Biochem Mol Biol Int
· 1998 Dec · PMID 9861442
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Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol d...Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH). The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase. Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation. The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+. The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM). Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C. These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.
Biochem Mol Biol Int
· 1998 Dec · PMID 9861441
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Endothelins; (ET1, ET2 and ET3) are a family of peptides which acts on different smooth muscle preparations inducing a slow long lasting contraction. We investigated the effects of ET 1 modulatory action on adrenergic, c...Endothelins; (ET1, ET2 and ET3) are a family of peptides which acts on different smooth muscle preparations inducing a slow long lasting contraction. We investigated the effects of ET 1 modulatory action on adrenergic, cholinergic and serotoninergic transmission on an isolated mouse's stomach with gastric nerves. The endothelin 1 stimulation of the mouse stomach tone was abolished by the specific serotonin antagonist methizergid. This study suggests that endothelin 1 plays a role in the regulation of nonvascular smooth muscle tone. The endothelin effect was dependent on free intracellular Ca++ which can be recruited from an extracellular solution as well as from intracellular stores. Complete reduction of Ca++ from the extracellular solution with a simultaneous depletion of calcium stores abolished endothelin 1 depolarization of BC3H1 cells.
Saso L, Valentini G, Giardino AM
… +4 more, Spadaro A, Riccieri V, Zoppini A, Silvestrini B
Biochem Mol Biol Int
· 1998 Dec · PMID 9861440
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Changes of glycosylation of serum proteins of patients with psoriatic arthritis were detected by lectin blotting and a new enzyme-linked lectin assay (ELLA) using concanavalin A (Con A). A good linear correlation was fou...Changes of glycosylation of serum proteins of patients with psoriatic arthritis were detected by lectin blotting and a new enzyme-linked lectin assay (ELLA) using concanavalin A (Con A). A good linear correlation was found between the total Con A-reactivity of serum and the serum levels of C-reactive protein and interleukin-6, which is known to regulate the glycosylation pattern of proteins upon inflammation. A good linear correlation was also observed between the immunoreactivity of alpha 1-antitrypsin, measured by ELISA, using a monoclonal antibody sensitive to glycosylation changes, and the erythrocyte sedimentation rate and the serum concentrations of soluble interleukin-2 receptor, an index of lymphocyte activation which correlated with some inflammatory parameters of disease activity. These protein changes, which are described here for the first time, deserve to be studied in further detail in view of their possible clinical applications.
Biochem Mol Biol Int
· 1998 Dec · PMID 9861439
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A study was undertaken to assess the ability of the erythrocyte to protect other tissues against oxidative damage. Radiolabelled (51Cr) human umbilical vein endothelial cells (HUVEC) were incubated with erythrocytes and...A study was undertaken to assess the ability of the erythrocyte to protect other tissues against oxidative damage. Radiolabelled (51Cr) human umbilical vein endothelial cells (HUVEC) were incubated with erythrocytes and neutrophils activated with phorbol myristate acetate (PMA). Damage to the endothelial cells was indicated by release of radioactivity into the suspending medium. We found that the co-incubation of HUVEC with an increasing range of erythrocyte concentrations resulted in a dose-dependent reduction in the release of radioactivity. When the ability of superoxide to cross the erythrocyte membrane or the glutathione systems was inhibited, the extent of endothelial cell damage increased. Inhibition of the catalase system did not affect results. It was concluded that the erythrocytes afforded some protection against oxidative damage to the endothelial cells by taking up and deactivating the superoxide ions. This protection depends upon intact erythrocyte antioxidant systems. These data support the hypothesis that erythrocytes can provide antioxidant protection to other tissues in vivo.
