[An autopsy case of primary biliary cirrhosis associated with progressive systemic sclerosis--a review of reported cases in Japan].
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Autoantibodies↗
Biomedical subjects
M Haraguchi
Publications and source records attributed to M Haraguchi.
The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function.
The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.
Animals↗
Structural characterization of thymidine phosphorylase purified from human placenta.
Human thymidine phosphorylase (dThdPase) is thought to be identical to an angiogenesis factor, platelet-derived endothelial cell growth factor (PD-ECGF). However, the whole amino acid sequence of dThdPase is still unknown. N-terminal amino acid sequencing of dThdPase isolated from human placenta gave the sequence Ac-AALMTPGTGAPPAPG. Comparison with the sequence predicted from the PD-ECGF cDNA reveals that residues 2-16 of dThdPase are identical to that of PD-ECGF. If dThdPase and PD-ECGF are derived from the same gene, the primary translational product of dThdPase would be processed one amino acid from the translation-initiating methionine residue and Ala-2 acetylated. Since placental and platelet PD-ECGF is reported to be processed at Thr-6 and Ala-11, respectively, and the N-terminal end is not blocked, further study is needed to clarify the reason for this discrepancy and whether the difference in N-terminal sequence affects the physiological function of these molecules.
Amino Acid Sequence↗
Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase.
Human thymidine phosphorylase (dThdPase) has been reported to be identical with the platelet-derived endothelial cell growth factor (PD-ECGF). To investigate whether the dThdPase activity of PD-ECGF/dThdPase is indispensable to its angiogenic activity, three PD-ECGF/dThdPase mutants, K115E (Lys-115-->Glu), L148R (Leu-148-->Arg) and R202S (Arg-202-->Ser) were made by site-directed mutagenesis. Although the expression level of the three mutant PD-ECGF/dThdPases in the COS-7 cells was similar to that of wild-type PD-ECGF/dThdPase, dThdPase activity was not detected in the COS-7 cells transfected with the mutant PD-ECGF/dThdPase cDNA. The lysates of COS-7 cells transfected with the wild-type PD-ECGF/dThdPase cDNA had angiogenic activity, but those transfected with the mutant PD-ECGF/dThdPase cDNAs did not. An inhibitor of dThdPase, 6-amino-5-chlorouracil, inhibited the angiogenic activity of purified dThdPase. These findings indicate that dThdPase activity is indispensable to the angiogenic activity of PD-ECGF/dThdPase.
Amino Acid Sequence↗
Substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells.
The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.
Animals↗
T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene.
cDNA clones of the mouse GM2/GD2 synthase (EC 2.4.1.92) gene were isolated, and their analyses revealed that the protein has a type II transmembrane structure with 533 amino acids, which was very similar to the human homolog except for the mRNA size. The mRNA level in thymocytes dramatically increased after treatment with anti-CD3 monoclonal antibody, whereas it was not elevated when treated with prostaglandin E2. In situ hybridization showed an elevation of mRNA levels in medullar thymocytes, suggesting that T cell receptor-mediated signaling induces up-regulation of the GM2/GD2 synthase gene in mature thymocytes.
Amino Acid Sequence↗
Absorption cytometric DNA analysis of sections of gastric carcinomas and a comparison with cytofluorometry of single-cell suspensions and flow cytometry.
Measurements were made of cellular deoxyribonucleic acid (DNA) content in paraffin-embedded materials from 44 gastric carcinomas, using three different methods; static cytometric light absorbance technique using tissue sections (SCM-abs), static cytometric fluorescence technique using single-cell suspensions (SCM-flu), and flow cytometry (FCM). The DNA ploidy determined by SCM-abs was grouped into low or high ploidy according to dispersion on the DNA histogram, and the DNA ploidy determined by SCM-flu and FCM was grouped into diploidy or aneuploidy, according to the peak DNA value. Accord between SCM-abs and SCM-flu or between SCM-abs and FCM was confirmed when low ploidy by SCM-abs showed diploidy by SCM-flu or FCM and high ploidy by SCM-abs showed aneuploidy by SCM-flu or FCM. An incomplete but good accord of DNA ploidy was obtained with these methods.
Absorption↗
Expression of alpha 2,8-sialyltransferase (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes.
