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Biomedical subjects

E M Tan

Publications and source records attributed to E M Tan.

At least 91 records · Page 5Linked to original sources
PubMed1993

Cloning of type XVII collagen. Complementary and genomic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BPAG2) predict an interrupted collagenous domain, a transmembrane segment, and unusual features in the 5'-end of the gene and the 3'-untranslated region of the mRNA.

Thus far, 16 distinct vertebrate collagens (types I-XVI) have been delineated. In this study, we have cloned a mouse collagenous protein, the 180-kDa bullous pemphigoid antigen (BPAG2). Isolation of over-lapping clones, together with 5' and 3' rapid amplification of cDNA ends, allowed delineation of the entire coding sequence. The 5' and 3' ends of the mRNA transcripts were confirmed by primer extension and anchored reverse transcription polymerase chain reaction analyses. The deduced polypeptide contained 1,433 amino acids, including a collagenous domain that consisted of 13 separate segments. Computer analysis of the deduced amino acid sequence demonstrated the presence of a membrane-associated segment. Examination of the 5' end of the BPAG2 gene revealed that the 295-base pair (bp) exon 1 contained two segments of (T)13AA and TT(A)11, whereas exon 2 was shown to contain the translation initiation codon. The 3' end of the mRNA transcript identified two 6-bp inverted repeat sequences that could form a stem for a 30-bp hairpin loop followed by a series of U residues. Comparison of mouse and human BPAG2 sequences demonstrated 86% homology and the unit of evolutionary period of 4.2 million years. In summary, we have cloned full-length mouse BPAG2 cDNA sequences that encode a collagenous polypeptide. We propose that this polypeptide be designated as the alpha 1-chain of type XVII collagen.

Amino Acid Sequence↗
PubMed1993

Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation.

Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.

Autoantigens↗
PubMed1993

Increasing titers and changing specificities of antinuclear antibodies in patients with chronic liver disease who develop hepatocellular carcinoma.

BACKGROUND: Patients with liver cirrhosis and chronic hepatitis are at high risk for development of hepatocellular carcinoma (HCC). In HCC and other malignant neoplasms, antinuclear antibodies (ANA) have been detected, but the clinical and biologic significance of these autoantibodies has not been established. This study documents changing ANA manifested as seroconversion from a negative to positive ANA status, increasing titers, and changing antibody specificities in patients in whom chronic liver disease has progressed to HCC. METHODS: Sera were collected from patients with chronic liver diseases and HCC in Japan. Indirect immunofluorescence was used to detect ANA, and Western blotting, two-dimensional immunoblotting, and enzyme-linked immunosorbent assay were used to characterize nuclear antigen-antibody systems. RESULTS: ANA were detected in 57 of 183 (31%) patients with HCC, a statistically higher frequency than in patients with the most common antecedent clinical conditions, liver cirrhosis (9 of 64 patients [14%]; P < 0.05) or chronic hepatitis (16 of 123 patients [13%]; P < 0.001). One patient with autoimmune hepatitis observed for 9 years had a decrease in ANA titer after therapy with corticosteroid and azathioprine and had a rebound increase in ANA titer associated with development of HCC. Immunologic assays demonstrated dramatic decreases in levels of autoantibodies to histones during therapy and the appearance of new autoantibodies coincident with HCC. Changes in ANA associated with the appearance of auto-antibodies of new specificities were documented in four other patients with chronic liver disease in whom HCC developed. CONCLUSIONS: Changes in ANA, especially alterations in autoantibody specificities, are seen infrequently in systemic autoimmune diseases. It is possible that in HCC such changes in ANA might reflect autoimmune responses to intranuclear antigens that are perturbed in cellular transformation.

Adult↗
PubMed1993

Decorin, versican, and biglycan gene expression by keloid and normal dermal fibroblasts: differential regulation by basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a multipotential heparin-binding factor that belongs to the fibroblast growth factor (FGF) family. The FGFs demonstrate a wide spectrum of biologic activities in vivo and in vitro. In this study, we investigated the potential of bFGF to regulate the expression of various dermal extracellular matrix proteoglycans and type I collagen mRNAs in cultured human fibroblasts from keloid, which is a prototype of dermal fibrosis, and normal skin tissue. We report that bFGF upregulates the expression of the decorin gene in normal and keloid fibroblasts. In contrast, the expression of biglycan is downregulated by bFGF. The mRNA steady-state level of versican, a large proteoglycan, is not altered by bFGF. Type I collagen gene expression is downregulated substantially in keloid and normal fibroblasts by bFGF. The results suggest that the expression of the proteoglycan genes are uncoordinately regulated and that the gene expression of type I collagen and biglycan is coordinately downregulated. The results also demonstrate that keloid fibroblasts respond similarly as do normal fibroblasts to bFGF in the regulation of proteoglycan and collagen expression.

