[Myofibroblastosis--importance of transformation of renal cells into myofibroblasts during the renal fibrosis process].
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Actins↗
Biomedical subjects
T Moriyama
Publications and source records attributed to T Moriyama.
Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL.
In chronic hepatitis C, Fas expression is up-regulated in the hepatocytes, especially near liver-infiltrating lymphocytes, and Fas ligand is expressed on the lymphocytes. The presence of hepatitis C virus (HCV)-specific CTLs has been demonstrated both in peripheral blood and among liver-infiltrating lymphocytes of patients with chronic hepatitis C. We studied the killing mechanisms of HCV-specific human CTLs using target cells that were sensitive or resistant to agonistic anti-Fas Abs and TNF-alpha. We show that HCV-specific CTL clones kill non-Ag-bearing bystander cells as well as Ag-bearing cells, although the bystander killing is less efficient than the specific target cell killing, and the efficacy of the bystander killing of anti-Fas- and soluble TNF-alpha-sensitive cells is greater than that of resistant cells. We also show that the killing of Ag-presenting, sensitive cells is mediated by Fas ligand and TNF-alpha as well as perforin, although the latter plays a major role in the killing at a low E:T ratio, and that the killing of sensitive bystander cells is primarily mediated by Fas ligand and TNF-alpha on CTLs expressed upon specific Ag stimulation, which may be relevant to the bystander lysis by HCV-specific CTLs of uninfected hepatocytes, in which Fas expression is up-regulated. Activated CTLs also kill bystander cells by the perforin-based mechanism, although it requires a high E:T ratio. The effective bystander killing requires a close intercellular contact between CTLs and target cells, although TNF-alpha released from the CTLs mediates lysis of the bystander cells without a close cell-cell contact.
Antigen Presentation↗
Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms.
Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O2-) production after a lag time. The rank order of efficiency of these PAs in triggering O2- production was PA8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)] > PA10:0 (1,2-didecanoyl-GP) > PA6:0 (1,2-dicaproyl-GP) > > PA12:0 (1,2-dilauroyl-GP). The O2- release from PMN stimulated with PA10:0 or PA12:0, but not with PA6:0 or PA8:0, was lowered by the addition of 1 mM extracellular Ca2+. Studies with various inhibitors showed that the mechanism of multiphasic O2- production induced by PA8:0 depended on its concentration: 1 and 3 microM PA8:0 induced O2- production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 microM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 microM TMB-8. Concentrations of 30, 60 and 100 microM PA first elicited transient O2- production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O2- production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [14C]PA8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O2- production via three different mechanisms described above.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope.
An epitope that acted as a weak agonist in the cytotoxicity assay was identified as part of the capsid protein of a hepatitis C virus (HCV) variant. In a low concentration, the variant epitope also had a weak antagonistic effect. When a minute amount of this variant epitope was added to the culture for induction, it selectively attenuated the expansion of major cytotoxic T cell populations and drastically reduced the cytotoxic responses against the wild-type epitope. Thus, antagonism to induction suppressed immune responses against both the wild type and the variant, thereby helping the persistence of not only variant itself but also the wild-type HCV. Because this variant was a weak agonist, most cytotoxic T cells induced with the wild-type epitope were cross-reactive with the variant and susceptible to the antagonism to induction. Only the T cells which were not cross-reactive with the variant and not susceptible to the antagonism survived the antagonism in induction. This implied that the specificity of the remaining immune response, if any, was directed exclusively to the wild-type epitope after the emergence of the variant. For viruses like HCV, being heterogeneous itself may contribute significantly toward persistent infection through antagonism to induction.
Antigenic Variation↗
Cytotoxic T lymphocyte response and viral load in hepatitis C virus infection.
