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E K Chan

Publications and source records attributed to E K Chan.

At least 19 recordsLinked to original sources
PubMed1995

52-kD SS-A/Ro: genomic structure and identification of an alternatively spliced transcript encoding a novel leucine zipper-minus autoantigen expressed in fetal and adult heart.

The 52-kD SS-A/Ro protein is one of the antigenic targets strongly associated with the autoimmune response in mothers whose children have manifestations of neonatal lupus. In addition to the cDNA clone we previously reported for the full-length 52-kD SS-A/Ro protein, an interesting MOLT-4 cDNA clone, p52-2, was found to have an internal deletion of 231 nucleotides including the domain encoding the leucine zipper motif. To further investigate the nature of this deletion, genomic DNA clones were isolated from a lambda FIXII library. The complete gene for the full-length 52-kD protein (alpha form, 52 alpha) spans 10 kb of DNA and is composed of seven exons. Exon 1 contains only the 5' untranslated sequence, while the translation initiation codon is located 3 kb downstream in exon 2, which also encodes the three zinc finger motifs. Exon 4 encodes amino acids 168-245, including the coiled coil/leucine zipper domain. Exon 7 is the longest and encodes the rfp-like domain and the 3' untranslated region. The cDNA p52-2 can now be accounted for as a product of alternative messenger RNA (mRNA) derived from the splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein (52 beta) with a predicted molecular weight of 45,000. An initial approach to identifying this alternatively spliced form in the human heart used a ribonuclease protection assay. Using an RNA probe corresponding to bases 674-964 of the full-length cDNA, two protected mRNA fragments were identified, a 290-bp fragment corresponding to expression of 52 alpha and a smaller fragment of 144 bp, the predicted size of 52 beta. Using reverse transcription followed by polymerase chain reaction, cDNAs from a 16-wk fetal heart, 24-wk heart, and adult heart were amplified with primers flanking exon 4. Two polymerase chain reaction products were observed in each tissue, one 1.0 kb likely representing 52 alpha and a second 0.78 kb, consistent with 52 beta. The 0.78-kb fragment identified in the 16-wk heart was cloned, and DNA sequencing confirmed the 52 beta type. Immunoprecipitation of in vitro-translated 35S-labeled 52 beta form was performed to evaluate the antigenicity of this novel form of 52-kD SS-A/Ro. 26 (87%) of 30 sera tested from mothers whose children were known to have neonatal lupus immunoprecipitated the 52 beta form.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1995

A cell-cycle nuclear autoantigen containing WD-40 motifs expressed mainly in S and G2 phase cells.

Recently a novel nuclear protein mainly expressed in S and G2 phase cells was characterized using autoantibodies from a cancer patient (Exp. Cell Res., 212, 255-261, 1994). cDNA clones were isolated by immunoscreening, and sequence analysis revealed that the cDNA clones encoded a 713-amino acid residue polypeptide which is provisionally named SG2NA (S/G2 nuclear antigen). In the carboxyl terminal half of SG2NA, there are 6 copies of WD-40 motifs characteristic of a large family of proteins of which the prototype is beta-transducin/enhancer of split (TLE). In addition, there are nuclear localization sequence and phosphorylation sites related to the TLE proteins. Autoantibodies occurring spontaneously in certain cancer patients have been useful reagents for identifying cellular proteins and this is the first report of a novel WD-40 protein which is the target of an autoimmune response.

Adenocarcinoma↗
PubMed1995

The effects of nicotinamide and glimepiride on diabetes prevention in BB rats.

Glimepiride is an oral sulfonylurea drug; nicotinamide is an inhibitor of poly (ADP-ribose) synthetase and a precursor of NAD. Three studies were carried out to determine whether glimepiride and/or nicotinamide could prevent diabetes in BB rats. In Study I, we administered glimepiride treatment 200 mg/kg/day orally from the 35th to 143rd day of age. The incidence of diabetes in the glimepiride group was lower than in the control group (32% vs 55%, p < 0.02). In Study II, the treatment period was from the 35th to 147th day of age, and rats received glimepiride combined with nicotinamide (500 mg/kg/day IP). The treatment group showed a 22% incidence of diabetes compared to 53 in controls (p < 0.03). In Study III, nicotinamide treatment alone (1000 mg/kg/day orally) and combined with glimepiride were compared to untreated controls. The treatment period was from the 35th to 167th day of age. Nicotinamide-treated rats showed a 42% incidence of diabetes compared to 60% in controls (p = NS). In the nicotinamide combined with glimepiride treatment group, a lower incidence (28%) was observed when compared to the controls (60%, p < 0.05). These findings suggest that glimepiride can prevent diabetes in BB rats; however, nicotinamide when used in young animals shows only a trend to lower the incidence of diabetes, and when combined with glimepiride, no significant effect is observed.

