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E K Chan

Publications and source records attributed to E K Chan.

At least 55 records · Page 3Linked to original sources
PubMed1998

Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: immunological relationship with hnRNP P.

Serum from a patient showing symptoms related to autoimmunity was found to contain autoantibodies to the nuclear mitotic apparatus (NuMA) protein and to several novel nuclear antigens with estimated molecular weights of 40, 43, 72, 74 and 82 kDa. Using this serum for screening a human cDNA expression library a 2.5 kb cDNA clone was isolated which encoded the complete sequence of a protein of 633 amino acids. Sequence analysis revealed a modular structure of the protein: an acidic N-terminal region of approximately 150 amino acids was followed by three adjacent consensus sequence RNA binding domains located in the central part of the protein. In the C-terminal portion a nuclear localization signal and an octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B were identified. This was followed by a glycine- and arginine-rich section of approximately 120 amino acids forming another type of RNA binding motif, a RGG box. Interestingly, three copies of a tyrosine-rich decapeptide were found interspersed in the RGG box region. The major in vitro translation product of the cDNA co-migrated in SDS-PAGE with the 82 kDa polypeptide that was recognized by autoantibodies. The structural motifs as well as the immunofluorescence pattern generated by anti-82 kDa antibodies suggested that the antigen was one of the proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Subsequently the 82 kDa antigen was identified as hnRNP R protein by its presence in immunoprecipitated hnRNP complexes and co-migration of the recombinant protein with this hitherto uncharacterized hnRNP constituent in two-dimensional gel electrophoresis. The concomitant autoimmune response to a hnRNP component of the pre-mRNA processing machinery and to NuMA, a protein engaged in mitotic events and reported to be associated with mRNA splicing complexes in interphase, may indicate physical and functional association of these antigens. Support for this notion comes from observations that concomitant or coupling of autoantibody responses to proteins which are associated with each other as components of subcellular particles are often found in autoimmune diseases.

Amino Acid Sequence↗
PubMed1998

Imaging of the irradiation of skin with a clinical CO2 laser system: implications for laser skin resurfacing.

BACKGROUND AND OBJECTIVE: Several published reports describe the benefits of using the carbon dioxide laser for cutaneous resurfacing. The mechanisms on which skin resurfacing work are still not completely understood. This study was performed to obtain quantitative and qualitative information describing the thermal response of skin during high-energy, short-pulsed CO2 laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: A Tissue Technologies TruPulse CO2 laser was used to irradiate an in vivo rat model. The laser parameters that were used were a 100-micros pulse width, a 1-Hz repetition rate, a 3 mm x 3 mm square spot size, and 2.4 J/cm2 and 3.9 J/cm2 radiant exposures. A 3-5 microm thermal camera was used to obtain temperature information during irradiation. Single spots were irradiated with one pulse, and the temperature-time history was obtained. In a different experiment, 15 pulses were applied to single spots, and both thermal and video images were obtained. RESULTS: Irradiation with one pulse at 2.4 J/cm2 and 3.9 J/cm2 led to peak temperatures >100 degrees C. The temperature relaxation time was approximately 25-150 ms. Multiple-pulse irradiation at 2.4 J/cm2 led to a slight rise in the peak temperature with each pulse. At 3.9 J/cm2, the peak temperature increased with successive pulses until pulse 10, after which the peak temperature oscillated between 300 and 400 degrees C. Video images showed concurrent burning events that occurred during pulses 10-15. CONCLUSION: Temperatures >100 degrees C were measured during CO2 laser irradiation of skin. Pulse stacking can lead to peak temperatures approaching 400 degrees C and to tissue charring with as few as three stacked pulses. It is crucial for the physician to manipulate the laser handpiece at parameters with which he or she can avoid pulse overlap.

Animals↗
PubMed1998

Laser assisted soldering: microdroplet accumulation with a microjet device.