Bovine spleen NAD+glycohydrolase, an ecto-enzyme closely related to CD38, catalyzes the conversion of NAD+ into ADP-ribose and cyclic ADP-ribose, a calcium-mobilizing metabolite. We have raised polyclonal antibodies agai...Bovine spleen NAD+glycohydrolase, an ecto-enzyme closely related to CD38, catalyzes the conversion of NAD+ into ADP-ribose and cyclic ADP-ribose, a calcium-mobilizing metabolite. We have raised polyclonal antibodies against the native enzyme which on immunoblots revealed, besides the 32 kDa monomer, the presence of a stable dimeric form. This dimerization was shown to result from a spontaneous oxidative process involving the formation of one or several disulfide bond(s) sensitive to reducing agents such as 2-mercaptoethanol. The homodimeric oxidized enzyme, which was not detected during the early steps of the enzyme purification procedure, was catalytically active. Our results underline the differences, in terms of oligomerization and reactivity towards thiols, between CD38/NAD+glycohydrolases depending on their origin.
Han BG, Ma XK, Meng L
… +4 more, Song XG, Peng SY, Wang JX, Ling SG
Biochem Mol Biol Int
· 1998 Nov · PMID 9844745
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In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the...In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3'-end of TRX gene a short DNA sequences encoding a linker peptide '-GSGSGVSQNYPIVQHHHHHH-', serving not only as a specific HIV-1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV-1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2-PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hIL6 with His6-tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni-NTA Superflow and ion-exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL-6-dependent cell line 7TD1 to be 2.1 x 10(8) unit/mg.
Matarese RM, Macone A, Fontana M
… +2 more, Duprè S, Cavallini D
Biochem Mol Biol Int
· 1998 Nov · PMID 9844744
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Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD), a member of a family of natural cyclic sulfur-containing aminoacids, recently detected in biological samples, protects low density lipoprotein (LDL) against cop...Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD), a member of a family of natural cyclic sulfur-containing aminoacids, recently detected in biological samples, protects low density lipoprotein (LDL) against copper-mediated oxidation, as assessed by monitoring the kinetics of conjugated diene formation, thiobarbituric acid-reactive substances production and LDL-tryptophan fluorescence quenching. Moreover, AECK-DD exerts a protective effect also against metal-independent, peroxyl radical-induced lipoprotein oxidation. It is of note that the concentrations exerting a protective effect against LDL oxidation are similar to those found in biological samples.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844743
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The cDNAs encoding six neurotoxin-like proteins were cloned from the total RNA isolated from one venom gland of Bungarus multicinctus multicinctus by reverse transcription-polymerase chain reaction. Although they shared...The cDNAs encoding six neurotoxin-like proteins were cloned from the total RNA isolated from one venom gland of Bungarus multicinctus multicinctus by reverse transcription-polymerase chain reaction. Although they shared a high degree of nucleotide sequence homology, the deduced proteins displayed a rather low extent of identical residues with the exception that the 21 residue signal peptides were highly conserved. Each mature protein consisted of 65, 66 or 68 amino acids with four or five disulfide bonds respectively. Comparative sequence analyses showed that they were structurally related to neurotoxin homologues. Phylogenetic analyses implied that they might represent two different evolutionary branches.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844742
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From pink soluble fractions prepared from cells cultured in a copper-free medium, active methanol dehydrogenase (MDH) and two soluble c-type cytochromes (c-I and c-II) were purified homogeneously. The green fractions fro...From pink soluble fractions prepared from cells cultured in a copper-free medium, active methanol dehydrogenase (MDH) and two soluble c-type cytochromes (c-I and c-II) were purified homogeneously. The green fractions from cells grown on a medium containing 1.0 mg/l of copper had inactive MDH, cytochrome c-II, and blue copper protein. The amount of copper retained in the blue copper protein increased with cultivation time. The oxidized blue copper protein was similar to the classical type I blue copper proteins since it had the novel absorption peak at 625 nm. However, when the blue protein was reduced with MDH or dithionite, it showed the same spectrum as ferrocytochrome c-I. The isoelectric points of cytochrome c-I, blue copper protein and cytochrome c-II were 9.08, 9.08 and 6.52, respectively. These results suggest that the identity of the purified blue copper protein is cytochrome c-I, and copper ions bind to the cytochrome as methanol is depleted in the culture medium. In addition, MDH activity was not detected at all in the methanol-depleted condition. The data suggest that blue copper protein acts as a negative regulator for MDH. The electrons were transferred as follows: MDH-->cytochrome c-II-->cytochrome c-I (blue copper protein). It was also revealed that the initial 'docking' of MDH and cytochrome c-II is accompanied by electrostatic interactions between lysine or arginine residues on the alpha-subunit of MDH and carboxyl groups on the cytochrome c-II.
