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Biomedical subjects

M Kitamura

Publications and source records attributed to M Kitamura.

At least 163 records · Page 9Linked to original sources
PubMed1997

c-Jun/AP-1, but not NF-kappa B, is a mediator for oxidant-initiated apoptosis in glomerular mesangial cells.

Oxidant stress is a trigger of cell death in various cell types. Hydrogen peroxide (H2O2) induced mesangial cell death with nuclear condensation and DNA fragmentation typical of apoptosis. To explore molecular mechanisms involved in this process, redox-sensitive transacting molecules, activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B), have been brought into focus. Northern blot analysis and transient transfection assays using reporter plasmids showed that H2O2 activated both AP-1 and NF-kappa B. Downregulation of c-Jun/AP-1 using a transdominant negative mutant of c-jun, an antisense c-jun, or a pharmacologic inhibitor curcumin inhibited the H2O2-initiated apoptosis. In contrast, inhibition of the NF-kappa B activation using a transdominant negative mutant of the p50 NF-kappa B subunit did not affect the H2O2-triggered cellular death. These data elucidated that c-Jun/AP-1, but not NF-kappa B, is involved in the oxidant-initiated cell death program in glomerular mesangial cells.

Animals↗
PubMed1997

Cloning and expression of the dog mast cell alpha-chymase gene.

Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.

Animals↗
PubMed1997

Cytokines promote glomerular mesangial cell survival in vitro by stimulus-dependent inhibition of apoptosis.

Resolution of glomerular inflammation requires the removal of proliferating resident glomerular mesangial cells, but excessive loss of glomerular cells is a feature of postinflammatory scarring. Because apoptosis regulates mesangial cell number in glomerular inflammation, we have studied the exogenous control of apoptosis triggered in cultured mesangial cells by stimuli likely to be important in vivo. Apoptosis could be induced by serum deprivation to model decreased availability of survival factors, by etoposide as an example of DNA-damaging agents, by ligation of mesangial cell Fas, and by protein synthesis inhibition by cycloheximide. Insulin-like growth factor I (IGF-I), IGF-II, and basic fibroblast growth factor were each able to suppress apoptosis induced by serum deprivation, whereas TGF-beta 1, epidermal growth factor, and platelet-derived growth factor had no effect. IGF-I and IGF-II (but not basic fibroblast growth factor) were also able to protect cells from apoptosis induced by etoposide or cycloheximide. However, Fas-mediated apoptosis was resistant to suppression by all three cytokines. None of the cytokines tested caused a change in the levels of expression of Bcl-2, Bax, Bcl-x, or Bak proteins. The survival-promoting properties of serum-free medium conditioned by mesangial cells was abrogated by neutralizing IGF-I Ab. These experiments are the first to define cytokines that inhibit apoptosis and thereby promote survival of mesangial cells, and the data indicate a paracrine survival signaling role for IGF-I. Finally, the data show that Fas ligation can override cytokine survival signaling, emphasizing a candidate role for this molecule in the undesirable apoptotic loss of mesangial cells during the progression of glomerular scarring.

Animals↗
PubMed1997

Creation of an In vivo cytosensor using engineered mesangial cells. Automatic sensing of glomerular inflammation controls transgene activity.

Automatic control over exogenous gene expression in response to the activity of disease is a crucial hurdle for gene transfer-based therapies. Towards achieving this goal, we created a "cytosensor" that perceives local inflammatory states and subsequently regulates foreign gene expression. alpha-Smooth muscle actin is known to be expressed in glomerular mesangial cells exclusively in pathologic situations. CArG box element, the crucial regulatory sequence of the alpha-smooth muscle actin promoter, was used as a sensor for glomerular inflammation. Rat mesangial cells were stably transfected with an expression plasmid that introduces a beta-galactosidase gene under the control of CArG box elements. In vitro, the established cells expressed beta-galactosidase exclusively after stimulation with serum. To examine whether the cells are able to automatically control transgene activity in vivo, serum-stimulated or unstimulated cells were transferred into normal rat glomeruli or glomeruli subjected to anti-Thy 1 glomerulonephritis. When stimulated cells were transferred into the normal glomeruli, beta-galactosidase expression was switched off in vivo within 3 d. In contrast, when unstimulated cells were transferred into the nephritic glomeruli, transgene expression was substantially induced. These data indicate the feasibility of using the CArG box element as a molecular sensor for glomerular injury. In the context of advanced forms of gene therapy, this approach provides a novel concept for automatic regulation of local transgene expression where the transgene is required to be activated during inflammation and deactivated when the inflammation has subsided.

