Biomedical subjects

A Kondo

Publications and source records attributed to A Kondo.

At least 73 records · Page 4Linked to original sources

PubMed2000

Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol.

Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3-52 trp1Delta2 his ade 2-1 can1-100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.

Journal Article↗

PubMed2000

Spectroscopic and electrochemical properties of the Met86Gln mutant of Achromobacter cycloclastes pseudoazurin.

The mutant replacing the Met86 ligand of Achromobacter cycloclastes pseudoazurin (Ac-pAz) with Gln has been prepared and spectroscopically and electrochemically characterized. Ac-pAz has four ligands (2His, Cys, and Met) and donates one electron to its cognate Cu-containing nitrite reductase (Ac-NIR). The mutant ([Met86Gln]pAz) shows the electronic absorption and CD spectra considerably similar to those of zucchini mavicyanin (Mv) and lacquer and cucumber stellacyanins (St) having 2His, Cys, and Gln. The EPR signal of the mutant has an axial character, although those of Mv and St show rhombic signals. The findings indicate that the Cu site having Gln might be a distorted trigonal geometry. The half-wave potentials (E(1/2)) of [Met86Gln]pAz and the intermolecular electron-transfer rate constant (kET) from the mutant to Ac-NIR were determined by cyclic voltammetry at pH 7.0 and 25 degrees C. The E(1/2) is +134 mV (versus NHE) and the coordination of Gln instead of Met negatively shifts the E(1/2) of Ac-pAz (+260 mV (versus NHE)). The kET of [Met86Gln]pAz (1.2x10(6) M(-1) s(-1)) is larger than that of the recombinant Ac-pAz (7.5x10(5) M(-1) s(-1)).

Alcaligenes↗

PubMed2000

Preparation of high-activity whole cell biocatalysts by permeabilization of recombinant yeasts with alcohol.

Recombinant yeast cells intracellularly overexpressing three different enzymes were permeabilized with alcohol under various conditions. The effects of enzyme stability in alcohol and enzyme molecular weight on the activities of permeabilized cells and enzyme leakage during incubation were examined. Saccharomyces cerevisiae YPH250 overexpressing glyoxalase I (GloI), S. cerevisiae MT8-1 overexpressing isocitrate lyase (ICL), and Pichia pastoris GS115 overexpressing beta-galactosidase (beta-gal) were used as model recombinant yeast systems. In all cases, the percentage of alcohol used for the treatment significantly affected the activity of permeabilized whole cell biocatalysts; cells showed high activity when treated with 40% isopropyl alcohol. The activity of whole cell biocatalysts was also significantly affected by the stability of the enzyme in alcohol solution; permeabilized yeast cells overexpressing ICL, which had low stability, showed rather low activity. Although the enzyme leakage from permeabilized cells was rather low in all cases, the molecular weight of the enzyme appeared to affect the extent of enzyme leakage during incubation. Permeabilized cells of P. pastoris overexpressing beta-gal (540 kDa) retained particularly high activity during incubation and could be used as an immobilized whole cell biocatalysts.

Journal Article↗

PubMed2000

The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope.

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Galbeta1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Galbeta1-4GlcNAcbeta1-4Manalpha1-3 branch.

Animals↗

PubMed2000

Transcriptional and post-transcriptional regulation of monocyte chemoattractant protein-3 gene expression in human endothelial cells by phorbol ester and cAMP signalling.

Monocyte chemoattractant protein-3 (MCP-3) is one of the most broadly active chemokines, potentially inducing chemotaxis of all leucocytic cells. In the present study, we examined the regulation of MCP-3 mRNA and protein production in endothelial cells by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) and cAMP signalling. On stimulation of endothelial cells with 10 nM PMA, MCP-3 mRNA increased to 300-fold the basal level at 3 hr and rapidly declined to 0.2-fold the basal level at 24 hr. PMA-induced MCP-3 mRNA and protein production of human endothelial cells were partially inhibited by pretreatment with the adenylate cyclase activator, forskolin, or membrane-permeable cAMP derivative. The PMA-induced MCP-3 mRNA increase was almost abrogated when cells were pretreated with cycloheximide (CHX). Forskolin inhibited the transcription of PMA-induced MCP-3 gene expression. Following PMA stimulation for 3 hr, subsequent addition of actinomycin D suppressed the rapid decay of PMA-induced MCP-3 mRNA. These results suggest that PMA induces the transcriptional activation of the MCP-3 gene through de novo protein synthesis and the rapid decay of PMA-induced MCP-3 mRNA through de novo synthesis of adenosine/uridine (AU)-rich element binding proteins and cAMP signalling inhibits the PMA-induced transcriptional activation of the MCP-3 gene expression.

