Biomedical subjects

T Koji

Publications and source records attributed to T Koji.

At least 55 records · Page 3Linked to original sources

PubMed1999

Analysis of Fas system in pulmonary injury of graft-versus-host disease after rat intestinal transplantation.

The lung is one of the primary targets of acute graft-versus-host disease (GVHD), which is the principal complication that occurs after allogeneic intestinal transplantation. The purpose of this study is to investigate the involvement of Fas/Fas ligand system in pulmonary injury after rat semi-allogeneic intestinal transplantation. The lungs were serially harvested from LEW x BN F1(LBNF1) recipients of either LEW heterotopic intestinal allografts or LBNF1 isografts, on days 1, 3, 5, 9, and 13 posttransplant. In light microscopy, pulmonary injury became apparent on day 13 in the allogeneic combination, showing a thickening of the alveolar septa. The incidence of apoptosis, examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling, was observed to increase steadily in the alveolar cells accompanied by a progression of GVHD. In an immunohistochemical study, Fas was constitutively expressed in the lung, although Fas ligand was expressed most extensively on day 9. The immunoreactivity of both Fas and Fas ligand were observed in alveolar cells, in addition to leukocytes. An analysis by reverse transcription polymerase chain reaction also revealed that the expression of Fas mRNA was constitutive without any significant change, although that of Fas ligand mRNA increased substantially and peaked on day 9, which was significant compared to the isogeneic combination. In conclusion, transcriptionally up-regulated Fas ligand and increased number of apoptosis suggests that the Fas system may play a role in the pathophysiology of GVHD-induced pulmonary injury.

Animals↗

PubMed1999

Analysis of the Fas system and Bcl-2 in rat liver allograft rejection.

BACKGROUND: Apoptosis is involved in the mechanism of cell death observed in liver allograft rejection. The liver cells are sensitive to Fas-mediated apoptosis; however, little is known about the involvement of the Fas system in liver allograft rejection. We used rat models to investigate the expression of Fas/Fas ligand and apoptosis-related proteins during liver allograft rejection. MATERIALS AND METHODS: DA rats to Lewis, and Lewis to Lewis orthotopic liver transplantation were performed; liver samples were collected on days 1, 3, 5, 7, and 9 postoperatively (each n = 3). Apoptosis was monitored by TUNEL and electron microscopy. The expression of Fas, FasL, bcl-2, and bax was examined at the mRNA level and by means of immunohistochemistry. RESULTS: The TUNEL index in the allografts and isografts on day 7 was 20.1 +/- 1.5 and 7.7 +/- 2.6/1000 cells, respectively. Fas and bax mRNA were constitutively expressed in both of the groups. The expression of Fas ligand mRNA in the allografts which rose on day 5 was 10 times stronger compared to that in the isografts. On the other hand, bcl-2 mRNA was generally expressed in the isografts while it decreased in the allografts. The immunohistochemical analysis also showed an increased reactivity of Fas ligand on day 5 in the allograft, which was observed both in parenchymal and nonparenchymal cells. CONCLUSIONS: These results strongly suggest that Fas/Fas ligand interaction mediates the liver injury during allograft rejection. In addition, other regulatory factors of apoptosis, such as bcl-2, might also be involved in this pathogenesis.

Animals↗

PubMed1999

A rat RuvB-like protein, TIP49a, is a germ cell-enriched novel DNA helicase.

We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg2+ for the helicase activity were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1999

Reciprocal changes in the expression of Bcl-2 and Bax in hypoglossal nucleus after axotomy in adult rats: possible involvement in the induction of neuronal cell death.

Numerous studies of neonatal neuronal development in mammals have revealed that neuronal cell death following axotomy is apoptotic in nature. In adult animals, however, neuronal cell death following axonal injury may or may not exhibit features of apoptosis. Bcl-2 and Bax have been identified as inhibitor and promoter proteins, respectively, of apoptosis. To investigate the relationship between these proteins and neuronal cell death following axotomy in adult animals, we performed axotomy of the right hypoglossal nerve in adult male Wistar rats, and sacrificed the rats at various intervals after axotomy. We analyzed the expression of Bcl-2 and Bax immunohistochemically in the hypoglossal nuclei of the adult rats following axotomy. Our analysis showed an increase in the percentage of Bax-positive motoneurons relative to the total number of motoneurons in the hypoglossal nucleus on the axotomy side at three days after axotomy. In contrast, a low percentage of Bcl-2-positive motoneurons to the total number of motoneurons was noted at the same time interval after axotomy. Quantitative analysis of the signal intensity for Bcl-2 and Bax in individual neurons showed that Bax immunostaining significantly increased 7 days after axotomy, while the intensity of Bcl-2 immunostaining decreased in most of Bcl-2-positive neurons. Our results confirmed the occurrence of motoneuron cell death in adult rats after axotomy, and that a close temporal relationship exists between the reciprocal changes in Bcl-2/Bax expression and the loss of motoneurons. These results indicate the possible involvement of the Bcl-2/Bax system in the induction of neuronal cell apoptosis after axotomy in adult rats.