Salvolini E, Lucarini G, Cester N
… +2 more, Arduini D, Mazzanti L
Biochem Mol Biol Int
· 1998 Nov · PMID 9844741
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Growth retardation and low birth weight represent an important factor associated with the high risk of mortality and morbidity, particularly in twin pregnancy, since twins are frequently characterised by fetal growth ret...Growth retardation and low birth weight represent an important factor associated with the high risk of mortality and morbidity, particularly in twin pregnancy, since twins are frequently characterised by fetal growth retardation and sometimes by a discordant growth between the twins. The present work sets out to elucidate the role of growth discordance between twins in the behaviour of human umbilical vein endothelial cells; biochemical, ultrastructural and immunohistochemical data obtained from a discordant twin pregnancy are discussed. Endothelial cells were obtained from umbilical cord of 5 singleton pregnancies and from 5 dichorionic twin pregnancies, among which was one discordant twin pregnancy. Membrane fluidity was assayed using a fluorescent probe and each sample was immunohistochemically processed employing specific monoclonal antibodies. An increased fluidity was observed in endothelial cells from the smaller twin as compared with the larger one. As concerns electron microscopy, the features of endothelial cells from the smaller twin were similar to those of twins with similar growth, while endothelial cells from the larger one were more similar to those from singleton pregnancies. Our findings confirm the presence of the feto-fetal transfusion as a pathogenetic mechanism involved in twin growth discordancy.
Hirakura Y, Satoh Y, Hirashima N
… +3 more, Suzuki T, Kagan BL, Kirino Y
Biochem Mol Biol Int
· 1998 Nov · PMID 9844740
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The beta-amyloid peptide (beta AP) is a major proteinaceous component of senile plaques and cerebrovascular amyloid deposits found in the brain of patients with Alzheimer's disease. beta AP is reported to be neurotoxic o...The beta-amyloid peptide (beta AP) is a major proteinaceous component of senile plaques and cerebrovascular amyloid deposits found in the brain of patients with Alzheimer's disease. beta AP is reported to be neurotoxic only when it forms beta-sheet structure and aggregates. In the present study, we report that the neurotoxic core of beta AP, beta AP-25-35 (beta 25-35), perturbs liposome membranes, induces membrane current, and exhibits hemolytic activity only in a buffer condition where the peptide forms beta-sheet structure and spontaneously aggregates. In contrast, beta 25-35 in its monomeric random coil structure does not perturb lipid membranes significantly, and exhibits no hemolytic activity. Also, the membrane current was inhibited by Congo Red. The ability of beta 25-35 to interact with membranes highly correlates with its neurotoxicity reported previously. These results suggest that membrane perturbation by aggregated beta 25-35 constitutes the molecular basis of the peptide's neurotoxicity.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844739
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A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced. Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be ide...A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced. Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be identical to that of the cDNA, indicating the absence of intron in its protein coding region. The sequence of 90 amino acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52% identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844738
