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M Chun

Publications and source records attributed to M Chun.

At least 37 records · Page 2Linked to original sources
PubMed1995

Caveolin isoforms differ in their N-terminal protein sequence and subcellular distribution. Identification and epitope mapping of an isoform-specific monoclonal antibody probe.

Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (alpha-isoform) and a faster migrating 21-kDa species (beta-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the alpha-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that alpha- and beta-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these "tagging" experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the alpha-isoform contains a complete N terminus while the beta-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the approximately 3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between alpha- and beta-caveolin.

Amino Acid Sequence↗
PubMed1995

Evidence for a regulated interaction between heterotrimeric G proteins and caveolin.

Caveolae are flask-shaped plasma membrane specializations. A 22-kDa protein, caveolin, is a principal component of caveolar membranes in vivo. As recent evidence suggests that caveolae may participate in G protein-coupled signaling events, we have investigated the potential interaction of caveolin with heterotrimeric G proteins. Using cell fractionation techniques, we found that mutational or pharmacologic activation of Gs alpha prevents its cofractionation with caveolin. In a second independent approach, we directly examined the interaction of G proteins with caveolin. For this purpose, we recombinantly expressed caveolin as a glutathione S-transferase fusion protein. Using an in vitro binding assay, we found that caveolin interacts with G protein alpha subunits (Gs, Go, and Gi). Mutational or pharmacologic activation (with guanosine 5'-O-(thiotriphosphate)) of G alpha subunits prevents this interaction, indicating that the inactive GDP-bound form of G alpha subunits preferentially interacts with caveolin. This G protein binding activity is located within a 41-amino acid region of caveolin's cytoplasmic N-terminal domain (residues 61-101). Further functional analysis shows that a polypeptide derived from this region of caveolin (residues 82-101) effectively suppresses the basal activity of purified G proteins, apparently by inhibiting GDP/GTP exchange. This caveolin sequence is homologous to a region of the Rab GDP dissociation inhibitor, a known inhibitor of GDP/GTP exchange for Rab proteins. These data suggest that caveolin could function to negatively regulate the activation state of heterotrimeric G proteins.

Amino Acid Sequence↗
PubMed1995

Endothelin-induced endocytosis of cell surface ETA receptors. Endothelin remains intact and bound to the ETA receptor.

We demonstrate unusual features of the intracellular processing of endothelin-1 (ET-1) and its receptor ETA, the receptor subtype that mediates contraction of vascular smooth muscle cells. First, we show that in stably transfected CHO cells expressing ETA, binding of an ET-1 ligand induces rapid endocytosis of cell surface ETA. Receptor endocytosis was measured both by immunofluorescence and by radioiodinated antibodies specific for ETA. Second, we demonstrate that ET-1 remains intact for up to 2 h after endocytosis and, as judged by co-immunoprecipitation, internalized 125I-ET-1 remains bound to ETA receptors. We hypothesize that internalized ET-1, bound to ETA receptors, continues to activate a signal-transducing G protein, thus accounting for the prolonged period of contraction induced in smooth muscle cells by a single administration of ET-1.

Animals↗
PubMed1995

Apolipoprotein E and Alzheimer's disease: ethnic variation in genotypic risks.

The presence of the apolipoprotein epsilon 4 (apo epsilon 4) allele significantly increases the risk of Alzheimer's disease. Whether this is due to biological effects of the apo epsilon 4 protein or reflects linkage disequilibrium with an as yet unidentified Alzheimer's disease susceptibility gene is of critical importance. In a community study in northern Manhattan we found a fivefold increase in the risk of Alzheimer's disease among African-Americans, Hispanics, and whites homozygous for apo epsilon 4. Overall, the risk between Alzheimer's disease and apo epsilon 4 heterozygosity was also increased by twofold, but the association was somewhat weaker for African-Americans than for Hispanics and whites. In contrast, the apo epsilon 2/epsilon 3 genotype was associated with an eightfold increased risk of Alzheimer's disease in African-Americans but it was associated with reduced risk in whites. Variability in the strength and type of association between Alzheimer's disease and the apo E polymorphisms in the three ethnic groups could not be fully explained by age differences. The allelic frequency of apo epsilon *4 was significantly higher in patients than control subjects in all ethnic groups at age 70 or younger, reflecting the higher proportion of apo epsilon 4 homozygotes, but this difference diminished with increasing age. The allelic frequency of apo epsilon *2 for African-Americans and Hispanics, but not whites, was significantly higher in patients than control subjects, but only after age 70.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Evaluation of the gel test for antibody screening in a tertiary hospital in Hong Kong; insensitivity for some cold antibodies that are reactive at 37 degrees C by conventional indirect antiglobulin tests.

