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Biomedical subjects

K Soda

Publications and source records attributed to K Soda.

At least 109 records · Page 6Linked to original sources
PubMed1995

Manifestations of cancer cachexia induced by colon 26 adenocarcinoma are not fully ascribable to interleukin-6.

In order to further clarify the role of interleukin 6 (IL-6) in the pathogenesis of cachexia, recombinant human IL-6 (hIL-6) was administered s.c. by osmotic pump for 9 days at a dose of 1 or 10 micrograms/day into CDF1 mice inoculated with a non-cachexia-inducing subclone of colon 26 adenocarcinoma (clone 5), or with a cachexia-inducing subclone (clone 20) of this malignancy. The serum level of IL-6 in non-cachectic mice with clone-5 tumors was 35% lower than in cachectic mice bearing clone 20 of colon 26 adenocarcinoma on the 19th day after tumor inoculation. IL-6 administration induced anemia, thrombocytosis and visceral organ hypertrophy not only in mice with clone-5 tumors but also in control mice with no tumor burden. Lipolysis and proteolysis became conspicuous when a large dose (10 micrograms/day) of IL-6 was infused into mice with clone-5 tumors. However, IL-6 supplementation did not induce loss of body weight, a decline in food intake or lymphocytopenia, which were characteristically observed in cachectic mice with clone-20 tumors. In conclusion, IL-6 appears to be a permissive factor for the development of cachexia but, while it can induce some of the symptoms typical of cachexia, it cannot in itself induce the full cachectic syndrome.

Adenocarcinoma↗
PubMed1995

Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

Alanine↗
PubMed1995

Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9.

A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43,000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermophilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Oshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056-9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity; the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.

Amino Acid Oxidoreductases↗
PubMed1995

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10.

The gene of NAD(+)-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.

Amino Acid Sequence↗
PubMed1995

Minute malignant islet cell tumor of the pancreas: report of a case.

We present herein the case of a 73-year-old Japanese man in whom a minute malignant islet cell tumor, 7 mm in maximal diameter, was discovered. The patient was admitted to our hospital to undergo a gastrectomy for gastric cancer, at which time preoperative ultrasonography (US) revealed a minute hypoechoic mass located in the head of the pancreas. Endoscopic retrograde pancreatography, angiography, computed tomography, and magnetic resonance imaging failed to reveal the features or location of the tumor; however, these details were obtained by endoscopic and intraoperative US. Although intraoperative fine-needle aspiration cytology of the tumor enabled a diagnosis of islet cell tumor to be made, it failed to provide enough material to evaluate the grade of malignancy, which was confirmed by histologic examination of the enucleated tumor, allowing the appropriate surgical procedure to be decided.

Aged↗
PubMed1995

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL.

The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.

Cloning, Molecular↗
PubMed1995

Splenectomy before tumor inoculation prolongs the survival time of cachectic mice.

The effects of splenectomy on the development of cachexia, tumor growth and animal survival were studied in tumor-bearing CDF1 mice. Mice were inoculated with two subclones of colon 26 adenocarcinoma, clone 20 (with a potent capacity to induce cachexia) and clone 5 (without such activity), and underwent splenectomy before or after tumor inoculation. Splenectomy significantly prolonged the survival of mice bearing clone 20 when it was performed prior to tumor inoculation, although the progression of cachexia and tumor growth were not affected. The survival rate was higher in splenectomized than it was in nonsplenectomized mice 20-40 days after tumor inoculation. Such effects on survival were not observed, however, in mice splenectomized after inoculation with clone 20 or in mice that underwent splenectomy either before or after inoculation with clone 5. The decrease of peripheral blood lymphocyte count observed in mice bearing clone 20 was magnified when splenectomy was performed before tumor inoculation, but the serum levels of tumor necrosis factor and interleukin-6 were comparable. These results indicate that cancer death from cachexia is not directly attributable to enhanced catabolism. The mechanism by which splenectomy ameliorates the survival of cachectic mice remains to be studied, although several changes observed in the splenectomized mice after inoculation, including decreases in the peripheral blood L3T4+ cells and Lyt-2+ cells on the 9th day and 15th day respectively, and increase in the L3T4+/Lyt-2+ cell ratio on the 15th day suggest the involvement of the modified host's immune response.