A cytotoxic T lymphocyte (CTL) response to the hepatitis C virus (HCV) nucleoprotein residues 88-96 that are the minimal and optimal epitope for human leukocyte antigen (HLA) B44-restricted CTLs was assessed in 27 HLA B44-positive patients with chronic HCV infection. Serum HCV RNA concentration and the amino acid sequence of the residues 81-100 were also determined. Three patients were infected with HCV with uncommon amino acid substitutions within the epitope. One was infected with HCV with an amino acid substitution in the flanking residues of the epitope. To stimulate CTLs in the peripheral blood, 9-mer peptides that corresponded to the residues 88-96 of the individual patients were synthesized and used. Seven of the 27 patients demonstrated a CTL response to the residues 88-96 with specific cytotoxic activities higher than 20%. The CTL activities were significantly higher in patients with a low titer of serum HCV RNA than in those with a high titer of serum HCV RNA (P = .0006). Some of the patients that demonstrated a CTL response to the residues 88-96 also demonstrated a CTL response to a newly identified HLA B44-restricted CTL epitope or a known HLA A11-restricted CTL epitope or both. No apparent association was observed between the CTL response and the stage of disease, or between the CTL response and the grade of necroinflammatory activity. The results suggest that the HLA B44-restricted CTLs together with other HCV-specific CTLs may inhibit the outgrowth of HCV and that high-titer infection with HCV may suppress the CTL responses.
Adult↗
Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomerular epithelial cells in vivo.
The aim of this study was to seek a promoter, transactivated selectively in renal cells in vivo by using transgenic (tg) mouse technology. We generated two kinds of tg mouse lines carrying a green fluorescence protein (GFP) cDNA driven either by cytomegalovirus enhancer and beta-actin/beta-globin promoter (CX-GFP) or by elongation factor 1alpha promoter (EF-GFP), and investigated the expression of GFP in the kidney. Microscopic examination of the renal tissues in CX-GFP-tg mice revealed that GFP was expressed only in glomeruli, mainly epithelial cells, but not in tubules, arteries and interstitium. Moreover, in situ hybridization demonstrated that GFP mRNA expression was localized in the glomerular cells. In contrast, GFP was not detectable in the kidney in any of the lines of EF-GFP-tg mouse. To exclude the possible involvement of the GFP cDNA as an enhancer, we constructed tg mice carrying the CX promoter driving a human CD4 cDNA. It was confirmed that the expression patterns of human CD4 in the kidney were quite similar to those of GFP in the kidney of CX-GFP-tg mice. These results strongly suggest that CX promoter could be transactivated in glomerular epithelial cells in vivo.
Actins↗
Expression of c-Met correlates with grade of malignancy in human astrocytic tumours: an immunohistochemical study.
AIMS: Recent studies suggest the involvement of hepatocyte growth factor/scatter factor (HGF/SF) in glioma cell invasion and tumour progression. We investigated the distribution and rate of tumour cells that express c-Met protein, which is the cell-surface receptor for HGF/SF, in astrocytic tumours. The type of cells that express c-Met in tumour tissues was also identified. METHODS AND RESULTS: c-Met expression was screened immunohistochemically in a total of 43 astrocytic tumours, including 14 low-grade astrocytomas (A), 13 anaplastic astrocytomas (AA) and 16 glioblastoma multiforme (GBM), c-Met reactivity was demonstrated predominantly in the cytoplasm of tumour cells. Bizarre large tumour cells tended to stain intensely. Higher c-Met expression levels (> or = 2+, more than 25% cells were positive) were noted in 21.4% of (A) vs. 53.8% in (AA) and 87.5% in (GBM) (P < 0.001), indicating a clear relationship between c-Met protein staining and higher grade astrocytic tumours. Moreover, c-Met immunoreactivity was also shown in tumour microvasculature, reactive astrocytes, and neurones in the cortex infiltrated by glioma cells. In 85.7% of cases containing infiltrated cortex, neurones were positive vs. no neurones in non-neoplastic regions (P < 0.002). CONCLUSIONS: This evidence suggests that c-Met expression in the brain could be associated with astrocytoma progression and also reactive process. Immunohistochemical determination of c-Met-expressing cell types helps to understand possible roles of c-Met in tumour tissues.
Adolescent↗
Functions of characteristic Cys-Gly-His-Cys (CGHC) and Gln-Glu-Asp-Leu (QEDL) motifs of microsomal ER-60 protease.
The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b(+) T7 promoter. The recombinant ER-60 protease was obtained in a water-soluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue(s) of the CGHC motifs may constitute the active site(s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue(s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.
