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Biomedical subjects

N Gotoh

Publications and source records attributed to N Gotoh.

At least 91 records · Page 5Linked to original sources
PubMed1993

Inhibition of glutathione synthesis increases the toxicity of oxidized low-density lipoprotein to human monocytes and macrophages.

Macrophages are thought to play an important role in the pathogenesis of atherosclerosis by mediating the oxidation of low-density lipoprotein (LDL). However, it is known that these cells show elevated glutathione levels after exposure to oxidized LDL. Here we demonstrate that this increase in the level of intracellular glutathione is due to synthesis de novo stimulated by oxidized LDL. Furthermore, inhibition of glutathione synthesis renders oxidized LDL cytotoxic to both monocytes and macrophages at a concentration well tolerated by untreated cells. The stimulation of cholesterol esterification in macrophages by low, non-toxic, concentrations of oxidized LDL is enhanced under conditions where glutathione synthesis is inhibited. These results suggest that the glutathione status of macrophages in the artery wall could be important in both controlling foam-cell formation and the detoxification of oxidized LDL.

Antimetabolites↗
PubMed1993

Dynamics of the oxidation of low density lipoprotein induced by free radicals.

Although the oxidation of low density lipoprotein (LDL) has been studied extensively, its dynamics have received much less attention. The present study was carried out aiming to elucidate the rates and products of the oxidation of LDL induced by free radicals generated in three different ways by hydrophilic and lipophilic azo radical initiators and copper. The oxidations were followed by measuring the formation of lipid oxidation products, the uptake of oxygen, consumption of vitamin E, and the fragmentation and acquisition of negative charge by apolipoprotein B within a single oxidation reaction. LDL was oxidized by a free radical chain mechanism independent of the manner of initiating free radical generation to give phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH) as major primary products. When the radicals were generated within the LDL compartment, CEOOH was formed almost exclusively and quantitatively at the initial stage. The kinetic chain length for the oxidation of cholesteryl ester was larger than that for phosphatidylcholine in the presence and absence of vitamin E even when the radicals were generated initially in the aqueous phase. PCOOH and CEOOH accumulated with increasing extent of oxidation, but then decreased at the later stage of oxidation. The decrease in hydroperoxides was more significant in copper-induced oxidations. The thiobarbituric acid reactive substances also increased with time, but they accounted for less than 10% of total oxygen uptake. Oxidative modification of apo B was also observed. The modification of apo B, that is, fragmentation and increase in negative charge correlated well with the oxidation of lipids independent of the manner of chain initiation. It was also suggested that some radicals formed in the aqueous phase attacked apo B directly. These results suggest the importance of chain propagation in the oxidative modification of LDL and also the lipophilic, chain-breaking antioxidants within LDL in its inhibition.

Apolipoproteins B↗
PubMed1993

Inhibition of oxidative modification of low density lipoprotein by antioxidants.

Oxidation of LDL induced by free radicals proceeds by a chain mechanism to give phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides as the major primary products. In addition, apolipoprotein B100 is also oxidized. Various antioxidants suppress the oxidative modification of LDL. Water-soluble radical-scavenging antioxidants such as vitamin C and uric acid act as the first defense to suppress the chain initiation. Lipophilic radical-scavenging antioxidants in LDL such as vitamin E and ubiquinol scavenge radicals attacking from outside and also within the LDL. The overall importance and potency of antioxidants depend not only on chemical reactivity but also on the physical factors such as location and mobility at the microenvironment in LDL.

Antioxidants↗
PubMed1992

Antioxidant activities of probucol against lipid peroxidations.

The antioxidant activities of probucol were measured in the oxidations of methyl linoleate in homogeneous solution and soybean phosphatidylcholine liposomal membranes and also of low-density lipoproteins. When an excess amount of probucol was reacted with galvinoxyl, the EPR spectrum of galvinoxyl disappeared and a new triplet EPR signal was found: g = 2.0058 and aH(2H) = 0.14 mT. The identical EPR spectrum was observed when probucol was reacted with tert-butoxyl radical generated from di-tert-butylperoxy oxalate. This EPR signal disappeared rapidly when reacted with either alpha-tocopherol or 6-O-palmitoyl-ascorbic acid. Probucol suppressed the free-radical-mediated oxidations of methyl linoleate in hexane and in acetonitrile, in a dose-dependent manner. Its antioxidant activity was 17.5-fold less than that of alpha-tocopherol in hexane. Probucol incorporated into soybean phosphatidylcholine liposomes suppressed its oxidation. The antioxidant activity of probucol was less than that of alpha-tocopherol, but the difference between the two antioxidant activities was smaller in the membranes than in homogeneous solution. Probucol also suppressed the oxidation of low-density lipoprotein. Interestingly, probucol suppressed the oxidation of LDL as efficiently as alpha-tocopherol, implying that physical factors as well as chemical reactivity are important in determining the overall activity of antioxidant in low-density lipoprotein.

Antioxidants↗
PubMed1992

A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor.

