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Biomedical subjects

K Kawahara

Publications and source records attributed to K Kawahara.

At least 19 recordsLinked to original sources
PubMed1992

Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei.

The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether. The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid. The growth conditions did not affect the electrophoresis profile and chemical composition of LPS. 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P. cepacia, which has close taxonomic relationship with P. pseudomallei.

Burkholderia pseudomallei↗
PubMed1992

Stabilization of basic fibroblast growth factor with dextran sulfate.

Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule.

Amino Acid Sequence↗
PubMed1992

Activation of calcium channel by shear-stress in cultured renal distal tubule cells.

Using the patch-clamp technique and the fura-2 fluorescence measurement, we found that flow of a normal solution simultaneously increased both the inward cation (Ca) currents and the cytosolic Ca activity (Cai) in cultured renal distal tubule cells (A6 cells). The activation of these signals was voltage-independent and required a lag period of about 30 s. Flow of a Ca free solution (plus 0.1-0.5 mM EGTA) failed to increase these signals. The Ca current increased and saturated with increasing extracellular Ca concentrations (apparent Km, 1 mM Ca; maximum Ca current, 43 pA). Ni (1 mM) and La (1 mM) inhibited the flow-induced Cai-increase, but nicardipine (50 microM) did not. These results strongly suggest that in A6 cells flow increases Ca-influx through a shear-stress activated Ca-channel and may regulate the cellular transport functions.

Animals↗
PubMed1992

Wortmannin, a microbial product inhibitor of myosin light chain kinase.

We have found that a fungal strain, Talaromyces wortmannin KY12420, produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product, designated as MS-54, was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain, which was disclosed by partial tryptic digestion, was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses, preincubation studies, and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min, 0.3 microM wortmannin inhibited the activity of MLCK, while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore, wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl, which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor.

Adenosine Triphosphate↗
PubMed1992

Marrow transplantation for paroxysmal nocturnal hemoglobinuria.

Between 1971 and 1990, nine patients ranging in age from 14-38 years received marrow transplants for paroxysmal nocturnal hemoglobinuria (PNH). Six were transplanted for aplastic complications of PNH. Four of these were from HLA-identical siblings, and the patients were conditioned with cyclophosphamide. One graft was form a syngeneic twin without conditioning, and one from a two HLA-antigen nonidentical father after conditioning with cyclophosphamide and total body irradiation. Three of the four recipients of allogeneic marrow developed acute and two chronic graft-versus-host disease (GVHD). Five of six transplanted for severe aplastic anemia are long-term survivors with follow-up ranging from more than 6.2 to more than 19.1 years. The HLA nonidentical transplant recipient experienced graft rejection and died of a pulmonary hemorrhage. Three patients were transplanted for nonaplastic complications of PNH consisting of life threatening recurrent thromboses or refractory hemolysis. Two of these patients received marrow grafts from HLA-identical siblings after conditioning with busulfan and cyclophosphamide. They are surviving with normal hemograms greater than 2.2 and greater than 2.5 years and had mild chronic GVHD which resolved, although one has biochemical evidence of PNH in 15% of the red cells. One received a syngeneic marrow graft without conditioning but reverted to PNH. He is alive greater than 8.6 years after transplantation. Marrow transplantation for aplastic complications of PNH is successful, well tolerated, and compatible with long-term survival when an HLA-identical sibling or a syngeneic donor is available. For patients without aplasia, one must weigh the complications of transplantation with the life threatening nature of thrombotic episodes and hemolysis.

Adolescent↗
PubMed1992

Upper airway obstruction from a benign goiter.

Acute upper tracheal obstruction from a huge benign goiter developed in an 80-year-old woman. Computed tomography showed severe stenosis of the cervical trachea by extrinsic compression. A left hemithyroidectomy was performed and the tracheal lumen returned to normal size. However, the tracheal wall was malacic due to the prolonged compression by the goiter and a silastic T-tube insertion was necessary for a two month period. This type of tracheal obstruction is rare. The importance of evaluating the upper trachea in patients with prolonged benign goiter is stressed and the diagnosis as well as the management of this type of complication are discussed.