Actins↗
PubMed1997

IL-1beta depresses expression of the 70-kilodalton heat shock protein and sensitizes glomerular cells to oxidant-initiated apoptosis.

The 70-kDa heat shock protein (hsp70) is induced by several physical stimuli, whereas little is understood about the regulation and function of this molecule during inflammation. We found that the proinflammatory cytokine IL-1beta depressed hsp70 expression in glomerular mesangial cells and that IL-1-pretreated cells were more susceptible to apoptotic death triggered by oxidant stress. To examine whether the altered expression of hsp70 causes the effect of IL-1beta on apoptosis, rat mesangial cells were stably transfected with a hsp70 cDNA under the control of a constitutively active regulatory element. Compared with mock-transfected cells, the established cells overexpressing hsp70 showed resistance to the effect of IL-1beta. The effects of IL-1beta on hsp70 expression and apoptosis were also examined in isolated rat glomeruli. Consistent with the results from mesangial cells, IL-1beta repressed the expression of hsp70 and enhanced the oxidant-initiated apoptosis in the glomerulus. The relationship between glomerular hsp70 and apoptosis was further investigated using an experimental model of anti-glomerular basement membrane glomerulonephritis in which IL-1 and oxidants play crucial roles. Compared with normal expression, the expression of hsp70 was significantly reduced in the inflamed glomeruli. Furthermore, the nephritic glomeruli exhibited oligonucleosomal DNA fragmentation typical of apoptosis. These findings suggested a novel mechanism by which IL-1 may induce glomerular injury. IL-1beta has the potency to affect intrinsic cytoprotective machinery and thereby sensitizes glomerular cells to oxidant-initiated apoptosis. We identified hsp70 as a potential molecular target in this process.

Animals↗
PubMed1997

In vivo transfer of engineered macrophages into the glomerulus: endogenous TGF-beta-mediated defense against macrophage-induced glomerular cell activation.

Communication between resident glomerular cells and infiltrating macrophages plays a crucial role in the pathogenesis of glomerular disease. Using matrix metalloproteinase-9 (MMP-9) as an indicator molecule, we examined the interaction between mesangial cells and macrophages. Mesangial cells cocultured with activated macrophages or exposed to macrophage-conditioned media produced abundant MMP-9. We identified the stimulator secreted by macrophages as IL-1 because mesangial cells overexpressing IL-1 receptor antagonist protein showed a blunted expression of MMP-9 in response to the macrophage-conditioned medium. In contrast, culture supernatants of mesangial cells inhibited MMP-9 production by macrophages in a dose-dependent fashion. This inhibitor was identified to be TGF-beta1, since neutralization of TGF-beta1 abrogated the inhibitory effect of the mesangial cell-conditioned medium. To investigate whether activated macrophages induce glomerular MMP-9 expression, and if so, how endogenous TGF-beta1 modulates the induction, stimulated reporter macrophages were transferred into normal rat glomeruli or glomeruli in the regeneration phase of acute anti-Thy-1 glomerulonephritis. In the normal glomeruli, MMP-9 expression was up-regulated in resident cells after the transfer of activated macrophages. This induction was substantially repressed in the regenerating glomeruli that produced active TGF-beta1. These results point to potential mechanisms involved in glomerular control of MMP-9. Based upon the in vitro evidence, TGF-beta1 was identified as an endogenous "defender" that attenuates certain actions of infiltrating macrophages in the glomerulus.

Animals↗
PubMed1997

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1.

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.

Animals↗
PubMed1997

Cloning and expression of the rubredoxin gene from Desulfovibrio vulgaris (Miyazaki F)--comparison of the primary structure of desulfoferrodoxin.

A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.

Amino Acid Sequence↗
PubMed1997

Direct measurement of doxorubicin concentration in the intact, living single cancer cell during hyperthermia.

