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Biomedical subjects

Y Shibuya

Publications and source records attributed to Y Shibuya.

At least 91 records · Page 5Linked to original sources
PubMed1994

Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules.

A bovine Hageman factor cDNA was cloned from a liver cDNA library. The nucleotide sequence was analyzed and the amino-acid sequence was deduced. The sequence deduced was consistent with the partial amino-acid sequences of bovine Hageman factor protein. The sequences for three portions including the amino terminal had been previously reported (Fujikawa et al. (1977) Biochemistry 16, 2270-2278). In comparison with the primary structures of human and guinea pig Hageman factors, the putative domain structures were totally conserved. Each domain possessed high sequence homology with the human molecule (66-88%) and the guinea pig one (63-81%) except for the proline-rich region (less than 10%) which connects the amino-terminal five domains with a serine proteinase portion. Significant heterogeneities were observed among the three species around the essential cleavage sites for the conversion to the activated Hageman factors. Bovine Hageman factor has no suitable amino-acid sequence as the substrate for the trypsin-type proteinases at the proline-rich region in difference from the human and guinea pig molecules. Probably this is the reason why the beta-form activated Hageman factor (the proteinase moiety) is not liberated in the activation of the bovine molecule with trypsin or plasma kallikrein.

Amino Acid Sequence↗
PubMed1994

Genetic analysis of HIV-1 during rapid progression to AIDS in an apparently healthy man.

We encountered a case of HIV-1 infection in a previously healthy man, which was characterized by rapid progression to AIDS and death within 7 months in association with high levels of antigenemia throughout the clinical course and no humoral immune response for at least 6 months. Genetic changes of the third variable domain (V3) of the envelope gene of HIV-1 in serum samples were analyzed at four time points during his rapid clinical course. The nucleotide changes were confined to a maximum of three substitutions among 105 nucleotides of the V3 region. A major population of the viral clones in this patient showed one amino acid substitution from aspartic acid (a negatively charged amino acid) to lysine (a positively charged amino acid) at position 30 from the first cysteine of the V3 loop. This substitution was thought to be associated with phenotypic changes, and viruses with this sequence in the V3 region had a strong syncytium-inducing ability in MT-4 cells. It appears that the lack of a humoral immune response accelerated disease progression in our patient and a genetic change that appeared to produce a phenotypic change occurred at an early stage of the disease.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Alpha-2-macroglobulin as the major defence in acute pseudomonal septic shock in the guinea-pig model.

An intravenous injection of 1.2 mg/kg of Pseudomonas aeruginosa elastase induces immediate lethal shock in guinea-pigs. In the present study, alpha-2-macroglobulin (alpha 2M) was shown to be the major factor in guinea-pig plasma that inhibits the enzymatic activity of elastase in vitro. Depletion of circulating alpha 2M by injecting anti-guinea-pig alpha 2M rabbit IgG F(ab')2 rendered the animals sensitive to a dose of elastase of 0.05 mg/kg. When the alpha 2M-depleted guinea-pigs were reconstituted with human alpha 2M, this sensitivity was reversed. Lethal shock did not occur in alpha 2M-depleted animals even at an elastase dose of 0.2 mg/kg when Hageman factor was simultaneously depleted, indicating that elastase induces shock through activation of the Hageman factor-dependent system. Similar results were obtained when the culture supernatants of an elastase-producing strain, IFO-3455, were used instead of the purified elastase, whereas no cardiovascular changes occurred, even in the alpha 2M-depleted guinea-pigs, when the culture supernatants were pretreated with an elastase specific inhibitor (zincov) or when the culture supernatants of an elastase non-producing strain, PA-103 were used.

Acute Disease↗
PubMed1994

[A case of primary pulmonary cryptococcosis showing infiltrative shadows in the unilateral lung field].

A case of primary pulmonary cryptococcosis is reported. A 76-year-old man, without underlying disease or immunological abnormalities, was admitted to the hospital. Chest X-ray revealed diffuse infiltrative shadows considered at first to be community-acquired pneumonia. A diagnosis of pulmonary cryptococcosis was performed using fluconazole, miconazole and transbronchial inhalation of amphotericin B. Cryptococcal antigen detection was performed by two latex agglutination methods: Crypto-test and Sero-direct test (Eiken-test). Comparing the results of the two tests, sensitivity was superior with the Sero-direct test, but the correlation between the titers of the two tests was good.