BACKGROUND AND OBJECTIVE: We investigated the feasibility of a microjet to dispense protein solder for laser assisted soldering. STUDY DESIGN: Successive micro solder droplets were deposited on rat dermis and bovine intima specimens. Fixed laser exposure was synchronized with the jetting of each droplet. After photocoagulation, each specimen was cut into two halves at the center of solder coagulum. One half was fixed immediately, while the other half was soaked in phosphate-buffered saline for a designated hydration period before fixation (1 hour, 1, 2, and 7 days). After each hydration period, all tissue specimens were prepared for scanning electron microscopy (SEM). RESULTS: Stable solder coagulum was created by successive photocoagulation of microdroplets even after the soldered tissue exposed to 1 week of hydration. CONCLUSIONS: This preliminary study suggested that tissue soldering with successive microdroplets is feasible even with fixed laser parameters without active feedback control.

Animals↗
PubMed1998

Genetic and physical mapping of the Lps locus: identification of the toll-4 receptor as a candidate gene in the critical region.

On the basis of 2093 meioses analyzed in two separate intraspecific backcrosses, the location of the mouse Lpsd mutation was circumscribed to a genetic interval 0.9 cM in size. A total of 19 genetic markers that lie in close proximity to the mutation were examined in mapping. Most of these were previously unpublished polymorphic microsatellites, identified by fragmentation of YAC and BAC clones spanning the region of interest. Lpsd was found to be inseparable from the microsatellite marker D4MIT178, and from three novel polymorphic microsatellites identified near D4MIT178. The mutation was confined between two novel microsatellite markers, herein designated "B" and "83.3." B lies centromeric to the mutation, and was separated by four crossovers in a panel of 1600 mice; 83.3 lies distal to the mutation and was separated by three crossovers in a panel of 493 mice. 66 BAC clones and one YAC clone were assembled to cover > 95% of the critical region. Estimates based on pulsed field gel electrophoresis and fluorescence in situ hybridization indicate that the The B-->83.3 interval is about 3.2 Mb in length. A minimal area of zero recombinational distance from Lpsd was also assigned, and found to occupy approximately 1.2 Mb of physical size. To identify gene candidates, nearly 40,000 sequencing runs were performed across the critical region. Selective hybridization and exon trapping were also employed to identify genes throughout the "zero" region. Only a single intact gene was identified within the entire critical region. This gene encodes the Toll-4 receptor, a member of the IL-1 receptor family.

Animals↗
PubMed1998

Immunological characterization of heterochromatin protein p25beta autoantibodies and relationship with centromere autoantibodies and pulmonary fibrosis in systemic scleroderma.

Anticentromere antibodies (ACA) are immunological markers for the subset of systemic scleroderma with the symptoms calcinosis cutis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (CREST). In Western blotting, some ACA-positive sera also recognize a doublet of 23 kDa (p23) and 25 kDa (p25) in addition to centromere protein antigens A (17 kDa), B (80 kDa), and C (140 kDa). Two forms of p25 have been shown to be human homologues of Drosophila heterochromatin-associated protein HP1. One form of p25 (p25beta) which was recently cloned in this laboratory was used to evaluate anti-p25beta antibody response in scleroderma sera. Of 318 scleroderma sera 42 had ACA (13.2%), and 16 of the 42 sera (38%) had anti-p25beta antibodies. On the other hand, 5 of 276 ACA-negative sera (1.8%) showed anti-p25beta antibody response, demonstrating that anti-p25beta antibody is significantly associated with the ACA response (P < 10[-8]). Clinically the anti-p25beta response was significantly associated with the CREST syndrome. Fourteen (36.8%) of 38 CREST patients compared to seven (2.5%) of 280 patients with other forms of scleroderma were anti-p25beta antibody positive (P < 10[-8]). The 14 CREST patients with anti-p25beta antibodies had significantly more interstitial lung disease than those without anti-p25beta antibodies (P < 0.003). There was also a tendency to increased liver involvement. Two dominant autoepitopes in p25beta were determined by Western blotting using p25beta recombinant fragments. In immunofluorescence C-terminal specific antibodies showed staining of heterochromatin, but N-terminal specific antibodies showed no staining. Interestingly, the majority of sera reacted preferentially with one or the other of the two dominant autoepitopes.

Autoantibodies↗
PubMed1998

Estrogenic xenobiotics increase expression of SS-A/Ro autoantigens in cultured human epidermal cells.