Amino Acid Sequence↗
Osmotic regulation of amino acids and system A transport in Madin-Darby canine kidney cells.
The effects of hypertonicity on the intracellular amino acid content and system A transport activity were studied in Madin-Darby canine kidney (MDCK) cells. Total content of 20 amino acids increased from 274 to 689 nmol/mg protein after 8 h of hypertonicity (500 mosmol/ kg), remaining almost constant until after 6 days of hypertonicity. The content of neutral amino acids increased from 77 to 307 and 395 nmol/mg protein after 8 h and 6 days of hypertonicity, respectively, accounting for 73% of the increased amount of total amino acids. In the hypertonic MDCK cells, system A transport activity, measured by Na+-dependent 2-(methylamino)isobutyric acid (MeAIB) uptake, increased approximately 60-fold relative to the uptake in isotonic cells. MeAIB was taken up primarily on the basal side in the isotonic MDCK cells cultured on permeable supports. Extracellular hypertonicity stimulated the MeAIB uptake predominantly on the basal side. These results indicated that amino acids, especially neutral amino acids, can function as volume-regulating osmolytes and that the stimulation of system A activity appears to contribute to the accumulation of neutral amino acids in hypertonic MDCK cells.
Amino Acids↗
Effects of inhibition of myo-inositol transport on MDCK cells under hypertonic environment.
To investigate the role of myo-inositol under hypertonic conditions, we examined the effects of inhibition of myo-inositol transport in Madin-Darby canine kidney (MDCK) cells using an analog of myo-inositol, 2-O,C-methylene-myo-inositol (MMI). We first characterized the inhibitory effects of MMI on myo-inositol transport in MDCK cells. The Na+-dependent component of [3H] myo-inositol uptake was inhibited by MMI in a concentration-dependent manner, although MMI did not inhibit the activities of the betaine transporter and system A neutral amino acid transporter. We found decreased affinity for myo-inositol in the presence of MMI, whereas the maximal velocity (Vmax) of the transporter did not change. Thus MMI behaves as a competitive inhibitor of myo-inositol transport with a relatively high inhibition constant (K(i)) value (1.6 mM). Myo-inositol content in hypertonic MDCK cells was markedly reduced in the presence of 5 mM MMI, but MMI itself did not accumulate in these cells. The hypertonic cells began to detach in the presence of MMI 3 days after increasing medium osmolality, whereas MMI did not affect the cells in isotonic medium. We also examined the effects of MMI on colony-forming efficiency of MDCK cells. MMI decreased colony-forming efficiency in a concentration-dependent manner, and addition of myo-inositol returned the efficiency to the value without MMI. Addition of betaine also increased colony-forming efficiency in the presence of MMI. These results indicate that myo-inositol plays an important role in survival and growth under hypertonic environment.
Animals↗
Optical recording of azimuth representation in guinea pig auditory cortex.
The frequency dependency of directional sensitivity of the auditory cortex of barbiturate-anesthetized guinea pigs in response to tone bursts was studied by an optical equipment and a voltage sensitive dye (RH795). Tone bursts at 4, 8 and 14 kHz were presented from loudspeakers placed at the azimuths from contralateral 90 degrees to ipsilateral 90 degrees in 30 degrees steps on a front-horizontal semi-circular board (100 cm in radius). The maximum responses were observed at around contralateral 30 degrees (best azimuth). The onset and peak latencies were the shortest at the best azimuth. The best azimuth was the same for all these frequencies, but the azimuth selectivity became broader for the lower frequencies. These results indicate that the azimuth selectivity observed in the guinea pig auditory cortex is mainly associated with pinna characteristics.
Animals↗
Responses of bat inferior collicular neurons to recorded echolocation pulse trains.
Under free field stimulation conditions, we studied the responses of inferior collicular neurons of the big brown bat, Eptesicus fuscus, to echolocation pulse trains which were recorded during the entire process of hunting. The entire series of recorded echolocation pulse trains was edited in different sequences according to echolocation pulses of different hunting phases. When stimulated with all edited sequences of echolocation pulse trains, the temporal characteristics and the relative position of the echolocation pulses of a specific hunting phase affect the number of impulses of each inferior collicular neuron studied. When stimulated with two different intensities, more than 59% of inferior collicular neurons studied discharged selectively to the echolocation pulses of a specific hunting phase such that the number of impulses discharged to the echolocation pulses of the most and least preferred hunting phases differed by at least 50%. Passible mechanisms for this selective response are discussed.