Adenocarcinoma↗
PubMed1995

In vivo effect of GroESL on the folding of glutamate racemase of Escherichia coli.

The overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells. The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form. We isolated the active enzyme and purified it effectively.

Amino Acid Isomerases↗
PubMed1995

Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.

We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.

Alanine↗
PubMed1995

Structural analysis of the L-methionine gamma-lyase gene from Pseudomonas putida.

The gene encoding L-methionine gamma-lyase from Pseudomonas putida was cloned and the primary structure of the enzyme was deduced from its nucleotide sequence. The L-methionine gamma-lyase gene was expressed in Escherichia coli. The amino acid sequences of BrCN-digested peptides agreed with the corresponding parts of the L-methionine gamma-lyase sequence determined from the gene structure. The polypeptide is composed of 398 amino acid residues with a calculated molecular weight of 42,626, corresponding to the subunit of the homotetrameric enzyme. The deduced amino acid sequence of L-methionine gamma-lyase only showed extensive homology with other well known alpha,gamma-elimination and/or gamma-replacement pyridoxal 5'-phosphate-dependent enzymes, such as cystathionine gamma-lyase, cystathionine gamma-synthase, and O-acetylhomoserine O-acetylserine sulfhydrylase, that participate in the biosynthesis of sulfur amino acids. However, the deduced essential cysteine residue of L-methionine gamma-lyase was not conserved in these enzymes. We confirmed the presence of a part of an open reading frame in the 3'-flanking region of the L-methionine gamma-lyase gene, which showed high homology with the N-terminal region of pyruvate dehydrogenase (lipoamide) from E. coli, suggesting that it participates in the degradative pathway for L-methionine together with L-methionine gamma-lyase.

Amino Acid Sequence↗
PubMed1995

Comprehensive site-directed mutagenesis of L-2-halo acid dehalogenase to probe catalytic amino acid residues.

L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration. Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis. The roles of these residues are discussed.

Amino Acid Sequence↗
PubMed1995

Thermostable ornithine aminotransferase from Bacillus sp. YM-2: purification and characterization.

Thermostable L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine: 2-oxo-acid 5-aminotransferase) [EC 2.6.1.13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82,000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to alpha-ketoglutarate, producing L-glutamate and L-glutamate gamma-semialdehyde, which is spontaneously dehydrated to L-delta 1-pyrroline-5-carboxylate, and the enzyme is most active at 70 degrees C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0 M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mol of the vitamin B6 compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine delta-aminotransferases.

Amino Acid Sequence↗
PubMed1995

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

Carbon Isotopes↗
PubMed1995

[Analysis of cause of recent rise in number of foreigners reported to HIV/AIDS surveillance in Japan].

HIV/AIDS surveillance in Japan experienced a sharp and transient rise in 1991-92 in the number of foreign HIV positives but not of AIDS cases, for reasons of which remain unclear. Using the national HIV/AIDS surveillance data base the cause of the increase was studied by comparing the trends of foreign cases diagnosed in 1991-92 with those in 1985-90 in terms of gender, age, nationality, clinical stage, possible route of infection, possible place where the case contracted HIV and the place where the case was identified. Present analysis revealed: (1) In 1991-92 there was a large increase in the number of heterosexually-infected female HIV positive aged below 30 years reported from the areas surrounding Tokyo, of which the majority was of Asia origin. (2) A similar but moderate change was also seen in foreign males infected heterosexually but not in those infected homosexually. These were predominantly of U.S./Europe origin, aged > or = 30 and reported from Tokyo. (3) Although most of the foreign cases reportedly contracted HIV outside of Japan, those infected in Japan began being reported in 1991-1992. These results suggest that change in foreign cases seen in 1991-1992 was not only in number but also in gender, age, nationality, route of infection and geographical distribution. This should be taken into consideration in considering the HIV/AIDS prevention strategy in Japan.

Acquired Immunodeficiency Syndrome↗