Epidermal growth factor receptor (EGF-R) is a widely expressed ligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R corresponds to Y416 of v/c-src kinase, which is highly conserved and functionally important in many tyrosine kinases. To clarify the functional role of Y845, we constructed a mutant human EGF-R in which this tyrosine was replaced with phenylalanine and transfected it to NIH3T3 cells. EGF-R F845 induced EGF-dependent cellular transformation and revealed tyrosine-autophosphorylation of a 170 kDa protein, and initiated DNA synthesis similar to the wild-type EGF-R. We conclude here that Y845 is dispensable in the above mentioned functions of EGF-R tyrosine kinase.

3T3 Cells↗
PubMed1992

Rates of interactions of superoxide with vitamin E, vitamin C and related compounds as measured by chemiluminescence.

The rate constants for the interactions of superoxide with vitamin E (alpha-tocopherol), vitamin C (ascorbic acid) and their related compounds have been measured by a chemiluminescence method. A strong chemiluminescence of a constant intensity was observed when xanthine oxidase was added to an aqueous solution of hypoxanthine and a Cypridina luciferin analog, 2-methyl-6-phenyl-3-7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA). Vitamin E, vitamin C and their related compounds competed with CLA to react with superoxide and reduced the chemiluminescence intensity. From a kinetic analysis of the effect of addition of these compounds on the chemiluminescence intensity, the rate constants for their interactions with superoxide were measured at 25 degrees C and pH 7.8. The rate constants were obtained as 3.3 x 10(5) and 1.7 x 10(4) M-1 s-1 for ascorbate and 2-carboxy-2,5,7,8-tetramethyl-6-chromanol, respectively, and also as 4.9 x 10(3) and 4.5 x 10(3) M-1 s-1 for alpha-tocopherol incorporated into soybean and dimyristoyl phosphatidylcholine liposomal membranes, respectively. It has been shown that this method is a sensitive and a quick method which can be applied for measurement of the reactivities of various natural and synthetic compounds toward superoxide. In addition it has been shown that this method can also be applied to the heterogeneous system as well as homogeneous solution, which makes it more versatile and useful for the study in biochemistry.

Ascorbic Acid↗
PubMed1992

Permeability of the outer membrane of Moraxella catarrhalis for beta-lactam antibiotics.

The susceptibility of ten clinical isolates and a standard reference strain, ATCC25238, of Moraxella catarrhalis to 22 beta-lactam antibiotics was examined and compared with that of Escherichia coli strain B. All the strains of M. catarrhalis tested, especially the non beta-lactamase-producing strain ATCC25238, were more susceptible to a rang of structurally unrelated beta-lactam antibiotics, including small Mr carbapenems, than E. coli B. The permeability of the M. catarrhalis outer membrane to beta-lactam antibiotics was examined by the swelling technique with proteoliposomes reconstituted from outer membranes. The diffusion rate of beta-lactams through the liposome membrane was inversely related to their Mr, a relationship which might be expected for entry by diffusion through a porin.

Anti-Bacterial Agents↗
PubMed1990

Decreases of the susceptibility to low molecular weight beta-lactam antibiotics in imipenem-resistant Pseudomonas aeruginosa mutants: role of outer membrane protein D2 in their diffusion.

Decreased susceptibilities to two low Mr beta-lactam antibiotics, CS-533 (a carbapenem; Mr, 339) and CGP31608 (a penem; Mr, 262), were found in imipenem-resistant mutants of Pseudomonas aeruginosa PAO. The diffusion rates of several beta-lactam antibiotics including imipenem, CS-533 and CGP31608 across the proteoliposome membrane reconstituted from the outer membrane of the wild type strain or its imipenem-resistant mutants were determined by the liposome swelling technique. Diffusion rates of imipenem, CS-533 and CGP31608 in the proteoliposomes from outer membranes of the imipenem-resistant strain were found to be 27, 20 and 47%, respectively, of the diffusion rates in proteoliposomes reconstituted from outer membranes of the sensitive parent strain. The SDS-polyacrylamide gel electrophoretogram of outer membrane proteins of the imipenem-resistant strains indicated deletion of protein D2. These results suggested that decreased susceptibilities to imipenem, CS-533 and CGP31608 were due to decreased outer membrane permeability, and that D2 is a protein fraction constituting pores for diffusion of these antibiotics through the P. aeruginosa outer membrane.

Anti-Bacterial Agents↗
PubMed1990

Resistance of Pseudomonas aeruginosa to cefsulodin: modification of penicillin-binding protein 3 and mapping of its chromosomal gene.

Spontaneous cefsulodin-resistant mutants of Pseudomonas aeruginosa PAO4089 were isolated on agar impregnated with 3 mg/l of cefsulodin. This strain does not produce any chromosomal beta-lactamase. The MICs of cefsulodin for the parent and its mutants were 0.78 and 12.5 mg/l, respectively. Complete cross-resistance between cefsulodin and seven other antipseudomonal beta-lactams was noted in the mutants. The mutant gene, designated as pbpB, was mapped by FP5 plasmid-mediated conjugation and found to be near to cys-59 on the PAO chromosome, the gene order being pur-67, oruI, pbpB and cys-59. There were no detectable differences between the parent and its mutants in their outer membrane protein profiles. Penicillin-binding protein assay, by the competition method, with cefsulodin or carbenicillin showed a significant reduction in affinity of PBP3 for these beta-lactams. This PBP is the primary target for cefsulodin in P. aeruginosa. The genetic mechanism by which the cefsulodin-resistant clinical isolates of P. aeruginosa have emerged is discussed.

