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E K Chan

Publications and source records attributed to E K Chan.

At least 109 records · Page 6Linked to original sources
PubMed1993

Pancreatic islet function in nondiabetic and diabetic BB rats.

A decreased acute insulin response to glucose in islet cell antibody positive humans predicts diabetes. Because the dominant mechanism leading to decreased in vivo acute insulin response to glucose remains unclear, perifused islets were examined before and after diabetes onset in BB rats to assess the role of glucose sensitivity on insulin secretion in individual islets. Islets from normal WF rats, diabetes-prone rats without inflamed islets, diabetes-prone rats with inflamed islets, and diabetic rats were studied at 2.0, 8.3, and 16.7 mM glucose. Immunoreactive insulin from WF islets at 16.7 mM glucose was 0.15 +/- 0.02 ng.0-7 min-1 x islet-1 for the first phase and 1.00 +/- 0.05 ng.7-20 min-1 x islet-1 for the second phase of biphasic secretion, compared with basal secretion of 0.10 +/- 0.03 ng.20 min-1 x islet-1 at 2 mM glucose. Diabetes-prone noninflamed islets showed a 0.20 +/- 0.03 ng first-phase secretion, a 1.32 +/- 0.13 ng second-phase secretion after 16.7 mM glucose, and 0.093 +/- 0.02 ng.20 min-1 x islet-1 at 2 mM glucose, indicating no intrinsic BB rat strain secretion abnormality. Diabetes-prone inflamed islets had secretions of 0.35 +/- 0.02 ng during the first phase (P < 0.05 vs. WF) and 1.78 +/- 0.29 ng during the second phase (P < 0.05 vs. WF) after 16.7 mM glucose, with 0.24 +/- 0.08 ng.20 min-1 x islet-1 at 2 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Natural history of atherosclerosis and hyperlipidemia in heterozygous WHHL (WHHL-Hh) rabbits. I. The effects of aging and gender on plasma lipids and lipoproteins.

Plasma lipid and lipoprotein levels are influenced by physiologic changes during the life span of mammalian organisms. In the present study, the effects of aging and gender on plasma lipids and lipoprotein cholesterol fractions in heterozygous Watanabe heritable hyperlipidemic (WHHL-Hh) rabbits were examined. For 3 years the rabbits were fed regular rabbit chow and were free from any experimental manipulation. Male WHHL-Hh rabbits (n = 17) had a 55% increase in total plasma cholesterol level (p < 0.005) between the ages of 7.5 and 34 months. This increase was due to significant increases in very low-density lipoprotein cholesterol (VLDL-C) (28%, p < 0.05), in low-density lipoprotein cholesterol (126%, p < 0.005), and in high-density lipoprotein cholesterol (28%, p < 0.05). Female WHHL-Hh rabbits (n = 11) did not show significant differences in lipid plasma values with aging. However, their plasma values, with the exception of the VLDL-C, were significantly (p < 0.05) higher than those of the male rabbits. Pregnancy and increased weight in the older female rabbits may have accounted for these differences. Significant plasma lipid changes occur as a function of heterozygosity, age, and gender in WHHL-Hh rabbits. The lipoprotein changes seen in male but not in female WHHL-Hh rabbits during the aging process are similar to those observed in human subjects. Pregnancy in WHHL-Hh rabbits, like in human beings, may induce hyperlipidemia, which is opposite to the effects observed in normal rabbits or homozygous WHHL rabbits.

Aging↗
PubMed1992

Laboratory evaluation of patients with Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterized by lymphocytic infiltration of the lacrymal and salivary glands, leading to severe dryness of eyes (keratoconjunctivitis sicca) and mouth (xerostomia). SS may exist as a primary disorder (1 degree-SS) or in association with other autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus or progressive systemic sclerosis (scleroderma). Diagnosis of 1 degree-SS is confirmed by minor salivary gland biopsy and the presence of circulating autoantibodies. Minor salivary gland biopsies exhibit focal lymphocytic infiltrates that are present in the majority of lobules. Incorrect methods of biopsy and failure to determine the average focus score are common causes for false-positive and false-negative biopsies. SS patients frequently have a positive antinuclear antibody test due to presence of SS-A (Ro) and SS-B (La) autoantibodies. Molecular analysis has revealed multiple "SS-A" proteins (60 kd, 54 kd, 52 kd) that react with sera from SS patients, as well as a 48 kd SS-B protein. Rheumatoid factor (anti-IgG Fc antibody) in 1 degree-SS patients exhibits restriction in its light chain-associated idiotype, in contrast to RA patients where no restriction of idiotype was detected. Other autoantibodies found in a subpopulation of SS patients include anti-ADP ribose polymerase, anti-cardiolipin, anti-mitochondrial, anti-mitotic spindle apparatus, anti-parietal cell, and anti-thyroid associated antibodies. Due to the high frequency of dryness syndromes in patients due to other causes (ranging from drug side effects to normal aging processes), the use of strict criteria for diagnosis of SS will lead to improved cost-efficient medical care avoiding needless anxiety in the patient.