Adenylyl Cyclases↗

PubMed2000

Accumulation of N-acetyl-L-aspartate in the brain of the tremor rat, a mutant exhibiting absence-like seizure and spongiform degeneration in the central nervous system.

The tremor rat is a mutant that exhibits absence-like seizure and spongiform degeneration in the CNS. By positional cloning, a genomic deletion was found within the critical region in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N-acetyl-L-aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency. Interestingly, direct injection of NAA into normal rat cerebroventricle induced 4- to 10-Hz polyspikes or spikewave-like complexes in cortical and hippocampal EEG, concomitantly with behavior characterized by sudden immobility and staring. These results suggested that accumulated NAA in the CNS would induce neuroexcitation and neurodegeneration directly or indirectly.

Amidohydrolases↗

PubMed2000

A new paxillin-binding protein, PAG3/Papalpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration.

Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, PAG3 (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. PAG3 bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Papalpha, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of PAG3 with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of PAG3. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.

ADP-Ribosylation Factors↗

PubMed2000

p53 interacts with the DNA mismatch repair system to modulate the cytotoxicity and mutagenicity of hydrogen peroxide.

This study focused on the question of how the DNA mismatch repair (MMR) system and p53 interact to maintain genomic integrity in the presence of the mutagenic stress induced by hydrogen peroxide (H(2)O(2)). The cytotoxic and mutagenic effects of H(2)O(2) were compared in four colon carcinoma sublines: HCT116, HCT116/E6, HCT116+ch3, and HCT116+ch3/E6, representing MMR(-)/p53(+), MMR(-)/p53(-), MMR(+)/p53(+), and MMR(+)/p53(-) phenotypes, respectively. Loss of p53 in MMR-proficient cells did not significantly alter cellular sensitivity to H(2)O(2), but disruption of p53 in MMR-deficient cells resulted in substantial resistance to H(2)O(2) (IC(50) values of 203.8 and 66.2 microM for MMR(-)/p53(-) and MMR(-)/p53(+) cells, respectively). The effect of loss of p53 and MMR function on sensitivity to the mutagenic effect of H(2)O(2) paralleled the effects on cytotoxic sensitivity. In MMR-deficient cells, loss of p53 resulted in a 3.5- and 2.2-fold increase in the generation of 6-thiogunaine and ouabain-resistant clones, respectively. Loss of MMR in combination with loss of p53 synergistically increased the frequency of frameshift mutations in the CA repeat tracts of the out-of-frame shuttle vector pZCA29 and further promoted instability of microsatellite sequences under H(2)O(2) stress. Flow cytometric analysis showed that H(2)O(2) treatment produced a G(l) and G(2)/M phase arrest in MMR(+)/p53(+) cells. Loss of MMR did not alter the ability of H(2)O(2) to activate either checkpoint; loss of p53 in either the MMR-proficient or deficient cells resulted in impairment of the G(l) arrest and a more pronounced G(2)/M arrest. H(2)O(2) caused a greater and more longed increase in p53 protein levels in MMR-proficient than in the MMR-deficient cells. The results demonstrate that the effect of disabling p53 function is modulated by the proficiency of the MMR system (and vice versa) and that there is an overlap between the functions of p53 and the MMR system with respect to the activation of apoptosis and mutagenesis after an oxidative stress.

Base Pair Mismatch↗

PubMed2000

Oral supplementation with gamma-linolenic acid extracted from Mucor circinelloides improves the deformability of red blood cells in hemodialysis patients.