The antiglobulin gel test (DiaMed) was compared with conventional IAT methods in pretransfusion antibody screening in a tertiary hospital in Hong Kong. Antibody titrations showed that the gel system was more sensitive than the tube IAT method in detecting anti-D, anti-S, anti-s, but slightly less sensitive with anti-E. The gel tests detected all clinically significant warm reacting allo-antibodies detected by conventional IAT methods except one case of weak anti-E. It detected a case of anti-C missed in conventional screening. The number of serological investigations due to false positive screens, non-specific antibodies and clinically insignificant cold antibodies (anti-P, and anti-Lewis) were reduced. The gel system was fast and easy to use. It would reduce delays and inconvenience caused to patients due to false positive screens and non-specific antibodies without compromising detection of clinically significant antibodies. However, of concern was the failure to detect several cases of anti-Le(a) and anti-P1 that were reactive at 37 degrees C by conventional IAT and one case of anti-H in a patient with Parabombay A.

Blood Grouping and Crossmatching↗
PubMed1995

Radiation therapy of external iliac lymph nodes with lateral pelvic portals: identification of patients at risk for inadequate regional coverage.

PURPOSE: To determine whether "standard" lateral pelvic radiation therapy portals provide adequate margins for treating the external iliac lymph nodes (EILNs). MATERIALS AND METHODS: With computed tomographic (CT) data and a computerized algorithm, the course of the EILN chain was reconstructed in 48 patients. The marginal distance between a consistently localized portion of the EILNs and the anterior field border that was placed at the front of the pubic symphysis was recorded for each patient. RESULTS: The EILNs were covered adequately in only 50% of both men and women. More adequate EILN margins were observed in (a) women older than 68 years, (b) women with small anteroposterior separations, (c) men older than 70 years, and (d) men and women with a low central obesity index. Less adequate margins were observed in men who were obese or men with large anteroposterior separations. In the study population, no correlation was observed for sex, race, or tumor site. CONCLUSION: The authors recommend portals that are designed specifically for patients rather than standard portals that may inadequately cover the intended targets.

Adult↗
PubMed1995

Synergistic effects of traumatic head injury and apolipoprotein-epsilon 4 in patients with Alzheimer's disease.

The apolipoprotein-epsilon 4 allele increases the risk of Alzheimer's disease (AD), but cerebral deposition of beta-amyloid with age, a genetic mutation, or head injury may contribute to the pathogenesis of this disease. We examined the risks of AD associated with traumatic head injury and apolipoprotein-epsilon 4 in 236 community-dwelling elderly persons. A 10-fold increase in the risk of AD was associated with both apolipoprotein-epsilon 4 and a history of traumatic head injury, compared with a two-fold increase in risk with apolipoprotein-epsilon 4 alone. Head injury in the absence of an apolipoprotein-epsilon 4 allele did not increase risk. These data imply that the biological effects of head injury may increase the risk of AD, but only through a synergistic relationship with apolipoprotein-epsilon 4.

Aged↗
PubMed1994

Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin.

Caveolae are small invaginations of the plasma membrane 50-100 nm in diameter. Since calcium channels, inositol 1,4,5-trisphosphate receptors, and heterotrimeric GTP-binding proteins (G proteins) are localized in caveolae, they may participate in signal transduction by G protein-coupled receptors. Here we show that the G protein-coupled endothelin receptor subtype A (ETA) and its bound endothelin ligand are found in plasma membrane caveolae. ETA and its bound ligand coimmunoprecipitate with caveolin, a structural component of caveolae, in extracts of cells expressing transfected ETA receptors. Confocal fluorescence microscopy shows colocalization of ETA receptors and caveolin in micropatches at or near the plasma membrane, in the absence of endothelin ligands. These observations demonstrate a functional role for plasma membrane caveolae in signal transduction by this G protein-coupled receptor.