BACKGROUND: It is well known that the effect of doxorubicin on cancer cells is enhanced by hyperthermia. The mechanism of this phenomenon is not fully understood. METHODS: Two esophageal squamous cell carcinoma cell lines, TE-2 and TE-6, were used; these cell lines have different sensitivities for doxorubicin. The cells were exposed to 1 microgram/mL of doxorubicin for 30 minutes. With a confocal laser scanning microscope and a transparent warming plate, doxorubicin concentration was measured continuously in the intact, living single cancer cells, and the two-dimensional distribution of the drug during hyperthermia (43 degrees C) was analyzed. RESULTS: A doxorubicin sensitivity difference was confirmed between TE-2 and TE-6 cells by colonogenic assay (P < 0.05). Hyperthermia increased the sensitivity of both cell lines to the drug (P < 0.05) and eliminated the sensitivity difference. Doxorubicin accumulated in the nuclei in both cell lines 30 minutes after exposure to the drug in a time-dependent manner (P < 0.05). Without hyperthermia, the doxorubicin concentration in the nuclei of the TE-2 cells (4.8 +/- 0.3 micrograms/mL) was higher than in the nuclei of the TE-6 cells (2.3 +/- 0.5 micrograms/mL) (P < 0.05). With hyperthermia, there was no significant difference in doxorubicin concentration between the nuclei of the TE-2 cells (20.8 +/- 1.3 micrograms/mL) and the nuclei of the TE-6 cells (16.5 +/- 3.9 micrograms/mL). CONCLUSIONS: Hyperthermia increased the uptake of doxorubicin in the nuclei of cancer cells. Thus, the authors concluded that hyperthermia increases the cells' sensitivity to the drug.

Antibiotics, Antineoplastic↗
PubMed1997

Laparoscopic repair of a paraesophageal hiatus hernia without fundoplication.

We treated a case of paraesophageal hiatus hernia by laparoscopic repair. The procedure included a reduction of the gastric fundus and duodenal bulbus, closure of the diaphragmatic defect, mesh wrapping of the closure, gastropexy to the diaphragm, and a gastrostomy. Preoperative monitoring of the pH for 24 h showed no reflux. Intraoperative intraluminal manometry of the esophagus after hernia reduction showed the pressure of the lower esophageal sphincter to be normal, and thus an antireflux procedure was not deemed to be necessary. The patient was put on a soft diet from postoperative day 2. A postoperative upper gastrointestinal series showed no gastroesophageal reflux. No complications or recurrence of the hiatus hernia have been observed in the 12 months since the operation. Laparoscopic repair of a paraesophageal hiatus hernia with normal pressure of the lower esophageal sphincter, so that fundoplication is not needed, is thus considered to be possible.

Aged↗
PubMed1997

Mediastinal extraadrenal myelolipoma: report of a case.

We herein report a case of surgically resected mediastinal extraadrenal myelolipoma. Myelolipoma is an uncommon tumor composed of adipose tissue and normal hematopoietic elements, and is most often found in the adrenal glands. We could find only five such cases of mediastinal myelolipoma in the English literature.

Adipose Tissue↗
PubMed1997

Genetic manipulation of the kidney.

Successful gene transfer into specific renal structures allows for evaluation of in vivo effects of certain molecules on the structure and function of the kidney. It would also be useful for therapeutic intervention in renal diseases by introducing "beneficial" genes into the affected sites. Towards achieving these goals, several gene transfer approaches have been developed using retrovirus, adenovirus and liposome. By introducing these gene transfer vectors via particular access routes, it is feasible to selectively manipulate the function of certain renal structures. Through the renal circulation, exogenous genes can be targeted to the vasculature and glomerulus, and possibly to the proximal tubules. Using a retrograde approach via the urinary tract, access to the collecting ducts can be gained. Implantation of genetically modified cells under the capsule of the kidney allows for diffusion of transgene products into the interstitium. Transplantation of embryonic metanephric tissues also provides a biological window for genetic manipulation. Furthermore, utilisation of fertilised eggs or embryonic stem cells would enable the creation of "transgenic kidneys" or "gene knockout kidneys". This article summarises the current experience with gene transfer to the kidney and addresses the potential strategies in vivo.