BACKGROUND: The development of abnormalities in red blood cell (RBC) deformability in patients undergoing hemodialysis remains a major problem, because it is related to peripheral microcirculation, oxygen supply, and various complications in such patients. gamma-Linolenic acid (GLA; 18:3n-6), one of the polyunsaturated fatty acids and a precursor of prostaglandin E(1), is reported to have a favorable effect on the deformability of circulating blood cells in diabetic patients. METHODS: In order to clarify the efficacy of GLA on RBC deformability in 7 patients undergoing maintenance hemodialysis, we examined in a pilot study the changes in the deformability of RBC and the changes in the phospholipid fatty acid composition in both plasma and RBC membrane before and after high-dose oral supplementation with GLA derived from Mucor circinelloides for 12 weeks. RESULTS: Before supplementation, the micropore passage time of RBC suspension, which is an indicator of RBC deformability, in these patients was markedly longer than that in healthy control subjects. After administering GLA, the prolonged passage time of the patients both rapidly and steadily decreased and nearly reached control levels. Light microscopic observations of RBCs using Giemsa stain revealed a decreased number of poikilocytes after supplementation. An analysis of the fatty acid composition before treatment and 8 weeks after starting the treatment showed the dihomo-gamma-linolenic acid (DGLA; 20:3n-6) level in the plasma to have increased (p < 0.05), while the arachidonic acid (AA; 20:4n-6) concentration in the RBC membrane decreased (p < 0.05). The level of DGLA in the RBC membrane, the level of GLA, and the ratio of GLA + DGLA/AA in plasma and RBC membrane did not change significantly; however, these all tended to increase. CONCLUSION: The results of this pilot study indicate that the oral supplementation of GLA extracted from M. circinelloides improves the poor RBC deformability in hemodialysis patients, partly by inducing changes in the composition of fatty acids in plasma and RBC membrane.

Adult↗

PubMed2000

Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins.

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

Journal Article↗

PubMed2000

Very-long-chain fatty acid metabolism in adrenoleukodystrophy protein-deficient mice.

X-linked adrenoleukodystrophy (X-ALD) is characterized by progressive mental and motor deterioration, with demyelination of the central and peripheral nervous system. Its principal biochemical abnormality is the accumulation of very-long-chain fatty acids (VLCFAs) in tissues and body fluids, caused by the impairment of peroxisomal beta-oxidation. The authors have generated a line of mice deficient in ALD protein (ALDP) by gene targeting. ALDP-deficient mice appeared normal clinically, at least up to 12 mo. Western blot analysis showed absence of ALDP in the brain, spinal cord, lung, and kidney. The amounts of C26:0 increased by 240% in the spinal cord. VLCFA beta-oxidation in cultured hepatocytes was reduced to 50% of normal. The authors investigated the roles of ALDP in VLCFA beta-oxidation using the ALDP-deficient mice. Very-long-chain acyl-CoA synthetase (VLACS) is functionally deficient in ALD cells. The impairment of VLCFA beta-oxidation in the ALDP-deficient fibroblasts was not corrected by over-expression of VLACS only, but was done by co-expression of VLACS and ALDP, suggesting that VLACS requires ALDP to function. VLACS was detected in the peroxisomal and microsomal fractions of the liver from both types of mice. Peroxisomal VLACS was clearly decreased in the ALDP-deficient mouse. Thus, ALDP is involved in the peroxisomal localization of VLACS.

ATP Binding Cassette Transporter, Subfamily D, Mem↗

PubMed2000

Hypertriglyceridemia characterized by low-density lipoprotein phenotype and lipoprotein lipase gene mutation.

A high serum triglyceride (TG) concentration is associated with an increased serum concentration of small, dense low-density lipoprotein (LDL). To further characterize the hypertriglyceridemic condition, we examined sera from 240 subjects for small, dense LDL using non-denaturing polyacrylamide gradient gel electrophoresis. We focused on determining the frequency of the pattern B, which is characterized by a higher proportion of small, dense LDL, among hypertriglyceridemic individuals. The subjects were divided into four groups: a control group (TG < or = 1.65 mmol/l, high-density lipoprotein cholesterol (HDL-C) > or =1.17 mmol/l; n = 71), a high TG group (TG > 1.65 mmol/l, HDL-C > or = 1.17 mmol/l; n = 36), a group with high TG and low HDL-C (TG > 1.65 mmol/l, HDL-C < or = 0.91 mmol/l; n = 106), and a low HDL-C group (TG < or = 1.65 mmol/l, HDL-C < or = 0.91 mmol/l; n = 27). We found that pattern B occurs at a high frequency mainly in individuals with high TG and low HDL-C levels. We also observed an increased percentage of LDL within the 20.0 nm to 25.5 nm particle diameter range in this group. Analysis of the lipoprotein lipase gene in this group showed that some mutations seem to be associated with small, dense LDL, resulting in LDL pattern B.