Aged↗
PubMed1993

Pseudomonal elastase injection causes low vascular resistant shock in guinea pigs.

An intravenous injection of culture supernatants obtained from an elastase producing strain (IFO-3455) of Pseudomonas aeruginosa exhibited immediate fall of mean arterial blood pressure from 63.8 +/- 1.62 to 35.6 +/- 2.31 mmHg (P < 0.001), increased heart rate from 249.6 +/- 3.86 to 272.6 +/- 2.18 beats/min (P < 0.05), and increased respiratory rate from 44.8 +/- 2.33 to 68.6 +/- 1.60/min (P < 0.01) within 5 min in the anesthetized guinea pigs. In contrast, culture supernatants obtained from an elastase non-producing strain (PA-103) did not cause the cardio-respiratory alterations, even though the same dose of endotoxin was contained in the supernatants. Intravenous or intracardiac injection of purified Pseudomonas aeruginosa elastase (1.2 mg/kg) but not endotoxin (up to 2.0 mg/kg) reproduced the immediate shock followed by death within 45 min in anesthetized or in conscious guinea pigs. Consistently, the shock-inducing ability of pseudomonal elastase was prevented by pretreatment with anti-pseudomonal elastase rabbit F(ab')2 antibodies or with a synthetic inhibitor of pseudomonal elastase. Furthermore, intravenous injection of a non-lethal dose of pseudomonal elastase (0.8 mg/kg) immediately decreased peripheral vascular resistance when estimated from a change of perfusion pressure at hindquarter circulation from 74.0 +/- 1.00 to 52.6 +/- 1.76 mmHg (P < 0.05) in association with fall of arterial blood pressure and of cardiac output which was estimated from a change of regional aortic flow. The same low-resistant shock was also observed in rats. We speculate, therefore, that bacterial proteinases may play an important role in human septic shock.

Animals↗
PubMed1993

Role of Hageman factor/kallikrein-kinin system in pseudomonal elastase-induced shock model.

The role of the Hageman factor dependent pathway in pseudomonal elastase-induced shock was investigated in guinea pigs. Presence of a bradykinin B2 receptor antagonist [D-Arg0,Hyp3,Thi5,8,D-Phe7]-bradykinin (200 nM) in the circulation prevented shock caused by an intrajugular injection of pseudomonal elastase (0.8 mg/kg body weight). During the lethal shock caused by elastase (1.2 mg/kg), a significant consumption of components of the Hageman factor/kallikrein-kinin system was observed such as 45.7 +/- 2.20% consumption of Hageman factor, 100 +/- 0% of prekallikrein, and 85.1 +/- 2.50 of high-molecular-weight kininogen. More striking evidence for the participation of this system was demonstrated in depletion experiments with monospecific F(ab')2 antibodies against the components of the system. After depletion of any one of the components, guinea pigs exhibited unresponsiveness to the same lethal dose of pseudomonal elastase in regard to the cardio-respiratory alterations. In vitro, pseudomonal elastase (60 micrograms/ml) possessed a capacity to generate substantial amount of bradykinin in undiluted plasmas of humans (300.0 +/- 32.16 ng/ml) as well as guinea pigs (460.2 +/- 20.67 ng/ml) at 37 degrees C but not in those deficient in Hageman factor or prekallikrein. These results strongly suggested a pathological role of elastase in pseudomonal sepsis through activation of the Hageman factor dependent pathway.

Amino Acid Sequence↗
PubMed1993

Difference between human and guinea pig Hageman factors in activation by bacterial proteinases: cleavage site shift due to local amino acid substitutions may determine the activation efficiency of serine proteinase zymogens.

