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Biochemistry And Molecular Biology International[JOURNAL]

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A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9 cells.

Kwon HJ, Kim DS

Biochem Mol Biol Int · 1998 Dec · PMID 9891860 · Publisher ↗

We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease a... We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.

Influence of a melatonin implant on the free radical load in avian thyroid and its relation with thyroid hormonogenesis.

Prakash P, Laloraya M, Kumar P

Biochem Mol Biol Int · 1998 Dec · PMID 9891859 · Publisher ↗

The primary aim of this study was to evaluate the effect of melatonin on the oxiradical load in avian thyroid. The superoxide free radicals have been spin trapped by EPR spectroscopy in the thyroid gland of Indian rock p... The primary aim of this study was to evaluate the effect of melatonin on the oxiradical load in avian thyroid. The superoxide free radicals have been spin trapped by EPR spectroscopy in the thyroid gland of Indian rock pigeon Columbia livia following melatonin implantation for two weeks. Melatonin implantation resulted in augmentation in the levels of superoxide radical in the thyroid gland of pigeons with a concomitant decrease in the levels of the total superoxide dismutase activity. This was also associated with increased lipid peroxidation. Melatonin implantation caused a significant increase in plasma levels of glucose. Plasma levels of thyroxine (T4) and triiodothyronine (T3) were lower in the melatonin-treated pigeons. However, the T3/T4 ratio was higher following melatonin implantation. Since iodination of tyrosine is an H2O2-dependent phenomenon, the inhibition in the activity of SOD could lead to impaired thyroid hormone synthesis.

Inhibitory effect of the leaf extract of Ginkgo biloba L. on oxidative stress-induced platelet aggregation.

Akiba S, Kawauchi T, Oka T … +2 more , Hashizume T, Sato T

Biochem Mol Biol Int · 1998 Dec · PMID 9891858 · Publisher ↗

The effect of the leaf extract of Ginkgo biloba L. on platelet aggregation induced by oxidative stress was studied. The extract caused a dose-dependent inhibition of platelet aggregation stimulated with tert-butyl hydrop... The effect of the leaf extract of Ginkgo biloba L. on platelet aggregation induced by oxidative stress was studied. The extract caused a dose-dependent inhibition of platelet aggregation stimulated with tert-butyl hydroperoxide (t-BHP) and Fe2+. Similar inhibitory activity was observed when platelets were exposed to H2O2 and Fe2+. Synergistic aggregation induced by a combination of t-BHP and Fe2+ or H2O2 and Fe2+ in association with suboptimal concentration of collagen or U46619, was prevented by the extract. However, the extract failed to inhibit aggregation in response to collagen, thrombin or U46619. Ginkgolides A, B and C inhibited platelet-activating factor-induced aggregation, but not oxidant-induced aggregation. These data suggest that the suppressive effect of the extract is specific on platelet aggregation stimulated by oxidative stress, and that this effect is involved in the mechanism related to its protective effect upon cerebral or myocardial injuries.

The age-associated decrease in the amount of amplifiable full-length mitochondrial DNA in human skeletal muscle.

Kovalenko SA, Kopsidas G, Islam MM … +5 more , Heffernan D, Fitzpatrick J, Caragounis A, Gingold E, Linnane AW

Biochem Mol Biol Int · 1998 Dec · PMID 9891857 · Publisher ↗

There has been a continuous evolution in our concept [1] that mtDNA undergoes a range of mutations with age and that such alterations lead to a decline in mitochondrial bioenergy capacity. Here we report that a wide rang... There has been a continuous evolution in our concept [1] that mtDNA undergoes a range of mutations with age and that such alterations lead to a decline in mitochondrial bioenergy capacity. Here we report that a wide range of deletion mutations accumulate with age and the amount of full-length mtDNA (FLmtDNA) amplifiable by extra-long PCR (XL-PCR) markedly decreases with age. An analysis of single human quadriceps muscle fibres reveals a close correlation between the decrease in FLmtDNA and the decline in cytochrome c oxidase activity, an exemplifier of mitochondrial bioenergy. However, Southern blotting analysis of unamplified genomic DNA shows that there is little decrease in FLmtDNA in aged quadriceps. The results are interpreted to indicate that while there is little change in the total mtDNA with age, nonetheless a significant proportion of this mtDNA is extensively damaged such that it cannot be amplified by XL-PCR. The amplifiable FLmtDNA, which putatively represents the functional component of the mtDNA, decreases markedly with age.