Administration, Oral↗
PubMed1995

Glimepiride-induced prevention of diabetes and autoimmune events in the BB rat: revised.

The purpose of this study was to determine whether Glimepiride, an oral sulfonylurea drug, prevents the onset of diabetes in diabetic prone BB rats. S750181, a sulfonylurea drug that has minimal in vivo glucose metabolic effects, was also tested. In addition, the shortest period of sulfonylurea treatment required for prevention was determined. Eighty rats were studied for all treatment periods with 40 receiving a daily oral gavage dosage of glimepiride and 40 receiving a daily oral gavage dosage of vehicle solution. Diabetes onset was monitored by glycosuria and blood glucose levels. In study I, with a treatment period of 35-142 days of age, Glimepiride-treated rats showed a 32% incidence of diabetes, whereas control rats had a diabetes incidence of 55% (p < 0.04). In study II, with a treatment period of 60-140 days of age, Glimepiride-treated rats showed a 29% incidence of diabetes compared to 54% in controls (p < 0.03). Further, comparing the time of diabetes onset between the Glimepiride and control groups showed that Glimepiride delays diabetes onset (p < 0.02). In study III, with a treatment period of 60-100 days of age, Glimepiride-treated rats showed a 17% overall diabetes incidence at 170 days, whereas the controls were 43% (p < 0.01). In study IV, with a treatment period of 60-140 days of age, S750181-treated rats showed a 38% diabetes incidence and the control group showed a 43% diabetes incidence. There was no significant delaying or prevention effect observed in the S750181 group. To determine if Glimepiride affected autoimmune events, the severity of islet inflammation was examined. In study I, islet histology from total and nondiabetic animals indicated that Glimepiride-treated rats had a lower severity of islet inflammation than that of the control rats (p = 0.023). These studies show that a) Glimepiride has diabetes preventive effects, b) shorter treatment periods of only 40 days can be effective and c) Glimepiride decreases the severity of islet inflammation.

Animals↗
PubMed1995

Formation of nuclear bodies in hepatocytes of estrogen-treated roosters.

As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.

Animals↗
PubMed1995

Non-muscle myosin as target antigen for human autoantibodies in patients with hepatitis C virus-associated chronic liver diseases.

Three patients with hepatitis C virus (HCV)-related chronic liver disease were shown to have autoantibodies strongly reacting with cytoskeletal fibres of non-muscle cells. The heavy chain of non-muscle myosin microfilament was the main target for those autoantibodies, as determined by (i) cell and tissue immunofluorescence studies showing colocalization with an anti-myosin antibody prototype; (ii) primary reactivity in immunoblotting with a 200-kD protein, using either MOLT-4 cells, human platelets, or affinity-purified non-muscle myosin as antigen extract; and (iii) immunoblotting of similar immunoreactive fragments in papain-digested MOLT-4 cell extracts, by using those human sera and antibody prototype. Autoantibodies to non-muscle myosin heavy chain were not previously reported in patients with chronic liver diseases, especially in those associated with HCV infection.

Aged↗
PubMed1995

Psyllium in hypercholesterolemia.

