Search PubMed⌕ Search

Biomedical subjects

E M Tan

Publications and source records attributed to E M Tan.

At least 73 records · Page 4Linked to original sources
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

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↗
PubMed1995

Expression of extracellular matrix genes in adult human dermal microvascular endothelial cells and their regulation by heparin and endothelial cell mitogens.

BACKGROUND: Microvascular alterations are prominent features of systemic sclerosis (SSc) and often precede the appearance of clinically detectable fibrosis. The mechanism leading to selective microvascular injury in SSc is not known; however, microvascular endothelial cell (EC) activation has been demonstrated in SSc skin and is considered to be an early event in the pathogenesis of SSc. EXPERIMENTAL DESIGN: The expression of genes encoding extracellular matrix (ECM) proteins was examined in adult human dermal microvascular EC (HDMVEC), human iliac vein EC (HIVEC), and human umbilical vein EC (HUVEC) using indirect immunofluorescence (IIF) and Northern hybridization analysis. The effects of heparin and the endothelial cell mitogens, endothelial cell growth factor (ECGF) supplement and acidic and basic fibroblast growth factors (aFGF and bFGF), on the expression of ECM genes by these cells were also studied. RESULTS: Abundant transcripts for collagen types I, IV, VI, and fibronectin (FN) and weak expression of the type III collagen gene were detected in HDMVEC cultures in the absence of ECGF and heparin. In contrast, in the presence of these factors, no mRNA for types I, III, and VI collagens and marked down-regulation (more than twofold) of mRNA levels for collagen type IV and FN were observed. These results were confirmed at the protein level by IIF staining. In contrast to HDMVEC, HIVEC and HUVEC did not show expression of genes encoding types I, III, and VI collagens under any culture conditions examined. Next we studied the separate effect of heparin and aFGF or bFGF on the expression of ECM genes in HDMVEC. In contrast to the maximal expression of types I and VI collagens and FN detected in the absence of growth factors, aFGF decreased mRNA levels by 43% for type I collagen, by 52% for type VI collagen, and by 47% for FN. The decreases in mRNA levels caused by bFGF were 37, 41, and 36%, respectively. Heparin alone decreased the mRNA levels for these genes by 60, 77, and 65%, respectively; however, FGF potentiated the negative effect of heparin on ECM gene expression. CONCLUSIONS: These results demonstrate that HDMVEC display a unique pattern of expression of ECM genes that is different from that displayed by EC from medium and large vessels. The data also demonstrate that heparin, ECGF supplement, aFGF, and bFGF regulate ECM gene expression in HDMVEC in vitro and suggest that these growth factors may modulate the expression of matrix genes in vivo. Altered expression of ECM genes by HDMVEC may play an important role in diseases affecting the microvasculature, such as SSc.

Adult↗
PubMed1995

Differential expression of the versican and decorin genes in photoaged and sun-protected skin. Comparison by immunohistochemical and northern analyses.

BACKGROUND: Chronic sun exposure induces numerous changes in exposed skin; the most striking histopathologic change is the massive accumulation of material with the staining characteristics of elastin, termed solar elastosis, in the superficial dermis. Previous studies have identified elastic fibers within areas of solar elastosis, as well as a decrease in collagen content. Recently, the large chondroitin sulfate (CS) proteoglycan, versican, has been identified in the dermis in association with elastic fibers, and the smaller CS proteoglycan, decorin, has been shown to codistribute with collagen fibers. Thus, changes in expression of these CS proteoglycans might be expected in photoaging and may help to explain the clinical alterations of chronically sun-exposed skin. EXPERIMENTAL DESIGN: Immunohistochemical staining and confocal laser scanning microscopy for versican and decorin was performed on paired tissue samples from photoaged and non-sun-exposed skin taken from the same individuals. To investigate versican and decorin mRNA expression, Northern analysis was performed on paired fibroblast cultures derived from tissue explants of photoaged and non-sun-exposed skin. RESULTS: Immunohistochemical staining and confocal laser scanning microscopy revealed a massive accumulation of versican localized to the abnormally large fibers comprising solar elastosis in the superficial and mid-dermis of photoaged specimens. Decorin staining was greatly decreased within the area of solar elastosis. Similarly, changes in mRNA were measured from fibroblast cultures, with a significant increase in versican mRNA in cultures derived from photoaged skin, whereas decorin mRNA levels were significantly decreased in photoaged skin. CONCLUSIONS: This study provides further evidence for the close association of versican with elastic fibers and decorin with collagen fibers, even in the situation of abnormal fiber deposition occurring in photodamaged skin. In addition, changes in versican and decorin immunostaining are accompanied by similar alterations in gene expression.

Aged↗
PubMed1995

Basic fibroblast growth factor regulates type I collagen and collagenase gene expression in human smooth muscle cells.

Basic fibroblast growth factor (bFGF) is a multifunctional peptide well known for angiogenic, neurotropic, and mesoderm-inducing effects. In the present study, we have investigated the effects of bFGF on collagen and collagenase gene expression in human iliac arterial smooth muscle cells. We report that bFGF inhibits type I collagen gene expression and collagen biosynthesis, with concomitant stimulation of collagenase gene expression. The smooth muscle cells incubated with human recombinant bFGF decreased the mRNA steady state levels of pro-alpha 1(I) type I collagen by as much as 72%. [3H]Hydroxyproline synthesis was also suppressed by 59% compared with untreated control cultures. Indirect immunofluorescence confirmed corresponding changes at the protein level. In contrast to the down-regulation of type I collagen gene expression, collagenase gene expression was found to be up-regulated severalfold by bFGF. The data suggest that bFGF is capable of regulating collagen and collagenase gene expression divergently in human smooth muscle cells and that the effects appear to be mediated at a pretranslational level.