Animals↗
PubMed1992

Glipizide-induced immunomodulation: inhibition of human mononuclear cell stimulation and macrophage-mediated islet cell killing in the BB rat.

Intensive insulin treatment, when combined with sulfonylurea drugs, may enhance remissions in new-onset type I diabetic patients. These clinical data suggest that sulfonylurea drugs may have immunosuppressive actions in addition to insulin secretory and sensitivity actions. Thus, studies were conducted in humans to determine if glipizide was immunomodulatory in vitro. Five, 10, and 15 micrograms/mL of phytohemagglutinin (PHA) and concanavalin A (Con A), and 0.5, 1.0, and 1.5 micrograms/mL of pokeweed mitogen (PWM) were incubated with normal human peripheral blood mononuclear cells. Maximum stimulatory indices were 52, 39, and 30 for PHA, Con A, and PWM, respectively. Additional incubations were performed in the presence of 1, 10(1), 10(2), 10(3), 10(4), and 10(5) ng/mL glipizide. Glipizide concentrations inhibiting mitogen stimulation approximately 50% (P less than .01 v nonglipizide control) were 1.0 ng/mL for PWM, 10 ng/mL for Con A, and 10(4) ng/mL for PHA. At higher glipizide levels, inhibition was 90% to 100%. To determine if glipizide immunomodulates diabetes-associated reactions, bio-breeding (BB) diabetic rat splenic macrophage-mediated islet killing was studied. Glipizide at 10(3) ng/mL inhibited islet killing by 60% (P less than .001). These preliminary data suggest that glipizide may have immunomodulatory actions not previously appreciated, and may be related to this drug's putative action in islet cell restoration.

Adult↗
PubMed1992

Glutathione S-transferase-mediated metabolism of glyceryl trinitrate in subcellular fractions of bovine coronary arteries.

The possible role of glutathione S-transferases (GTSs) in vascular glyceryl trinitrate (GTN) metabolism was investigated. GTN degradation to form its dinitrate metabolites (GDNs) in the 9000g (9k) supernatant fraction of bovine coronary arteries (BCA) was examined. BCAs were homogenized with a 3x volume of phosphate buffer, and the 9k fraction was obtained by centrifugation. GTN (40 ng/ml; 1.76 x 10(-7) M) was incubated for 2 hr in the 9k fraction of BCA in the presence of reduced glutathione (2 x 10(-3) M). Samples were taken at 10, 20, 40, 60, and 120 min. GTN was observed to degrade readily, exhibiting a half-life of 26 min in the incubate. While both 1,2- and 1,3-GDNs were generated from GTN, formation of 1,3-GDN was predominant (GDN ratio, as 1,2/1,3-GDN, = 0.7-0.8). Coincubation with 2 x 10(-5) M concentrations of two GST inhibitors, sulfobromophthalein (SBP) and ethacrynic acid (ECA), decreased the rate of GTN loss. The GTN half-lives in SBP- and ECA-treated incubations were 66 and 84 min, respectively. In addition, the pattern of GDN formation was also altered. The resultant GDN ratios exceeded unity in the presence of these inhibitors, indicating that 1,3-GDN formation was attenuated to a greater extent than that of 1,2-GDN. These data suggest that vascular GTN metabolism in BCA is carried out by cytosolic GST isozymes which possess a preference for C-2 denitration of GTN.

Animals↗
PubMed1992

Antinuclear antibodies in Sjögren's syndrome.

The definition and characterization of the reactivity of ANA in patients with SS are greatly improved with the current advances in cellular and molecular biology. Based on the characteristic autoantibody profiles in different systemic rheumatic diseases and the nature of the defined autoantigens, it has been proposed that the polyclonal autoimmune response is induced and maintained through an antigen-driven mechanism. The autoantigens are presented in the cell as components of large particles or structures composed of protein-protein or RNA-protein complexes; however, the role of these autoantibodies in the etiology and immunopathogenesis of SS remains to be determined.

Amino Acid Sequence↗
PubMed1991

Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor.

In dividing eukaryotic cells, nucleoli disperse before mitosis and reform in daughter cells at sites of ribosomal RNA (rRNA) gene clusters that are at the secondary constrictions of chromosomes, called nucleolus organizer regions (NORs). In this study, cDNA clones for a NOR autoantigen (NOR-90) were selected using a specific human autoantibody probe and were subsequently identified to encode an alternative form of the reported human upstream binding factor (hUBF). Results from immunoprecipitation showed that anti-NOR-90 antibodies recognized both forms of hUBF/NOR-90. Our data therefore showed that UBF, a critical factor in the regulation of rRNA transcription, was tightly bound to NOR during mitosis even when rRNA synthesis was thought to be minimal. Furthermore, we identified a nucleolar transcription factor as a novel target for human autoimmune response.

