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Biomedical subjects

M Miwa

Publications and source records attributed to M Miwa.

At least 109 records · Page 6Linked to original sources
PubMed1996

First evidence for accumulation of protein-bound and protein-free pyrraline in human uremic plasma by mass spectrometry.

Glucose-derived advanced glycation end products (AGEs) cross-link proteins and cause various biological tissue damage. One of them, pyrraline [epsilon-2-(formyl-5-hydroxymethyl-pyrrol-1-yl) -L-norleucine], has been demonstrated by utilizing antibody to accumulate in plasma and sclerosed matrix of diabetic individuals, suggesting responsibility for diabetic complications. To elucidate the involvement of pyrraline in uremia, we examined the pyrraline levels in patients with chronic renal failure by a mass spectrometric approach. Here we show that protein-free pyrraline as well as pyrraline with binding protein are significantly increased in non-diabetic uremic plasma compared to healthy subjects. Our results suggest that circulating pyrraline could be a substance contributing to complications in uremia.

Blood Proteins↗
PubMed1996

An improved isolation method for murine migratory cutaneous dendritic cells.

Dendritic cells are highly specialized accessory cells for the initiation of primary immune responses. They occur as trace populations in non-lymphoid and lymphoid organs. Therefore, the isolation and enrichment of primary dendritic cells is difficult and time-consuming. This applies also to dendritic cells from skin, i.e. epidermal Langerhans cells and dermal dendritic cells. Recently introduced skin organ cultures serve as a convenient source for primary cutaneous dendritic cells. We report here a refinement of such cultures in which cutaneous dendritic cells emigrate spontaneously into the culture medium. Murine ear skin is cultured for a total of 3 days in three sequential 24 h steps. This simple modification doubles or triples the yields of dendritic cells that can be obtained and up to 30,000 dendritic cells can be recovered from one ear half. This represents 50-70% (range 30-80%) of all viable cells. The cells are mature dendritic cells that possess potent T cell stimulatory function. Compared to the classical methods of preparing epidermal Langerhans cells by trypsinization this technique is easier and quicker; it does not require enzymes such as trypsin, and it yields similar numbers of mature dendritic cells. It should prove useful for further studies of dendritic cells of the skin.

Animals↗
PubMed1996

Interaction of platelet-activating factor with rat hepatocytes: uptake, translocation, metabolism, and effects on PAF-acetylhydrolase secretion and protein tyrosine phosphorylation.

In the present study, the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Neuronal and vascular pathology produced by verocytotoxin 2 in the rabbit central nervous system.

To study the pathogenesis of the central nervous system (CNS) involvement associated with verocytotoxin-producing Escherichia coli infection, we developed an animal model by administering verocytotoxin 2 to rabbits either intravenously or intrathecally. After an interval of 2-9 days, the rabbits became paralyzed in a dose-dependent manner and in the absence of renal impairment. The minimal intravenous and intrathecal doses that produced these neurological signs were 250 and 0.4 ng/kg, respectively. After intravenous administration, most of the toxin was cleared from the serum within 24 h, with concomitant transition of a small amount into the cerebrospinal fluid. Pathological examination revealed that neurons in various CNS regions showed atrophy, cytoplasmic hyperchromasia and nuclear pyknosis as early as 6 h after administration. The distribution of affected neurons was constant and irrespective of the route of administration. Abnormalities of the blood vessels, such as the thickening of arterioles walls, were noted from 2 days after administration. The vascular lesions became more prominent after the intrathecal injection, which caused thrombosis and multiple infarction. Selective deposition of the toxin on the vessel walls was demonstrated immunohistochemically. Thus, the pathological manifestations of verocytotoxin 2 neurotoxicity consisted essentially of two types of lesions, early neuronal and late vascular, both of which might have developed under the influence of the toxin that had entered the CNS by crossing or circumventing the blood-brain barrier.

Animals↗
PubMed1996

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells.

The regulation of platelet-activating factor (PAF) synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation in HDL, did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of 125I-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyltransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Alternate repetition of short fore- and backfiltrations reduces convective albumin loss.

An effective therapeutic means to remove relatively large polypeptide uremic toxins seems to be a hemofiltration (HF) or hemodiafiltration (HDF) employing a larger-pore membrane, that is, a protein-permeable membrane. With either method, however, a significant amount of albumin will be lost into the ultrafiltrate or dialysate. Now, repetition of alternate short fore- and backfiltrations may prevent the development of the ultrafiltration-induced higher albumin concentration on the membrane surface (protein concentration polarization), where a single forefiltration time is shorter than the time needed for completion of protein concentration polarization. Since the albumin concentration on the protein-permeable membrane surface will be one of the determinants of albumin loss by convection, such HDF treatment may reduce protein loss into the dialysate. To examine this assumption, we alternately repeated short and rapid fore- and backfiltrations (push/pull HDF) through a protein-permeable membrane, each less than 1 second in duration and at each filtration volume of 15 ml, where a pyrogen-free dialysate was supplied. The present results indicated that the albumin amount lost by push/pull HDF was approximately one-third of that by conventional HDF. Nevertheless, the reduction rates of beta 2-microglobulin and myoglobin were significantly greater by push/pull HDF than by conventional HDF.