Densitometry↗

PubMed2000

Prolapsing gyrus rectus as a cause of progressive optic neuropathy.

The pathogenesis of optic neuropathy caused by neurovascular compression or by similar mechanisms is unclear. Thin-slice magnetic resonance (MR) imaging was performed in 69 patients with optic neuropathy without demonstrable ophthalmological lesions (57.0 +/- 17.1 years of age) and 102 normal subjects (57.7 +/- 13.9 years of age). The MR imaging features were classified into "no compression" by the internal carotid artery (ICA), "compression" by the ICA, "no contact" with the anterior cerebral artery (ACA) or the gyrus rectus, "contact" with either or both, "compression" by the ACA, and "compression" by the gyrus rectus. The Spearman correlation coefficients were calculated between patients or controls, the MR classification, and the age, and the number of patients in each MR classification were evaluated by the chi 2 test. Five of the 69 patients with rapidly progressive symptoms were operated on via the frontotemporal approach. The MR imaging feature of "compression" by the gyrus rectus was the best predictor of optic neuropathy (Spearman correlation coefficients rho = -0.23646, p < 0.0018). This MR imaging feature was observed in 38 of 69 patients and in 32 of 102 controls (p = 0.002). Compression of the nerve by the gyrus rectus or the ACA was confirmed in all five operated cases. Decompression of the nerve was fully achieved in four of the five patients, and their symptoms have not progressed since then. Optic neuropathies due to compression by the prolapsing gyrus rectus are not well understood. Such neuropathies may be detected by MR imaging.

Adolescent↗

PubMed2000

Growth inhibitory effects of ATP and its derivatives on human fibroblasts immortalized with 60Co-gamma rays.

In our previous study (Katayama B et al, Int J Mol Med 2: 603-606, 1998), cell growth inhibition caused by ATP added to cultures was found to be greater in immortalized human fibroblasts than in the normal human fibroblasts. Since it has been reported that ATP affects cells via P2-purinergic receptors, growth inhibitory effects of ATP and its derivatives on immortalized human fibroblasts were investigated in the present study in order to learn what type of receptors are involved in ATP cytotoxicity. The ATP derivatives used in this study were: ATP, ADP, beta, gamma-methyleneadenosine 5'-triphosphate (MeATP), 2' & 3'-o-(4-benzoylbenzoyl) adenosine, triethylammonium salt (BzATP), adenosine 5'-o-(3-thiotriphosphate) (ATPgammaS), 2-methylthioadenosine 5'-triphosphate (2-MeSATP) and UTP. The extent of cytotoxicity induced by these drugs was found to be in the order of: ATP=ADP>ATPgammaS>MeATP=BzATP. On the other hand, neither 2-MeSATP nor UTP showed any cytotoxicity. These findings indicate that ATP may exert the cell growth inhibition by certain kinds of signal transduction via P2x or P2y purinergic receptors which affect intrinsic channels/pores of cell membrane and/or G protein activation. As a result, intracellular elevation in the concentrations of ions such as calcium and potassium, membrane depolarization, loss of endogenous ions/metabolites, and activation of inositol phospholipid-specific phospholipase C may occur. Actually, a dihydropyridine calcium channel blocker, nifedipine, and an ATP-sensitive K+-channel blocker, glybenclamide, reduced the growth inhibitory effects of ATP on the cells to some extent. The growth inhibition caused by ATP was not due to apoptosis or induction of a cyclin/CDK kinase inhibitor, P21.

Adenosine Triphosphate↗

PubMed2000

Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil.

