Biochem Mol Biol Int
· 1998 Nov · PMID 9844730
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The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we descri...The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we describe a simple, semi-quantitative and single tube multiplex RT-PCR assay for simultaneous and relative expression analysis with an application to all the six members of Arabidopsis calmodulin multigene family. In the multiplex primer set, individual gene specific primers were derived from 3'-untranslated region of the genes and a single common primer from the conserved exonic region. Transcriptional activation of all the members of the calmodulin gene family in response to touch was monitored. The results demonstrate that two of the genes are not regulated by touch; however, the other four that are induced by touch show a differential response including their kinetics of induction.
Cabrini L, Bergami R, Fiorentini D
… +3 more, Marchetti M, Landi L, Tolomelli B
Biochem Mol Biol Int
· 1998 Nov · PMID 9844729
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We have evaluated the effects of a diet containing normal amounts of lipids and a marginal content of vitamin B6 on lipid peroxidation. Pyridoxal phosphate concentrations of plasma and liver indicated that an initial def...We have evaluated the effects of a diet containing normal amounts of lipids and a marginal content of vitamin B6 on lipid peroxidation. Pyridoxal phosphate concentrations of plasma and liver indicated that an initial deficiency state was reached. Vitamin B6 deficiency led to peroxidative stress: TBARS production was higher in the liver (+18.6%) and even more in the heart (+61%) of deficient rats as compared with controls. Furthermore, significant stimulation of glutathione-dependent enzymes occurred in both heart and liver of deficient rats: glutathione peroxidase activity increased in heart (+144%) and liver (+505%); glutathione reductase increased in heart (+54.9%) and liver (+15.5%). No difference in the total glutathione content of the organs of the two groups was observed. The reduced glutathione/oxidized glutathione ratio was significantly lower in deficient rats. Although the activity of glutathione-dependent enzymes was significantly greater in deficient rats than in controls, this stimulation was only partially able to counteract the peroxidative damage due to vitamin B6 deficiency.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844728
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The effect of arachidonic acid (AA) on insulin-stimulated glucose transport by 3T3-L1 adipocytes was examined in the presence of cycloheximide. We found that AA acted synergistically with cycloheximide to suppress insuli...The effect of arachidonic acid (AA) on insulin-stimulated glucose transport by 3T3-L1 adipocytes was examined in the presence of cycloheximide. We found that AA acted synergistically with cycloheximide to suppress insulin-stimulated glucose transport, although it alone was without effect. Similar phenomena were observed while protein synthesis inhibitors other than cycloheximide were employed. Immunoblot analysis indicated that the increase in plasma membranes of the insulin-regulated glucose transporter (GLUT4) in response to insulin was decreased in cells pretreated with cycloheximide for a prolonged time, while total amount of GLUT4 was not altered. Simultaneous presence of AA with cycloheximide had no further effect on the amount of GLUT4 in either total or plasma membranes. Thus the present study suggests that AA in the presence of a protein synthesis inhibitor seems to decrease the intrinsic activity of GLUT4.
Ayabe T, Park SK, Nagahama H
… +6 more, Maruyama H, Sumida M, Takenaka H, Takenaka O, Onitsuka T, Hamada M
Biochem Mol Biol Int
· 1998 Nov · PMID 9844727
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Site-directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C-terminal segment (Val182, V186, and Leu193). Five mutants [(H36T, K55G, V182G, V186S, and L193Stop (deletion of...Site-directed mutagenesis of human adenylate kinase (AK) was carried out on residues His36, Lys55, and the C-terminal segment (Val182, V186, and Leu193). Five mutants [(H36T, K55G, V182G, V186S, and L193Stop (deletion of residues 193-194)] were generated and analyzed by steady-state kinetics. H36T, K55G, and L193Stop mutants showed an increase of K(m) values (19.8-, 19.7-, and 11.3-fold) for AMP2- compared to that for the wild-type enzyme, and these residues appeared to interact with AMP2-. V182G showed an increased K(m) value (7.4-fold) for MgATP2-. Therefore, V182 may be essential for interaction with MgATP2-. V186S increased the K(m) value (7.0- and 7.5-fold) for MgATP2- and AMP2-. V186 may thus interact with both substrates. The C-terminal domain of AK appears to be essential for MgATP2- and AMP2- binding.