Blood Proteins↗
PubMed1996

Mutation analysis of gonadotropin receptor and G protein genes in various types of human ovarian tumors.

The heterotrimeric guanine-nucleotide-binding proteins (G proteins) and G protein-coupled hormone receptors including gonadotropin receptors have been suggested to play a role in ovarian tumorigenesis. However, no functional significance of gonadotropin receptors and G proteins in this process has been demonstrated. To investigate this issue, we examined point mutations in these genes in various types of ovarian tumors by polymerase chain reaction-single strand conformation polymorphism analysis and direct sequencing. Among 37 tumors (20 epitherial, 8 sex cord-stromal, and 9 germ cell tumors) and 5 carcinoma cell lines examined, no mutational sequence of G protein-interaction domains of luteinizing hormone receptor and follicle-stimulating hormone receptor, or "hot spots" of the alpha subunit of adenylyl cyclase-stimulating G protein and -inhibitory G protein was observed. Although this analysis was performed on only a limited number of tumors and cell lines, and on limited gene loci, the results suggest that mutational activation in gonadotropin receptors and G proteins is not crucial for ovarian tumorigenesis.

Female↗
PubMed1996

Advantage of monitoring skin vibration as an index of arteriovenous fistular blood flow.

Monitoring of the skin vibration is disturbed little by environmental noise. Therefore, the skin vibration is more relevant than bruit on the skin by means of monitoring the fistular blood flow. Frequency analysis of the skin vibration generated by an internal arteriovenous fistula indicated two peaks of power spectral density (PSD), one in the frequency range of 4-10 Hz and the other from 100-300 Hz. The analysis of the skin vibration generated by an external fistula, however, showed only one peak in the range of 4-10 Hz and a moderate flat PSD level in the range of 100-300 Hz. The PSD level in the range of 100-300 Hz decreased dramatically or disappeared when the fistular blood flow diminshed or ceased. Therefore, the optimal frequency for monitoring skin vibration may be the range of 100-300 Hz.

Arteriovenous Fistula↗
PubMed1995

Relationship between susceptibility to apoptosis and Fas expression in peripheral blood T cells from uremic patients: a possible mechanism for lymphopenia in chronic renal failure.

Chronic renal failure (CRF) is often complicated by lymphopenia, which may be partly responsible for immune deficiency. We hypothesized that lymphopenia in CRF might result from apoptosis of T cells in vivo. To elucidate the involvement of Fas antigen which mediates apoptosis, we analyzed Fas expression on peripheral blood T cells in uremic non-dialyzed (non-HD) patients and hemodialysis (HD) patients. T cells from both uremic groups expressed Fas with higher intensity than control T cells. When two uremic groups were compared, Fas intensity on T cells was significantly higher in non-HD patients than in patients on HD. Moreover, uremic T cells were shown to undergo accelerated apoptosis when cultured in vitro, in correlation with Fas expression. Our results suggest that T cells in CRF may undergo apoptosis by the Fas system and that hemodialysis treatment has beneficial effects in the light of the inhibition of T cell apoptosis.

Apoptosis↗
PubMed1995

Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers.

p16Ink4 and p15Ink4B are cyclin-dependent kinase 4 inhibitors and link to the regulation of cell cycle in mammalian cells. The genes encoding these inhibitors are located at 9p21, which is a frequent site of allelic loss in various types of tumors. Twenty-five primary biliary tract cancers were examined for somatic mutations in p16Ink4/CDKN2, p15Ink4B/MTS2, p53, and K-ras genes and allelic loss of 9p21 by microsatellite analysis. Four biliary tract cancer cell lines were analyzed for homozygous deletions and point mutations. We found frequent homozygous deletions in p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in the biliary tract cancer cell lines. Each cancer cell line had alteration of either p16Ink4/CDKN2, p15Ink4B/MTS2, or p53 genes. In primary tumors, 16 of 25 (64%) biliary tract cancers had point mutations in the p16Ink4/CDKN2 gene. These include 14 missense and 2 silent mutations. The frequency of mutations in gall bladder cancer and hilar bile duct cancer were 80% (8 of 10) and 63% (5 of 8), respectively. Each of codons 1, 80, and 111 was changed in two cases of these cancers. One of three intrahepatic bile duct cancers, one of two common bile duct cancers, and one of two ampullary cancers had mutations in the p16Ink4/CDKN2 gene. In contrast, no mutation in the p15Ink4B/MTS2 gene, one base change in the K-ras gene, and one loss of heterozygosity at the IFN alpha locus in 25 cancers and one base change in the p53 gene in 19 cancers were observed. These results suggest that p16Ink4/CDKN2, rather than p15Ink4B/MTS2 or p53 genes, and its inactivation may be important in biliary tract carcinogenesis.

Base Sequence↗
PubMed1995

Analysis of biological function of poly(ADP-ribosyl)ation in Drosophila melanogaster.