Aged↗
PubMed1992

Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin.

An accurate and reproducible colorimetric assay was established to determine the concentration of basic fibroblast growth factor (bFGF) or bFGF-like activity in culture media and biological fluids. Fetal bovine heart endothelial cell line ATCC CRL 1395 was used as the bFGF-dependent cell line. The proliferation-stimulating activity of bFGF was determined by measuring the amount of formazan formed by the mitochondrial enzymes from a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), instead of counting the viable cell numbers or measuring the incorporation of [3H]-thymidine. The addition of 250 ng/mL of heparin to the culture medium resulted in about a tenfold increase in the proliferation-stimulating activity of bFGF and allowed the detection of as low as 10 pg/mL of bFGF. Heparin also resulted in much smaller inter- and intraassay variations. The bFGF concentrations determined by this colorimetric assay correlated well with those determined by both the [3H]-thymidine incorporation assay using BALB/c 3T3 fibroblast cells (r = 0.998) and the cell number count assay (r = 0.996). This assay can be adapted to quantify bFGF or bFGF-like activity in tissue culture media and biological fluids such as plasma and organ extracts.

Animals↗
PubMed1992

Serum resistance and aerobactin iron uptake in avian Escherichia coli mediated by conjugative 100-megadalton plasmid.

A total of 115 strains of Escherichia coli isolated from chickens with colisepticemia in Japan were examined for chicken lethality and virulence factors. It was found that serum resistance and aerobactin-mediated iron uptake are the most prevalent characteristics in these strains. Among them, S-20, a representative virulent strain of serotype O2, was further studied. S-20 harbored a conjugative 100-megadalton (Mdal) plasmid, designated pKI100. Curing and reintroduction experiments showed that pKI100 encodes both serum resistance and aerobactin-mediated iron uptake, and the diminished virulence of the pKI100-cured strain was fully restored by the reintroduction of the plasmid. These results demonstrated that pKI100 is the virulence plasmid of the S-20 strain, and that serum resistance and aerobactin-mediated iron uptake are the virulence factors in E. coli strains which cause avian colibacillosis.

Animals↗
PubMed1992

Identification of the cross-reacting antigen among Actinobacillus pleuropneumoniae strains of serotype 1, 9 and 11 by use of monoclonal antibodies.

Two monoclonal antibodies (MAbs), lMAb-1 and lMAb-5, against Actinobacillus pleuropneumoniae serotype 1 were obtained. In enzyme-linked immunosorbent assay-inhibition tests with whole cell antigens obtained from serotype 1 to 12 strains of A. pleuropneumoniae, lMAb-1 reacted to only a serotype 1, strain 4074. The epitope recognized by lMAb-1 was a carbohydrate sensitive to periodate oxidation and resided on capsular polysaccharide (CP) of A. pleuropneumoniae serotype 1. On the other hand, lMAb-5 reacted with serotype 1, 9 and 11 strains at the same degree and its epitope was found to be located on O-polysaccharide of serotype 1, 9 or 11 lipopolysaccharide (LPS). These results showed that CP was one of the serotype-specific antigens of A. pleuropneumoniae, and that O-polysaccharide of LPS obtained from serotype 1, 9 or 11 strain was the cross-reacting antigen among these strains.

Actinobacillus pleuropneumoniae↗
PubMed1992

[Ion channels in the kidney: molecular biological aspects].

Ion channels in renal tubules are involved in maintenance of the volume and ion composition of the body. In the kidney these channels are expressed at the apical and/or basolateral cell membranes and are responsible for vectorial ion transport. Structure and function of the renal ion channels differs from those of previous channels which were cloned in excitable tissues. In this review I outline the various types of ion channels in renal and non-renal cells and give examples that emphasize the physiological roles of these channels in the nephron.

Animals↗
PubMed1992

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.

Actinobacillus pleuropneumoniae↗
PubMed1992

[Effect of meropenem on fecal flora in children].