Seok JH, Kim JB, Hong JH
… +4 more, Sung JY, Hur GM, Lim K, Lee JH
Biochem Mol Biol Int
· 1998 Nov · PMID 9844726
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The changes of Na,K-ATPase activity and its regulation have been investigated in the renal cortex of 1-clip-1-kidney hypertensive rat. Ouabain-sensitive Na,K-ATPase activity (Emax) and [3H]ouabain-binding site (Bmax) in...The changes of Na,K-ATPase activity and its regulation have been investigated in the renal cortex of 1-clip-1-kidney hypertensive rat. Ouabain-sensitive Na,K-ATPase activity (Emax) and [3H]ouabain-binding site (Bmax) in the hypertensive rat were slightly increased than those in the control. The levels of Na,K-ATPase alpha 1- and beta 1-subunit mRNA of the renal cortex in hypertensive rat were more increased than those in the control. Their increases were repressed by actinomycin-D, but not altered or more increased by cycloheximide. These results suggest that the increase of Na,K-ATPase activities and ouabain binding sites in 1-clip-1-kidney hypertensive rat may be correlated with the increases of gene expression in transcription level and/or of mRNA stability of Na,K-ATPase.
Horecká T, Perecko D, Kutejová E
… +2 more, Mikulásová D, Kollárová M
Biochem Mol Biol Int
· 1998 Nov · PMID 9844725
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Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide-containing substrate. Thioredoxin reductase from Streptomyces...Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide-containing substrate. Thioredoxin reductase from Streptomyces aureofaciens 3239 has been purified to homogeneity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on 2'5'-ADP-Sepharose 4B. Molar mass determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 69 kDa for the native protein and 34.8 kDa for the enzyme subunit. The isoelectric point determined by isoelectric focusing gel electrophoresis was 4.3. TrxR effectively catalyzed the reduction of DTNB in the presence of S. aureofaciens thioredoxin-1. TrxR activity in the presence of S. aureofaciens thioredoxin-2 was only 1/4 of the activity with thioredoxin-1 (1). The activity of pure TrxR decreased drastically in the presence of NADPH, while NADP+ as well as Streptomyces aureofaciens thioredoxin-1 protected the enzyme from inactivation. These results indicate that thioredoxin reductase activity in bacteria could be modulated by the redox status of NADP+/NADPH and thioredoxin pools.
Saso L, Leone MG, Sorrentino C
… +7 more, Giacomelli S, Silvestrini B, Grima J, Li JC, Samy E, Mruk D, Cheng CY
Biochem Mol Biol Int
· 1998 Nov · PMID 9844724
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Prostaglandin D synthetase (PGD-S; prostaglandin-H2 D-isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as beta-trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to app...Prostaglandin D synthetase (PGD-S; prostaglandin-H2 D-isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as beta-trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to apparent homogeneity from human cerebrospinal fluid (CSF) using a two-step procedure involving HPLC on a Vydac C8 reversed-phase column and high performance electrophoresis chromatography (HPEC) using a 10% T SDS-polyacrylamide gel. The purity of PGD-S isolated from CSF was confirmed by silver stained SDS-polyacrylamide gel and direct protein microsequencing (NH2-APEAQVSVQPNFQ). A highly specific polyclonal antibody was prepared against this protein for immunoassay development. Using an ELISA, it was found that the concentration of PGD-S in CSF did not alter significantly in different pathological conditions of the central nervous system (CNS). These include dementia (n = 9), hydrocephalus (n = 4), neuropathy (n = 11), optic neuritis (n = 4), multiple sclerosis (n = 11), and demyelinating syndrome (n = 11), when compared to normal individuals (n = 12); however, the level of PGD-S in the CSF obtained from patients with brain tumor (n = 11), was reduced by as much as 2-fold when compared to control samples (n = 12) illustrating PGD-S is a potentially useful marker for brain tumor.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818102
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Using differential hybridization technique, cDNA that was a partial sequence of rat ribosomal protein L41 was isolated from fos- and src-transfected rat fibroblast 3Y1. The cDNA was named #31. Subsequently, the human cou...Using differential hybridization technique, cDNA that was a partial sequence of rat ribosomal protein L41 was isolated from fos- and src-transfected rat fibroblast 3Y1. The cDNA was named #31. Subsequently, the human counterpart(named hm3168) was cloned from a human melanoma cell line(M14) and the amino acid sequence deduced from the cDNA proved to be identical to that of HG-12. The gene expression of hm3168 was inducible by serum-stimulation. It reveals the possible up-regulation of the transcription via fos-mediated signal transduction. Northern-blot and Western-blot analyses showed a large expression of the #31 gene in the rat transformed cell lines.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818101
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Decrease in intracellular thiols leads to oxidative stress and thus may cause alterations in the activity of redox-sensitive enzymes required for signal transduction. Here, we report that, N-ethylmaleimide and phenylarsi...Decrease in intracellular thiols leads to oxidative stress and thus may cause alterations in the activity of redox-sensitive enzymes required for signal transduction. Here, we report that, N-ethylmaleimide and phenylarsine oxide, which are known to oxidize free thiols as well as protein thiols, induced phosphatidyl ethanol generation in the micromolar range suggesting activation of phospholipase D in vascular smooth muscle cells. These agents also induced significant phosphatidic acid and diacylglycerol generation without causing protein kinase C activation. Phenylarsine oxide and N-ethyl maleimide induced phospholipase D activation is protein kinase C independent as it was not inhibited by compound-3 and bisindolylmaleimide, potent protein kinase C inhibitors. Tyrosine kinase inhibitor herbimycin A by itself activated PLD, but inhibited the phospholipase D activation by phenylarsine oxide and N-ethylmaleimide. These results suggest that oxidation of the cellular thiols activates phospholipase D independent of protein kinase C.