SS-A/Ro autoantibodies are detected at high levels in patients with autoimmune diseases such as systemic lupus erythematosus. It has been reported that natural estrogen is capable of inducing cell surface expression of SS-A/Ro autoantigens in human epidermal keratinocytes. In this study, we analysed, by reverse transcriptase polymerase chain reaction and immunohistochemistry, the effects of estrogenic xenobiotics (i.e. environmental estrogens) on the expression of 52-kDa SS-A/Ro autoantigen in cultured keratinocytes. At a concentration of 10 microM, various estrogenic xenobiotics derived from plants, insecticides, or detergents induced up to a 3-fold increase in 52-kDa SS-A/Ro mRNA levels in human keratinocytes compared with untreated cells. The immunohistochemistry results paralleled the reverse transcriptase polymerase chain reaction results. These findings suggest that environmental stimulation can induce the expression of autoantigens such as SS-A/Ro.

Adult↗
PubMed1998

The roles of pH and concentration in lactic acid-induced stimulation of epidermal turnover.

BACKGROUND: Alpha hydroxy acids such as lactic and glycolic acids (AHAs) accelerate epidermal turnover and exfoliate the stratum corneum. The roles of pH and concentration in these antiaging effects of AHAs is unclear, but a lower (more acidic) pH and higher concentration of acid are thought to be more effective. OBJECTIVE: To examine the effects on skin renewal rates of lactic acid 10% at pHs of 2.0, 3.0, and 4.0, and of 5%, 10% and 15% lactic acid at a pH of 3.0. METHODS: Twenty-six female subjects participated in the double-blind, randomized, placebo-controlled study. The dansyl chloride stain technique was used to measure epidermal renewal times. RESULTS: Both pH and concentration are critical in the lactic acid effect. At a fixed lactic acid concentration, the desquamative effect was highly pH dependent. At a fixed pH, the turnover rate of skin was concentration dependent. CONCLUSION: The desquamative and proliferation-stimulating effects of lactic acid are very pH and concentration dependent, suggesting the "free acid" concentration is the active moiety.

Adult↗
PubMed1997

Molecular cloning of the RNA polymerase I transcription factor hUBF/NOR-90 (UBTF) gene and localization to 17q21.3 by fluorescence in situ hybridization and radiation hybrid mapping.

The 90-kDa nucleolus organizer region autoantigen (NOR-90) was previously shown to be identical to human upstream binding factor (hUBF), which has two molecular mass forms of 89 and 93 kDa, respectively. hUBF/NOR-90 is a member of the HMG-box DNA-binding protein family and is known to bind to enhancer regions upstream of the ribosomal RNA genes, which are clustered at NORs. The smaller version of UBF lacks an internal 111-bp region corresponding to 37 amino acids in the second HMG-box of the larger form. We isolated human genomic clones from a phage library and localized one of them by fluorescence in situ hybridization to chromosome 17q21.3. DNA sequence analysis showed that the 111-bp region represented a single exon, consistent with the previous notion that the two isoforms were products of alternative pre-mRNA splicing of a single gene in human. Radiation hybrid mapping placed this STS with very high probability (LOD > 19) to chromosome 17, approximately 3.77 cR distal to MIT framework marker UTR-9641. The order of the markers (a partial list) from this region was UTR-9641, SGC30031, WI-17308, D17S930, NOR53/33, WI-16100, D17S920, WI-16913, and WI-6808.

Alternative Splicing↗
PubMed1997

Controlled temperature tissue fusion: Ho:YAG laser welding of rat intestine in vivo. Part two.