Human and guinea pig Hageman factors have been subjected to the action of pseudomonal elastase and serratial E15 proteinase. The pseudomonal elastase cleaved 22-24% of the human molecule at Arg353-Val354, and the remainder at Gly357-Leu358 resulting in the generation of about 20% of potential activity as activated Hageman factor, compared with trypsin activation, while it hydrolyzed Arg340-Ile341 bond in guinea pig molecule and generated about 75% of activity as activated Hageman factor. The serratial proteinase did not hydrolyze the essential cleavage site (Arg353-Val354) of the human zymogen but Gly356-Gly357 (30%) and Gly357-Leu358 (70%) bonds. Both products showed no activity. The guinea pig zymogen, in contrast, was cleaved mostly at Arg340-Ile341 (70%) and less abundantly at Gly344-Leu345 (30%), generating about 85% of the whole potential activity as activated Hageman factor. From the high correspondence between the proportions of activation and of hydrolysis at the essential cleavage site in activation, it was concluded that hydrolysis of the bonds different from the essential bond did not cause activation, even when the spatial separation was only 3 or 4 residues. Considering the amino acid differences between human and guinea pig Hageman factors, -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val-Ala360- and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val-Ala347-, respectively, it was realized that even the minor amino acid substitutions caused the cleavage site shift which resulted in significant differences in activation efficiency of the proteinase zymogens.

Amino Acid Sequence↗
PubMed1993

Preliminary findings on the variation of serum apolipoprotein levels in neural degenerative disorders.

We measured six apolipoproteins (AI, AII, B, CII, CIII, and E) in the serum of patients with several kinds of neural diseases [diabetic neuropathy and neural degenerative disorders (motor neuron degenerative disorders, spinocerebellar degeneration, Parkinson's disease)], comparing them to the age-matched healthy controls using the immunoturbidimetric method. Statistically significant decreases of serum apo-AI, apo-A-II and increases of apo-CIII, apo-E were observed in neural degenerative diseases; and, particularly, higher apo-B and apo-CII concentrations were observed in diabetic neuropathy. Most neural degenerative disease showed lower apo-AII. However, in motor neuron degenerative disorders, higher apo C-II and apo-E were seen. Lower apo-AI was seen in spinocerebellar degeneration, and lower apo-AII was seen in Parkinson's disease. Higher apo-B, CII, and E levels were observed in females with spinocerebellar degeneration and Parkinson's disease, and lower apo-AII was seen in males with spinocerebellar disease.

Aged↗
PubMed1993

Corneoscleral ulceration and corneal perforation after pterygium excision and topical mitomycin C therapy.

A 78-year-old woman complained of ocular pain in the right eye. Thirty-three months before, the patient had a pterygium excised and mitomycin C, 0.04%, was instilled 3 times a day postoperatively for 10 days in the right eye. The same surgery was performed and the same drug was used for a course of 5 days in the left eye. Corneoscleral ulceration and corneal performation occurred in the right eye. The lesions were successfully treated with a lamellar patch graft with reserved corneosclera. We believe that it may not be safe to instill 0.04% mitomycin C 3 times a day postoperatively for 10 days.

Administration, Topical↗
PubMed1993

Hormonal regulations of alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase activities in adult rat hepatocytes cultured in serum-free medium on collagen gel.

Primary hepatocytes were cultured on collagen gel in serum-free, alpha-modified Eagle's minimum essential medium containing 0.1 microM insulin, 0.1 microM dexamethasone, 10 mM pyruvate and supplements such as glucagon, epinephrine or growth hormone. The activities of alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase were assayed in cell extracts prepared from the cultures. All three enzyme activities were induced by glucagon, epinephrine or dibutyryl cAMP. The maximally effective concentration of glucagon was 5-10 nM for both alkaline phosphatase and 5'-nucleotidase and 100 nM for gamma-glutamyltransferase. Only alkaline phosphatase activity was suppressed by growth hormone, which caused marked suppression at about 1 microU (0.25 ng)/ml. Taurocholate also induced both alkaline phosphatase and gamma-glutamyltransferase activities at 1 mM.

5'-Nucleotidase↗
PubMed1993

Various fundus manifestations in a Japanese family with Best's vitelliform macular dystrophy.

Recently we examined 7 members in one Japanese family with Best's vitelliform macular dystrophy. Five family members in three generations had abnormal electro-oculographic (light peak/dark trough ratio) and normal electroretinographic responses, but their fundus manifestations varied. The proband (a girl) had yellowish macular lesions in both eyes and a neovascular membrane and subretinal hemorrhage in the left macula. The proband's grandfather had mottled macular lesions bilaterally and a mottled extramacular lesion in the left eye. The proband's father had yellowish macular lesions in both eyes and an extramacular lesion in the right eye. The proband's sister had ophthalmoscopically normal fundi. The proband's brother had yellowish macular lesions bilaterally. We believe that finding a variety of fundus manifestations of Best's vitelliform macular dystrophy in one family, as reported in this study, may be uncommon.

