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Biomedical subjects

T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 253 records · Page 14Linked to original sources
PubMed1997

The origin of an EPR signal observed in dithiocarbamate-loaded tissues. Copper(II)-dithiocarbamate complexes account for the narrow hyperfine lines.

We examined the electron paramagnetic resonance spectra of some copper(II)-dithiocarbamate complexes in various media at liquid nitrogen temperature. It is demonstrated that Cu(II)-dithiocarbamate complexes exhibit atypical spectra with narrow hyperfine splitting, and that the dithocarbamates forming a water-insoluble Cu(II) complex are permeable in tissues, while those yielding water-soluble complexes are not.

Animals↗
PubMed1997

Distribution of intramembranous particle size in the muscle plasma membrane of the mdx mouse.

We investigated whether the reduced intramembranous particles (IMP) in the muscle plasma membrane in mdx mice reflects a preferential depletion of a particular size of the IMP. The experiments were performed using the freeze-fracture method to analyze the frequency distribution of the size of IMP, the density of orthogonal array and caveolae in the extensor digitorum longus (EDL) and soleus (SOL) muscles obtained from mdx and control mice. We detected a reduced density of IMP and orthogonal array, and the increased density of caveolae in EDL muscle but not in SOL in the mdx mouse compared with those of the same muscles in control animals. The reduction of IMP was, however, not limited to any specific size of IMP. Our results suggest that the dystrophin associated glycoprotein present in the membrane does not reflect a specific size of IMP. Therefore, our findings indicate that the mechanism of reduced IMP in dystrophinopathy may be different from that of diminished dystrophin binding glycoprotein associated with dystrophin deficiency in Duchenne muscular dystrophy.

Animals↗
PubMed1997

Determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related compounds in biological fluids of infants with cholestasis by gas chromatography-mass spectrometry.

A method has been developed for the determination of 3-oxo-delta4- and 3-oxo-delta4,6-bile acids and related bile acids in biological fluids of infants by gas chromatography-mass spectrometry (GC-MS) of the methyl ester-dimethylethylsilyl ether-methoxime derivatives. The 7alpha-hydroxylated 3-oxo-delta4-bile acids were partially dehydrated to give the 3-oxo-delta4,6-bile acids by trimethylsilyl or dimethylethylsilyl derivatization and other pretreatments under acidic or alkaline conditions for GC-MS analysis. To prevent dehydration, the 3-oxo-delta4-bile acids were derivatized to the oximes by treatment with O-methylhydroxylamine prior to pretreatments such as solid-phase extraction, enzymatic solvolysis and hydrolysis of the conjugates, and silylation with dimethylethylsilylimidazole. Calibration curves for the bile acids were linear over a range of 5-250 ng and the detection limit was 100 pg for each 3-oxo-delta4-bile acid. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free urine and serum ranged from 94.2 to 105.9% of their added amounts. The bile acids in urine and serum of four patients with severe cholestatic liver disease were measured by the analytical method, and the 3-oxo-delta4-bile acids were determined to be the major bile acids (59-68%) in the urines associated with 3-oxo-delta4-steroid 5beta-reductase deficiency or dysfunction.

Bile Acids and Salts↗
PubMed1997

Antibody to Mac-1 or monocyte chemoattractant protein-1 inhibits monocyte recruitment and promotes tumor growth.

Human tumors are frequently infiltrated by numerous monocytes/macrophages, which can be found within the tumor mass (intratumoral) or surrounding the tumor (peritumoral). The functional role that these monocytes/macrophages play in tumor growth is controversial. To address this issue we inhibited intratumoral monocyte/macrophage recruitment with mAbs that either blocked integrin function or neutralized a tumor-produced chemotactic protein. Both treatments significantly increased tumor formation and accelerated tumor growth. Surprisingly, the same results were obtained when recruitment of peritumoral or intratumoral monocytes/macrophages was blocked. Our findings are contrary to one of the purported roles of monocytes/macrophages, particularly in the peritumoral area, since we found no evidence for monocyte/macrophage-supported tumor growth. These results provide direct evidence that intratumoral as well as peritumoral monocytes/macrophages act to limit tumor size in the early stages following tumor inoculation and provide a mechanism that accounts for monocyte/macrophage recruitment to human tumors.