BACKGROUND AND OBJECTIVE: Temperature feedback control (TFC) during laser-assisted tissue welding was implemented to eliminate exponential increases in the rate of denaturation associated with rapidly increasing temperatures. This study was undertaken to investigate and compare the weld strengths and healing responses of laser welded enterotomies with and without TFC using a cw Ho:YAG laser and to examine the effects of wavelength on weld strength and histology. The Ho:YAG experimental results were compared with a similar study using cw argon ion laser irradiation. STUDY DESIGN/MATERIALS AND METHODS: An automated system was developed for temperature feedback controlled laser irradiation. An experimental device incorporating co-aligned laser delivery and temperature detection was used to perform cw Ho:YAG laser-welded enterotomies (with and without TFC). The weld strength and histology of laser welded and control sutured enterotomies were compared in an in vivo rat model (Ho:YAG, n = 42; argon, n = 41). Animals were sacrificed at 1, 3, 7, and 21 days postoperatively and the anastomotic site was removed for bursting/leaking pressure measurements and histological examination. RESULTS: Argon and Ho:YAG laser-welds with and without TFC and the control sutured anastomoses healed comparably, although wound abscesses were more prevalent in the Ho:YAG group leading to delay in mucosal healing. Laser-welded anastomoses without TFC were associated with more spontaneous ruptures and leaks (argon: 4/6 ruptures; Ho:YAG: 1/4 leak, 2/4 ruptures, & 1/3 stenosis) during the survival period than those with TFC (argon: 1/3 leak; Ho:YAG: 1/5 rupture). Bursting pressures of the Ho:YAG welds were weaker at 1 week than the argon welds, but by 3 weeks, laser welds and suture anastomoses were equally strong. CONCLUSION: From the spontaneous failure rates encountered, it is believed that TFC improves the quality and stability of laser-assisted enterotomy closures in surviving animals. However, TFC does not provide a satisfactory method to identify completion of a weld.

Anastomosis, Surgical↗
PubMed1997

Cardiac expression of 52beta, an alternative transcript of the congenital heart block-associated 52-kd SS-A/Ro autoantigen, is maximal during fetal development.

OBJECTIVE: Congenital heart block (CHB), associated with antibodies to SS-A/Ro and SS-B/La, is most often detected between 18 and 24 weeks of gestation, yet the maternal heart is unaffected. We recently described an alternatively spliced 52-kd SS-A/Ro messenger RNA (mRNA) derived from the skipping of exon 4 which encodes a smaller protein, 52beta (MW 45 kd), recognized by CHB maternal antisera. This study was designed to identify whether cardiac expression of 52beta and full-length 52alpha relates to the development of CHB. METHODS: Reverse transcriptase-polymerase chain reaction was performed using primers flanking exon 4 and mRNA from 22 human fetal hearts (age 11-25 weeks) and 3 adult hearts. The brain, kidney, liver, lung, and spleen were similarly evaluated in a 15-week, an 18-week, and a 24-week fetus. RESULTS: Expression of 52beta was greatest and 52alpha lowest between 14 and 16 weeks of gestation. In fetal hearts ages 22-25 weeks and adult heart, the 52beta transcript was markedly diminished and 52alpha clearly dominated. The 52beta mRNA was observed in a 15-week brain, kidney, lung, and spleen; however, its expression relative to 52alpha was greatest in the heart. CONCLUSION: Since expression of the alternative product 52beta is maximal at the time of cardiac ontogeny when maternal antibodies gain access to the fetal circulation, just prior to the clinical detection of bradyarrhythmia, a role for 52beta in the development of CHB is implicated. Although other fetal tissues express 52beta, there may be differences in accessibility of antigen or regenerative capacities.

Adult↗
PubMed1997

The 52-kd protein as a target of intermolecular spreading of the immune response to components of the SS-A/Ro-SS-B/La complex.