The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated. Methanolysis progressed much faster when Novozym 435 was preincubated in methyl oleate for 0.5 h and subsequently in soybean oil for 12 h. The initial reaction rate of methanolysis catalyzed by both the non-treated and preincubated enzyme decreased significantly with increasing water content. The initial reaction rate increased with increasing methanol content, showed a maximum, and thereafter decreased when the methanol content was increased further. The variation of the initial reaction rate with the methanol content was therefore analyzed using a Michaelis-Menten-type equation with substrate inhibition. Based on this equation, a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired methanol content was determined. When preincubated Novozym 435 was used, the ME content reached over 97% within 3.5 h by stepwise addition of 0.33 molar equivalent of methanol at 0.25-0.4 h intervals.

Journal Article↗

PubMed1999

The expression of transforming growth factor-beta1 is significantly correlated with the expression of vascular endothelial growth factor and poor prognosis of patients with advanced gastric carcinoma.

BACKGROUND: Transforming growth factors beta (TGFs beta) are involved in a variety of important cellular functions, including cell growth and differentiation, adhesion, migration, extracellular matrix formation, and immune function. Moreover, it has been reported that TGFs beta are correlated with angiogenesis. However, the role of TGF-beta as an angiogenic factor in gastric carcinoma is still unclear. METHODS: TGF-beta1 expression was determined in 101 patients with gastric carcinoma by immunohistochemical procedures, and this expression was compared in the current study with both the expression of vascular endothelial growth factor (VEGF), which is thought to be the most potent angiogenic factor, and microvessel density, to evaluate the effect of TGF-beta1 on the angiogenesis of gastric carcinoma tissues. RESULTS: TGF-beta1 expression was detected in 23 tumors (22.8%). TGF-beta1 expression was more frequent in differentiated than in undifferentiated gastric carcinoma. Furthermore, TGF-beta1 expression was significantly correlated with the depth of invasion and the stage of disease. There was a close correlation between TGF-beta1 expression and VEGF expression. There was no correlation between TGF-beta1 expression and microvessel density, whereas VEGF expression was significantly correlated with microvessel density. With regard to prognosis, the 5-year survival rate was 55.9% for patients with TGF-beta1 positive tumors and 67.0% in patients with TGF-beta1 negative tumors. Accordingly, the prognosis for patients with TGF-beta1 negative tumors was significantly better than that for patients with TGF-beta1 positive tumors. Multivariate analysis indicated that lymph node metastasis, tumor size, and TGF-beta1 expression were independent prognostic factors. CONCLUSIONS: These results suggest that TGF-beta1 might be associated with tumor progression by indirectly stimulating angiogenesis through the up-regulation of VEGF expression in gastric carcinoma.

Adenocarcinoma↗

PubMed1999

Involvement of nitric oxide and biopterin in proinflammatory cytokine-induced apoptotic cell death in mouse osteoblastic cell line MC3T3-E1.

We previously demonstrated that the addition of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) caused induction of mRNAs for inducible nitric oxide (NO) synthase and GTP cyclohydrolase I, a rate-limiting enzyme for 5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis, and produced their end-products, NO and BH4, in osteoblastic cells. In the present study, we examined whether NO and BH4, biologically active substances produced in response to proinflammatory cytokines, are involved in the effect of these cytokines on cell viability and apoptotic cell death involving DNA fragmentation. Cytokines as well as S-nitroso-N-acetyl-d,l-penicillamine, an NO generator, decreased cell viability, whereas sepiapterin, which was converted intracellularly to BH4, increased it. The examination of cytotoxicity measured in terms of lactate dehydrogenase release and apoptotic cell death assessed by flow cytometric analysis showed that cytokine-induced reduction of cell viability may be based upon cell death by apoptosis, but not lytic death as in necrosis. In the presence of sepiapterin, cytokine treatment resulted in a statistically pronounced reduction in the amount of DNA fragmentation. Furthermore, this fragmentation could be blocked by 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide, an NO scavenger. These results suggest that cytokine-induced apoptotic cell death is attributed to NO and is protected by BH4, and that osteoblastic cells in response to proinflammatory cytokines operate both a stimulatory process resulting in NO production and an inhibitory one resulting in BH4 production for apoptotic cell death. Cytokine-induced apoptotic cell death may be a consequence of the predominance of the stimulatory process over the inhibitory process.

3T3 Cells↗

PubMed1999

Production of S-lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S-lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S-lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4 degrees C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70-80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S-lactoylglutathione production rates of these permeabilized cells were 1.5-2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