In summary, within a controlled study situation, psyllium seems to be effective in lowering total and LDL cholesterol by 4-8% and 6-13%, respectively. Compared with conventional antilipidemic agents such as lovastatin, which lowers total cholesterol 30%, LDL cholesterol 40%, and triglycerides 20%, the effect of psyllium on lipids is minimal, and its use as a form of drug therapy in patients with moderate-to-severe hypercholesterolemia is not recommended. As an adjunct to AHA step 1 diet therapy, however, psyllium can be useful. The NCEP recently has stressed diet therapy as a first-line primary intervention in patients not a high risk from multiple risk factors or very high LDL cholesterol concentrations. Men ( < or = 45 y) and premenopausal women with no other risk factors and moderately high LDL cholesterol concentrations (4.1-5.7 mmol/l) are at relatively low risk for coronary events in the near future, and would therefore be suitable for diet therapy combined with adjunctive psyllium therapy. Psyllium is well tolerated with minor transient adverse effects (abdominal distention, excessive gas, flatulence) and has a good compliance record ( > or = 90%). It would be a useful adjunct to dietary intervention in low-risk patients with mild-to-moderate hypercholesterolemia. Further research is needed to evaluate psyllium's effect in women and its efficacy in long-term use.

Cholesterol, LDL↗
PubMed1995

p80-coilin: a component of coiled bodies and interchromatin granule-associated zones.

We investigated at the electron microscope level the fate of the three intranuclear structures known to accumulate snRNPs, and which correspond to the punctuate immunofluorescent staining pattern (the coiled bodies, the clusters of interchromatin granules and the interchromatin granule-associated zones) after exposure to either a low salt medium which induces a loosening and partial spreading of nucleoprotein fibers or a high ionic strength salt medium and subsequent DNase I digestion, in order to obtain DNA-depleted nuclear matrices. The loosened clusters of interchromatin granules and the coiled bodies could no longer be distinguished from surrounding nucleoprotein fibers solely by their structure, but constituents of the clusters of interchromatin granules could be detected by in situ hybridization with both U1 and U2 DNA probes, and constituents of the coiled bodies were detectable mainly with the U2 DNA probe. The interchromatin granule-associated zones, the electron-opacity and compactness of which were preserved despite the loosening treatment, remained labeled with the U1 DNA probe only. In DNA-depleted nuclear matrices, the snRNA content of the coiled bodies, the clusters of interchromatin granules and their associated zones, which were all easily recognizable within the residual nuclear ribonucleoprotein network, was unmodified. The data indicate, therefore, that the loosening procedure as well as the high salt extraction procedure preserve the snRNA content of all three spliceosome component-accumulation sites and reveal that interchromatin granule-associated zones are elements of the nuclear matrix. The p80-coilin content coiled bodies was also preserved whatever the salt treatment used. An intriguing new finding is the detection of abundant p80-coilin within the interchromatin granule-associated zones, both before and after either low or high salt treatment of cells. Therefore, p80-coilin is an integral constituent of the interchromatin granule-associated zones.

Cell Nucleus↗
PubMed1994

Structure, expression and chromosomal localization of human p80-coilin gene.

Coiled bodies (CBs) are non-capsular nuclear bodies with a diameter of 0.3-1 micron and appear to be composed of coiled fibrils. Human autoantibodies to CBs recognize an 80-kD nuclear protein highly enriched in CBs, and this protein has been named p80-coilin. CBs are known to assemble and disassemble during the cell cycle, with the highest number of CBs occurring at mid to late G1 where p80-coilin is assembled into several small nuclear body-like structures. In S and G2 phases, CBs become larger and their number decreases and often they are undetectable during mitosis. Using a human autoantibody as a probe for expression cloning, we initially isolated a partial cDNA encoding p80-coilin. In this report, the 5' end of the complete cDNA for p80-coilin was obtained using the 5'-RACE (rapid amplification of cDNA ends) methodology. The size of the reconstructed full-length cDNA corresponds to the 2.7-kb mRNA detected in Northern blot analysis. The complete p80-coilin protein consists of 576 amino acids with a predicted molecular mass of 62,608. A putative p80-coilin pseudogene was also detected during the rescreening of p80-coilin cDNA. To confirm the validity of the cDNA sequence, three overlapping genomic DNA clones representing the human p80-coilin gene were selected for further analysis. The complete gene for p80-coilin contains 7 exons spanning approximately 25kb. Sequence analysis of exons 1 and 2 in genomic DNA clones confirmed the accuracy of the 5' cDNA sequence derived from the 5'-RACE procedure. Furthermore, the human p80-coilin gene was localized to chromosome 17q22-23 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

Autoantibody responses to the "native" 52-kDa SS-A/Ro protein in neonatal lupus syndromes, systemic lupus erythematosus, and Sjogren's syndrome.