Han B, Meng L, Song X
… +4 more, Chen Q, Wang H, Ling S, Ma X
Biochem Mol Biol Int
· 1998 Oct · PMID 9818100
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A chimera HIV-1/HIV-2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV-1 envelope protein (HIV-1 IIIB: env482-518 + env548-675) and the HIV-2 gp36 immunodominant epitope (env592-603), was c...A chimera HIV-1/HIV-2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV-1 envelope protein (HIV-1 IIIB: env482-518 + env548-675) and the HIV-2 gp36 immunodominant epitope (env592-603), was constructed and directly over-expressed in E. coli by using a prokaryotic translation initiation sequence contained within the gene of HIV-1 envelope. The recombinant product was purified and applied in antibody-screening assay. The purified chimera antigen reacted with all the thirty-eight HIV-1 positive serum samples, the two HIV-2 serum samples, and had no cross-reaction with all the eighty-eight normal healthy serum sample. The results indicated that this recombinant chimera HIV-1/HIV-2 envelope protein could be useful for diagnostic purposes of HIV infection.
Kubareva EA, Vasilenko NL, Vorobjeva OV
… +4 more, Volkov EM, Oretskaya TS, Korshunova GA, Nevinsky GA
Biochem Mol Biol Int
· 1998 Oct · PMID 9818099
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Interaction of different oligodeoxyribonucleotides (oligos), their analogs and oligonucleopeptide with uracil-DNA glycosylase (UDG) from human placenta was investigated. It is shown that there is no considerable contribu...Interaction of different oligodeoxyribonucleotides (oligos), their analogs and oligonucleopeptide with uracil-DNA glycosylase (UDG) from human placenta was investigated. It is shown that there is no considerable contribution of heterocyclic bases of DNA to UDG-substrate binding but the UDG interaction with some DNA phosphate groups is necessary for enzyme-substrate recognition. However the phosphate group adjacent to single dU from the 3'-end in oligo is not involved into the electrostatic contact with UDG. It is found that UDG has the high affinity to its reaction product. An oligonucleotide containing a single 2'-deoxy-2'-aminouridine is a non-hydrolyzable substrate analog for UDG.