Meropenem (MEPM, SM-7338), a novel parenteral carbapenem antibiotic, was examined for its effect on intestinal flora in children. Seven children with infectious diseases (3 male and 4 female children of age's ranging from 4 months to 8 years and 9 months weighing from 7.3 to 23.0 kg) were treated with MEPM at doses ranging 10.3 to 40.5 mg/kg 3 or 4 times a day for 6 to 12 days. Before, during and after the treatment, identities and numbers of various bacteria contained in 1 g of feces were determined and fecal beta-lactamase activity and Clostridium difficile D-1 antigen were also assayed. Changes in fecal flora during MEPM treatment was somewhat different depending on cases. Regarding Enterobacteriaceae among aerobes, all of 7 cases exhibited moderate or pronounced reductions in Escherichia coli. Some of the cases exhibited the tendency to increase in Klebsiella oxytoca. Enterobacter cloacae and Citrobacter freundii. E. coli which was reduced during the treatment increased rapidly after the treatment in 5 out of 7 cases, and the initial bacterial counts were restored. Diverse strains were observed within the genus Enterococcus, while the overall bacterial counts of this genus exhibited the tendency to increase during the treatment. As a result, no significant change in total aerobe count was observed in any case except 1 case where Enterococcus count was somewhat reduced. Among anaerobes, major bacteria such as Bacteroides, Bifidobacterium, Eubacterium and Peptococcaceae exhibited tendencies to decrease in some cases during the antibiotic treatment. Two infants and 1 child exhibited significant decreases in total anaerobe counts. In most of the cases, such changes in major anaerobes were transient and bacterial counts recovered to their initial values rapidly after completion of the treatment. In no cases, glucose non-fermentative Gram-negative bacilli or fungus became predominant. Although C. difficile D-1 antigen was observed in 4 cases, its changes had no relationship with characteristics of feces. C. difficile was not detected in any of the cases. MEPM was detected in feces in 4 cases being treatment, in concentrations ranging from 0.35 to 66.0 micrograms/g. Fecal MEPM levels were very low except in 1 case in which beta-lactamase was negative. From these results, effects of MEPM on intestinal flora in children were relatively minor compared to other new beta-lactam drugs. However, a care should be taken to minimize diarrhea and bacterial turnover when a prolonged use of the antibiotic, was practiced because of potential significant effects on intestinal flora.

Age Factors↗
PubMed1992

[The influence of cefdinir on the intestinal bacterial flora].

The influence of cefdinir (CFDN), a new oral cephalosporin, on the intestinal bacterial flora was studied in tetra-contaminated mice and in pediatric patients. CFDN in fine granules was administered at a dose of 10 mg/kg once a day for 5 consecutive days to mice contaminated with 4 different species of organism: Escherichia coli, Enterococcus faecalis, Bacteroides fragilis and Bifidobacterium breve. No remarkable changes were observed in the fecal viable cell counts except that decreases in E. coli counts were observed on the day 3 to 5 after starting administration. The subjects in pediatric study were 7 children with infections, 3 boys and 4 girls, with their ages from 6 months to 12 years 7 months. Their body weights ranged from 5.5 to 29.2 kg. CFDN fine granules was administered at each dose of 3.0 mg/kg to 3.7 mg/kg, 3 times a day for 4 to 14 days. During the administration of CFDN, some variations were observed in the pattern of changes in the fecal bacterial flora between subjects. Although Enterobacteriaceae and total counts of anaerobes were markedly decreased in 2 cases, total counts of aerobes were unchanged in the 2 cases, whereas main aerobes and anaerobes except enterococci hardly varied in the other cases. There was no case in which glucose non-fermenting Gram-negative rods and fungi became predominant species continually. Although Clostridium difficile and C. difficile D-1 antigens were detected in 1 and 4 cases, respectively, no relationship was found between the number of C. difficile and the characteristics of the feces. With regard to the drug sensitivities of bacteria isolated from feces before and after administration of CFDN, higher levels of resistance were found in some bacteria such as Enterococcus and Bacteroides during or after administration than before administration. CFDN was detected in fecal samples from 2 cases during administration with concentrations ranging between 0.99-254 micrograms/g. High value of CFDN was found in a case with low beta-lactamase activity in feces, in which marked decrease of Enterobacteriaceae and total counts of anaerobes was observed. The above results suggest that CFDN is considered to be a drug with relatively small influence on the intestinal bacterial flora. But as high concentrations of drugs were detected in feces under some circumstances, our attention will be required. Particular care is also required for the occurrence of diarrhea and microbial replacement during continuous, long-term administration of the drug.