Biglycan↗
PubMed1993

The natural history of interstitial cystitis: a survey of 374 patients.

A survey directed at determining the natural history of interstitial cystitis was conducted at our clinic. Information on demographics, risk factors, symptoms, pain and psychosocial factors was elicited from 374 patients who satisfied the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria for interstitial cystitis and had all been diagnosed as having interstitial cystitis by a urologist. With regard to demographics, patients were predominantly female (89.8%) and white (94.1%), with a mean age of 53.8 +/- 0.7 years (standard error) and age at the first symptoms of 42.5 +/- 0.8 years. Information on 25 potential risk factors included 44.4% of the women reporting hysterectomy, 38.2% of the patients having strong sensitivities or allergic reactions to medication and only 2.7% being diabetic. With regard to interstitial cystitis symptoms, frequency and urgency were reported by 91.7% and 89.3% of the patients, respectively, while pelvic pain, pelvic pressure and bladder spasms were reported by more than 60% of respondents and burning by 56%. Location and degree of pain were also reported. Urination relieved or lessened interstitial cystitis pain for 73.6% of the patients and medication was effective for 46.8%. Other behaviors (for example hot baths, heating pads, lying down or sitting) were less effective. Conversely, stress, constrictive clothing and intercourse increased interstitial cystitis pain in more than 50% of the patients. In addition, acidic, alcoholic or carbonated beverages, and coffee or tea increased interstitial cystitis pain in more than 50% of the patients. More than 60% of the patients were unable to enjoy usual activities or were excessively fatigued and 53.7% reported depression. Travel, employment, leisure activities and sleeping were adversely affected in more than 80% of the patients. Pain location and degree differed significantly between patients with and without ulcers in the bladder. In addition, there was an apparent plateau in the frequency and urgency among patients after approximately 5 years with symptoms.

Adult↗
PubMed1993

Autoantibodies in viral hepatitis-related hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) develop autoantibodies to nuclear and nucleolar antigens (ANAs) which can be readily detected by immunofluorescence on cell substrates. The frequency of ANAs in HCC is 31% (57/184). The identity of three autoantigens was established as: NOR-90, nucleolus organizer region (doublet) polypeptides involved in RNA polymerase I transcription; fibrillarin, a component of nucleolar U3 RNP involved in pre-ribosomal RNA processing, and nucleophosmin/protein B23, a nucleolar protein involved in ribosome maturation and cell proliferation. Changes in ANAs were observed in some patients during transition from chronic liver disease to HCC and were manifested as seroconversion from ANA-negative to ANA-positive status by an increase in titers and changes in ANA specificities. Serum from a patient during this transition period was used to isolate a cDNA clone encoding a novel nuclear protein with structural motifs characteristic of a family of splicing factors. These observations support the notion that ANA responses in HCC might be driven by intracellular events related to transformation from the stage of chronic injury to the stage of malignancy. Changes in ANA profiles which were observed to precede clinically diagnosed HCC in some patients might be early markers of transformation.

Autoantibodies↗
PubMed1993

Novel nuclear autoantigen with splicing factor motifs identified with antibody from hepatocellular carcinoma.

A patient with liver cirrhosis who progressed to hepatocellular carcinoma was found to develop novel antinuclear antibodies. The serum was used to isolate full-length cDNA clones encoding related proteins of 530 amino acids (representative clone HCC1.4) and 524 amino acids (representative clone HCC1.3). Affinity-purified antibodies eluted from recombinant proteins recognized a 64-kD nuclear protein in Western blotting and decorated the nucleoplasm in a speckled-network fashion in immunofluorescence, colocalizing with antibodies to pre-mRNA splicing factor SC35 and uridine-rich small nuclear RNAs. The deduced amino acid sequence contained an arginine/serine-rich (RS) domain and three-ribonucleoprotein consensus sequence domains, two classes of motifs present in several splicing factors. A repeating octapeptide of Arg-Ser-Arg-Ser-Arg(Lys)-Glu(Asp)-Arg-Lys(Arg) was present in RS region of HCC1. This octapeptide sequence called RS-ERK motif was also found in splicing factors U2AF 35- and 65-kD proteins and 70-kD U1 small nuclear ribonucleoprotein. The molecular features and immunolocalization data suggest that the HCC1 autoantigen may be associated with splicing activities and are consistent with observations that autoantibody responses frequently target molecules involved in important cellular biosynthetic functions.