Adult↗
PubMed1992

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.

Amino Acid Sequence↗
PubMed1992

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase. II. Kinetic analysis and identification of scissile bond of prekallikrein in the activation.

Activation of human plasma prekallikrein by a bacterial metalloendopeptidase, Pseudomonas aeruginosa elastase, was reported (Shibuya et al. (1991) Biochim. Biophys. Acta 1097, 23-27). Details of the activation process were presently studied. The activation accompanied limited proteolysis of a peptide bond inside of a disulfide bridge of prekallikrein molecule. Amino acid sequencing analysis of the newly generated amino-terminal revealed that the cleavage site was Arg371-Ile372 bond which is the scissile bond in the activation of prekallikrein with trypsin-type proteinases. A pentapeptide substrate, 2-aminobenzoyl-Ser-Thr-Arg-Ile-Val-4- nitrobenzylamide, which contained the amino acid sequence identical to that around the scissile bond of prekallikrein was synthesized. Pseudomonal elastase, indeed, hydrolyzed the substrate at Arg-Ile bond with the kinetic parameters of Km = 118 microM, kcat = 1.56/s and kcat/Km = 1.33.10(4)/s M. These results indicated that the Arg371-Ile372 bond was sensitive not only to trypsin-type serine proteinases, but also a bacterial metalloproteinase. Kinetic analysis of the prekallikrein activation by pseudomonal elastase, however, revealed that the activation rate was slow, though the Km values was good enough to expect an occurrence of this activation in vivo (Km = 248 nM, kcat = 6.8.10(-4)/s, and kcat/Km = 2.7.10(3)/s M). The activation rate of prekallikrein by pseudomonal elastase in Hageman factor deficient plasma was remarkably improved when the plasma was reconstituted with purified Hageman factor molecule. From the results, a biological significance of the proteinase cascade in the plasma kinin generation was also indicated. The present in vitro study might support the hypothesis that the Hageman factor/kallikrein-kinin system plays an important role in bacterial infection including the pseudomonal one.

Amino Acid Sequence↗
PubMed1992

Effect of calcium concentration on survival, proliferation and activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase of adult rat hepatocytes cultured in serum-free medium.

Mature rat hepatocytes were cultured on collagen coated dishes in serum-free alpha-modified Eagle's minimum essential medium containing 0.1 microM insulin, 0.1 microM dexamethasone, 10 mM pyruvate and Ca2+ at concentrations of 0-2 mM. Survival of nondivided cells was best in medium containing 2 mM Ca2+. Proliferation during 5-day culture was greatest with 0.4 mM Ca2+, but DNA synthesis was scarcely affected by the concentration of Ca2+. Both the activities of alkaline phosphatase, 5'-nucleotidase, gamma-glutamyltransferase and lactate dehydrogenase and the number of cell nuclei of cultures in 0.1 mM and 2 mM Ca2+ media were assayed over a 5-day period, and their activities were calculated as enzyme activities per unit number of cell nuclei. Alkaline phosphatase activity increased rapidly during the first day of culture in both media, and its activity in 0.1 mM medium was higher than that in 2 mM medium after culture for 3 days. The activity of 5'-nucleotidase became higher in 0.1 mM medium than in 2 mM medium from day 2 and was maximal on day 3 in both media. gamma-Glutamyltransferase activity increased and lactate dehydrogenase activity decreased with time in culture, both activities showing no appreciable difference in the two media.

5'-Nucleotidase↗
PubMed1992

[Identification and differentiation of the human herpes virus group using the PCR method].

Six kinds of human herpes viruses have been identified and classified on the basis of structure and characteristics. We studied the identification and classification of these types using PCR to amplify the virus-specific DNA sequences. This method showed higher sensitivity than the conventional method of virus isolation and culture for HSV and CMV detection. For each positive control, the viral DNA was amplified only when the complementary primers themselves were used. PCR apparently detects only the activated virus, because normal controls were negative when this method was used. Therefore, the present method is thought to closely reflect viral activation and infectious diseases in patients with latent infections.

Base Sequence↗