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Biomedical subjects

T Kokubu

Publications and source records attributed to T Kokubu.

At least 37 records · Page 2Linked to original sources
PubMed1994

Differences in cardiac function changes between diabetic and non-diabetic hemodialysis patients.

The main cause of death in chronic hemodialysis patients is heart failure. We studied cardiac function prospectively in diabetic and non-diabetic patients who had recently been started on hemodialysis. We analyzed the mechanocardiograms and echocardiograms in addition to routine blood chemistry tests, chest X-rays, and electrocardiograms of ten hemodialysis patients at our hospital from 1989 to 1991. Ejection fraction (EF) and fractional shortening (FS) were not significantly different between diabetic patients and non-diabetic patients at the start of hemodialysis. After starting hemodialysis, EF and FS declined in diabetic patients. In contrast, non-diabetic patients did not show any changes in EF and FS during hemodialysis. The results demonstrated a poor prognosis in cardiac systolic function in hemodialysis patients with diabetes mellitus.

Adult↗
PubMed1993

Immobilization of dihydrofolate reductase by engineered cysteine residue attached to its C-terminal end.

A cysteine residue with a short amino acid chain spacer was attached to the C-terminal end of mutant dihydrofolate reductase [DHFR(C152E)] by a recombinant DNA technique. By using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a SH reagent, it was determined that the reactivity of the SH group of the introduced C-terminal cysteine was much higher than that of cysteines with the wild-type DHFR (C85 and C152), probably due to an increase of the solvent accessible surface area of the SH group. By utilizing the increased reactivity of the SH group of the C-terminal cysteine, the engineered DHFR was effectively immobilized to a thiopropyl-Sepharose gel. The amount of immobilized DHFR was estimated to be ca. 6 mg/g of dried gel, and the activity of the bound DHFR was comparable to that of the free enzyme. Thus, the attachment of the cysteine residue to the C-terminal was extremely useful for immobilization of the enzyme.

Amino Acid Sequence↗
PubMed1993

Immunological application of the dihydrofolate reductase handle carrying a small peptide, leucine enkephalin.

The "dihydrofolate reductase (DHFR) handle" [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45; Iwakura, M. & Tanaka, T. (1992) J. Biochem. 111, 638-642] fused with a pentapeptide, leucine enkephalin (LEK), has been applied in immunoassays for LEK and for the preparation of anti-LEK monoclonal antibody. DHFR fused with LEK (DHFR-LEK) was first utilized as an immobilized antigen in an enzyme-linked immunosorbent assay for LEK. By using a commercially available anti-LEK and peroxidase-linked anti-IgG, LEK could be quantified in the range between 0.1 ng/ml and 10 micrograms/ml. By using a commercially available anti-LEK and the DHFR-LEK as an enzyme-labeled antigen, LEK was quantified in the range between 0.1 ng/ml and 1 microgram/ml by monitoring the recovery of the DHFR activity from the immuno-precipitates. By using the DHFR-LEK as an immunogen, three mouse monoclonal antibodies against LEK, but not DHFR, were isolated. All three monoclonal antibodies were of IgG1 kappa type. The large-scale preparation of two of these monoclonal antibodies, designated as anti-LEK-36 and anti-LEK-74, was carried out and their recognition specificities were studied by competitive binding assays. The IC50 values of LEK for the anti-LEK-36 and anti-LEK-74 were 3.74 x 10(-6) and 4.66 x 10(-6) M, respectively. The competitive binding assays showed that recognition specificities of the two monoclonal antibodies were high and restricted to LEK and leucine-enkephalin (sulfated form). These results strongly suggest that the DHFR handle is useful in several immunological applications.

Amino Acid Sequence↗
PubMed1993

Isolation of reovirus type 3 from dogs with diarrhea.

Three virus strains were isolated in DK cell cultures inoculated with fecal specimens of dogs manifesting diarrhea. The isolates were identified as reoviruses on the basis of their biological and physico- chemical properties. They possessed reovirus type 3 antigenic specificity revealed by hemagglutination-inhibition and neutrarization tests with the reovirus prototype strains. It was suspected that the isolates were participated in this diarrheic cases.

Animals↗
PubMed1992

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.

Amino Acid Sequence↗
PubMed1992

Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.

Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.

Acetates↗
PubMed1992

Increased proportions of peripheral blood gamma delta T cells in patients with pulmonary tuberculosis.

There is a small population of peripheral T cells bearing the gamma delta T-cell receptor, which may be involved in the defense against invading microorganisms and tumor cells. The present study was designed to evaluate the levels of gamma delta T cells in patients with pulmonary tuberculosis, bacterial pneumonia, chronic lower respiratory tract infection, lung cancer, and normal control subjects with or without old tuberculous lesion. The results showed that only patients with tuberculosis had significantly increased proportions of peripheral blood gamma delta T cells. This study suggests that the increased proportions of gamma delta T cells in tuberculosis could be related to T-cell activation by Mycobacterium tuberculosis, although it remains to be investigated which components of mycobacteria are the major ligands for gamma delta T cells.