Amino Acid Sequence↗
PubMed1993

Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis.

We have employed human autoantibodies to characterize a novel cell cycle-regulated nuclear protein, provisionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was concentrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predominantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet reproduced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of 'chromosomal passenger' proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transition. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.

Autoantibodies↗
PubMed1993

Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts.

The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.

Collagen↗
PubMed1993

Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies.

BACKGROUND: Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) are increasingly used for evaluation of cell proliferation especially in diagnostic pathology. To understand the different staining characteristics of such antibodies, we found it important to map the reactive epitopes in detail. EXPERIMENTAL DESIGN: Overlapping, synthetic 15-mer peptides encoding the entire PCNA amino acid sequence were analyzed for reactivity with 6 anti-PCNA monoclonal antibodies using enzyme-linked immunosorbent assay and flow cytometric competition analysis. One immunodominant region was studied using a set of peptides with single amino acid substitutions. Further epitope localization was obtained by immunoblotting of fusion protein constructs. RESULTS: The epitopes recognized by the antibodies could be assigned to specific peptides. Five monoclonal antibodies (19A2, 19F4, TO17, TO30, PC10) reacted with the same protein region (aa 111-125) whereas one antibody (TOB7) recognized a separate region of the protein (aa 181-195). The reactivity pattern with 8 recombinant PCNA-fusion proteins agreed with the peptide data. The immunodominant aa 111-125 peptide reactive antibodies differed concerning immunofluorescence patterns. These differences could be attributed to epitope microheterogeneity within this peptide as determined by reactivity with Ala-substituted peptides. The 19A2, 19F4 and PC10 antibodies showed nuclear fluorescence and similar but not identical peptide recognition patterns. Compared with 19A2 and 19F4, the PC10 antibody was directed against a simpler epitope. TO17 and TO30 gave cytoplasmic filamentous staining and their epitopes were different from those defined by the other monoclonal antibodies. CONCLUSIONS: The results indicate that induced anti-PCNA antibodies recognize well defined linear epitopes, in contrast to PCNA autoepitopes which are strongly dependent on the protein conformation.

Amino Acid Sequence↗
PubMed1993

Type VII collagen gene expression in human umbilical tissue and cells.

BACKGROUND: Type VII collagen is a minor collagen found in anchoring fibrils. It is expressed predominantly by keratinocytes. In this study, we report the localization and spatial distribution of type VII collagen gene expression in the human umbilical cord, a fetal-derived tissue. EXPERIMENTAL DESIGN: Human umbilical cords were examined in indirect immunofluorescence studies, employing a mouse monoclonal anti-human type VII collagen antibody. Endothelial cells were cultured from the vein and grown on chamber slides for the detection of type VII collagen epitopes. In addition, cultured human umbilical vein cells were analyzed by Northern transfer analysis and by polymerase chain reaction for the expression of the corresponding gene. Fibroblast-like cells were isolated from the Wharton's jelly and were analyzed similarly for type VII collagen expression as well. RESULTS: We demonstrate that type VII collagen is expressed by human umbilical tissue and cells. Indirect immunofluorescence studies demonstrate the presence of type VII collagen epitopes in the epithelium surrounding a connective tissue region known as Wharton's jelly. In addition, there was low but detectable immunofluorescence signal associated with endothelial cells of blood vessels within the umbilical cord. In vitro, the fibroblast-like cells cultured from the Wharton's jelly showed prominent type VII collagen signal. This result was supported by the finding of high level of type VII collagen mRNA in these cells. The human endothelial cells from the vein demonstrated weak but detectable staining for type VII collagen, and the corresponding gene expression was shown by polymerase chain reaction analysis of the mRNA of the endothelial cells. CONCLUSIONS: The results show that umbilical tissue and cells, specifically those from the Wharton's jelly, are relatively enriched in type VII collagen. There is differential spatial localization of this collagen in the fetal tissue. The novel finding is that cells, other than epithelial cells such as keratinocytes, are able to express the type VII collagen gene.

