma membrane sphingomyelin to generate ceramide via a sphingomyelinase. Ceramide serves as a second messenger stimulating a cascade of kinases and transcription factors that activate a final common pathway of programmed cell death. The extent to which this signaling system is used in apoptosis induced by other toxic modalities is not known, but accumulating evidence suggests that it is a commonly employed pathway that could be exploited therapeutically.Key words: sphingomyelin, apoptosis, tumor necrosis factor, Fas, ionizing radiation.
Changes in clusterin expression associated with light-induced retinal damage in rats
Authors: Wong P. , Kutty R. K. , Darrow R. M. , Shivaram S. , Kutty G. , Fletcher R. T. , Wiggert B. , Chader G. , Organisciak D. T.
Published: December 2009

Prolonged periods of high-intensity visible light exposure lead to photoreceptor cell degeneration, but the mechanism of damage is not understood. Increased clusterin mRNA levels have been found in several models of apoptosis, as well as in neurodegeneration. We report here that changes in clusterin mRNA levels are also associated with light-induced retinal damage in adult male albino rats. Animals previously maintained in darkness or a weak cyclic light environment were exposed to intense visible green light for up to 24 h. Some rats were pretreated with a synthetic antioxidant, dimethylthiourea (DMTU), which reduces photoreceptor cell degeneration. Clusterin mRNA steady-state levels increased with the duration of light exposure in both cyclic light and dark reared animals, suggesting that an apoptotic mechanism may be involved. Animals pretreated with DMTU showed a delay in the initial increase in clusterin mRNA levels, suggesting that oxidative damage is involved in the damage mechanism. However, the incomplete suppression of increasing steady-state clusterin mRNA levels by DMTU suggests that either oxidative damage triggers a second pathway or multiple damage mechanisms are induced in the retina by light exposure.Key words: retinal degeneration, clusterin, apoptosis, retinitis pigmentosa.
Expression of clusterin in cell differentiation and cell death
Authors: Ahuja Harleen Singh , Tenniswood Martin , Lockshin Richard , Zakeri Zahra F.
Published: December 2009

Clusterin, originally isolated as testosterone-repressed prostate message-2 from regressing rat ventral prostate, has been identified with the process of active cell death (ACD). The clusterin gene product is a glycosylated dimer consisting of α and β subunits, resulting from the 70-kilodalton preprotein. To determine its relationship with ACD, we have examined clusterin expression via in situ hybridization and immunohistochemistry. Clusterin message is detected in the supporting cells in both testes and ovaries and the protein surrounds the mature germ cells. The highest level of expression was found in the head region of the epididymis. Clusterin message is also detected in selected cells of uterine glands and ducts both in the normal and pregnant uterus. The expression of clusterin in the developing embryo is most abundant in the choroid plexus, inner ear, and epithelium of the eye. None of the cells in the testes, epididymis, or embryo that express clusterin are undergoing ACD. The expression of clusterin appears to correlate with cell remodelling or differentiation that occurs during these periods of development. However, in the female reproductive system, we found clusterin to be expressed in both differentiating as well as dying cells. These results suggest that clusterin may provide support for cells undergoing specific biochemical and (or) physical changes. Our results are consistent with the hypothesis that clusterin is an antiinflammatory agent.Key words: clusterin, apoptosis, testis, epididymis, ovary, uterus, embryo.