Animals↗
Establishment and characterization of a new human glioblastoma cell line (MGM-1) with highly motile phenotype.
A new cell line MGM-1 was established from a primary tumor of the left temporal lobe with histological diagnosis of glioblastoma multiforme, removed from a 64-year-old Japanese male. The patient died of recurrence and unusual extracranial metastases of the tumor 7 months after the surgery. The cultured MGM-1 cells are spindle or polygonal in shape. After serial passages, glial fibrillary acidic protein became negative immunocytochemically in vitro. The modal chromosome number was 61-64. Doubling time and soft agar colony forming efficiency were 42.9h and 0.4%, respectively (at 25th passage). MGM-1 is a highly motile cell line in vitro and its serum-free conditioned medium is chemotactic and chemokinetic for other glioma cells. Secretion of gelatinases (probably MMP-2/72-kDa type i.v. collagenase) and MMP-9/92-kDa type i.v. collagenase) and urokinase-type plasminogen activator were also investigated. MGM-1 would therefore be useful for studying the mechanisms regulating glioma-cell motility and invasion. The MGM-1 cell line has been propagated continuously by serial passages (more than 100 passages) during the past 4 years.
Brain Neoplasms↗
TCV-116 inhibits interstitial fibrosis and HSP47 mRNA in rat obstructive nephropathy.
Unilateral ureteral obstruction (UUO) is a well established disease model leading to fibrosis of the obstructed kidney. In this model, involvement of enhanced renin-angiotensin system in the pathogenesis of interstitial fibrosis has been demonstrated. A 47-kDa heat-shock protein (HSP47) was originally identified as a collagen-binding stress protein, and is currently considered to be a collagen-specific molecular chaperone that plays a pivotal role during the biosynthesis and secretion of procollagen from endoplasmic reticulum. To test if HSP47 is involved in interstitial fibrosis in UUO, we examined the expression of HSP47 mRNA in rat UUO kidneys after 12 hours. 1, 4, 7 days of obstruction. HSP47 mRNA expression was significantly increased as early as 12 hours after obstruction and was sustained at the increased level until seven days. Type I collagen mRNA significantly increased after four days of UUO. Fibrotic changes of interstitium appeared in Masson's trichrome stained section after four days. To explore the possible involvement of angiotensin II (Ang II) in HSP47 induction, the effect of Ang II receptor antagonist (TCV-116) and angiotensin converting enzyme inhibitor (lisinopril) was tested. TCV-116 or lisinopril was given to the animals orally once a day at the dose of 10 mg/kg. TCV-116 or lisinopril significantly ameliorated the fibrotic change of interstitium seven days after obstruction. HSP47 and type I collagen mRNA levels in the TCV-116- or lisinopril-treated groups were reduced to about 60% of untreated UUO. A possible involvement of HSP47 in the pathogenesis of interstitial fibrosis in UUO is suggested; however, further investigation is required to identify the signals involved in the induction of HSP47 in UUO.
Angiotensin II↗
Hydrolases acting on glycosidic bonds: chromatographic and electrophoretic separations.
We describe analyses of unusual human alpha-amylase, performed in our laboratory and review available methods for amylase study. Electrophoretic and chromatographic methods provide an effective means for the analysis of amylase isoenzymes and unusual amylase. The recent identification of a selective inhibitor and a monoclonal antibody to amylase isoenzyme contributes to rapid routine clinical assays of amylase isoenzymes. However unusual amylases such as variants, macroamylasemia and sialyl salivary-type amylasemia cannot be detected by those conventional methods. The unusual amylases can only be detected by electrophoresis and can be easily characterized by combination study with chromatographic methods. Electrophoretic and chromatographic methods are universal means to validate unusual amylases found in patient sera. Further basal studies are needed to define the roles of salivary amylase in exocrine fluids using those separation techniques.