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The adult rat liver is normally in a state of growth arrest. However, cell loss such as partial hepatectomy can induce the proliferation of the hepatocytes. Early after partial hepatectomy, the concentration of p53 mRNA...The adult rat liver is normally in a state of growth arrest. However, cell loss such as partial hepatectomy can induce the proliferation of the hepatocytes. Early after partial hepatectomy, the concentration of p53 mRNA increases during the prereplicative phase. In this study, we identified the cis-regulatory element involved in the induced transcription of the rat p53 gene by DNase I footprinting assay. This element had a partial homology to the AP1 recognition motif, but the competition study with AP1 oligonucleotide showed that this element was not the AP1 recognition motif. The molecular weight of the binding protein to this motif was determined as 39 kDa by southwestern blotting analysis. In vitro transcription assay with the competitor containing the binding motif showed that the 39 kDa protein binding to the element was required for the induced transcription of the rat p53 gene during the liver regeneration.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844737
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A change in the regulation of the beta-amyloid precursor protein (beta APP) gene may be a factor for Alzheimer's disease (AD). We have screened a rhesus monkey genomic library and pulled out a approximately 17 kb genomic...A change in the regulation of the beta-amyloid precursor protein (beta APP) gene may be a factor for Alzheimer's disease (AD). We have screened a rhesus monkey genomic library and pulled out a approximately 17 kb genomic DNA region which contains the 5'-flanking region (promoter), first exon and intron of the beta APP gene of the Rhesus monkey (rh beta APP). We have partially characterized the genomic clone by selective restriction enzyme digestion followed by Southern blotting against the beta APP gene-specific DNA probes. The identity and authenticity of the different bands of the clone were also confirmed by Southern blot analysis of the rhesus monkey genomic DNA. Functional identification of the genomic fragment was determined by a promoter assay using the chloramphenicol acetyltransferase (CAT) enzyme as a reported gene. Our results showed that a 1.2 kb fragment from the 5'-flanking region of the rh beta APP gene possessed strong promoter activity. The isolation of this genomic clone will enable characterization of the structure and function of the promoter of the rh beta APP gene. Our initial results indicate a high degree of homology between the structure of the rhesus monkey and human beta APP promoter.
Renugopalakrishnan V, Piazzolla P, Tamburro AM
… +1 more, Lamba OP
Biochem Mol Biol Int
· 1998 Nov · PMID 9844736
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The secondary structure of cucumber mosaic virus (CMV) was investigated in solution using Fourier transform infrared (FT-IR) spectroscopy. The amide I region of intact CMV revealed a doublet at 1671 cm-1 and 1653 cm-1, r...The secondary structure of cucumber mosaic virus (CMV) was investigated in solution using Fourier transform infrared (FT-IR) spectroscopy. The amide I region of intact CMV revealed a doublet at 1671 cm-1 and 1653 cm-1, respectively. In order to isolate the IR bands arising from the protein backbone of CMV, the FT-IR spectra of the RNA component, isolated by phenol-SDS treatment of purified CMV and subsequent precipitation by ethanol, was obtained separately and digitally subtracted from the intact CMV spectra. After digital subtraction, the amide I region contained two bands at 1682 cm-1 and 1644 cm-1. The former band was ascribed to beta-sheet structures, while the later band occurs in the region between alpha-helix and "unordered" structures. Resolution enhancement of the finger print amide I region was accomplished using Fourier self-deconvolution of the digitally subtracted FT-IR spectrum of CMV which further confirmed the presence of anti-parallel beta-sheet structure in the protein coat of CMV. Chou-Fasman predictions on the the coat protein also revealed the presence of beta-sheet structure in agreement with FT-IR studies.