Genomic structure of the human ribosomal protein L41 gene.

Go H, Miyado K, Taniguchi S

Biochem Mol Biol Int · 1998 Dec · PMID 9891856 · Publisher ↗

Using PCR technique, genomic DNA encoding the gene of the human ribosomal protein L41 was amplified. Subsequent sequencing showed that the gene was constituted with 4 exons and 3 introns. Because the amplified gene conta... Using PCR technique, genomic DNA encoding the gene of the human ribosomal protein L41 was amplified. Subsequent sequencing showed that the gene was constituted with 4 exons and 3 introns. Because the amplified gene contained introns and whole the open reading frame, we concluded the fragment corresponded to the functional gene.

Role of reducing co-factor in cerulenin-insensitivity of 6-hydroxymellein synthase in carrot cell extract.

Kurosaki F, Togashi K, Arisawa M

Biochem Mol Biol Int · 1998 Dec · PMID 9891855 · Publisher ↗

The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to th... The activity of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, was not inhibited by cerulenin in the presence of NADPH. However, cerulenin showed a marked inhibitory activity to the synthase if the reducing co-factor was omitted from the assay mixture. The synthase was also sensitive to the antibiotic even in the presence of NADPH when the acyl condensation site and the reducing domain at the reaction center of the enzyme were dissociated under the high ionic strength condition. In addition, the synthase activity was appreciably inhibited when NADH was employed instead of NADPH. These observations strongly suggest that a phosphate group attached to 2'-position of adenosyl moiety of NADPH molecule plays an important role in the apparent insensitivity of 6-hydroxymellein synthase toward cerulenin.

Characterization and cloning of long neurotoxin homolog from Naja naja atra.

Lin SR, Huang HB, Wu BN … +1 more , Chang LS

Biochem Mol Biol Int · 1998 Dec · PMID 9891854 · Publisher ↗

The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequ... The cDNA encoding a long neurotoxin homolog was constructed from the cellular RNA isolated fom the venom glands of Naja naja atra (Taiwan cobra) by reverse transcription-polymerase chain reaction. BLAST searches for sequence similarity in the GenBank databases reveal that the cDNA sequence of the long neurotoxin homolog is not highly homologous with long and short neurotoxins. Although the long neurotoxin homolog exhibited an activity to inhibit acetylcholine-induced muscle contractions as Naja naja atra cobrotoxin, the degree of inhibition caused by the addition of long neurotoxin homolog was only approximately 35% of that observed with the addition of cobrotoxin. Moreover, the primary structure of the long neurotoxin homolog did not fulfill the characteristic features of long or short neurotoxins. Together with long neurotoxin homologs from other snake species, they probably represent an evolutionary divergence between long and short neurotoxins.

Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon.

Chen WS, Chiu CC, Liu HY … +5 more , Lee TL, Cheng JT, Lin CC, Wu YJ, Chang HY

Biochem Mol Biol Int · 1998 Dec · PMID 9891853 · Publisher ↗

In order to obtain transgenic fusarium wilt resistant watermelon plants, squash DNA was introduced into the ovaries of watermelon plants via the pollen-tube pathway. The introduction of foreign genes into ovaries was acc... In order to obtain transgenic fusarium wilt resistant watermelon plants, squash DNA was introduced into the ovaries of watermelon plants via the pollen-tube pathway. The introduction of foreign genes into ovaries was accomplished using co-transformation with the CaMV35S-GUS as a marker. Transformed watermelon plants contained integrated copies of the GUS activity and the seeds of transformed progeny produced a blue color when stained with 5-bromo-4-chloro-3-indolyl glucuronide, whereas seeds from untransformed control plants did not. Of 200 transformed seedlings, ten were wilt resistant. The presence of the GUS activity in the genome of stable transgenic seedlings was confirmed by Southern blot analysis. Furthermore, the generation of random amplified polymorphic DNA (RAPD) fingerprints using primers with embedded restriction sites showed amplification products unique to these transgenic plants. Primers OPA-1 and OPA-9 gave distinct band patterns of genomic DNA using the polymerase chain reaction.

