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Biomedical subjects

K Shirabe

Publications and source records attributed to K Shirabe.

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

An in-frame deletion of codon 298 of the NADH-cytochrome b5 reductase gene results in hereditary methemoglobinemia type II (generalized type). A functional implication for the role of the COOH-terminal region of the enzyme.

The nucleotide sequence was determined for the gene of NADH-cytochrome b5 reductase of a patient of type II hereditary methemoglobinemia found in Yokohama, Japan. An in-frame deletion of 3 base pairs corresponding to codon 298 (TTC) was identified in the patient. The patient was homozygous for the mutation as shown by hybridization experiments using allele-specific oligonucleotides. The mutation causes deletion of Phe-298, which is the third to the COOH-terminal residue, indicating that in this mutant enzyme the sequence of this region has changed from -Cys-Phe-Val-Phe-COOH to -Cys-Val-Phe-COOH. The mutant enzyme, whose Phe-298 was deleted (F298 delta), was prepared by means of a bacterial expression system and site-directed mutagenesis. The kcat/Km value (NADH) of the enzyme was 5.7 s-1 M-1, which corresponds to 0.4% of that of the wild type. Moreover, the enzyme was much less thermostable than the wild type. To examine further the role of the COOH-terminal portion of the enzyme, various mutant enzymes were also prepared and characterized. The enzymatic properties of F298L, F300L, and F298L/F300L were essentially the same as that of the wild type. The kinetic properties of F298A, and F300A were not greatly affected, but the stability of the enzymes was somewhat impaired. Since Val-299 is naturally Ala in steer enzyme, no specific residues in the carboxyl-terminal region (298-300) are essential to the enzyme function. The instability of the F298/F300A double mutant indicates that the hydrophobicity of the carboxyl-terminal region of the enzyme might be important to maintain the conformation of the enzyme. high impairment of the activity of the F298 delta, F298stop, and F300stop mutants might be caused by the loss of the residue(s) in the carboxyl-terminal portion. These results indicate that the hydrophobicity, but not the specific amino acid residues, of the carboxyl-terminal portion of the enzyme is important for the stability of the enzyme.

Adolescent↗
PubMed1994

Cloning and nucleotide sequence of a cDNA of the human erythrocyte NADPH-flavin reductase.

A cDNA of human erythrocyte NADPH-flavin reductase was cloned from a lambda gt 11 human reticulocyte cDNA library by polymerase chain reaction using degenerate primers and followed by a plaque hybridization. The nucleotide sequence of the cDNA contains an open reading frame of 621 base pairs which encodes 206 amino acid residues including an initial methionine. The amino acid sequence deduced from the base sequence coincided well with peptide sequence determined for the purified human erythrocyte NADPH-flavin reductase. A homologous sequence to the FMN-binding site of flavodoxins was found at the amino-terminal region.

Amino Acid Sequence↗
PubMed1994

Detection of hepatitis C virus RNA in the ultrasonic dissector irrigating solution used in liver surgery.

Hepatitis C virus (HCV) RNA was detected in the irrigating solution aspirated by the ultrasonic dissector system used in liver surgery with a reverse transcription polymerase chain reaction technique and was then compared with HCV RNA in the serum to clarify the risk of viral transmission to hospital personnel. The positivity of HCV RNA in the aspirated irrigating solution was approximately equal in all patients whose serum was positive for HCV RNA. However, viral RNA was not detected in the aspirated irrigating solution of any patient who was seronegative for HCV RNA. These results indicate that it is necessary to be cautious regarding the transmission of HCV during liver surgery when using the ultrasonic dissector in HCV RNA seropositive patients, because the irrigating solution aspirated by this device appears to be highly infectious.

Aged↗
PubMed1994

The prediction of portal pressure: a multivariate analysis of clinical data and intraoperative portal pressure.

Portal pressures were estimated non-invasively in 100 patients who underwent hepatic resection and completely fulfilled the 21 variables evaluated. Ten variables were selected from among all those in the univariate analysis, and a stepwise discriminant analysis revealed four independent significant variables, namely: The indocyanine green dye retention test at 15 min (ICGR15); the prothrombin time index; the platelet count; and the globulin fraction. An equation to estimate the portal pressure was made using the coefficients in the analysis, the reliability of which was confirmed (r = 0.70484, P = 0.0001). The univariate analysis revealed ten significant variables to discriminate portal hypertension, defined as a portal pressure of over 200 mmH2O. A multiple logistic regression analysis of these variables revealed two independent variables, being ICGR15 and the platelet count. Thus, we consider that our equation for estimating portal pressure is potentially useful, and that the platelet count and ICGR15 are the most significant parameters in discriminating between the presence or absence of portal hypertension. Moreover, a platelet count of less than 120 x 10(3)/mm3 and an ICGR15 value of more than 15% correlated well with portal hypertension.