Kilic F, Handelman GJ, Traber K
… +3 more, Tsang K, Packer L, Trevithick JR
Biochem Mol Biol Int
· 1998 Oct · PMID 9818098
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In previous studies stereospecific protection against lens opacity was consistent with specific reduction of R-alpha-lipoic acid(R-alpha-LA) in mitochondria of the vulnerable cells at the lens equator where the first glo...In previous studies stereospecific protection against lens opacity was consistent with specific reduction of R-alpha-lipoic acid(R-alpha-LA) in mitochondria of the vulnerable cells at the lens equator where the first globular degeneration is seen in glucose cataract. In this study two further possible explanations of this effect were investigated: (1) increased glucose uptake by the lens, leading to increased glycolysis and release of lactate into the incubation medium and/or (2) maintenance of glutathione levels by the R-alpha-LA. The data did not support 1, but was consistent with 2, after 24 hr incubation. The concentrations of glutathione in normal lenses or lenses incubated with R- or racemic alpha-LA were not significantly different, but the concentration of glutathione in lenses incubated with S-alpha-LA was significantly lower than the R-alpha-LA-incubated lenses.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818097
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The aim of this study was to examine lipid peroxidation and activities of key antioxidant enzymes in kidneys of rats with streptozotocin (STZ)-induced diabetes and the effect of aminoguanidine on diabetes-induced alterat...The aim of this study was to examine lipid peroxidation and activities of key antioxidant enzymes in kidneys of rats with streptozotocin (STZ)-induced diabetes and the effect of aminoguanidine on diabetes-induced alterations. Three groups, 6 rats each, were studied: control animals, not treated diabetic rats and rats treated with aminoguanidine (AG; 1 g/liter of drinking water). After 6 and 12 weeks the animals were sacrificed and lipid peroxidation products and activities of antioxidant enzymes were determined in their kidney homogenates. Malondialdehyde (MDA) content was significantly elevated and activities of SOD and catalase decreased in the kidneys of STZ-diabetic rats. AG treatment attenuated the increase in MDA content and diminutions of activities of SOD and catalase in the kidneys of diabetic rats. These results confirm oxidative stress in the kidney of rats with STZ diabetes and point to an antioxidant effect of AG in experimental diabetes.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818096
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Changes in the activities of alkaline phosphatase (ALP), lactate dehydrogenase(LDH) and glutamate dehydrogenase (GDH) in the heart following the consumption of diets deficient in zinc and essential fatty acids (EFA) by r...Changes in the activities of alkaline phosphatase (ALP), lactate dehydrogenase(LDH) and glutamate dehydrogenase (GDH) in the heart following the consumption of diets deficient in zinc and essential fatty acids (EFA) by rats were investigated. The study was a 2 x 2 factorial design in which rats were fed diets deficient in zinc (5 micrograms/g) or EFA (4% hydrogenated coconut oil) or both. The control diet was adequate in both zinc (100 micrograms/g) and EFA (4% soybean oil). The experimental diets were fed for forty-two days after which time the activities of the enzymes were terminally determined in the heart of the rats. The result showed that the activity of ALP was markedly elevated in rats consuming either zinc or EFA deficient diet and slightly in the group deficient in both EFA and zinc. LDH and GDH activities were significantly depressed by EFA only. An interactive effect of zinc and EFA was indicated only for ALP activity. The data indicate a resistance of cardiac tissue to moderate zinc deficiency. However, the effects of EFA were apparent.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818095
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Both pre-activated and phorbol ester tetradecanoyl phorbol myristate acetate (TPA) activated reactive oxygen species (ROS) generation were inhibited by dexamethasone in vivo. Time kinetics on influence of dexamethasone o...Both pre-activated and phorbol ester tetradecanoyl phorbol myristate acetate (TPA) activated reactive oxygen species (ROS) generation were inhibited by dexamethasone in vivo. Time kinetics on influence of dexamethasone on cytosolic phosphoprotein phosphatase activity revealed that, when compared to phosphatase activity in cytosol of control Ehrlich ascites tumor (EAT) cells, a 5-fold increase in specific activity is seen in the cytosol of EAT cells treated (in vivo, 0-90 min. 1 mg/kg body weight) with dexamethasone. Dexamethasone induced phosphatase was partially purified by conventional ion-exchange and gel filtration column chromatographic techniques. Purified phosphatase had a molecular weight of 70 KDa by SDS-PAGE. A dose-dependent inhibition of TPA activated ROS generation by partially purified phosphatase in permeabilized EAT cells suggested that dephosphorylation is a major regulatory mechanism in "switching off" of the respiratory burst. Anti-phosphatase antibodies were raised, purified and were used to quantitate cytosolic phosphatase by ELISA, which revealed that dexamethasone induces 6-fold increase in expression of phosphatase in EAT cells by 120 min. The expression of phosphatase in EAT cell cytosol was further confirmed by immunostaining using anti-phosphatase antibodies, the results of which showed intense blue staining on development with BCIP/NBT.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818094
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To identify candidate cis-acting regulatory regions involved in regulation of the Msx2 gene in anterior limb mesenchyme and calvarial osteoblasts, DNase I hypersensitive sites (DHSs) from -6.1 kb to +8.4 kb relative to t...To identify candidate cis-acting regulatory regions involved in regulation of the Msx2 gene in anterior limb mesenchyme and calvarial osteoblasts, DNase I hypersensitive sites (DHSs) from -6.1 kb to +8.4 kb relative to the translation start site of the chicken Msx2 gene were identified in anterior and posterior limb mesenchyme, calvarial osteoblasts, and embryonic fibroblasts. A total of 12 DHSs were detected. Except for the DHS in the basal promoter region, none of the other DHSs were present in all four tissues, suggesting that the chromatin structure in the Msx2 gene locus is differently organized in these four cell types. One DHS was unique to Msx2 expressing cells and a second site to nonexpressing cells. Anterior and posterior limb mesenchyme had similar patterns of DHSs that were much more complex than observed in calvarial osteoblasts possibly reflecting differences in the complexity of Msx2 regulation in these two tissues.