The free radical-generating function of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant.

Kim SM, Eum WS, Kwon OB … +1 more , Kang JH

Biochem Mol Biol Int · 1998 Dec · PMID 9891852 · Publisher ↗

The free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type... The free radical-generating functions of the D90A Cu,Zn-superoxide dismutase (SOD) associated with Swedish familial amyotrophic lateral sclerosis (FALS) patients are investigated. The results show that both the wild-type and mutant enzymes have identical dismutase activity, while the free radical-generating activity of the D90A mutant is enhanced relative to that of the wild-type enzyme. The studies suggest that the active channel of the D90A mutant is larger than that of the wild-type enzyme. A higher free radical-generating activity of the mutant enzyme led to the release of copper ions from the damaged protein. The generation of strand breaks in plasmid DNA was enhanced more effectively by the D90A mutant Cu,Zn-SOD than by the wild-type enzyme. The results suggest that the pathology of FALS may be attributed to oxidative damage caused by the gain-of-function of FALS Cu,Zn-SOD mutant.

Influence of age on enzyme activities of pyrimidine metabolism in the chicken heart.

Wegelin I, Pane G, Orlandini G … +1 more , Clò C

Biochem Mol Biol Int · 1998 Dec · PMID 9891851 · Publisher ↗

The effect of the aging on the activities of enzymes involved in UMP-CMP metabolism were evaluated in the heart of newborn (1-day-old), young (20-day-old), adult (12-month-old), and aged (30-month-old) chickens. In newbo... The effect of the aging on the activities of enzymes involved in UMP-CMP metabolism were evaluated in the heart of newborn (1-day-old), young (20-day-old), adult (12-month-old), and aged (30-month-old) chickens. In newborn animals, UMP metabolism proceeds preferentially towards cytidine compounds rather than to breakdown. In addition, two pathways different from those involved in de novo synthesis may contribute to the synthesis of UMP: one, through cytosine deaminase that shows its maximal activity; the other, by uridine kinase, the main "salvage" enzyme of pyrimidine nucleotides. In young chickens, pyrimidine metabolism regards especially UMP. In fact, the lower activities of cytidylate phosphatase and cytosine deaminase, together with the remarkable increase of uridine kinase indicate that the metabolic flux converges on the main salvage pathway. In adult chickens, pyrimidine catabolism is enhanced, as supported by the maximal activity of the enzymes involved in UMP-CMP breakdown. On the contrary, the remarkable reduction of the anabolic enzymes suggests a limited resort to the salvage pathways. Finally, in aged chickens a reduced pyrimidine catabolism and a greater utilization of the salvage pathways appear to take place, thus contributing to the maintenance of pyrimidine nucleotide pool.

Vitamin E protects the brain against oxidative injury stimulated by excessive aluminum intake.