Amino Acid Sequence↗
PubMed1991

Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin.

Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex.

About 50% of patients with the polymyositis/scleroderma (PM-Scl) overlap syndrome are reported to have autoantibodies to a nuclear/nucleolar particle termed PM-Scl. The particle is composed of several polypeptides of which two have been identified as autoantigens. In this report, human cDNA clone coding for the entire 75-kD autoantigen of the PM-Scl particle (PM-Scl 75) was isolated from a MOLT-4 lambda gt-11 library. The deduced amino acid sequence of the cDNA clone represented a protein of 355 amino acids and 39.2 kD; the in vitro translation product of this cDNA migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at approximately 70 kD. The aberrant migration of the polypeptide in SDS-PAGE was shown to be related to the COOH half that was rich in acidic residues. Authenticity of the cDNA coding for PM-Scl 75 was shown by immunoreactivity of PM-Scl sera with in vitro translation products and recombinant fusion proteins encoded by the cDNA. In addition, rabbit antibodies raised to recombinant fusion protein reacted in immunofluorescence, immunoblotting, and immunoprecipitation with the characteristic features displayed by human anti-PM-Scl sera.

Amino Acid Sequence↗
PubMed1991

Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies.

Studies with human autoimmune sera identified auto-antibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.

Animals↗
PubMed1991

Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen.

Serum SS-A/Ro autoantibodies are commonly found in patients with Sjogren's syndrome, systemic lupus erythematosus, neonatal lupus, and subacute cutaneous lupus. Two proteins of 60 and 52 kD have been described as targets for these autoantibodies. To define the 52-kD component unambiguously, cDNA clones were isolated from human HepG2 and MOLT-4 cell cDNA libraries. The identity of cDNA was established by (a) the specificity of the antibody affinity purified from the recombinant protein, (b) the reactivity of the purified recombinant protein with prototype SS-A/Ro sera in immunoblot and ELISA, and (c) two-dimensional gel comigration of MOLT-4 cell 52-kD protein and the recombinant protein. A 1.9-kb cDNA encoded the complete 52-kD protein containing 475 amino acids (Mr 54,082). Putative zinc-finger domains and a leucine zipper motif were identified in the amino-terminal half of the 52-kD protein, implicating its possible association with DNA/RNA. Sequence homology detected between the 52-kD protein and human ret transforming protein, and mouse T cell gene expression down-regulatory protein rpt-1, may provide leads to the functional role of the 52-kD protein in addition to the possibility that these proteins might constitute members of a subfamily of finger proteins.

Amino Acid Sequence↗
PubMed1990

Effective separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide by altering conditions of polyacrylamide gel electrophoresis.

A method is described for the separation of the 52 kDa SSA/Ro polypeptide from the 48 kDa SSB/La polypeptide. These two proteins anomalously co-migrate with the same relative mass under conditions of conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis according to Laemmli using a stock solution of acrylamide: bisacrylamide 30:0.8, ratio = 37.5. A higher ratio of monomer to cross-linker, ratio = 172.4 used in a 15% acrylamide solution, readily separates the two peptide chains. This method facilitates the detection of the 52 kDa SSA/Ro component which otherwise might have been incorrectly assigned as a 48 kDa SSB/La polypeptide.

Autoantigens↗
PubMed1990

Visualization of dynamic subcutaneous vasomotor response by computer-assisted thermography.

Infrared thermography is a noninvasive and nonionizing imaging modality which detects thermally significant subcutaneous blood vessels as linear heat patterns projected onto the skin surface. In clinical thermography, pseudo-colors are typically used to represent isothermal regions. However, pseudo-colors destroy the connectivity of vascular patterns since the intravenous temperature of a subcutaneous blood vessel varies along its length. This representation also confounds estimates of vessel boundary location since boundary information is rendered by temperature gradients, and not by isotherms. This paper describes two computer-assisted methodologies for the visualization of peripheral subcutaneous vasomotor events. The first approach, which utilizes a three-stage segmentation strategy based on edge detection, can visualize temperature differences of approximately 3.5 degrees C between the subcutaneous vessel boundaries and surrounding tissue. The second approach requires user interaction with an adaptive filtering algorithm that selectively enhances vascular patterns in the thermogram while decreasing background noise artifacts. The user interactively selects decision thresholds used by the algorithm to develop symbolic, axiomatic models of homogeneous and bimodal local contrast regions. The result of this trained filter is then employed in a technique called digital subtraction thermographic venography for the extraction of subcutaneous venous patterns. This second approach shows less ambiguity and higher sensitivity than the edge detection approach in resolving subtle temperature differences of approximately 1.2 degrees C between the vessel and surrounding tissue. Computer-processed frames from both of these approaches are used for the dynamic visualization of normal and pathological vasomotor responses to thermal challenges, thereby providing diagnostic visual cues which are unavailable in the original thermograms.