Adult↗
PubMed1994

The role of prostaglandins in hepatic resection.

The role of prostaglandins (PGs) and the efficacy of PGE1 were investigated in hepatic resection. Patients who underwent hepatic resection were randomly assigned to two groups consisting of a control (n = 5) and a PGE1 treatment group (n = 6). Then the amount of 6-keto PF1 alpha, thromboxane B2 and (TXB2) and PGE2 were serially measured both before and after hepatic resection. Regarding changes in the PGs, a remarkable increase in TXB2 during hepatectomy was demonstrated in both groups. In the control group, both 6-keto PF1 alpha and PGE2 showed only a slight increase both during and after the operation. Regarding the efficacy of PGE1 on liver damage, no significant difference in postoperative liver function was found, however the lipoperoxide level at postoperative day 3 in the PGE1 treatment group (1.9 +/- 0.4 nmol/ml) was significantly lower than that in the control (2.6 +/- 0.5 nmol/ml: P < 0.05). Judging from the remarkable increase of TXB2, a stable metabolite of TXA2 during hepatectomy, it may be suggested that TXA2 plays an important role in the development of liver damage during hepatic resection.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

Strategies for reducing blood transfusions in hepatic resection.

A comparison of 60 blood transfused and 71 nonblood transfused hepatic resection patients was done to evaluate strategies for reducing blood transfusions during hepatic surgery. There were no significant differences between the two groups with regard to preoperative laboratory data, except for prothrombin time and hematocrit value. The mean operative blood loss was 1990 ml and 760 ml in the blood transfused and nonblood transfused groups, respectively. A multivariate analysis suggested that the patient's body weight, preoperative prothrombin time, and operative blood loss independently predicted the need for intraoperative blood transfusion. Major postoperative complications developed more frequently in the blood transfused group than in the nonblood transfused group (31.7 vs. 11.3%, p < 0.005). These results suggest that the difference in operative blood loss between the two groups was related to the prolonged prothrombin time and a susceptibility for blood transfusion was found to exist particularly in patients with a lower hematocrit value as well as a lower body weight. Thus, the improvement of these preoperative laboratory data combined with avoiding the use of the hematocrit value as a determining factor for intraoperative transfusion could correspond to a reduction in operative blood loss, while curtailing the demands on blood bank facilities, and lowering the risk of postoperative complications.

Adult↗
PubMed1994

Indications for major hepatectomy in cirrhotic liver.

In an investigation of the indications for major hepatic resection of the cirrhotic liver, the records of 152 consecutive patients who had undergone a right hepatic resection between April 1985 and January 1991 were reviewed. A comparison of right hepatic lobectomy and right partial hepatectomy of the liver with no cirrhotic changes, revealed that postoperative values of serum glutamic pyruvic transaminase were significantly higher after right partial hepatectomy than after right lobectomy, despite the fact that there were no significant differences with respect to preoperative laboratory data, and there was a greater blood loss and total weight of the resected liver in patients receiving a right lobectomy as compared with those undergoing partial hepatectomy. These results suggest that in order to enable a more favorable recovery from hepatic resection, it is essential to avoid both mechanical damage and ischemic injury to the residual liver during hepatic surgery. A total of 77 patients underwent a partial hepatectomy of a cirrhotic liver, and among these patients, 16 patients had values of the indocyanine green test of less than 20%, as well as a portal pressure of less than 200 mm saline. Compared with these 16 cirrhotic patients and those patients who underwent right lobectomy, there were no significant differences with regard to the pre-operative laboratory data and portal pressure. These results therefore suggest that major hepatic lobectomy could be performed on selected patients with cirrhotic livers.

Adult↗
PubMed1994

Effect of nafamostat mesilate on coagulation and fibrinolysis in hepatic resection.

The effect of nafamostat mesilate on coagulation and fibrinolysis was investigated in a study of 22 patients with hepatocellular carcinoma who underwent a hepatic resection. The patients were divided into two groups: group 1, control (n = 11) and group 2, those with the intraoperative and postoperative use of nafamostat mesilate (0.2 to 0.4 milligram per kilogram per hour, n = 11). Nafamostat mesilate tended to suppress the coagulation expressed by thrombin-antithrombin III complex and fibrinopeptide A both during and immediately after operation. Moreover, nafamostat mesilate significantly suppressed the fibrinolysis expressed by euglobulin lysis activity both during and after operation. With regard to the initial stage of the fibrinolytic system, such as tissue-type plasminogen activator and plasminogen activator inhibitor-1, there was no difference between the groups. Therefore, the suppression of the euglobulin lysis activity may be caused by the inhibition of plasmin activity. There was no difference between the groups regarding operative blood loss. However, the rate of blood transfusion in group 2 was lower than that in group 1, and no fresh frozen plasma was required for the patients who lost over 2,000 milliliters of blood. Nafamostat mesilate can suppress euglobulin lysis activity both intraoperatively and postoperatively, and thus decrease the amount of blood transfusion needed. Therefore, at present, nafamostat mesilate seems to be one of the most useful agents for stabilizing the coagulant and fibrinolytic systems in hepatic resection.