OBJECTIVE: To determine whether immunization of healthy non-autoimmune mice with 52-kd SS-A/Ro induces a secondary antibody response to other components of the 48-kd SS-B/La-60-kd SS-A/Ro RNP complex and vice versa, since anti-52-kd antibodies have been invariably linked to these antigens in patients with Sjogren's syndrome and in mothers whose children have neonatal lupus. METHODS: Female BALB/c mice were immunized with 100 microg of 6xHis recombinant human 48-kd SS-B/La, 52-kd SS-A/Ro, or 60-kd SS-A/Ro proteins, or the 6xHis polypeptide control, each purified by Ni2+ affinity chromatography. Mice subsequently received booster injections with 50 microg of the same antigen every 10-21 days. Immune responses were measured by enzyme-linked immunosorbent assay (ELISA), immunoblotting of recombinant antigens, and immunoprecipitation of 35S-methionine-labeled in vitro translation products. RESULTS: Immunization with 48-kd SS-B/La resulted in anti-48-kd SS-B/La antibodies within 45 days, followed 10 days later by a secondary response to 52-kd SS-A/Ro, as measured by ELISA. Antibody spreading to 60-kd SS-A/Ro was not detected. Immunization with 52-kd SS-A/Ro resulted in rapid high-titer anti-52-kd SS-A/Ro responses within 27 days. Spreading to 48-kd SS-B/La occurred in only 1 mouse and 60-kd SS-A/Ro was detected in a minority of the mice after prolonged antigen exposure. Immunization with 60-kd SS-A/Ro led to anti-60-kd SS-A/Ro responses within 37 days, followed 3 months later by low-titer anti-48-kd SS-B/La and anti-52-kd SS-A/Ro antibodies. All primary immune responses were confirmed by immunoblotting and immunoprecipitation. While immunoblotting of the recombinant proteins revealed reciprocal intermolecular spreading in the majority of mice, immunoprecipitation clearly demonstrated that predominant spreading was generated after immunization with 48-kd SS-B/La, which consistently resulted in antibodies to 52-kd SS-A/Ro. CONCLUSION: The murine responses observed in the present study, demonstrating reciprocal intermolecular spreading to 48-kd SS-B/La, 52-kd SS-A/Ro, and 60-kd SS-A/Ro, support the linkage of 52-kd SS-A/Ro with the other proteins, despite their as-yet-undetected association in vivo. The marked recruitment of anti-52-kd SS-A/Ro responses elicited by 48-kd SS-B/La may provide a lead to exploring the physical interaction, direct or indirect, of 52-kd SS-A/Ro with the SS-A/Ro-SS-B/La RNP particle and its presentation to the immune system. These data should facilitate the establishment of a murine model of neonatal lupus.

Animals↗
PubMed1997

Molecular cloning of a novel 97-kd Golgi complex autoantigen associated with Sjögren's syndrome.

OBJECTIVE: To identify a Golgi complex autoantigen bound by Sjögren's syndrome (SS) autoantibodies. METHODS: Serum from a patient with secondary SS and anti-Golgi antibodies was used as a probe to isolate a complementary DNA (cDNA) insert from a HeLa cDNA library. RESULTS: A 3.7-kb cDNA encoding a 56-kd recombinant protein was immunoprecipitated by the human anti-Golgi serum and immune rabbit serum. Western blot analysis showed that the immune rabbit sera recognized a protein of 97 kd (golgin-97), suggesting that the isolated clone contained a partial cDNA. The 5' upstream sequence was obtained by rapid amplification of the cDNA ends. The complete cDNA contained 4,860 basepairs, encoding a protein with a calculated Mr of 88 kd. Antibodies to golgin-97 were found in 12 (20%) of 60 sera known to have anti-Golgi autoantibodies, and the majority of these sera (8 of 12, or 75%) were from patients who had secondary SS. CONCLUSION: Golgin-97 is a unique Golgi complex antigen that appears to be a target of SS autoantibodies.

Adult↗
PubMed1997

Immune response to cyclin B1 in hepatocellular carcinoma.

Proteins expressed by plasmids encoding human cyclins and cyclin-dependent kinase 2 (CDK2) were used as antigens in immunoblotting. Fifteen of 100 patients with hepatocellular carcinoma (HCC) were found to have autoantibodies reactive with cyclin B1 and with a 40-kd degradation product of cyclin B1-glutathione-S-transferase (GST) fusion protein. Only one serum was found to react with cyclin A and another single serum with CDK2 but no antibodies were detected to cyclin D1 and E. The basis for autoimmune responses to cyclin B1 in HCC are unknown at the present time but the possibilities might include aberrations in cyclin B1 regulation leading to altered product or its expression which resulted in stimulation of immune reactions.

Animals↗
PubMed1997

Heterochromatin protein HP1Hsbeta (p25beta) and its localization with centromeres in mitosis.