Animals↗
PubMed1997

Simultaneous expression of ferredoxin, ferredoxin reductase and P450 in COS7 cells.

cDNA fragments encoding mouse ferredoxin and ferredoxin reductase were simultaneously introduced into COS7 cells by using an expression vector, pUC-SR alpha plasmid. When using the mitochondrial fraction prepared from the transfected cells, cytochrome-c reductase activity was detected. This activity was highest when 7.5 micrograms of the ferredoxin expression plasmid (pSR alpha F) and 2.5 micrograms of the ferredoxin reductase expression plasmid (pSR alpha FR) were transfected into COS7 cells. In this system, NADPH could be replaced by NADH as a cofactor for the reduction of cytochrome-c although the cytochrome-c reductase was more dependent on NADPH than NADH at a low concentration. When CYP24 expression plasmid was transfected into COS7 cells along with both pSR alpha F and pSR alpha FR, the transfected cells revealed a 3-fold higher 25-hydroxyvitamin D3-24-hydroxylase activity than COS7 cells transfected with CYP24 expression plasmid.

Animals↗
PubMed1997

The role of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of collagen-induced arthritis in rats.

Collagen-induced arthritis was produced in rats by intradermal immunization with type II collagen and the expression and production of monocyte chemoattractant protein-1 (MCP-1) were examined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Northern blot analysis. Two to three weeks after the immunization, the hindfeet showed swelling and redness, followed by the development of severe arthritis, particularly in the ankle joints. During this period, prominent infiltration of neutrophils and macrophages was observed. Sandwich ELISA and Northern blot analysis revealed that MCP-1 concentrations in the joint lavages and MCP-1 mRNA levels in the joint tissues both peaked at 2 weeks after the immunization. By immunohistochemistry, various types of cells, particularly neutrophils, macrophages, synovial cells, and vascular endothelial cells, stained positively for MCP-1. Finally, injection of a neutralizing monoclonal antibody against rat MCP-1 significantly decreased the number of exudate macrophages in the lesions and reduced the ankle swelling by about 30 per cent compared with controls. These results suggest that MCP-1 plays a critical role in this model in the recruitment of monocytes and in the development of arthritis.

Animals↗
PubMed1997

False aneurysm of the axillary artery caused by graft infection: report of a case.

A 77-year-old man presented to our hospital following the sudden onset of left subclavicular swelling, 8 years after undergoing subtotal removal of an axillofemoral bypass. Computed tomography revealed a rupture of the left axillary artery with the formation of a false aneurysm. Emergency surgery was performed in the form of an extraanatomic, axillo-axillary bypass with resection of the false aneurysm. Prior to removal of the false aneurysm, the subclavian artery was ligated via a left thoracotomy to minimize blood loss. The pathogen was identified as Proteus mirabilis, which is a rare causative organism for prosthetic graft infection. This case report demonstrates that total removal of the graft with revascularization should be performed for graft infections.

Aged↗
PubMed1997

A rapid, useful and quantitative method to measure telomerase activity by hybridization protection assay connected with a telomeric repeat amplification protocol.

Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. TRAP (the telomeric-repeat amplification protocol) developed by Kim et al. is a sensitive method to detect telomerase activity. Telomerase activity is detected by TRAP in most malignant cells in vivo and in vitro, but it is not found, or found only in very low amounts, in normal somatic cells and tissues. TRAP and its modified protocols are, however, not always suitable for measuring the activity of a large number of clinical samples to diagnose cancer, because they generally require a time-consuming detection step such as gel electrophoresis with radioactive materials. To improve the procedure for mass diagnosis, we applied a hybridization protection assay (HPA) to replace the detection step. HPA, which employs an acridinium-ester-labelled probe, is radioactivity-free, easy to handle without electrophoresis, quick, and applicable to a quantitative format. In this work we have established and demonstrated the advantages of TRAP/HPA. The telomerase activity of various primary and established cells, differentiating cancer cells, and normal and tumour colorectal and liver tissues was quantitatively analysed by TRAP/HPA. The results indicate that HPA combined with TRAP is a rapid and simple method, easy to handle and quantify, for the clinical diagnosis of cancer.