Acyltransferases↗
PubMed1989

Ultrastructural aspects of fragility of Pseudomonas aeruginosa outer membrane devoid of protein F.

The protein F-deficient cells of Pseudomonas aeruginosa were previously found to be more susceptible to osmotic shock than the sufficient cells (Gotoh et al., J. Bacteriol., in press). The protein F-deficient cells were observed by the thin-section method of electron microscopy to determine the effects of osmotic shock. The osmotic shock induced breakage of the protein F-deficient outer membrane, while it had no effect on the protein F-sufficient outer membrane. These results suggested that the cells lost their viability by the osmotic shock caused by fragility of the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1989

Supersusceptibility to hydrophobic antimicrobial agents and cell surface hydrophobicity in Branhamella catarrhalis.

To clarify the cause of the supersusceptibility of Branhamella catarrhalis to macrolide antibiotics, which are well-known to be inactive to most Gram-negative bacteria, we determined its cell surface hydrophobicity by the partition experiment between water and hydrocarbons. Its cell surface was found to be markedly more hydrophobic than that of Escherichia coli or Pseudomonas aeruginosa cells. This suggested that the outer membrane of B. catarrhalis plays no role as a diffusion barrier towards hydrophobic agents.

Anti-Bacterial Agents↗
PubMed1989

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane.

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.

Bacterial Outer Membrane Proteins↗
PubMed1988

In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa.

Determination of the rates of saccharide diffusions by the proteoliposomes showed that the outer membrane of Pseudomonas aeruginosa only possesses small diffusion pores and that protein F might have not been involved in the pore formation. Proteoliposomes containing stachyose or Dextan T-10 showed the same relative diffusion rates as measured by the liposome swelling method. Slopes of the lines, diffusion rate vs saccharide Mr, in the liposomes made of the P. aeruginosa and E. coli B outer membranes appeared to be -7.4 and -3.5, respectively. Intercepts of the lines with x-axis in the liposomes containing the P. aeruginosa and E. coli B outer membrane appeared to be about Mr, 220 and 320, respectively. Relative diffusion rates of saccharides through the liposome membranes reconstituted from the protein F-deficient outer membrane were superimposable with that of the protein F-sufficient outer membrane.

Cell Membrane↗
PubMed1987

Encephalocele, polycystic kidneys, and polydactyly with other defects. A necropsy case of Meckel syndrome and a review of literature.

Meckel syndrome, which is diagnosed by 2 of 3 main congenital malformations such as a occipital encephalocele, polycystic kidneys, and polydactyly, is an autosomally inherited recessive disease. We have experienced a case of Meckel syndrome and performed necropsy. Necropsy findings revealed multiple congenital malformations with occipital meningo-encephalocele and agenesis of the cerebellum, 6 digits on the hands and feet, polycystic kidneys. The criteria of Meckel syndrome is still unclear. We propose that the diagnosis of this syndrome may be accompanied by the presence of all triad of main malformations. Ninety four cases satisfying this criteria have been reported in the world literature. Several discussion were made from a review of the literature.

Abnormalities, Multiple↗
PubMed1983

Effects of nucleotides and nucleosides on the activity of cyclic AMP phosphodiesterase from rat brain.

Cyclic nucleotides phosphodiesterase (PDE) was prepared from cerebrum of male rats and its kinetic properties were studied. The phosphodiesterase preparation exhibited two Michaelis constants, 8.7 microM and 83.3 microM. Adenine derivatives such as adenosine, 5'-AMP and 2'(3')-AMP inhibited the PDE activity at concentrations exceeding 7 X 10(-3)M, and 2'-deoxyadenosine inhibited the activity at lower concentrations (Ki = 1.8 X 10(-3)M); its inhibiting efficacy was almost the same as that of theophylline (Ki = 1.9 X 10(-3)M). Guanine derivatives, on the other hand, showed several different effects. Guanosine and 3',5'-cyclic GMP activated the PDE at 10(-5) M and inhibited at concentrations higher than 10(-4)M. 2'(3')-GMP showed no effect, but 5'-GMP activated markedly at concentrations of 10(-3) to 10(-2)M. Thymidine showed slight inhibitory effect, but cytidine or 2'-deoxycytidine had no effect. Uracil derivatives such as uridine, 5'-UMP, 3',5'-cyclic UMP and 2'(3')-UMP activated the PDE at concentrations exceeding 3 X 10(-3) M. These results indicate that individual nucleosides and nucleotides exhibit structure-activity relationship with PDE.

3',5'-Cyclic-AMP Phosphodiesterases↗