Adolescent↗
PubMed1991

Kidney renin gene expression after renin inhibition in the marmoset.

1. The effects of renin inhibitor ES-8891 on renin synthesis and its secretion by the kidney were investigated in normotensive sodium-depleted marmosets. We measured plasma renin activity, plasma immunoreactive renin concentration, plasma angiotensin II concentration and kidney renin mRNA content after oral administration of ES-8891 (60 mg day-1 kg-1) for 1 week. 2. The mean blood pressure was significantly decreased (P less than 0.01) on day 7 after oral administration of ES-8891. There was no significant change in heart rate during the administration. 3. Oral administration of ES-8891 for 1 week markedly decreased the plasma renin activity, the plasma immunoreactive renin concentration and the plasma angiotensin II concentration (to 18%, 41% and 24% of the corresponding control values; P less than 0.05 for each, n = 5). 4. The kidney renin mRNA content in ES-8891-treated marmosets was significantly lower than that in normal controls (4.2 +/- 3.5 versus 12.8 +/- 5.5 pg/micrograms of total RNA, means +/- SD, P less than 0.05, n = 5). 5. Oral administration of the renin inhibitor ES-8891 for 1 week not only inhibited plasma renin activity but also decreased renin synthesis and its secretion by the kidney.

Angiotensin II↗
PubMed1991

Clinical evaluation of delapril in Japan. Report from the Japan Study Group on Delapril.

Delapril, a new angiotensin converting enzyme (ACE) inhibitor discovered in the laboratory of Takeda Chemical Industries, Ltd., is the result of drug design based on the structure-activity relationships of ACE inhibitors. Delapril is an antihypertensive agent with a relatively long duration of action and no SH moiety in its structure. Following administration, it is converted into two active metabolites. Delapril effectively lowered blood pressure in 73% of 1,008 patients with hypertension during clinical trials in Japan. Efficacy rates were 73% for essential hypertension, 85% for renal hypertension, and 80% for renovascular hypertension. Excellent hypotensive response was observed in all age groups, from young to elderly patients. Side effects during administration of delapril, based on subjective evidence, were reported in 80 out of the 1,008 cases (7.9%). The main symptoms included orthostatic dizziness (1.7%), dizziness (1.3%), and nausea (1.1%). Dry cough, which has attracted attention in recent years as a side effect of ACE inhibitors, was reported at a low incidence of 1.1%. In a double-blind, controlled study in patients with mild to moderate essential hypertension in which captopril served as a positive control, delapril showed superior hypotensive effect and greater safety. Data derived from the Japan Study Group on Delapril indicate that this ACE inhibitor has excellent hypotensive effects and a high level of safety. It is suitable as a first-line drug in both monotherapy and combined therapy.

Adolescent↗
PubMed1991

Characteristics of systolic time intervals in patients with pheochromocytoma.

The effect of chronic catecholamine excess on cardiac function was assessed in 8 patients with surgically proven pheochromocytoma and the results compared with data obtained from normal controls and essential hypertensives. Major findings in systolic time intervals (STI) in patients with pheochromocytoma were a marked shortening of electromechanical systole and left ventricular ejection time (ET), but pre-ejection period (PEP) remained within normal limits. These findings were not altered by correction for heart rate. The ET/PEP ratio was very low (1.87 +/- 0.31) due to the remarkable shortening of ET. The ET/PEP ratio in essential hypertensives was also low (1.77 +/- 0.38), but this was mainly due to a remarkable prolongation of PEP. Low cardiac index, low stroke index and high total peripheral resistance index were preoperative characteristics in patients with pheochromocytoma, but returned to normal after operation. These results suggest that chronic excessive production of catecholamines from pheochromocytoma has deleterious effects on the heart, and that wide differences in STI exist between patients with pheochromocytoma and those with essential hypertension.

Adrenal Gland Neoplasms↗
PubMed1991

Clinical efficacy of carvedilol in severe hypertension.