GD3 Synthase (alpha 2,8sialyltransferase) (EC 2.4.99.8) cDNA has been cloned by eukaryotic cell expression cloning. Using this cDNA as a probe, the expression level of the gene in human cancer cell lines was analysed by Northern blotting and RT-PCR, then correlated with the ganglioside expression and enzyme activity. Melanoma cell lines showed extremely strong bands in Northern blot and RT-PCR/Southern analysis. The enzyme activity was also very high in melanomas as expected. Neuroblastoma and astrocytoma lines showed relatively low levels of the gene expression, whereas they expressed high levels of GD2. Although the mRNA level of the GD3 synthase gene and enzyme activity in individual cell lines correlated positively, some cell lines showed much higher activity than expected from the mRNA level. Among leukaemia lines, adult T cell leukaemia-associated (HTLV-I+) lines showed fairly high levels of the mRNA. On the other hand, T-ALL lines showed very low levels. In addition, GD3 and GD2 expression and mRNA level of the gene during T lymphocyte activation were analysed. Only GD3 expression was induced by any of the stimulatory reagents used, and corresponding up-regulation of the GD3 synthase gene was shown in RT-PCR/Southern analysis.
Base Sequence↗
Cytophotometric study of nuclear DNA content in scirrhous gastric carcinoma.
We made a cytophotometric DNA analysis using 21 scirrhous gastric carcinomas (SGC), and compared the results with those of 12 undifferentiated type early gastric carcinomas (EGC) located in the gastric body. Nuclear DNA content of tumor cells was measured in the mucosa of SGC (SGCm), serosa of SGC (SGCs) and mucosa of EGC (EGCm). DNA distribution pattern was high ploid in 18 SGCm (86%), 16 SGCs (71%) and 4 EGCm (33%). Mean DNA content was 4.02c in SGCm, 3.91c in SGCs and 3.26c in EGCm. Percentage of tumor cells with a DNA content over 3c was 77.4% in SGCm, 69.1% in SGCs and 49.5% in EGCm. Statistically significant differences in these 3 factors were present between SGCm and EGCm, but absent between SGCm and SGCs. Furthermore, DNA distribution pattern was not different between the mucosa and serosa in 19 SGC (90%). The results suggest that most scirrhous gastric carcinomas are high ploid in origin and do not change their biologic behaviour throughout the tumor progression.
Adenocarcinoma, Scirrhous↗
Synchronous multicentric development of hepatocellular carcinoma.
Recently, the multicentric origin of hepatocellular carcinoma (HCC) has been recognized, but its clinical importance has still not yet been clarified. The histological characteristics of small hyperechoic HCCs coexisting in 44 consecutively resected Japanese patients whose main HCCs were < 5.0 cm in size were studied. Twelve small hyperechoic HCCs were found and classified into the following two groups: eight nodules in seven patients (15.9%) were early-stage HCC, and four nodules in four patients (9.1%) were more advanced HCC. Thus, early-stage HCC comprised 66.7% of the small echogenic HCCs. Eight HCCs detected as small hyperechoic lesions (found in 15.9% of the patients) showed varying degrees of fatty change yet proved to be well differentiated and retained the preexisting liver structure of either associated liver cirrhosis or chronic hepatitis. Moreover, the histologic characteristics of the eight early-stage HCCs were different from those of the main HCCs. In conclusion, approximately 15% of HCCs in Japanese patients may have a synchronous multicentric origin, and small hyperechoic lesions should be carefully evaluated. However, in the United States or other areas where the occurrence of fatty liver is common, that advice for small hyperechoic lesions may be overly cautious.
Adult↗
Adenosquamous carcinoma of the gall-bladder with gastric foveolar-type epithelium.
An 80 year old Japanese man had adenosquamous carcinoma of the gall-bladder characterized by an adenocarcinoma (AC) in the gall-bladder lumen and a squamous cell carcinoma (SCC) in the invaded region of the liver. In the AC, the tumor cells consisted of atypical columnar epithelium with pseudostratification, mimicking gastric foveolar epithelium, while atypical signet-ring cells were scattered within the SCC. There was an abrupt transition between the AC and SCC areas. The tumor cells in the AC area were intensely positive for galactose oxidase-Schiff staining, and paradoxical concanavalin A staining revealed these tumor cells to have Class II mucins. Immunohistochemically, the tumor cells in foveolar-type adenocarcinoma were diffusely positive for cathepsin D. Flow cytometrical analysis of DNA content showed the AC area to be diploid and the SCC area to be aneuploid. The S-phase fraction of the SCC area (46.9%) was larger than that of the AC area (19.5%). The positive rate of immunostaining for proliferating cell nuclear antigen in the SCC area (mean 50.627%) was larger than that of the AC area (mean 3.048%, P < 0.01). These results suggest that the AC area of this tumor, histochemically and immunohistochemically, showed gastric foveolar-type characteristics, the SCC component was squamous cell metaplasia of the pre-existing AC, and that the SCC area had a greater proliferating capacity than the AC area.