Abd el-Fattah AA, al-Yousef HM, al-Bekairi AM … +1 more , al-Sawaf HA

Biochem Mol Biol Int · 1998 Dec · PMID 9891850 · Publisher ↗

The effect of feeding groups of mice with a diet containing 2000, 4000 and 6000 micrograms aluminum (Al3-/g) for two weeks (subacute) or 2000 and 4000 micrograms Al3+/g for eight weeks (subchronic) as well as the coadmin... The effect of feeding groups of mice with a diet containing 2000, 4000 and 6000 micrograms aluminum (Al3-/g) for two weeks (subacute) or 2000 and 4000 micrograms Al3+/g for eight weeks (subchronic) as well as the coadministration of vitamin E (alpha-tocopherol) 500 micrograms/g with Al3+, on the status of glutathione (GSH) and lipid peroxides as thiobarbituric acid reactive substances (TBARS) in whole brain tissues were evaluated. Changes in TBARS were further evaluated in vitro following the incubation of brain homogenates of the Al(3+)-fed mice in the presence of 50 microM FeSO4. The results of subacute experiments revealed that the brain levels of GSH were significantly decreased only in the group of mice that received 6000 micrograms Al3+/g diet (P < 0.05) and this effect was partially ameliorated when vitamin E was coadministered with Al3+. TBARS were significantly increased in vitro only in the presence of free iron ions and depended on the concentration of Al3+ in the diet. The effect was opposed by the vitamin E intake. Following subchronic Al3+ intake, the GSH content of the brain was significantly decreased only in the group of mice that received 4000 micrograms Al3+/g diet (P < 0.01), while TBARS were significantly increased in the brain tissues in vivo as well as in the presence of free iron ions in vitro. However, coadministration of vitamin E with Al3+ for eight weeks preserved GSH levels and decreased TBARS in the brain of mice in vivo and in the presence of free iron ions in vitro. It is concluded that the long term administration of vitamin E may prevent Al3(+)-stimulated oxidative injury in the brain.

Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/pseudogenes.

Chen A, Mannen H, Li SS

Biochem Mol Biol Int · 1998 Dec · PMID 9891849 · Publisher ↗

Mouse SMT3A and SMT3B cDNAs encoding ubiquitin-like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced. The sequence of the first 92 amino acids (ending with the conserved Gly-Gly) of mouse SMT... Mouse SMT3A and SMT3B cDNAs encoding ubiquitin-like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced. The sequence of the first 92 amino acids (ending with the conserved Gly-Gly) of mouse SMT3A exhibited two differences at amino acid no. 38 and 76 in comparison with that of human SMT3A. The C-terminal 18 amino acid sequence of mouse SMT3A was completely different from the C-terminal 11 amino acid sequence of human SMT3A. Mouse and human SMT3B were identical for a sequence of 95 amino acids. Mouse SMT3A genomic DNAs were amplified by polymerase-chain-reaction and sequenced. The nucleotide sequence of a PCR-amplified SMT3A genomic DNA fragment was found to be identical to that of SMT3A cDNA, indicating the absence of intron(s) in its protein coding region. Another genomic DNA fragment of 1,531 nucleotides, containing 7% differences from that of cDNA, is unable to encode a functional protein, and thus, it is a SMT3A processed pseudogene. Three mouse SMT3B processed pseudogenes were cloned and sequenced. The genuine mouse SMT3B gene has not yet been isolated. Mouse SMT3A transcript of 1.8 kb was predominantly expressed in most tissues, while SMT3B transcript of 1.0 kb was abundantly present in all tissues analyzed. A family of ubiquitin-like proteins was recently discovered. One distinguishing feature of ubiquitin and ubiquitin-like proteins is the capacity to conjugate with other proteins post-translationally. The ubiquitin-like proteins are cleaved endoproteolytically after a diglycine sequence, corresponding to the C-terminal Gly75-Gly76 of ubiquitin. The cleavage activates the molecule for conjugation. The yeast SMT3 gene was originally identified as a suppressor of mutations in MIF2 gene, which encodes an essential protein binding to the A+T-rich CDEII region of centromere DNA (1). Studies using temperature-sensitive mutants showed that the loss of yeast Mif2 protein function results in chromosome missegregation, mitotic delay, and aberrant microtubule morphologies (2). The yeast Mif2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C, an integral component of active kinetochores (3, 4). Human SMT3A cDNA was identified from the genome sequencing project of chromosome 21 (5). We have cloned human SMT3B (formerly designated as HSMT3) cDNA (6). Human SMT3C protein was independently isolated by several groups and denoted as SUMO-1 (7), GMP1 (8), PICI (9), UBL1 (10), sentrin (11). SUMO-1/GMP1 was found to be covalently linked to the Ran GTPase-activating protein RanGAP1, and attachment of SUMO-1 targets the otherwise cytosolic RanGAP1 to the nuclear pore complex. The modified form of RanGAP1 also appeared to associate with the mitotic spindle apparatus during mitosis (7, 8). PIC1 was shown to interact with the PML component of nuclear multiprotein complex that is disrupted in acute promyelocytic leukemia (9). UBL1 was found to associate with human RAD51/RAD52 proteins involved in DNA recombination and DNA double-strand break repair (10). Sentrin was shown to interact with Fas/APO-1 or the TNF receptor 1 death domain, and the overexpression of sentrin provided protection against both anti-Fas/APO-1 and TNF-induced cell death (11). Here we report the characterization of mouse SMT3A and SMT3B cDNAs, gene/pseudogenes, and mRNA expression.