Abs to the 52-kDa SS-A/Ro protein are found in high prevalence in patients with Sjogren's syndrome (SS) and mothers whose children have the neonatal lupus syndrome (NLS). This study further defines the specificity of this response. By ELISA, 97% of 59 mothers of offspring with NLS had Abs to the 52-kDa recombinant protein compared with 80% in 132 non-NLS sera with anti-SS-A/Ro Abs (p < 0.004). Antigenic regions on the 52-kDa protein were evaluated by immunoprecipitation of [35S]-radiolabeled in vitro translation products. Ninety-five percent of 99 sera that contained anti-52-kDa Abs by ELISA reacted with a large fragment spanning amino acids (aa) 1-291. Two antigenic regions were identified, aa169-291 containing the leucine zipper that was recognized by 83% of the anti-52-kDa sera tested and aa1-78 containing the zinc finger domains that was recognized by only half the sera. No sera immunoprecipitated this N-terminal fragment exclusively. Recognition of one or both regions was not unique to any clinical subset of patients; however, a greater number of sera from patients with SS contained both specificities, whereas asymptomatic mothers whose children had NLS comprised the only clinical group in which the majority recognized the central region of the molecule. Reactivity with both epitopes was demonstrated significantly more often in sera with high titers of Abs to the 60-kDa rSS-A/Ro protein by ELISA in association with the anti-52-kDa response compared with anti-52-kDa responses associated with low titers of anti-60-kDa Abs (p < 0.04). Eighty-one percent of 16 sera that recognized the N-terminal epitope were from patients with the combination of HLA-DRB1*0301, DQA1*0501, and DQB1*0201 alleles, compared with 30% of 10 that recognized only the central epitope (p < 0.02). In summary, this study demonstrates that there are at least two antigenic determinants on the 52-kDa SS-A/Ro protein, one "immunodominant" and the other recognized by a more "restricted" subset of anti-52-kDa SS-A/Ro Abs.

Autoantibodies↗
PubMed1994

Is the coiled body involved in nucleolar functions?

Coiled bodies (CBs) are structural constituents observed in nuclei of most eukaryotic cells. They usually occur in the nucleoplasm as well as in contact with the nucleolar surface. In this work we studied the hepatocyte nuclei of hibernating dormice in order to investigate possible modifications of CBs along the seasonal cycle. CBs were abundant during hibernation and rapidly disappeared upon arousal from hibernation. Moreover, CBs were frequently found to be integrated into the nucleolar body. Immunocytochemical analyses showed that CBs contain nucleoplasmic as well as nucleolar RNA-processing factors, suggesting an "ambiguous" role for this organelle in the nuclear functions.

Animals↗
PubMed1994

Immunocytochemical characterization of human NOR-90 (upstream binding factor) and associated antigens reactive with autoimmune sera. Two MR forms of NOR-90/hUBF autoantigens.

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to the human upstream binding factor (hUBF) and composed of two Mr forms. In this study, thirteen human anti-NOR-90/hUBF autoimmune sera were used to further characterize NOR-90/hUBF and its associated autoantigens. Nucleolar and nucleoplasmic staining of interphase cells and NOR staining in mitosis were observed with all sera by immunofluorescence. All sera showed equal reactivity with both high and low Mr forms in Western blotting and immunoprecipitation, suggesting that the cellular content and distribution for both Mr forms were approximately equal. Using extracts of [35S]methionine- and [32P]orthophosphate-labeled cells, phosphorylated and nonphosphorylated NOR-90/hUBF were identified for both Mr forms and these two populations were recognized by human autoantibodies. In immunoprecipitation analyses, the nonphosphorylated population was readily extracted while the phosphorylated population was tightly bound. Clinical data were available for 8 patients in whom anti-NOR-90/hUBF autoantibodies were present. They had diverse diagnoses including SLE, rheumatoid arthritis and malignancies. Although only one patient was diagnosed as scleroderma, Raynaud's phenomenon was observed in 4 of the 8 patients. Interestingly, one NOR-90/hUBF serum was shown to contain additional antibodies to RNA polymerases I and II.