Some autoimmune sera containing anticentromere autoantibodies also recognize a doublet of Mr 23 000 (p23) and 25 000 (p25) in addition to CENP (centromere protein)-A (Mr 19 000), -B (Mr 80 000), and -C (Mr 140 000). A p25 antigen (HP1(Hsalpha)) has been shown to be a human homolog of Drosophila HP1 (heterochromatin protein 1). We have isolated a cDNA clone encoding another form of p25 (HP1(Hsbeta ) or p25beta) from a lambdaZap HepG2 library using human autoimmune serum. The deduced amino acid sequence of the clone contained a conserved chromodomain (chromatin modifier domain) in the N-terminal region and a heterochromatin binding domain in the C-terminal region. In immunofluorescence experiments, only affinity purified antibodies reactive with the C-terminal (amino acids 70-185) domain showed nucleoplasmic and heterochromatin staining, whereas N-terminal (amino acids 1-115) specific antibodies were nonreactive. In metaphase chromosome spreads, the C-terminal domain antibody was also localized to the centromeric regions of chromosomes. Association with centromeres was most prominent at anaphase and changed to a more generalized association with whole chromosomes in telophase. The cooccurrence of autoantibodies to centromere proteins and HP1 in certain autoimmune diseases might be a reflection of coordinated immune responses to these closely associated sets of proteins.

3T3 Cells↗
PubMed1997

Analysis of an exon 1 polymorphism of the B2 bradykinin receptor gene and its transcript in normal subjects and patients with C1 inhibitor deficiency.

The B2 bradykinin receptor (B2BKR) mediates most of the inflammatory actions of bradykinin. To evaluate its potential role in allergic diseases, we assessed the structure of the human B2BKR gene. Screening a human placenta genomic DNA library identified only clones containing exons 2 and 3. Human placenta and colon tissues were used for 5' rapid amplification of complementary DNA ends to identify nine exon 1 clones, each containing one 9 bp and two 1 bp deletions compared with published sequences. Exon 1 genomic polymerase chain reaction of human leukocyte DNA revealed two distinct products, which were shown to differ by the presence or absence of the 9 bp deletion. Alleles with the 9 bp deletion were designated as (-)21-29, whereas alleles without the deletion were designated as (+)21-29. Genomic polymerase chain reaction in 39 Caucasian, 31 African-American, and 32 Asian normal subjects revealed a highly significant difference in the allelic frequency of the two genotypes, primarily because of an absence of the (+)21-29 allele in Asian subjects. Analysis of steady-state B2BKR messenger RNA levels by reverse-transcription polymerase chain reaction in heterozygous normal subjects revealed consistently higher expression of (-)21-29 transcripts. To investigate the potential clinical significance of the exon 1 polymorphism, 21 patients with angioedema and C1 inhibitor deficiency were genotyped. None were homozygous for the (+)21-29 allele (p = 0.0088 compared with normal subjects). In contrast, two patients with immunochemical evidence of hereditary angioedema without history of clinical angioedema were (+)21-29 homozygous. These results suggest that the B2BKR genotype may influence clinical status in diseases characterized by involvement of bradykinin.

Adolescent↗
PubMed1997

Diversity of antinuclear antibody responses in hepatocellular carcinoma.

BACKGROUND/AIMS: The development of antinuclear antibodies (ANA) in malignancies has been described but its mechanism is still not understood. The aim of this study was to examine ANA specificities in hepatocellular carcinoma to further understand autoimmunity in cancer. METHODS: Two hundred and four hepatocellular carcinoma patients were compared with 68 chronic hepatitis C, with 126 chronic hepatitis B and with 30 alcoholic liver cirrhosis patients, as well as with 87 healthy donors. Indirect immunofluorescence, immunoblotting, and immunoprecipitation were used to study ANA reactivities. RESULTS: Hepatocellular carcinoma had a significantly higher frequency of ANA using HEp-2 cells as substrate (31%) than chronic hepatitis C (10%), chronic hepatitis B (9.5%), alcoholic liver cirrhosis (10%) or healthy donors (4.5%). A great diversity of ANA specificities was found in hepatocellular carcinoma. Three hepatoma sera had antibodies that co-localized with non-snRNP splicing factor SC35, suggesting that the antigenic targets might be involved in mRNA splicing. We identified antibodies to two known nuclear autoantigens: fibrillarin and p330d/CENP-F. These autoantigens are involved in the 5' processing of precursor ribosomal RNA transcripts and in mitotic functions, respectively. CONCLUSIONS: Diversity was found in the autoantibody specificity, in contrast to the specific subsets of autoantibodies seen in several systemic rheumatic autoimmune diseases. Our data suggest that immune response in hepatocellular carcinoma targets important proteins involved in cellular biosynthetic or proliferative functions.

Adult↗