Complementation of the growth defect of an rnpA49 mutant of Escherichia coli by overexpression of arginine tRNA(CCG).

Kim MS, Park BH, Kim S … +3 more , Lee YJ, Chung JH, Lee Y

Biochem Mol Biol Int · 1998 Dec · PMID 9891848 · Publisher ↗

We previously found that overexpression of arginine tRNA(CCG) from Brevibacterium albidum complements the rnpA49 mutation, which is responsible for the thermosensitivity of Escherichia coli RNase P function. In this pres... We previously found that overexpression of arginine tRNA(CCG) from Brevibacterium albidum complements the rnpA49 mutation, which is responsible for the thermosensitivity of Escherichia coli RNase P function. In this present work, we show that the E. coli homologue tRNA also complements the same mutation, but other tRNAs do not. These results suggest that the rnpA49 mutation causes a major cellular defect in an RNase P reaction to generate the mature arginine tRNA(CCG).

Hexokinase, glucose-6-phosphate dehydrogenase and antioxidant enzymes in diabetic reticulocytes: effects of insulin and vanadate.

Gupta BL, Baquer NZ

Biochem Mol Biol Int · 1998 Dec · PMID 9891847 · Publisher ↗

Experimentally induced diabetic rats were treated separately with insulin and vanadate. The activities of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6PDH) were increased in reticulocyte hemolysate isolated... Experimentally induced diabetic rats were treated separately with insulin and vanadate. The activities of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6PDH) were increased in reticulocyte hemolysate isolated from the diabetic rats and were restored to normal levels by insulin. The restoration was not detected in vanadate treated diabetic animals. The enzymes of glutathione metabolism namely glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) exhibited increases in their activities with diabetes and were restored to almost control values by insulin treatment. Vanadate given to diabetic animals further increased GPx, and GST. The level of superoxide dismutase(SOD) decreased in the reticulocytes of diabetic rats and catalase (CAT) was unchanged. Both CAT and SOD had normal values when the diabetic rats were treated with insulin and vanadate. It is proposed that vanadate may cause an increase in the activity of GR which may stimulate glucose transporters and glucose metabolism.

Analysis of common mutations and associated haplotypes in Chinese patients with glucose-6-phosphate dehydrogenase deficiency.

Li P, Thompson JN, Wang X … +1 more , Song L

Biochem Mol Biol Int · 1998 Dec · PMID 9891846 · Publisher ↗

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a rare disease in North China. In the present investigation, DNA samples from 17 patients with G6PD deficiency from Tianjin area in North China were studied for the... Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a rare disease in North China. In the present investigation, DNA samples from 17 patients with G6PD deficiency from Tianjin area in North China were studied for the two G6PD common mutations (R459L and R463H) and two single nucleotide polymorphisms (1311C/T and 1365-13T/C) using a dideoxy fingerprinting method. Five patients were positive for mutation R459L, and six patients were positive for mutation R463H. Further haplotype analyses using three flanking dinucleotide repeat polymorphism loci, DXS1123, DXS1113, and F8C(IVS13), were performed on 14 patient families and 16 control Chinese females. The results indicated that the two common mutations were from different haplotypes. Also, the data suggested a possible allelic association between the two G6PD common mutations and the F8C(IVS13) locus and a different allelic distribution for loci DXS1113 and F8C(IVS13) between Chinese and Caucasian populations.