Amylases↗
Effects of hepatocyte growth factor (HGF) on human glioma cells in vitro: HGF acts as a motility factor in glioma cells.
Expression of c-Met, the receptor for hepatocyte growth factor (HGF), and the biological roles of HGF were examined in cultured human glioma cells. All of the 5 glioma cell lines examined expressed c-Met protein as well as the c-met gene. Expression of the c-met gene was also confirmed in a glioblastoma tissue. Three cell lines (MGM-3, U251, KG-1-C) demonstrated chemotactic response to HGF in a dose-dependent manner. The response was not only chemotactic but also chemokinetic as judged by a checkerboard analysis. The amounts of c-Met mRNA and protein were abundant in the cell lines which showed a migratory response to HGF. Moreover, c-Met protein expression was highest in U251 with the highest migratory response to HGF. Among the cell lines, KG-1-C produced notable amounts of HGF protein as well as of c-Met, suggesting that HGF may act in an autocrine fashion in this case. HGF did not act as an apparent growth factor in the glioma cell lines examined. Furthermore, HGF stimulated the production of metalloproteinase, probably gelatinase A, in U251 cells.
Cell Division↗
Effects of dietary phosphatidylcholine on memory in memory deficient mice with low brain acetylcholine concentration.
Data concerning the effect of phosphatidylcholine (PCh) administration on the improvement of memory in senile dementia of Alzheimer type are inconsistent, probably due to the different conditions under which studies were conducted. Animal studies provide a good model, but data on this is limited. We studied the effect of PCh on memory in memory deficient mice (Dull mice) with low brain acetylcholine (ACh) concentration and normal mice. Mice were fed 24% casein diet (control) or this diet supplemented with 2 or 8% egg yolk PCh from gestation (Experiment 1) and after weaning (Experiment 2). Memory acquisition and retention were studied by step-down type passive avoidance performance at 8 and 10 weeks old, respectively. Control group of Dull mice had poorer memories than that of the normal mice in Experiments 1 and 2. On the 2% PCh diet, Dull mice improved memory acquisition and retention in Experiment 1 and retention in Experiment 2. On the 8% PCh diet in Dull mice there was improvement of memory and retention in Experiment 1, but no effect was observed in Experiment 2 (P > 0.05). In the normal mice, the 2% PCh diet did not affect memory acquisition and retention, however on the 8% PCh diet, there was no or adverse effect. These results suggest that dietary supplementation of egg yolk PCh improves memory of Dull mice, particularly when given from gestation and that the 2% PCh diet elicits better response than the 8% PCh diet.
Acetylcholine↗
Effects of dietary nucleoside-nucleotide mixture on memory in aged and young memory deficient mice.
Intestinal mucosa, bone marrow hematopoietic cells and brain have limited capacity for the de novo synthesis of nucleosides (NSs) and nucleotides (NTs). Whereas the role of dietary NS and NT in the former two tissues is known, it is not known in the brain. Therefore we studied the effect of dietary NS and NT mixture on memory in aged mice (Experiment 1) and young memory deficient mice (Experiment 2). Memory retention was studied by step-through type passive avoidance performance (maximum 180 seconds). In Experiment 1 aged (7 month old) senescence accelerated mice (SAM) were fed 20% casein diet (control) or this diet supplemented with 0.5% NS/NT mixture for 12 weeks. Memory was studied 1, 2 and 3 days after the electric shock (punishment). In Experiment 2, young (1 month old) memory deficient mice (Dull mice) and normal mice (ddY mice) were fed the same diets as those in Experiment 1 for 12 weeks. Memory retention was studied 1 and 3 days after the punishment. In the aged SAM the average time of avoidance and also the percentages of successful memory 2 and 3 days after the punishment were significantly higher in the NS/NT diet group than the control diet group (P < 0.05). In the Dull mice percentage of successful memory was higher in the NS/NT diet group than in the control group 3 days after the punishment, however, such an effect was not observed in the normal mice. These results suggest that insufficient endogenous supply of NSs and NTs may be responsible for the factor of memory deficiency with aging or of genetical memory deficiency, which can be improved by the dietary administration of NSs and NTs.
Aging↗