Cell Line↗
PubMed1997

Risk factors for methicillin-resistant Staphylococcus aureus (MRSA) infection in a Japanese geriatric hospital.

A case control study on MRSA infection was carried out, with the purpose of evaluating the effect of age, gender, hypoalbuminemia, the limitation of activities of daily living (ADL), the administration of antibiotics and the use of the new cephems which include third generation cephalosporins and monobactam and carbapenems, on the occurrence of MRSA infection among the inpatients in a geriatric hospital. From April 1991 to March 1994, 285 patients underwent a bacterial culture in the various clinical aspects. 118 patients were positive for MRSA, who were then used as cases while 167 patients who were negative for MRSA were used as controls. The level of serum albumin and the ADL score were lower in the MRSA group than in the non-MRSA group (P < 0.01) while the number of antibiotics administered before bacterial culture was greater in the MRSA group than in the non-MRSA group (P < 0.01). The third generation cephems were more commonly used in the MRSA positive patients than the negative patients (P < 0.01). Even after controlling for the other factors, hypoalbuminemia (OR = 1.73, 95% CI = 1.27-2.36), the limited ADL (partially limited vs without limitation: OR = 1.88, 95% CI = 1.19-2.96, completely limited vs without limitation: OR = 2.50, 95% CI = 1.64-3.82), the use of antibiotics other than the third generation cephems (vs without antibiotics: OR = 1.73, 95% CI = 1.20-2.50) and the administration of the third generation cephems (vs without antibiotics: OR = 3.12, 95% CI = 2.16-4.50) increased the risk of MRSA infection.

Aged↗
PubMed1997

Investigation of the mineralocorticoid and hypertensinogenic activity of 18-hydroxycortisol in conscious sheep.

The increased urinary excretion of 18-hydroxycortisol (18-OHF) in patients with primary aldosteronism has raised the possibility that 18-OHF is involved in the maintenance and/or pathogenesis of the associated hypertension. This study has investigated the mineralocorticoid, glucocorticoid, and hypertensinogenic activities of 18-OHF in the conscious sheep. Infusion of 18-OHF (400 micrograms/h i.v. 5 days; n = 5) alone had no effect on blood pressure or on fluid and electrolyte balance. Infusion of a combination of five adrenal steroids (aldosterone 3 micrograms/h, cortisol 5 mg/h, corticosterone 0.5 mg/h, 11-deoxycortisol 1 mg/h and deoxycorticosterone 25 micrograms/h, i.v. 5 days; n = 5) increased blood pressure by 14 +/- 1 mmHg (p < .001), but when 18-OHF was infused together with the five adrenal steroids, no additional increase in blood pressure was observed. In another group of sheep (n = 4) 18-OHF was infused at a range of doses (5, 50, 100, 200, 500, and 1000 micrograms/h i.v.), each for 2 h, into sodium-replete and sodium-deplete, adrenalectomized sheep. 18-OHF had no effect on the urinary sodium or potassium excretion or on the salivary Na/K ratio in either group as compared with vehicle infusion. To examine the renal effects of 18-OHF, a range of doses of 18-OHF (5, 50, 100, 200, 500, and 1000 micrograms/h) were infused directly into the renal artery of conscious sheep (n = 4). 18-OHF did not affect the renal blood flow nor the urinary sodium or potassium excretion compared with vehicle infusion. In summary, we could not demonstrate any mineralocorticoid, glucocorticoid, or hypertensiongenic effects of 18-OHF in conscious sheep at a dose of 400 micrograms/h. Thus, a cautious approach to interpreting the role that 18-OHF plays in the clinical manifestations of primary aldosteronism, is necessary.

Adrenalectomy↗
PubMed1997

Effects of cAMP-phosphodiesterase isozyme inhibitor on cytokine production by lipopolysaccharide-stimulated human peripheral blood mononuclear cells.