Age Factors↗

PubMed1999

Prognostic significance of beta-microseminoprotein mRNA expression in prostate cancer.

BACKGROUND: Human beta-microseminoprotein (beta-MSP or PSP94) is a small protein secreted by prostatic epithelial cells. We recently reported the presence of low levels of beta-MSP mRNA expression and protein in most prostate cancer tissues. METHODS: Beta-MSP and mRNA expression was examined by in situ hybridization in biopsy specimens obtained from 92 patients with prostate cancer. All tissue specimens were obtained by needle biopsies prior to treatment. All patients subsequently received endocrine therapy. To estimate the influence of beta-MSP mRNA expression and three possible prognostic factors, i.e., patient age, clinical stage, and Gleason score, on time to progression under endocrine therapy, univariate and multivariate analyses were performed using Cox's proportional hazards regression model. RESULTS: Multivariate survival analysis showed that clinical stage was the strongest prognostic factor (P = 0.006) and that beta-MSP mRNA expression was the second strongest factor (P = 0.038) in 92 patients with stage B-D disease. Analysis of only 51 patients with stage D disease showed that beta-MSP mRNA expression was the only significant prognostic indicator for progression under endocrine therapy (P = 0.003). CONCLUSIONS: The presence of cells that express the beta-MSP transcript may be a novel indicator of potentially aggressive prostate cancer.

Aged↗

PubMed1999

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.

3T3 Cells↗

PubMed1999

Life-long caloric restriction suppresses age-associated Fas expression in the Fischer 344 rat kidney.

It has been shown that the expression of Fas is substantially increased in the aging process in various organs, but its role in the aging kidney is not yet clear. In this study, the expression of Fas in the kidneys of 6- and 24-month-old male Fischer 344 rats fed ad libitum was studied by using quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. In addition, possible effects of life-long caloric restriction (30% as those of ad libitum fed group) in the expression of Fas were also studied in 6- and 24-month-old rat kidneys. Kidneys obtained from 24-month-old ad libitum fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old calorie-restricted rats, renal damage was remarkedly less than that noted in 24-month-old ad libitum fed rats kidneys. RT-PCR and immunohistochemical analysis showed an increased expression of Fas in both mRNA and protein level in 24-month-old rat kidneys; life-long caloric restriction significantly reduces renal expression of Fas. Our results suggest that increased expression of Fas is associated with age-related renal damage and that life-long diet-restricted alteration of its expression is associated with the modulation of age-associated renal structural damage.

Aging↗

PubMed1999

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections.

Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of alpha1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro alpha1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for alpha1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN.

Adolescent↗

PubMed1999

Cisplatin-induced apoptosis in human proximal tubular epithelial cells is associated with the activation of the Fas/Fas ligand system.

The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20-80 microM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process.

Apoptosis↗

PubMed1999

Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis.

Lymph node dissemination is a major prognostic factor in human cancer. However, the molecular mechanisms underlying lymph node metastasis are poorly understood. Recently, vascular endothelial growth factor-C (VEGF-C) was identified as a ligand for VEGF receptor-3 (VEGFR-3/Flt-4) and the expression of VEGFR-3 was found to be highly restricted to the lymphatic endothelial cells. In this report, we investigated the expression of VEGF-C and VEGFR-3 in human prostatic carcinoma tissue by using in situ hybridization and immunohistochemical staining respectively. Expression of VEGF-C mRNA in prostatic carcinoma was significantly higher in lymph node-positive group than in lymph node-negative group. In addition, the number of VEGFR-3-positive vessels was increased in stroma surrounding VEGF-C-positive prostatic carcinoma cells. These results suggest that the expression of VEGF-C in prostatic carcinoma cells is implicated in the lymph node metastasis.

Adenocarcinoma↗

PubMed1999

Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus.