Antibodies, Monoclonal↗
PubMed1992

Antinuclear autoantibodies in women with silicone breast implants.

Clinical syndromes resembling autoimmune diseases have been reported in women who have had breast augmentation procedures. To see whether there is a humoral immune response in these diseases that is similar to the immune response in their idiopathic counterparts, we assessed the immunological specificity of antinuclear antibodies (ANAs) and certain epidemiological features in 24 patients, all of whom (with 1 exception) had received silicone gel breast implants. ANA specificities were identified by indirect immunofluorescence, immunodiffusion, western blot analysis, and immunoprecipitation of radiolabelled intracellular proteins. Of 11 patients who had symptoms and signs that met criteria for defined autoimmune diseases, 7 had scleroderma or subsets of this disorder and the others had systemic lupus erythematosus, rheumatoid arthritis, or overlapping autoimmune diseases. High ANA titres were present in 10 of these 11 patients and the ANA specificities were similar to those found in the idiopathic forms of the corresponding autoimmune diseases. Trauma, with resultant rupture of implants, accelerated onset of symptoms. 13 other patients had autoimmune disorders of a less clearly defined nature and low titres of ANAs whose specificities could not be identified. ANAs are associated with the development of autoimmune complications in women with silicone breast implants. Further studies are needed to see whether this relation is one of cause and effect and whether ANAs might be early serological markers preceding development of autoimmune symptoms.

Academies and Institutes↗
PubMed1992

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.

Autoantibodies↗
PubMed1992

Antibodies in rabbits immunized with cationized IgG react with histones H3 and H4.

OBJECTIVE: Rabbits immunized with cationized rabbit IgG develop antinuclear antibodies. This study was aimed at identifying the reactive antigens. METHODS: Rabbits were immunized with homotypic or allotypic IgG that were physicochemically altered to produce positively charged dimethylpropanamide (polycationic) side chains. RESULTS: Seven of 11 rabbits injected with cationized IgG produced antinuclear antibodies detected by indirect immunofluorescence. By enzyme-linked immunosorbent assay, these were IgG antibodies reacting with histone (H3-H4)2 tetramer and with individual histone polypeptides H3 and H4. The antihistone antibodies were absorbed by cationized IgG but not by normal IgG. CONCLUSION: Cationized IgG appears to possess antigenic determinants of sufficient similarity to produce antibody responses cross-reactive with histones H3 and H4.

Animals↗
PubMed1992

Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies.

Patients with hepatocellular carcinoma (HCC), gastrointestinal, lung, and ovarian cancers were shown to have autoantibodies to nuclear and nucleolar antigens as detected by immunofluorescence on cell substrates. The frequency of antinuclear antibodies (ANAs) was significantly higher (P less than 0.001) in patients with HCC (57/184 = 31%) than in patients with chronic hepatitis or liver cirrhosis (25/187 = 13%). Although a range of fluorescence patterns was observed, a higher percentage of nucleolar fluorescence was detected in HCC, and three of these nucleolar antigens were identified. They were NOR-90, nucleolus organizer region doublet polypeptides of 93 and 89 kDa involved in RNA polymerase I transcription; fibrillarin, a 34 kDa protein of the nucleolar U3 ribonucleoprotein particle which is engaged in preribosomal RNA processing; and nucleophosmin/protein B23, a 37 kDa polypeptide which is associated with ribosome maturation and cellular proliferation. All these antigens are nucleolar components that are engaged in some aspect of ribosome biosynthesis. Since autoantibodies to these nucleolar antigens have also been found in systemic autoimmune diseases, they do not represent autoimmune reactions unique to cancer but might reflect reaction pathways related to immune responses that are antigen-driven. The ANA response in HCC appears to be dynamic reactions to this antigen-drive since some patients with chronic liver disease showed seroconversion to ANA positivity, marked increase in titer and/or change in antibody specificity preceding or coincident with clinical detection of HCC. These changes in ANA showed a close temporal relationship with transformation from long-established chronic liver disease to HCC.

Antibodies, Neoplasm↗