1. The effects of cAMP-phosphodiesterase (PDE) isozyme inhibitors on the production of tumor necrosis factor alpha (TNF-alpha), and interleukins 1 beta 8 (IL-1 beta and IL-8) by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) were evaluated. In addition, we investigated the effects of dibutyryl cAMP (dbcAMP) and beta-adrenergic receptor agonist on the production of these cytokines. 2. Type IV PDE inhibitors were more effective at inhibiting the production of TNF-alpha and IL-1 beta by LPS-stimulated PBMC than a nonselective, type III or type III/IV inhibitor. In contrast, these agents had no effect on IL-8 production. 3. Increasing concentrations of dbcAMP progressively reduced the production of TNF-alpha and IL-1 beta but not IL-8. 4. The addition of beta-agonist increased the inhibitory effect of PDE inhibitors tested on the production of TNF-alpha and IL-1 beta. 5. Type IV PDE inhibitors could be potent pharmacological agents for the treatment of diseases in which TNF-alpha and IL-1 beta are important etiological factors.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

In vivo ESR-CT imaging of the liver in mice receiving subcutaneous injection of nitric oxide-bound iron complex.

ESR measurements and ESR-CT imaging of subcutaneously (SC) injected nitric oxide (NO)-bound iron complexes were conducted on the upper abdomen of live mice. The use of NO-bound iron complex with N-(dithiocarboxy)sarcosine resulted in a clear ESR-CT image showing high intensity areas in the ventral regions, while other NO-bound iron complexes with N-methyl-D-glucamine dithiocarbamate or N,N-diethyl-dithiocarbamate were inappropriate because of low S/N ratios. To investigate the distributions of SC injected NO-bound iron complexes in the abdomens of mice, we measured ESR signals in resected abdominal organs. The signal amplitude was higher in the resected liver than in the resected gastrointestinal organs or the blood samples. The findings suggest that the high intensity areas in the ESR-CT images thus obtained correspond to the liver.

Animals↗
PubMed1997

Chemokine production by human vascular smooth muscle cells: modulation by IL-13.

1. The production of chemokines by vascular smooth muscle cells (SMC) is implicated in the pathogenesis of atherosclerosis, although the factors regulating chemokine production by these cells are incompletely characterized. 2. We describe the differential stimulation of interleukin-(IL)-8, monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T-cell expressed and secreted (RANTES) synthesis following treatment of human vascular SMC with IL-1alpha or tumour necrosis factor alpha (TNFalpha). Under basal conditions, cultured SMC release very low amounts of IL-8, MCP-1 and RANTES as assessed by specific ELISA. Concentration-response studies with IL-1alpha or TNFalpha revealed that each stimulus induced a similar amount of MCP-1. In contrast approximately three fold more IL-8 was induced by IL-1alpha than by TNFalpha whereas significant RANTES production was induced only by TNFalpha. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL-1alpha or TNFalpha stimulation. 3. The T-cell derived cytokines IL-10 and IL-13 were also found to have differential effects on chemokine production by SMC. IL-13, but not IL-10, significantly enhanced IL-8 and MCP-1 release in response to IL-1alpha or TNFalpha. This increase in chemokine release appeared to be accounted for by increased mRNA expression. 4. These findings provide support for the concept that smooth muscle cells can have an active role in a local immune response via the production of chemokines which can be selectively modulated by T-cell derived cytokines.

Cells, Cultured↗
PubMed1997

Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Amino Acid Sequence↗
PubMed1997

Cloning and expression of the glutamate racemase gene of Bacillus pumilus.

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.

Amino Acid Isomerases↗
PubMed1997

Mutation of arginine 98, which serves as a substrate-recognition site of D-amino acid aminotransferase, can be partly compensated for by mutation of tyrosine 88 to an arginyl residue.

D-Amino acid aminotransferase is the only aminotransferase that catalyzes the transamination of D-amino acids. We studied the role of the binding site for the alpha-carboxyl group of substrates, which is presumably crucial for the unique stereospecificity of the enzyme. The site-directed mutagenesis of Arg98, which is the putative carboxyl-binding site, as judged on the basis of X-ray crystallographic studies [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669], by replacement with methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, that of Tyr88, which is located near Arg98 in the spacial structure, by replacement with arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. These results suggest that Arg98 constitutes the carboxyl-binding site for the substrate, efficient catalysis by the enzyme being facilitated upon binding. The mutant enzymes are also relieved from inhibition by high concentrations of alpha-ketoglutarate, which is an inherent character of the wild-type enzyme. Therefore, Arg98 is also responsible for the inhibition by alpha-ketoglutarate.

Arginine↗