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Biomedical subjects

K Hiraga

Publications and source records attributed to K Hiraga.

At least 37 records · Page 2Linked to original sources
PubMed2000

[Effect of gargling the throat with Panaxylocaine as a lubricant for the laryngeal mask airway].

We used Panaxylocaine as a suitable lubricant with gargling before insertion of laryngeal mask for twenty preoperative breast cancer patients. Patients were divided into two groups; Control group (N = 10) received water-based lidocaine gel just before insertion of laryngeal mask, and Panaxylocaine group (N = 10) received Panaxylocaine as a premedication for breast cancer operation. Compared to water-based lidocaine gel, Panaxylocaine was more effective for oral discomfort after insertion of laryngeal mask. But there was no difference between two groups about oral paresthesia. We conclude that Panaxylocaine is a good premedication before insertion of laryngeal mask preventing oral and pharyngeal complications.

Anesthetics, Local↗
PubMed1999

Industrial application of fuzzy control to large-scale recombinant vitamin B2 production.

A fuzzy control system featuring the identification of culture phases by fuzzy logic was applied to the large-scale recombinant vitamin B2 (VB2) production at the Fukuroi factory of Nippon Roche, Fukuroi. Prior to actual operation, the knowledge base for fuzzy control was tuned and verified by a simulation system. Only four rules, formulated as IF approximately THEN instructions and membership functions for state variables, are employed to control the feed rate and pH of the fed-batch culture. By applying fuzzy control, total VB2 production and VB2 yield was increased by 6-16% and 4-11% compared with conventional control, respectively. Operating stability was also improved. The large-scale fermentor used for VB2 production has been successfully operated under the control of the fuzzy system for more than 2 years.

Journal Article↗
PubMed1999

The difference between delayed extubation and tracheostomy in post-operative sleep apnea after glossectomy or laryngectomy.

BACKGROUND: Patients with cancer of the tongue or larynx require glossectomies or laryngectomies and subsequent reconstruction. These procedures remove part of the patient's upper airway. In cancer of the tongue, the removed part of the airway is substituted by a flap of their skin. Post-operatively, it is possible that the patients have problems respiring comfortably. In addition to this, long surgical procedures may simply interfere with their circadian rhythm. To elucidate the possible change in their post-operative respiration, we monitored the patient's respiratory pattern with an apnea monitor. METHODS: We attached an apnea monitor to the patients and recorded their respiratory pattern and arterial oxygen saturation. The patients were monitored for a total of five days: three days prior to the operation, one day before the operation, the day of operation, two days after, and on the fourth day after the operation. The period of monitoring was from 8:00 p.m. to 6:00 a.m. the next morning. RESULTS: Sixteen patients completed this study. The patients whose tube was extubated after glossectomy showed frequent apnea, low mean oxygen saturation and low comfort score as compared to the patients with tracheostomy after laryngectomy. Because two failed cases of free skin flap were among the former, it is possible that the frequent apnea is a factor of failed free skin graft after glossectomy and laryngectomy. CONCLUSION: Further studies are required to improve the patient's respiration during their sleep after tracheal extubation in glossectomy.

Aged↗
PubMed1999

Mutational analysis of the reactive site loop of Streptomyces metalloproteinase inhibitor, SMPI.

Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.

Bacterial Proteins↗
PubMed1999

Isolation and some properties of a novel killer toxin-like protein produced by Streptomyces sp. F-287.

A killer toxin-like protein was found in the culture supernatant of a strain isolated from soil. The strain was classified and designated as Streptomyces sp. F-287. The molecular weight of the purified killer toxin-like protein was estimated to be 9,500 by SDS-PAGE. The purified protein was heat stable (100 degrees C, 5 min), pH stable (pH 6.0-9.0, 60 degrees C, for 30 min), and had a relatively wide action spectra. The SKLP showed a cytocidal effect on both budding yeast, Saccharomyces cerevisiae W303 (IC50 = 15.6 micrograms/ml) and on fission yeast, Schizosaccharomyces pombe SP870 (IC50 = 20.0 micrograms/ml). The SKLP also caused morphological changes on some sensitive yeasts and filamentous fungi. These characteristics are apparently different from known killer toxins. These results suggest that this is a novel killer toxin-like protein from Streptomyces sp. strain F-287.

Amino Acid Sequence↗
PubMed1998

NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure.

The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d. values of 0. 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved.

Amino Acid Sequence↗
PubMed1998

Elucidation of the mode of interaction of thermolysin with a proteinaceous metalloproteinase inhibitor, SMPI, based on a model complex structure and a structural dynamics analysis.

SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1') are closely located in the hydrophobic S1' pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase.

Bacterial Proteins↗
PubMed1998

Difficult tracheal intubation in patients with retinoblastoma caused by 13q deficiency.

Anesthetic management of retinoblastoma patients is unremarkable in most cases. Patients are most often babies and laryngoscopic procedures for intubation are usually easy. However, we recently experienced two cases with retinoblastoma with whom tracheal intubation was accomplished with difficulty. We report the two cases with a review of all 5-year records of retinoblastoma (rbl) in our institution. The cases we experienced recently were all patients with rbl with deletion of the long arm of thirteenth chromosome (13q-). In the 5-year review, the incidence of the difficult intubation was significantly higher (P < 0.05) in rbl with 13q- (4/11) than in rbl without 13q- (0/147). In our experience macroglossia was noted for the difficulty in the intubation. We assume that some patients with rbl will be cases of difficult intubation even to cannot ventilate-cannot intubate level when the results of their chromosomal analysis show a deletion on the thirteenth chromosome.