Miura A, Ishizuka T, Itaya S
… +5 more, Ishizawa M, Kanoh Y, Kimura M, Kajita K, Yasuda K
Biochem Mol Biol Int
· 1998 Nov · PMID 9844735
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This study examined the effect of glucose and 12-O-tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKC alpha and beta activation in the...This study examined the effect of glucose and 12-O-tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKC alpha and beta activation in the membrane fractions, respectively. Treatment with 5 mM and 20 mM glucose for 5 min and 20 min resulted in 6-7-fold increases in insulin secretion, and treatment with 1 microM TPA for 5 min also resulted in 3-fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKC alpha and beta immunoreactivities after treatment with 5 mM, 20 mM glucose and 1 microM TPA. Translocations of PKC alpha after treatment with glucose and TPA were greater than those of PKC beta in membrane fractions. These results suggest that TPA independently provokes insulin secretion via PKC activation and that PKC alpha and beta activation may be involved in insulin secretion in human insulinoma cells.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844734
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Immunoblot analysis showed that transferrin and human serum albumin were degraded in a similar rate on the basis of the protein weight by radical reactions using a solution containing each protein initiated by Cu2+ and h...Immunoblot analysis showed that transferrin and human serum albumin were degraded in a similar rate on the basis of the protein weight by radical reactions using a solution containing each protein initiated by Cu2+ and hydrogen peroxide. When plasma was treated with Cu2+ and hydrogen peroxide, the rate profile of the degradation of transferrin was similar to that of albumin based on immunoblot analysis in spite that the content of these proteins in plasma was considerably different. These results suggest that highly reactive hydroxyl radicals attack these proteins indiscriminately. Present observations demonstrate that immunoblot analysis is an effective method to analyze radical reactions of each protein in the presence of many other proteins like plasma.
Biochem Mol Biol Int
· 1998 Nov · PMID 9844733
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In an attempt to elucidate the mechanism of the membrane binding of protein kinase C-alpha (PKC-alpha), effects of pH and ionic strength were examined on Ca(2+)-dependent and diacylglycerol-induced association of this PK...In an attempt to elucidate the mechanism of the membrane binding of protein kinase C-alpha (PKC-alpha), effects of pH and ionic strength were examined on Ca(2+)-dependent and diacylglycerol-induced association of this PKC with rat liver plasma membrane. In the absence of diacylglycerol, the membrane-bound PKC-alpha increased according to the changes in pH from 6 to 8. However, these reactions were not seriously influenced by alterations in ionic strength (0-240 mM NaCl). In the presence of diacylglycerol, this pH-modulated binding of PKC-alpha to plasma membrane was more effective under lower Ca2+ concentration at pH 8. These results suggest that alkaline pH conditions seem to be important for preferable association of PKC-alpha with plasma membrane.
Fujikawa T, Kaneko H, Hibasami H
… +4 more, Sakaguchi K, Alam KS, Tanaka M, Nakashima K
Biochem Mol Biol Int
· 1998 Nov · PMID 9844732
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The major form of human growth hormone (22K hGH) stimulates the growth of T-47D human breast cancer cells in culture and in nude mice by binding to their receptors for growth hormone and prolactin. Another isoform of hGH...The major form of human growth hormone (22K hGH) stimulates the growth of T-47D human breast cancer cells in culture and in nude mice by binding to their receptors for growth hormone and prolactin. Another isoform of hGH having a smaller molecular mass (20K hGH) is known to show different binding affinities to these receptors. In this study, we have analyzed the effects of 20K hGH on the growth of T-47D cells in vitro and in vivo. 20K hGH (50 ng/ml) inhibited the proliferation and DNA synthesis of T-47D cells cultured in the presence and absence of 17 beta-estradiol (100 ng/ml), while 22K hGH (50 ng/ml) promoted the cellular growth. In estradiol-treated nude mice, 22K hGH (100 micrograms) remarkably promoted the growth of T-47D tumor, but 20K hGH again suppressed the tumor growth significantly. The results suggest the presence of different signal pathways for these two hGH isoforms and imply a possible clinical application for 20K hGH.
Masumizu T, Fujii K, Kohno M
… +5 more, Nagai S, Odagaki Y, Imanari M, Mori A, Packer L
Biochem Mol Biol Int
· 1998 Nov · PMID 9844731
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In vivo electron spin resonance imaging (ESR imaging) was applied to living mice after peroral administration of a nitroxide radical spin probe. A 3D ESR imaging procedure was applied in vivo in order to obtain the exact...In vivo electron spin resonance imaging (ESR imaging) was applied to living mice after peroral administration of a nitroxide radical spin probe. A 3D ESR imaging procedure was applied in vivo in order to obtain the exact distribution of the spin probe in a living animal. The imaging pictures demonstrated that the administered spin probe was firstly located in the stomach, then delivered to the liver, kidney and heart of the animal.