Animals↗
PubMed1992

[Management of airway obstruction using the neodymium-yttrium-aluminium-garnet laser].

The neodymium-yttrium-aluminium-garnet laser (Nd-YAG laser) has proved to be useful therapeutic tool for the management of endobronchial lesions. Between February 1983 and January 1991, 16 patients received endobronchial laser therapy at the Nagasaki University Hospital, 7 patients for tracheal stenosis, and 9 for endobronchial obstruction. In 4 patients with tracheal stenosis, the therapy was performed in an emergency. The type of lesions were lung cancer in 7 patients, thyroid cancer in 3, tracheal tumor (squamous cell carcinoma, and chondrosarcoma) in 2, esophageal cancer in 1, bronchial submucosal tumor in 1, and tracheobronchial tuberculosis in 2. Good symptomatic relief was obtained in 13 patients. One patient died from massive bleeding in the left main stem bronchus. Reobstruction had occurred in 2 patients with cicatricial lesion in right main stem bronchus. In patients with an unresectable malignant lesion, concurrent radiotherapy was needed to prevent the reobstruction.

Aged↗
PubMed1991

Retrovirus-mediated gene transduction into long-term repopulating marrow cells of dogs.

Amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (adenosine aminohydrolase, EC 3.5.4.4; ADA) were used to transduce canine marrow cells. In one approach, dogs were treated for 7 days with recombinant human granulocyte colony-stimulating factor to stimulate hematopoietic cell division. Bone marrow cells were collected and transduced by 24 hours of cocultivation on vector-producing cells followed by incubation in a vector-containing long-term marrow culture system for 4 days. Transduced autologous marrow (0.4 to 1.0 x 10(8) cells/kg) was infused into dogs administered otherwise lethal total body irradiation (TBI) of 920 cGy. Two of four dogs engrafted, and their marrows showed intermittently between 1% and 11% G418-resistant colony-forming unit granulocyte-macrophage (CFU-GM) colonies for up to 2 years after transplantation. In a different experimental approach, autologous marrow, obtained at the time of the PB neutrophil nadir 7 days after a single cyclophosphamide injection (40 mg/kg intravenously), was cocultivated for 24 hours on vector-producing cells and infused at doses of 0.06 to 0.18 x 10(8) cells/kg into dogs administered 920 cGy TBI. One of three dogs engrafted, and the marrow showed intermittently 1% to 10% G418-resistant CFU-GM colonies for at least 2 years. Culture results were confirmed by polymerase chain reaction (PCR) showing the presence of the neo gene in marrow cells, peripheral blood (PB) granulocytes, and PB and lymph node lymphocytes. Dilution experiments indicated that up to 10% of marrow, lymph node, and PB cells contained the neo gene, consistent with the culture results. Samples harboring the neo gene also contained the gene for human ADA. However, repeated analyses of PB and marrow cells for human ADA gene expression by starch gel electrophoresis were negative. PB samples of all dogs were free of helper virus, and no long-term side effects from the transduction were observed.

3T3 Cells↗
PubMed1991

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis.

Two novel glycosphingolipids were isolated from Sphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an alpha-D-Manp-[1----2)-alpha-D-Galp-(1----6)-alpha-D-GlcpN-(1 ----4)-alpha-D- GlcpA-R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue. Erythro-1,3-dihydroxy-2-amino-octadecane and erythro-1,3-dihydroxy-2-amino-cis-13,14-methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)-2-hydroxymyristic acid in amide linkage.

Glycosphingolipids↗