Amino Acid Sequence↗
PubMed1994

Cosegregation of the renin gene with an increase in mean arterial blood pressure in the F2 rats of SHR-WKY cross.

Using the restriction endonuclease, BgI I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats. This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F2 rats produced from F1 rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F2 rats of SHR and WKY cross. The mean arterial blood pressure of F2 rats homozygous with the SHR allele was significantly higher than F2 rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in these F2 rats. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F2 rats of SHR and WKY cross.

Alleles↗
PubMed1993

Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis.

Ser-49 located at the exposed surface loop of subtilisin J was replaced with Asp and Arg. Proteinase activity of the Asp-49 mutant was similar to that of wild-type, but the Arg mutant was inactivated. At 37 degrees C, mature subtilisin J protein of the Asp-49 mutant rapidly degraded, and specific breakdown products were accumulated. These proteins were analyzed by SDS-PAGE, and the N-terminal sequences were determined for the mature and deleted protein. We identified the autoproteolytic cleavage site in the mature Asp-49 mutant protein from sequencing data.

Amino Acid Sequence↗
PubMed1993

The distorted alpha 2-adrenoceptor genotype distribution in F2 populations of Dahl salt sensitive and resistant rats cross.

To examine the role of the alpha 2-adrenoceptor gene in the development of genetic hypertensive rats, we tested Dahl salt sensitive (S) and resistant rats (R) for the presence of a restriction fragment length polymorphism (RFLP) in that gene. An RFLP was found between the S and R rats with a human kidney cDNA alpha 2-adrenoceptor probe (alpha 2-C4) and Msp I restriction endonuclease. The alpha 2-C4 probe detected two alleles, S and R, of 3.0 and 2.8 kb in size. The two strains of rats were each homozygous for their corresponding allele. The inheritance of the alleles was investigated by crossbreeding S and R rats and subsequent brother/sister mating of F1 rats. Two hundred and fifteen F2 rats were produced by breeding 14 pairs of F1 rats. An atypical distribution of alpha 2-adrenoceptor genotypes was observed. We found a reduced number of the SS genotype in both males and females of the F2 generation. To investigate the mechanism of this distorted alpha 2-adrenoceptor genotype distribution in the F2 rats of S and R cross, we backcrossed the F1 rats to their S and R parents. The litter size and gender distribution were counted for each breeding colony. Analysis by chi-square test showed that there was no sex difference among the backcrosses. Also, there was no significant decrease in litter size. This excludes the possibilities of fetal demise of S homozygotes and intrauterine selection. Therefore the deficiency of SS genotype may be due to gene recombination or may not be due to the alpha 2-adrenoceptor gene itself, but to the effect of other genes closely linked to the alpha 2-adrenoceptor.

Animals↗
PubMed1993

Cosegregation of the renin gene with an increase in mean arterial blood pressure in the F2 rats of SHR-WKY cross.

Using the restriction endonuclease, Bgl I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats (1). This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F2 rats produced from F1 rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F2 rats of SHR and WKY cross. The mean arterial blood pressure of F2 rats homozygous with the SHR allele was significantly higher than F2 rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in F2 rats with different genotypes. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F2 rats of SHR and WKY cross.

Alleles↗
PubMed1992

Northern blot and ribonuclease protection study of alpha 2-adrenoceptor subtypes in cultured cell lines.