Aged↗
Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.
To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.
Animals↗
Signet ring cell carcinoma in hyperplastic polyp.
BACKGROUND: A polypectomy was performed on a 68-year-old woman who had three gastric hyperplastic polyps. A focal signet ring cell carcinoma was discovered in one of the polyps, which was 2.2 cm in diameter and was located in the antrum. METHODS: To study the carcinoma's phenotype expression, special mucin histochemical stainings were performed. RESULTS: The carcinoma infiltrated only into the mucosa. Intestinal metaplasia was scant. In the neoplasm neutral and foveolar mucin was predominant over acid mucin. The surrounding hyperplastic foveolar epithelium showed positivity for neutral mucin, and the intestinal metaplasia foci for acid mucin. CONCLUSIONS: This neoplasm has a mixed composition of mucins and shares histochemical characteristics with the surrounding hyperplastic foveolar epithelium.
Aged↗
Chemical and biological demonstration of the presence of monofluoroacetate in the leaves of Palicourea marcgravii St. Hil.
Cattle losses in Brazil have been attributed to Palicourea marcgravii St. Hil., a toxic plant for cattle. The crude extract from the leaves of P. marcgravii was successively fractionated using solvents with different polarities to determine whether monofluoroacetic acid and/or some other substance present in the leaves may be responsible for the acute symptoms caused by the plant. Authentic sodium monofluoroacetate (SMFA) was used for comparison. The only P. marcgravii fraction which induced seizures and death in intoxicated rats was water soluble. The signs and symptoms induced in the animals by the crude extract and water-soluble fraction were the same as induced by SMFA and included tonic seizures and other actions on the CNS. The dose-lethality and dose-latency to the 1st seizure curves constructed for the water-soluble fraction of the leaf extract (30-100 mg/kg) and SMFA (0.6-3.0 mg/kg) were parallel. Five animals per dose were used. The potency ratio of SMFA in relation to the water-soluble fraction of the leaf extract was 53.8 (dose-lethality curve) and 64.1 (dose-latency to the 1st seizure curve). The water-soluble fraction contained a substance with hRf = 20 which was the same as that of authentic SMFA. The 19F NMR spectra of authentic SMFA and the P. marcgravii water-soluble fraction were identical. These data demonstrate the presence of SMFA in the water-soluble fraction of P. marcgravii leaves and show that monofluoroacetate is the active principle responsible for the signs and symptoms of acute intoxication.
Animals↗
The acute toxicity of Riedeliella graciflora in laboratory animals.
Riedeliella graciflora is a plant mainly found in southeast South America and related to bovine mortality in Sao Paulo State, Brazil. Our laboratory showed that cattle intoxicated with 10 g R graciflora leaves/kg had organ congestion, edema and hemorrhage. The aim of this study was to determine which laboratory animal species would present the same lesions found in cattle. We also studied the effects of administering parts of the fruits or leaves po, sc, ip or im. All the laboratory species developed organ congestion, edema and hemorrhage and severe nephrosis regardless which plant part was given or the route of administration used. The effects were more intense in animals dosed with R graciflora fruits ip.
Animals↗
The acute toxicity of Riedeliella graciflora in calves.
Three calves received 10 g Riedeliella graciflora dry leaves/kg body weight by gavage. Blood samples were taken immediately before plant administration and at 2, 4, 6, 12 and 24 hours later; serum ALT, AST, AP, TB, urea and creatinine were determined. After R graciflora administration, the calves had anorexia, profound depression and recumbency prior to death. Creatinine levels increased markedly until death. Severe tubular nephrosis was consistently observed.
Alkaline Phosphatase↗
Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.
For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.
Amino Acid Sequence↗
Angiogenic activity of enzymes.
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Animals↗