Khan ZK, Chowdhary L, Gyanchandani A
… +3 more, Goswami M, Farooqui N, Ranade SA
Biochem Mol Biol Int
· 1998 Oct · PMID 9818093
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Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A. fumigatus dominates...Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A. fumigatus dominates the ubiquitous incidence of invasive or allergic broncho-pulmonary aspergillosis (ABPA). The definitive diagnosis of invasive aspergillosis is difficult. We have analyzed 24 strains of A. fumigatus recovered from ICP using the RAPD technique. The profiles generated with the 20 primers tested were mostly unique. These results may have a profound impact on the management of aspergillosis, especially in the ICP.
Biochem Mol Biol Int
· 1998 Oct · PMID 9818092
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The effect of diets enriched with fat containing different fatty acids on the activity and expression of the glucose-6-phosphate dehydrogenase (EC 1.1.1.49) of mesenteric lymph nodes lymphocytes and intraperitoneal macro...The effect of diets enriched with fat containing different fatty acids on the activity and expression of the glucose-6-phosphate dehydrogenase (EC 1.1.1.49) of mesenteric lymph nodes lymphocytes and intraperitoneal macrophages was examined. Measurements of the enzyme were also performed using spleen, thymus and liver for comparison. The following fat rich diets containing a variety of fatty acids were used: 1-standard chow (CC); 2-medium chain saturated fatty acids (MS)-coconut fat-oil; 3-long chain saturated fatty acids (LS)-cocoa butter; 4-monounsaturated fatty acids (MU)-canola oil (n-9); 5-polyunsaturated fatty acids (PU)-soybean oil (n-6). Of the fat-rich diets tested, MS had the least effect. The G6PDH activity of lymphocytes was reduced by all the fat-rich diets; 16% for MS, 38% for LS, and 54% for MU. Similarly, the enzyme activity was reduced in macrophages; 35%, 86%, and 73%, for LS, MU, and PU, respectively. In contrast, the fat-rich diets elevated G6PDH activity in the lymphoid organs; by 42% in the spleen due to LS and by 131%, 35%, and 56% in the thymus due to LS, MU, and PU, respectively. Fat-rich diets decreased the activity of G6PDH in liver; 42%, 68%, and 39% for MS, MU, and PU, respectively. Some of the changes in G6PDH activity induced by the fat-rich diets occur through the mechanisms of mRNA abundance.
Bartosz G, Janaszewska A, Ertel D
… +1 more, Bartosz M
Biochem Mol Biol Int
· 1998 Oct · PMID 9818091
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A simple spectrophotometric method of determination of peroxyl radical-trapping capacity (PRTC) of body fluids and food products is proposed. In this method, decomposition of 2,2'-azobis(2-amidopropane) hydrochloride (AB...A simple spectrophotometric method of determination of peroxyl radical-trapping capacity (PRTC) of body fluids and food products is proposed. In this method, decomposition of 2,2'-azobis(2-amidopropane) hydrochloride (ABAP) is the source of peroxyl and alkoxyl radicals which oxidize 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to a green cation radical. Antioxidant present in a sample inhibit the reaction; the induction time of the reaction is proposed as a parameter enabling determination of antioxidant content. Standard assay conditions are: 20 mM ABAP and 150 microM ABTS in 0.1 M phosphate buffer, pH 7.0, at 37 degrees C; absorbance is monitored at 414 nm. A 10-min assay allows for determination of the induction time of appropriately diluted sample. As examples of application of this method, PRTC values of several types of beverages are reported.
Kumar TK, Samuel D, Jayaraman G
… +2 more, Srimathi T, Yu C
Biochem Mol Biol Int
· 1998 Oct · PMID 9818090
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Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by prolin...Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.