In an open clinical study, the efficacy and safety of carvedilol was investigated in 26 severely hypertensive patients controlled inadequately on a diuretic [diastolic blood pressure (DBP) greater than 120 mm Hg at first visit and greater than 110 mm Hg following more than 1 week administration of a diuretic]. Following diuretic treatment all patients were initially administered 5 mg of carvedilol once daily. The dose was gradually increased to 10 mg and 20 mg until DBP was reduced below 100 mm Hg or until it was reduced by at least 10 mm Hg. Antihypertensive activity of carvedilol (5 mg) was sufficient in only three cases, but after 4 weeks (inpatients) or 8 weeks (outpatients) administration of carvedilol (10 mg or 20 mg), DBP/systolic blood pressure was significantly reduced from 176 +/- 6/117 +/- 3 to 145 +/- 3/94 +/- 2 mm Hg (p less than 0.001) in all patients. Overall, a sufficient antihypertensive effect was observed in 80% of the patients. Heart rate was significantly decreased from 76 +/- 2 to 67 +/- 2 beats/min, but no patient experienced bradycardia. Carvedilol was generally well tolerated. These findings suggest that 10-20 mg of carvedilol once daily, in combination with a diuretic, is an effective and safe treatment for patients with severe hypertension.

Adrenergic beta-Antagonists↗
PubMed1991

Effects of small myelinated afferent fiber stimulation on the threshold of tail flick reflex in the rat.

Effects of A-delta afferent fiber stimulation on the response threshold in the tail flick reflex were examined in rats anesthetized with thiamylal sodium. In a statistical analysis, the suppressive effect on the skin temperature at the time of the tail flick (tail flick temperature) was represented as a percent of maximal possible effect (%MPE) expressing more clearly the degree of change of the tail flick temperature. A-delta afferent fiber stimulation produced a reduction of EMG activity of the tail muscle, but no changes of both the tail flick latency and the tail flick temperature were observed. The results of the present study and the previously reported modulation system in the spinal cord dorsal horn are discussed.

Action Potentials↗
PubMed1990

Effect of SQ29,852, a new angiotensin converting enzyme (ACE) inhibitor with a phosphonic acid group, on the activity of angiotensin converting enzyme from human kidney.

1. An in vitro experiment was carried out to compare the inhibitory effect of SQ29,852 on human renal angiotensin converting enzyme (ACE) with those of captopril, enalapril and enalaprilat. 2. SQ29,852 strongly inhibited human renal ACE; its IC50 value was 1.5 x 10(-8) M. In terms of the IC50, SQ29,852's efficacy was about 1/10 of that of captopril and 1/28 of that of enalaprilat, but it was about 14 times more potent than enalapril. 3. SQ29,852 showed no inhibitory effects on cathepsin D, urinary kallikrein, renal renin, pepsin, trypsin and chymotrypsin. Its ACE-specificity was higher than that of captopril. 4. ACE inhibition by SQ29,852 was shown to be competitive, as revealed by Lineweaver-Burk plots. The affinity of SQ29,852 to ACE was shown to be high by a Ki value of 1.2 x 10(-8) M.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1990

Clinical significance of systolic time intervals in hypertensive patients.

This paper updates the current view on clinical significance of systolic time intervals (STI) in estimating the cardiac changes associated with hypertension. The following three intervals were measured as STI: (1) electromechanical systole (QS2 interval); (2) left ventricular ejection time (LVET) and (3) pre-ejection period (PEP). Firstly, the influences of changes in heart rate, preload, afterload and myocardial contractility upon each interval were reviewed; secondly, clinical applications of STI in various types of hypertension such as essential hypertension, hypertension with angina pectoris and pheochromocytoma were studied. In patients with essential hypertension, there was a good positive correlation between PEP and left ventricular mass, and a shortening of LVET was observed only at the decompensated stage. The changes in STI in angina pectoris with or without hypertension were similar and were different from those in essential hypertensives. STI in patients with pheochromocytoma were characterized by a marked shortening of QS2 and LVET with normal PEP. These findings indicate the usefulness of STI in detecting cardiac changes in various types of hypertension.

Adrenal Gland Neoplasms↗
PubMed1990

The effect of the renin inhibitor ES-1005 on the expression of the kidney renin gene in sodium-depleted marmosets.

The effect of the renin inhibitor ES-1005 or captopril on the expression of the kidney renin gene was investigated in sodium-depleted marmosets. We measured the level of kidney renin messenger RNA (mRNA) after continuous administration of ES-1005 (48 mg/kg per day) or captopril (2 mg/kg per day) intraperitoneally, via an osmotic mini-pump, for one week. The level of kidney renin mRNA was measured by densitometric Northern blot analysis using an alpha-32P-labelled human renin cDNA fragment as the hybridization probe. Captopril treatment markedly increased plasma renin activity and the level of kidney renin mRNA by 4.7-fold and 6.3-fold, respectively. ES-1005 treatment completely inhibited plasma renin activity and significantly decreased the level of kidney renin mRNA (46% of the normal control P less than 0.01). However, plasma immunoreactive renin concentration was significantly increased by the treatment with ES-1005 (P less than 0.05). These results suggest that the treatment with the renin inhibitor ES-1005 for one week has a paradoxical effect on kidney renin gene expression and renin release from the kidney in sodium-depleted marmosets.

Animals↗