Aged↗
PubMed1993

Role of Lys-110 of human NADH-cytochrome b5 reductase in NADH binding as probed by site-directed mutagenesis.

Lys-110 of human NADH-cytochrome b5 reductase was replaced by Ala, Met, or Arg by site-directed mutagenesis to evaluate the role of the residue. Km values of purified Lys-110-->Ala and Lys-110-->Met mutants for NADH were approximately 200-fold and 1,100-fold higher than that of the wild-type, respectively, while the value of the Arg mutant was almost the same as that of the wild-type. These results indicate that the positive charge at position 110 is important for NADH binding. The kcat value of Lys-110-->Ala was not affected, indicating that the residue only participates in the binding process in the reaction by forming an ionic interaction with phosphoryl group of NADH.

Base Sequence↗
PubMed1993

A comparative study of hepatic resection and transcatheter arterial embolization for the treatment of primary hepatocellular carcinoma.

BACKGROUND: Because transcatheter arterial embolization (TAE) has been shown to be effective in patients with unresectable hepatocellular carcinoma (HCC), the question arises whether TAE has an equivalent or superior effect on resectable HCC compared with surgery. To clarify this point, the authors compared the therapeutic results achieved by surgery and TAE in patients with early-stage HCC and well-preserved liver function, who were independently treated by two different policies in two separate departments of the same university hospital during the same period. METHODS: From 1983 to 1987, 67 patients with HCC underwent hepatic resection at the Department of Surgery of Kyushu University Hospital. During the same period, TAE was the treatment of first choice for HCC, and surgical resection was not chosen in the Department of Internal Medicine of the hospital. TAE was done in 68 patients, who were evaluated blindly in terms of liver function reserve and roentgenographic resectability of the tumor. Among the 68 patients, 20 were thought to have anatomically and functionally resectable disease. The therapeutic results obtained in the two groups were compared. RESULTS: The 1-year, 3-year, and 5-year cumulative survival rates for 67 patients undergoing surgery, including one operative death and four hospital deaths, were 89.1%, 74.6%, and 54.6%, respectively. However, the rates were 90.0%, 50.0%, and 17.5%, respectively, for the 20 patients treated with TAE; these differences were statistically significant (P < 0.05). CONCLUSIONS: Surgery can offer more favorable results in patients with early-stage HCC compared with TAE.

Carcinoma, Hepatocellular↗
PubMed1993

Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase.

A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu-148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

Adult↗
PubMed1993

Overproduction and purification of the colicin E1 immunity protein.

Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector. The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography. A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence. The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes. The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature.

Base Sequence↗
PubMed1993

Rapamycin: immunosuppression, hyporesponsiveness, and side effects in a porcine renal allograft model.

Rapamycin prolongs allograft survival and induces donor-specific tolerance in some small animal transplant models. Large animal studies, however, are limited. We studied rapamycin in a porcine renal allograft model. Donor-recipient combinations were chosen based on high response in pretransplant MLCs. Allografts were anastomosed to the aorta and vena cava and the native kidneys removed. There were 5 treatment groups: (a) no immunosuppression; (b) triple therapy (CsA, 1 mg/kg/day; AZA, 2-3 mg/kg/day; and PRED, 3-4 mg/kg/day); (c) rapamycin (0.75 mg/kg/day i.m.) in carboxymethylcellulose (CMC); (d) rapamycin (0.25 mg/kg/day i.m. in CMC); and (e) a vehicle (CMC) control. Serum creatinine levels were determined every other day. Most allografts were biopsied once a week. Immunosuppression was stopped after 30 days. Mean graft survival in nonimmunosuppressed recipients was 6.8 +/- 3.6 days. Mean graft survival in triple therapy recipients (n = 10) was 45.7 +/- 36 days vs. 59.6 +/- 11.4 days in rapamycin (0.25 mg/kg/day) recipients (n = 7) (P = 0.51). Both triple therapy and rapamycin improved renal allograft survival versus nonimmunosuppressed controls (P = 0.0025 and 0.001, respectively). Serum creatinine levels were significantly lower (P < 0.05) in rapamycin versus triple therapy recipients. We conclude that rapamycin is a potent immunosuppressant in a porcine renal allograft model and may avoid the elevated serum creatinine levels associated with CsA.

Animals↗