The goal of this study is to clarify whether the expression of metallothionein (MT) could affect the prognosis and the metastatic potential of squamous cell carcinoma (SCC) of the oesophagus. In paraffin-embedded specimens resected from 57 patients, MT mRNA and protein expressions were detected by in situ hybridization and immunohistochemistry respectively. The expression of MT was evaluated in respect of clinicopathologic variables and patients' survival. MT mRNA expression was significantly associated with the proportion of lymph node metastasis (71% in MT mRNA-positive tumours vs 42% in MT mRNA-negative tumours; P = 0.0343) and that of distant metastasis (29% in MT mRNA-positive tumours vs 5% in MT mRNA-negative tumours; P = 0.0452). In respect of MT protein expression, the frequency of distant metastasis was more common in MT-positive tumours than in MT-negative tumours (30% in MT-positive tumours vs 8% in MT-negative tumours; P = 0.0446). The survival rate of the patients with MT protein-negative tumours was significantly better than that of the patients with MT protein-positive tumours (P = 0.0340). There was a positive correlation between the expression of MT protein and that of proliferating cell nuclear antigen (P = 0.0018). Therefore, we conclude that MT expression, both at the mRNA and protein levels, may be a potential marker predicting metastatic and proliferative activities of oesophageal SCC.

Aged↗

PubMed1999

Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey.

Hepatitis E virus (HEV) has been considered to be the major cause of enterically transmitted non-A, non-B hepatitis in developing countries. However, little is known about viral replication and localization in the liver. The aim of this study was to examine the distribution of HEV-infected cells in experimentally infected animals. Seven captured wild rhesus monkeys were inoculated intravenously with faecal extract derived from a Myanmar strain of HEV. Animals were killed at different time-points of clinical illness: during early infection, during prehepatitis with viral-like particles in bile, during acute hepatitis and during convalescence. Intrahepatic localization of HEV was analysed using non-isotopic thymine dimer in situ hybridization (NITDISH). Both plus and minus strands of HEV RNA were found in hepatocytes during the early infection period. Staining in the submembranous cytoplasmic region of hepatocytes was observed. In the prehepatitis period, both plus and minus strand HEV RNAs appeared in the canalicular side of isolated bile epithelial cells. Subsequently, HEV RNA became universally distributed in the cytoplasm of medium-size bile epithelial cells. After recovery, HEV RNA disappeared.

Acute Disease↗

PubMed1999

Expression of thioredoxin and thioredoxin reductase in placentae of pregnant mice exposed to lipopolysaccharide.

We have previously shown that thioredoxin and thioredoxin reductase were immunohistochemically localized in cytotrophoblasts, decidua and stromal cells in the stem villi of human placenta and that the addition of exogenous thioredoxin and thioredoxin reductase to mitochondrial fractions from human placenta displayed a protective effect on fumarase activity against oxidative stress. In this study, to investigate further the roles of thioredoxin and thioredoxin reductase in protecting pregnancy against oxidative stress, we examined the effect of lipopolysaccharide (LPS), which induces a variety of cytokines and produces radical oxygen species, on the expression of thioredoxin and thioredoxin reductase in mouse placenta. We focused on the placental protective effect in the second trimester, when the onset of placental dysfunction might occasionally lead to a critical state for the fetus. Thus we analysed placentae from mice on day 13 of pregnancy at various time points after they were injected with LPS (50 microg/kg i.p.) or saline as a control. The expressions of thioredoxin and thioredoxin reductase were evaluated by Western blotting and immunohistochemistry. Western blot analysis revealed that LPS approximately quadrupled the expression of both thioredoxin and thioredoxin reductase in the placentae of pregnant mice. When both proteins were localized immunohistochemically, it was found that the decidua and the diploid trophoblasts in the basal zone were intensively stained. Furthermore, the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins, which are markers of oxidative stress, was enhanced in placenta by LPS. Our study suggests that the induced thioredoxin and thioredoxin reductase might protect the placenta from the stress induced by LPS.

Aldehydes↗

PubMed1999

[Recent advances in molecular histochemical techniques].

To understand better the physiological states of cells and tissues, it is essential to analyze the regulatory mechanism of cell-type or tissue-type specific gene expression in individual cells. For this purpose, in situ hybridization (ISH) has been regarded as a powerful molecular histochemical method. Over the past decade, we made lots of efforts to establish nonradioactive ISH protocol with thymine-thymine dimerized or digoxigenin-labeled synthetic oligo-DNA probes, including extensive optimization of pretreatment, evaluation of hybridizable RNA and new trials of signal amplification. Furthermore, we have originally developed a new method, Southwestern histochemistry, which localizes specific transcription regulatory factors and allows us to understand the fluctuation of mRNA levels detected by ISH at the level of transcription regulation of the gene in individual cells. Here, I will review the outlines of these methods with recent progress and will see what we can see by combining them.