Northern blot and ribonuclease protection assay were used to identify alpha 2-adrenoceptor subtypes in human colonic adenocarcinoma (HT29), neuroblastoma x glioma rat-mouse hybrid NG108-15 (NG108) and opossum kidney (OK) cell lines. Radioligand binding studies showed that the alpha 2-adrenoceptor expressed in HT29, NG108 and OK cells represent the pharmacological alpha 2A, alpha 2B and alpha 2C subtypes respectively. In our Northern blot analysis, hybridization of poly(A)+ RNA from HT29, NG108 and OK cells with human kidney alpha 2-adrenoceptor cDNA probe (alpha 2-C4) identified a single band of 4.4, 4.2 and 4.4 kb respectively in each cell line. Hybridization with a human platelet alpha 2-adrenoceptor genomic probe (alpha 2-C10) resulted in two bands for HT29 cells with the size of 4.4 kb and 3.9 kb. No bands were seen for HT29, NG108 and OK cells when hybridized with a third alpha 2-adrenoceptor human genomic DNA probe which is localized in chromosome 2 (alpha 2-C2). For the HT29 cells, the 3.9 kb band was seen only when using the alpha 2-C10 probe. Thus, this band probably represents alpha 2-C10 mRNA. To further characterize the alpha 2-adrenoceptor mRNA expressed in HT29, NG108 and OK cells, the sensitive ribonuclease protection assay was performed. A single band about 900 bp was protected when the poly(A)+ RNA from NG108 and OK cells was hybridized with an alpha 2-C4 RNA probe and digested with RNAases. Hybridization of mRNA from HT29 cells with alpha 2-C10 RNA probe and digestion with RNAases protected a 500 bp fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Comparative study of the expression of Rb and p53 genes in human colorectal cancers, colon carcinoma cell lines and synchronized human fibroblasts.

We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line WI38. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.

Actins↗
PubMed1990

Uterine thickness determination using real-time ultrasonography: a guide for intracavitary brachytherapy in the treatment of endometrial carcinoma.

Two patients with medically inoperable stage I endometrial carcinoma were treated with intracavitary implants alone using Simon capsules, tandems, and ovoids. In both cases, uterine thickness was measured during the implant procedure by realtime ultrasonography. Tumor doses in both patients were then calculated to the midmyometrium and to the serosal surface of the uterus. These estimates, rather than the usual milligram-hours or points A and B, were used to make treatment decisions.

Brachytherapy↗
PubMed1990

Stage II carcinoma of the cervix: analysis of the value of pretreatment extraperitoneal lymph node sampling and adjunctive surgery following irradiation.

This is a retrospective analysis of the results of treatment of 24 patients with bulky stage II carcinoma of the cervix treated with full course irradiation followed by adjunctive surgery between 1975 and 1980. A review of the surgical specimens following irradiation showed that 12 patients had no residual cancer, five had only microscopic foci of cancer, and five had extensive residual cancer. Two patients had unresectable persistent cancer. Six patients had histological evidence of lymph node metastases prior to irradiation. The surgical-pathological findings following irradiation had important prognostic implications. All five patients with extensive residual cancer in the surgical specimen recurred, 2 of 5 patients with only microscopic foci of residual cancer and, none of the 12 patients with no residual cancer in the resected specimens developed a recurrence. Lymph node involvement was not associated with an increased incidence of recurrence. Most patients with residual cancer following full course irradiation recurred locally. Thus the addition of adjunctive surgery following full course irradiation did not significantly improve the treatment results of patients with bulky stage II carcinoma of the cervix.

Brachytherapy↗
PubMed1989

Interstitial iridium-192 implantation for malignant brain tumours. Part II: Clinical experience.

The treatment results of 37 patients with malignant brain neoplasms treated with a computed-tomography-guided stereotactic iridium-192 implant are reviewed. Of these, 29 patients with high-grade gliomas (20 with glioblastoma multiforme (GBM), nine with anaplastic astrocytoma (AA] received an implant as part of their initial management. The median survival was 14.5 and 15.5 months in the patients with previously untreated GBM and AA, respectively. In those patients with recurrent tumour after external-beam irradiation, durable local control over a year was achieved with implantation. Increasing the total tumour dose from 120 to 160 Gy did not improve survival or local control. Karnofsky Performance Status (KPS) was used as an indicator of quality of life and was seen to decrease with a median interval of 8.5 months following treatment. No severe complications were noted in the entire group of patients treated with this implant procedure.

Adult↗