Algorithms↗
PubMed1990

In vitro posttranslational modification of lamin B cloned from a human T-cell line.

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.

Amino Acid Sequence↗
PubMed1989

Ribonucleoprotein SS-B/La belongs to a protein family with consensus sequences for RNA-binding.

Autoantibodies from systemic rheumatic disorders have become useful reagents in molecular biology. SS-B/La, a major target of autoantibodies in lupus and Sjogren's syndrome, has been identified as a 46 kDa protein component of a ribonucleoprotein (RNP) particle implicated in the maturation of RNA polymerase III transcripts. This report describes the complete sequences of human and bovine SS-B/La and the identification of RNA-binding protein consensus sequences RNP1 and RNP2 in the N-terminal region previously shown to be complexed with RNA in UV-crosslinking experiments. Segments of about 95 residues from the RNA-binding domain of SS-B/La and from 29 RNA-binding domains of several other proteins are analysed with respect to the frequency of amino acids and their hydrophobicity at each position. The data suggest that SS-B/La belongs to a large family of RNA-binding proteins which includes heterogeneous nuclear RNPs, nucleolin, mRNA polyadenylate binding protein, and small nuclear RNPs.

Amino Acid Sequence↗
PubMed1989

Sjögren's syndrome nuclear antigen B (La): cDNA cloning, structural domains, and autoepitopes.

SS-B/La is a major antigenic target for autoantibodies in patients with Sjögren's syndrome. Its transient association with nascent RNA polymerase III transcripts in the cell nucleus suggest a functional role of SS-B/La in RNA processing and maturation. Human SS-B/La autoantibodies recognize at least two distinct epitopes on two separate structural domains of the SS-B/La protein and these epitopes are conserved among mammalian species. In contrast, murine monoclonal antibodies produced though immunization with purified bovine SS-B/La recognize different epitopes. To elucidate these differences, cDNA sequences of SS-B/La were cloned from several mammalian species including human, bovine, and rabbit. Complete human SS-B/La cDNAs including the coding sequence (1227bp) and untranslated sequences were isolated. The complete bovine sequence was determined from two overlapping partial cDNA clones. Comparison of the complete protein sequences encoded by the human and bovine cDNAs revealed a high degree of conservation of amino acid sequence showing only 26 substitutions/deletions out of 408 residues. RNA blot analysis indicated the presence of two size species of transcripts in bovine and rabbit cells, 1.8 kb and greater than 2.5 kb, in contrast to a single 1.8 kb mRNA species in human cells. The results of cDNA cloning support our previous finding of SS-B/La as a two-domain protein, and the RNA-binding site is confirmed to be located within N-terminal domain designated X. Epitope mapping using recombinant SS-B/La fusion proteins confirmed the findings of at least two autoepitopes located on different domains.

Animals↗
PubMed1989

Viral genomes in lymphomas of patients with Sjögren's syndrome.

The recent isolation of a new member of the herpes virus family (Human Herpes Virus-6, HHV-6) from patients with lymphoproliferative diseases prompted us to examine biopsies from six patients with primary Sjögren's Syndrome (SS) who developed non-Hodgkin's lymphoma. Five SS patients developed B-cell lymphoma and one developed a T-cell lymphoma based on immunoglobulin and T-cell antigen receptor (TCAR) gene rearrangements. In two SS patients with B-cell lymphomas, viral DNAs were detected, including: (a) Epstein-Barr Virus (EBV) DNA that exhibited an unusual pattern of restriction fragment length polymorphisms (RFLP) of the Bam M viral DNA segment; and (b) HHV-6 DNA in a second SS patient's lymphoma, with an RFLP similar to recent viral isolates from patients with other lymphoproliferative diseases. Viral DNA was not detected in the other four SS lymphoma biopsies. Also, all six biopsies were examined for presence of other viral DNAs (including CMV, HTLV, HIV and adenovirus) and were negative. Antibody titers to EBV-associated early-diffuse-antigen (EA-D), as assessed by ELISA method, and antibody titers against HHV-6, as detected by immunofluorescence and radio-immunoprecipitation assays, were markedly elevated in several SS patients with lymphoma and pseudolymphoma. These results suggest a potential role of EBV or HHV-6 in the neoplastic transformation that occurs with increased frequency in SS patients.

Antigens, Viral↗