Adult↗
PubMed1994

Isolation and function of human and pig islets.

This report confirms reproducible methods to isolate and assess viability and function of pig and human islets. We processed 10 pig and five human pancreata. The pancreata were digested by a modification of Ricordi's automated method for islet isolation. The number of islet equivalents (150 microns diameter islets) was 335,190 +/- 79,345 islets per pig pancreas (5,146 +/- 1,274 islets/g pig pancreas) and 323,630 +/- 147,810 per human pancreas (6,252 +/- 2,572 islets/g human pancreas). The majority of islets were in the range of 50-200 microns diameter, and 20% of the islet population had a size distribution of 200 microns diameter in both porcine and human models. The purity of the final preparations exceeded 90%. The secretory response of perifused islets showed a biphasic insulin release pattern in both species. Perifused fresh pig islets released 2.5 pmol/L islet-1 min-1 at 2.0 mM glucose and 6.2 pmol/L islet-1 min-1 at 16.7 mM glucose. After 7 days culture at 37 degrees C, human islets released 1.32 pmol/L islet-1 min-1 at 2.0 mM and 12.24 pmol/L islet-1 min-1 at 16.7 mM. These results indicate that this procedure is useful to obtain pure, large, and functional islets from pig and human pancreata.

Adult↗
PubMed1994

Relationship between maternal and neonatal levels of antibodies to 48 kDa SSB(La), 52 kDa SSA(Ro), and 60 kDa SSA(Ro) in pregnancies complicated by congenital heart block.

OBJECTIVE: Since there is suggestive but, to date, indirect evidence that maternal anti-SSA(Ro) and SSB(La) antibodies are pathogenic in congenital heart block (CHB), we explored the hypothesis that binding to fetal tissues would result in selective depletion of autoantibodies from the neonatal circulation. METHODS: Maternal and umbilical cord levels of anti-48 kDa SSB(La), anti-52 kDa SSA(Ro) and anti-60 kDa SSA(Ro) antibodies were measured by ELISA and immunoprecipitation at parturition in 15 pregnancies complicated by CHB. A control group consisted of 13 pregnancies in which the mother was known to have antibodies to either SSA(Ro) and/or SSB(La) and the children did not have CHB. RESULTS: The ratios of maternal to cord serum levels of anti-48 SSB(La), anti-52 SSA(Ro) and anti-60 kDa SSA(Ro) antibodies ranged from 0.71 to 2.38 in both affected and unaffected pregnancies. The mean ratio obtained for each of the 3 autoantibodies was not significantly different between the 2 groups. Moreover these ratios did not significantly differ from the mean ratios obtained for total IgG levels in either group. CONCLUSION: These data demonstrate that maternal antibodies to all components of the SSA(Ro) SSB(La) system are efficiently transported across the placenta and are not selectively depleted in the circulation of neonates with CHB.

Antibodies, Antinuclear↗
PubMed1994

Cytochemical and immunocytochemical characterization of nuclear bodies during hibernation.

Brown adipose tissue and liver of hibernating, arousing and euthermic individuals of the dormouse Muscardinus avellanarius were studies using ultrastructural cytochemistry and immunocytochemistry with the aim to investigate possible fine structural modifications of the cell nucleus during the seasonal cycle. The general morphology of brown adipocyte and hepatocyte nuclei was similar in the three experimental groups. However, three nuclear structural constituents were identified only in hibernating individuals: coiled bodies (CBs) and amorphous bodies (ABs) were observed in hepatocytes and, together with bundles of nucleoplasmic fibrils (NF), were present in brown adipocytes of hibernating dormice. In arousing animals only some structural constituents suggestive of poorly structured CBs were found. The latter showed the same immunocytochemical features as CBs of hibernating individuals, suggesting that they are disappearing CBs. A possible involvement of CBs in storing and/or processing RNA which must be rapidly and abundantly released upon arousal is discussed. ABs similarly to CBs contain RNA and nucleoplasmic ribonucleoproteins (RNPs) and could also be involved in mRNA pathways. NF do not contain nucleic acids or RNPs and seem to be composed of protein-aceous material; their functional role in the nuclear metabolism of hibernating brown adipocytes remains unclear.

Adipocytes↗