Animals↗

PubMed1998

Stage-specific expression of testis-specific protein kinase 1 (TESK1) in rat spermatogenic cells.

TESK1 (testis-specific protein kinase 1) is a serine/threonine kinase, with a unique structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Northern blot analysis revealed that TESK1 mRNA is predominantly expressed in testicular germ cells. We present here evidence that expression of TESK1 mRNA and protein in the rat testes is developmentally regulated and increases after 20-22 postnatal days. To identify cells which express TESK1 mRNA and protein during male germ cell differentiation, in situ hybridization and immunohistochemistry were done using frozen sections of adult rat testes. Prominent expression of TESK1 mRNA and protein was detected in testicular germ cells at stages of late pachytene spermatocytes to round spermatids, but not in somatic cells such as Sertoli and Leydig cells. Expression of TESK1 mRNA and protein at specific stages of testicular germ cells suggests a role for this kinase in spermatogenesis, particularly at stages of meiosis and/or early spermiogenesis.

Animals↗

PubMed1998

Expression and localization of inducible nitric oxide synthase in the rat ovary: a possible involvement of nitric oxide in the follicular development.

In the present study, using Northern blot analysis, we demonstrated that the level of iNOS mRNA in the ovary of immature rats decreased after 6 h of pregnant mare serum gonadotropin administration and recovered gradually up to the untreated level 48 h after the administration. Both in situ hybridization and immunohistochemistry revealed that iNOS mRNA and protein was predominantly localized in granulosa cells in most of immature follicles, but not in mature follicles with an antrum, which was a consistent finding regardless of gonadotropin treatment. Furthermore, we found that cultured granulosa cells had the ability to express iNOS mRNA in the presence of cytokines such as tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, which are inherently detectable in the ovary. These results raise the possibility that locally produced NO synthesized by iNOS may be involved in the developmental status of ovarian follicles in concert with gonadotropin.

Animals↗

PubMed1998

Cellular expression of beta-microseminoprotein (beta-MSP) mRNA and its protein in untreated prostate cancer.

BACKGROUND: Previous studies have shown that beta-microseminoprotein (beta-MSP) may be used as a diagnostic marker for prostate cancer. However, the level of expression of beta-MSP in prostate cancer detected by immunohistochemistry (IHC) has varied from one study to another. METHODS: We analyzed the expression of both beta-MSP mRNA and its protein in a large sample of prostate tumors from 104 patients with untreated prostate cancer, using both nonradioactive in situ hybridization (ISH) and IHC. RESULTS: Our results showed that 72 and 96 of 104 specimens were negative for beta-MSP mRNA (69.2%) and beta-MSP (92.3%), respectively. Furthermore, a reduced expression of both beta-MSP mRNA and its protein was detected in all malignant epithelial tissues compared with benign epithelia. Not all malignant tissue samples negative for beta-MSP mRNA were negative for beta-MSP (6.7%), and vice versa (29.8%). Other tissue samples were either negative for both (62.5%) or positive for both (1.0%). CONCLUSIONS: Our results showed a lower level of expression of beta-MSP in prostate cancer tissue, compared with benign prostate tissue. This phenomenon may be mainly due to the presence of reduced levels of beta-MSP mRNA.

Aged↗

PubMed1998

Post-meiotic expression of the mouse dihydropyrimidinase-related protein 3 (DRP-3) gene during spermiogenesis.

The dihydropyrimidinase-related protein (DRP) family, originally identified in humans by their homology to dihydropyrimidinase, contains at least four members. Genes of this family, and their counterparts in other mammals and chickens, are expressed mainly in fetal and neonatal brain, suggesting that the encoded proteins have a physiological role in the development of the central nervous system. In addition, the DRP-3 gene is expressed in testis as a shorter mRNA than the brain form. As a first step in understanding the extra-neuronal function of DRP-3, the structure and expression of testis DRP-3 were examined. Testis DRP-3 cDNA showed the same sequence as brain DRP-3 cDNA, except for the 5'-terminal end, which encodes a 5'-untranslated region and the 11 N-terminal amino acid residues, indicating that the two forms of DRP-3 mRNA were transcribed from a single copy gene. Northern blotting analysis detected DRP-3 mRNA in 30-, 40- and 70-day-old, but not in 10- and 20-day-old testes. In situ hybridization analysis indicated that the expression of DRP-3 in testis is restricted to post-meiotic round spermatids. This is the first report of the expression of DRP genes in extra-neuronal cells.

Amino Acid Sequence↗