An inverted cAMP response element mediates the cAMP induction of the ovine beta 1-adrenergic receptor gene.

Tseng YT, Stabila J, McGonnigal B … +2 more , Nguyen TT, Padbury JF

Biochem Mol Biol Int · 1998 Dec · PMID 9891845 · Publisher ↗

We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies... We identified an inverted, functional cAMP response element (CRE) located at--1599 bp relative to the translation start site within the ovine beta 1-adrenergic receptor (beta 1 AR) gene promoter. In transfection studies with SK-N-MC cells, a 40-bp oligonucleotide containing the potential CRE, beta 1 AR-CRE, conferred a 3- to 4-fold increase in luciferase activity mediated by cAMP. The induction was mimicked by co-transfecting the cells with a vector overexpressing the alpha-catalytic subunit of the cAMP-dependent protein kinase (PKA) without treatment, and was blocked by overexpressing a PKA inhibitor (PKI). In electrophoretic mobility shift assays, a discrete binding pattern was shown in cell nuclear extract probed with the 40 bp beta 1 AR-CRE. The binding was shown to be specific and supershifted by addition of a CRE binding protein (CREB-1) antibody. These data demonstrate that cAMP mediates the induction of beta 1 AR gene expression by interacting with an inverted CRE within the promoter region. This is the first reported functional CRE among all beta 1 AR genes.

Age-related changes in plasma and tissue fatty acid composition in Fischer 344 rats.

Engler MM, Engler MB, Nguyen H

Biochem Mol Biol Int · 1998 Dec · PMID 9891844 · Publisher ↗

Advancing age is associated with increased risk of coronary artery disease. Changes in fatty acid metabolism affect important cellular membrane properties and functions which may contribute to the vascular pathophysiolog... Advancing age is associated with increased risk of coronary artery disease. Changes in fatty acid metabolism affect important cellular membrane properties and functions which may contribute to the vascular pathophysiology of aging. This study was designed to investigate the effects of aging on the fatty acid composition of the plasma, liver, aorta, and renal artery in 4-, 15-, and 24-month old Fischer 344 rats, an animal model for aging. With aging, the levels of total polyunsaturated fatty acids (PUFA) increased in the plasma, aorta, and renal artery. The major changes in the liver fatty acid profile were increases in the levels of 18:2n6 and 18:3n3 and a decrease in the levels of 20:3n6 and 20:5n3. The results indicate that significant shifts occur in the levels of n6 and n3 PUFA in the plasma, liver, and vasculature with aging. The alterations in the fatty acid composition may be a pathogenetic mechanism of the vascular changes associated with aging.

High-level expression of human interleukin-17 in the yeast Pichia pastoris.

Zhou L, Wang J, Peng S … +4 more , Duan J, Cai X, Zou M, Su Q

Biochem Mol Biol Int · 1998 Dec · PMID 9891843 · Publisher ↗

Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monome... Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer. The monomer of rhIL-17 stimulated mouse fibroblast 3T3 cells to secrete IL-6 and was specifically bound to its receptors on 3T3 cells.

The shortening of the N-terminus of myosin essential light chain A1 influences the interaction of heavy meromyosin with actin.

Efimova NN, Stepkowski D, Nieznańska H … +1 more , Borovikov YS

Biochem Mol Biol Int · 1998 Dec · PMID 9891842 · Publisher ↗

The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool... The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N-terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2 theta parameter of actin bound rhodamine-phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N-terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N-terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.

Use of prokaryotically expressed nucleocapsid protein as positive antigen in ELISA.

Kumar SS, Renji R, Saini M … +2 more , Goel AC, Sharma B

Biochem Mol Biol Int · 1998 Dec · PMID 9891841 · Publisher ↗

A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expre... A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expressed protein was found to be immunogenic in western blot with hyperimmune sera. It reacted with rinderpest and 'N' protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA). Prokaryotically expressed 'N' protein also gave precipitin band in counter immunoelectrophoresis test (CIE). The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.
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