Animals↗
PubMed1997

Effects of selective cyclooxygenase-2 inhibitors on alkaline secretory and mucosal ulcerogenic responses in rat duodenum.

Effects of the selective cyclooxygenase-2 (COX-2) inhibitors such as NS-398 and nimesulide on duodenal HCO3- secretory and ulcerogenic responses to mucosal acidification were examined in rats, in comparison with indomethacin, a nonselective COX inhibitor. Duodenal HCO3- secretion in anesthetized rats was increased in response to mucosal acidification. The increased HCO3- response to acid was significantly suppressed by pretreatment with indomethacin (10 mg kg(-1), s.c.), while both NS-398 and nimesulide (10 mg kg(-1), s.c.) had no effect on this response. The luminal release of prostaglandin E2 (PGE2) was increased during and after mucosal acidification, and this response was significantly inhibited by indomethacin but not NS-398 or nimesulide. Indomethacin provoked hemorrhagic lesions in the duodenum when acid hypersecretion was concomitantly induced by histamine (8 mg kg(-1) hr(-1), i.v.), while either NS-398 or nimesulide did not cause damage in the duodenum. Either of these drugs had no effect on histamine-induced acid secretion. On the other hand, both NS-398 and nimesulide showed a significant suppression against carrageenan-induced rat paw edema, similar to indomethacin. The present study supports a mediator role for endogenous PGs in duodenal HCO3- secretion in response to mucosal acidification and suggests that COX-1 but not COX-2 is a key enzyme in regulating this process and maintaining the mucosal integrity against acid in the duodenum.

Animals↗
PubMed1997

Possible association of infertility with sperm-specific abnormality of CD46.

Three infertile patients fulfilling normal or subnormal criteria on routine semen analysis showed abnormal sperm CD46 (membrane cofactor protein of complement) by SDS-PAGE/immunoblotting analysis using a panel of monoclonal antibodies. The sperm CD46 isoform has been reported to be associated with sperm-egg interaction. These three patients expressed normal CD46 isoforms on their lymphocytes and granulocytes. Sperm-specific abnormalities in these proteins thus parallel male infertility, suggesting a new category of infertility, probably due to aberrations in the molecules related to sperm-egg interaction.

Acrosome↗
PubMed1997

Transition of serum vitellogenin cycle in Sakhalin taimen (Hucho perryi).

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 micrograms/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E2). The appearance and levels of Vg in males treated with E2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 micrograms/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E2 levels were correlated with Vg levels. However, E2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years.

Animals↗
PubMed1997

Increase in the circulating level of hepatocyte growth factor in gastric cancer patients.

We measured serum concentrations of hapatocyte growth factor (HGF) in patients with gastric cancer and compared these with the histological findings and conventional tumour markers, including CEA, CA19-9 and CA125, for evaluation of the significance of serum HGF levels as a tumour marker. The HGF levels were measured by an enzyme-linked immunosorbent assay (ELISA) system. The average levels of serum HGF in 89 healthy control subjects, 104 patients with primary gastric cancer and 15 patients with recurrent gastric cancer were 0.31 +/- 0.11 ng ml(1), 0.42 +/- 0.50 ng ml(-1) and 0.92 +/- 0.39 ng ml(-1) respectively. The average level in patients with recurrent disease was significantly higher than in healthy control subjects and in primary cancer patients (P< 0.001 and P< 0.003 respectively). Of 104 patients with primary gastric cancer, 35 (33.7%) showed an aberrant increase in the circulating level of HGF. The increased HGF levels were significantly associated with the degrees of histological tumour invasion and venous invasion. Of 15 patients with recurrent gastric cancer, 14 (93.3%) showed an aberrant increase. No correlation was found between serum HGF levels and CEA levels, CA19-9 levels and CA125 levels. However, the rate of the aberrant increase in HGF levels was significantly higher than that of any other tumour markers, including CEA, CA19-9 and CA125, in primary gastric cancer patients. In conclusion, the circulating levels of HGF were elevated in approximately one-third of patients with primary gastric cancer, particularly in those with high grades of histological tumour invasion and venous invasion, and frequently in patients with distant metastases, suggesting that HGF might play important roles in the tumour progression of gastric cancer. Furthermore, serum HGF levels may be of value as a tumour marker in patients with gastric cancer.

Adult↗