Anesthesia↗
PubMed1998

Molecular cloning and expression of a gene encoding a novel sorbitol oxidase from Streptomyces sp. H-7775.

A gene encoding a novel intracellular sorbitol oxidase of a soil bacterium, Streptomyces sp. H-7775, was cloned and sequenced. The gene consists of an open reading frame of 1,260-bp encoding a protein of 420 amino acids with a molecular weight of 45,148. Deduced amino acid sequence of the gene has 25.3% identity and 68.1% similarity to that of rat L-gulonolactone oxidase at the overall amino acids. Nucleotide-binding motifs were not found in the deduced amino acid sequence of SOX protein. We succeeded in expressing recombinant sorbitol oxidase with covalently bound FAD in E. coli at about a 4,000-fold higher total enzyme activity than that of the Streptomyces sp. H-7775. The enzymatic properties of the recombinant SOX were similar to those of the enzyme from Streptomyces sp. H-7775. This is the first report of the cloning and expression of a newly categorized enzyme, sorbitol oxidase, from Streptomyces sp.

Amino Acid Sequence↗
PubMed1998

[Pharmacokinetics of PF-402, sustained-release morphine capsule, in cancer patients with pain].

A once-daily dose of PF-402 60 mg and twice-daily doses of sustained-release morphine sulfate tablets (MSC) 30 mg were repeatedly administered in cancer patients in a cross-over design. Their plasma concentrations were measured, and the pharmacokinetics of PF-402 and MSC were compared. A total of 7 subjects in the study were taking commercially sold MSC 60 mg daily (30 mg twice-daily) prior to the study and had "mild" or "no" pain at the start of the study. Plasma morphine concentrations of PF-402 were longer-lasting and showed smaller fluctuations than those of MSC. Repeated administration of the same daily doses of PF-402 and MSC produced similar plasma concentrations for periods of 24 hr and 12 hr. PF-402 administration produced a Tmax of 7.4 hr, and an MRT of 9.8 hr, all longer than those with MSC. Moreover, no significant difference was observed in AUC between PF-402 and MSC. These results indicate that the sustained-release characteristics of PF-402 are superior to those of MSC, and that the two drugs have a similar absorption pattern. Adverse drug reactions (ADRs) were observed in all 7 subjects and consisted of 6 incidences of constipation, 3 incidences of nausea, 2 incidences of itching, and 1 incidence each of vomiting and somnolence. Study drug administration was not discontinued in any case due to ADRs, and no symptoms indicating physical or psychic drug dependence were observed. No abnormal laboratory values related to study drug administration were observed. The above results indicate that once-daily administration of PF-402 is sufficient to maintain plasma concentrations obtained with twice-daily administration of MSC. As the safety of PF-402 is confirmed, the drug is considered to be a useful sustained release formulation in the treatment of cancer pain.

Adult↗
PubMed1997

Roles of hydrogen bonding residues in the interaction between the alpha and beta subunits in the tryptophan synthase complex. Asn-104 of the alpha subunit is especially important.

The interaction of the alpha subunit with the beta2 subunit of tryptophan synthase is known to be necessary for the activation of each subunit and for the catalytic efficiency of the alpha2beta2 complex. To elucidate the roles of hydrogen bonds in the interaction site between the alpha and beta subunits for subunit association, eight mutant alpha subunits at five hydrogen bonding residues (N104D, N104A, N108D, N108A, E134A, E135A, N157D, and N157A) were constructed, and the thermodynamic parameters of association with the beta subunit were obtained using a titration calorimeter. The N104D and N104A mutations remarkably decreased the stimulation activities, the association constants, and the association enthalpies. Although the association constant and the stimulation activities of E134A were reduced in the absence of salt, the change in the association enthalpy was relatively small, and the addition of salt could repair its defects. The substitutions at positions 135 and 157 did not affect the stimulation activity and decreased the Gibbs energy of association corresponding to the defect in 1 mol of hydrogen bond. The present results suggest that the alpha subunit which has a mutation at position 104 cannot fold into an intact conformation upon complex formation, resulting in reduced stimulation activities. The hydrogen bond with Asn-104, which is a conserved residue among 16 microorganisms, was especially important for alpha/beta interaction and mutual activation.

Escherichia coli↗
PubMed1997

Identification of reactive site of a proteinaceous metalloproteinase inhibitor from Streptomyces nigrescens TK-23.

Streptomyces metalloproteinase inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous metalloproteinase inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.

Bacterial Proteins↗
PubMed1997

Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor.

Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.

Binding Sites↗
PubMed1997

Isolation and some properties of sorbitol oxidase from Streptomyces sp. H-7775.

A sorbitol oxidase (SOX) was found in the cell-free extract of a strain isolated from soil. The strain was classified and designated as Streptomyces sp. H-7775. SOX is constitutively expressed in the cell. The molecular weight of SOX that purified from the cell-free extract was 45,000. The optimum pH and the K(m) for sorbitol were 6.5-7.5 and 0.26 mM, respectively. The prosthetic group was a covalently bound FAD. SOX catalyzed oxidation of D-sorbitol to glucose and hydrogen peroxide without any requirements of exogenous cofactors. SOX did not react with glucose, a reaction product of D-sorbitol. This feature is useful in its application for diagnosis.

Chromatography, Affinity↗