To understand the biological function of poly(ADP-ribosyl)ation of proteins, we have isolated and characterized the gene for poly(ADP-ribose) polymerase from Drosophila melanogaster. Two approaches were taken to analyze the function of the poly(ADP-ribosyl)ation reaction. The first is analysis of the homology of the amino acid sequences of poly(ADP-ribose) polymerase from phylogenetically different eukaryotes, namely human, mouse, bovine, chicken, Xenopus laevis and Drosophila melanogaster and elucidation of the conserved amino acid sequences that appear to be important for the function of poly(ADP-ribose) polymerase. Analysis of the recombinant poly(ADP-ribose) polymerase which had truncated or mutated motifs expressed in E coli would confirm the importance of the conserved amino acid sequence. The interaction of poly(ADP-ribose) polymerase with other proteins involved in DNA repair, replication, recombination and transcription will clarify the function of poly(ADP-ribosyl)ation. The second approach is to get the mutants which have disruption in the poly(ADP-ribose) polymerase gene and to analyse the phenotypes of these mutants. The characterization of these mutants will be discussed.

Amino Acid Sequence↗
PubMed1995

Monoclonal integration of HTLV-1 in pleural effusion cells in a seropositive patient with tuberculous pleuritis.

The pleural effusion of a 79-year-old man who was a carrier of human T-cell leukemia virus type 1 (HTLV-1) showed monoclonal growth of HTLV-1 infected T-cells. While the integration pattern of HTLV-1 was polyclonal in the peripheral blood mononuclear cells, it was monoclonal in the infiltrated cells of the pleural effusion. Morphologically atypical lymphoid cells were also found in the peripheral blood and in the pleural effusion. The pleural effusion disappeared after treatment with antituberculous drugs. The nature of the monoclonal proliferation of HTLV-1 infected T-lymphocytes is discussed.

Aged↗
PubMed1995

Werner's syndrome associated with basal cell epithelioma.

A Japanese female with Werner's syndrome associated with basal cell epithelioma is reported. Werner's syndrome is prone to malignancy, and many such cases have been reported. However, basal cell epithelioma is rare in Werner's syndrome. In the presented case, a black nodule about 2 cm in diameter with erosion developed on her abdomen. The histology revealed the findings of basal cell epithelioma.

Basal Cell Carcinoma↗
PubMed1995

Disposition of polaprezinc (zinc L-carnosine complex) in rat gastrointestinal tract and effect of cimetidine on its adhesion to gastric tissues.

The disposition of polaprezinc in the rat gastrointestinal tract was studied by a double tracer method using [14C]- and [65Zn]polaprezinc. At 0.5 h after oral administration of [14C]-,[65Zn]polaprezinc to rats, the 14C-radioactivity in the gastric contents was comparable with the 65Zn-radioactivity. However, a significant difference was observed in the time course of changes in gastric contents between 14C- and 65Zn-radioactivity over 1 h after administration, indicating that polaprezinc existed in complex form at 0.5 h after administration and was dissociated to L-carnosine and zinc in the gastrointestinal tract as a function of time. The adhesion of zinc to stomach mucosa after oral administration of polaprezinc to rats was significantly increased by treatment with cimetidine. These results suggest that the adhesion of zinc to gastric tissues is increased by inhibiting the dissociation of polaprezinc, and that H2-receptor antagonists, such as cimetidine, may increase anti-ulcer effects of polaprezinc.

Administration, Oral↗
PubMed1995

Intrauterine transmission of human T-cell leukemia virus type I in rats.

To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.

Animals↗
PubMed1995

Residence time of polaprezinc (zinc L-carnosine complex) in the rat stomach and adhesiveness to ulcerous sites.

Polaprezinc, an insoluble zinc complex of L-carnosine, exhibits anti-ulcer effects by acting directly on mucosal lesions. The disposition of polaprezinc in the stomach was studied to clarify the usefulness of its structure as an insoluble complex. The time courses of 14C-radioactivity in the gastric contents and gastric tissues were parallel to those of 65Zn after oral administration of a mixture of 14C-polaprezinc and 65Zn-polaprezinc (14C-, 65Zn-polaprezinc) to rats. The gastric contents of 14C-polaprezinc and 65Zn-polaprezinc were greater than those of 14C-L-carnosine and 65ZnSO4. Mean residence times (MRT) of 14C-polaprezinc and 65Zn-polaprezinc in the stomach were almost the same (ca. 2 hr), and they were double those of 14C-L-carnosine and 65ZnSO4. In gastric tissues, the area under the concentration curves (AUC0-8 hr) of 14C-polaprezinc and 65Zn-polaprezinc were 1.7 times greater than those of 14C-L-carnosine and 65ZnSO4, respectively. After administration of 14C-, 65Zn-polaprezinc to rats with acetic acid-induced ulcers, 14C and 65Zn-radioactivities in the ulcerous sites were very similar and greater than those of 14C-, 65Zn-polaprezinc dissolved in acid. In conclusion, polaprezinc is retained in the stomach longer and adheres to the ulcerous sites more than zinc or L-carnosine. The characteristics of this compound may arise from its insolubility and contribute to its strong pharmacological action.

Animals↗