Cells, Cultured↗
PubMed1994

Synthetic compound peptide simulating antigenicity of conformation-dependent autoepitope.

Proliferating cell nuclear antigen (PCNA), also known as auxiliary protein of DNA polymerase delta, is involved in DNA replication and repair. Certain patients with systemic lupus erythematosus (lupus) produce autoantibodies to PCNA and the autoantibody-defined epitopes of PCNA have been inferred to be conformation-dependent. Based on antigenic properties of continuous primary structure peptides, compound peptides composed of covalently linked discontinuous sequences were synthesized and used as immunogens in rabbits. One compound peptide induced an antibody response to a nuclear antigen which demonstrated a cell cycle-related pattern of nuclear immunofluorescence with maximum expression in the DNA synthesis phase of the cell cycle associated with other features which simulated the conformation-dependent properties of lupus defined auto-epitopes. Other rabbit antibodies raised against different compound peptides did not show such properties and recognized epitopes which were continuous primary sequence regions. These results show that from analysis of antigenic sites on a protein, it might be possible to construct synthetic compound peptides which can mimic conformation-dependent epitopes recognized by spontaneously occurring autoantibodies.

Amino Acid Sequence↗
PubMed1994

Current status of available standards for quality improvement of assays for detection of autoantibodies to nuclear and intracellular antigens.

Considerable progress has been made in the improvement of clinical assays for the detection of autoantibodies to nuclear and intracellular antigens with the use of available World Health Organization (WHO) and Arthritis Foundation/Centers for Disease Control (AF/CDC) standards. The ultimate goal of standardization is for various clinical laboratory test results to be interchangeable and for an exchange of data to be done with confidence. This report discusses the available standards. In addition, significant technical problems and variations in methodologies for the detection of autoantibodies to intracellular antigens noted during a 4-year study by a European Consensus Study Group are detailed. Currently, there is a need for a future generation of reference preparations and standards that will show specific antibody reactivity on sensitive enzymes and immunoblotting assays. Standardization efforts should be done to characterize specific nuclear and cellular antigen preparations that may be of natural or of recombinant technology origin.

Antibodies, Antinuclear↗
PubMed1994

Characterization of a DNA-binding nuclear autoantigen mainly associated with S phase and G2 cells.

During analysis of autoimmune sera showing reactivity to nuclear antigens a serum producing a previously undescribed cell cycle-specific nuclear immunofluorescence pattern was observed. By indirect immunofluorescence the serum generated a densely speckled nucleoplasmic staining pattern of varying intensity with relative sparing of the nucleoli. The staining pattern could be abolished by treating cells with DNase but was not affected by RNase treatment. By flow cytometric dual parameter analysis the antigen expression was shown to increase during S phase with a maximum in G2 cells, whereas G1 and M cells expressed low levels. The cell cycle-specific expression of the target antigen was similar in some aspects to the expression of cyclin A but clearly not identical. The serum recognized a 68/60 kDa doublet in immunoblotting of soluble MOLT-4 cell extract. Antibodies eluted separately from either of the two bands produced the cell cycle-specific staining pattern as well as the doublet in immunoblotting. DNA-cellulose was used for analysis of DNA-binding property and the higher band was found to be DNA-binding, whereas the lower band was not. In summary, this serum reacts with a DNA-binding and cell cycle-specific nuclear antigen which might be a novel protein involved in cell cycle progression.

Aged↗
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

Autoantibodies in interstitial cystitis.

We have identified and partially characterized autoantibodies from the sera of patients with interstitial cystitis. Our characterization included initial screening by antinuclear antibody testing on human HEp-2 cell substrate and mouse kidney/stomach tissue substrate, titering and subtyping of positive sera, and Western blotting to identify target autoantigens. Of 96 interstitial cystitis patients 35 (36%) were positive for antinuclear antibodies at titers of 1/40 or greater. Among the antinuclear antibody patterns observed 24 were dense fine nuclear speckles, 7 were nucleolar, 3 were mitochondrial and 1 was coarse nuclear speckles. All but 4 of the antinuclear antibody positive sera were exclusively of the IgG class. As determined by unique antinuclear antibody staining patterns and by specificities on Western blots, interstitial cystitis autoantibodies appear to recognize novel autoantigens not previously described in patients with systemic autoimmune diseases, such as lupus, scleroderma and Sjögren's syndrome.

Adult↗
PubMed1994

Antimyenteric neuronal antibodies in scleroderma.

The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.

Adolescent↗
PubMed1994

Coiled bodies in the nucleolus of breast cancer cells.

Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992) Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immunoelectron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.

Autoantigens↗
PubMed1993

Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin.

We have isolated and determined the sequence of a 1.1-kb cDNA from a murine WEHI-3 macrophage library which encodes the highly conserved, nucleolar protein, fibrillarin. The murine fibrillarin protein sequence displays 94.2% identity with human fibrillarin, 82.9% identity with amphibian fibrillarin and 74.0% identity with the yeast fibrillarin homolog, NOP1. Immunoprecipitation showed that anti-fibrillarin autoantibodies from human scleroderma sera and the monoclonal autoantibody 72B9 recognize the approx. 34-36 kDa in vitro transcribed and translated protein. Mouse fibrillarin contains a N-terminal glycine- and arginine-rich (GAR) domain which although conserved among the fibrillarins is not as strongly conserved as several regions in the carboxy tail of the protein. Specific amino acid residues in yeast NOP1 thought to be associated with the synthesis and maturation of ribosomes show strong conservation between the mouse, human, amphibian and yeast protein sequences.

Amino Acid Sequence↗