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Biomedical subjects

E Wang

Publications and source records attributed to E Wang.

At least 217 records · Page 12Linked to original sources
PubMed1996

The NF-kappa B inhibitor, tepoxalin, suppresses surface expression of the cell adhesion molecules CD62E, CD11b/CD18 and CD106.

Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.

Antibodies, Blocking↗
PubMed1996

[A study of suitable age for intraocular lens implantation in children according to ocular anatomy and development].

OBJECTIVE: To study the suitable age for intraocular lens (IOL) implantation in children. METHODS: 240 (240 eyes) normal children aged 3-13 years old were selected. Their corneal refraction, anterior chamber depth, lens thickness and ocular axial length were measured with Roden-Stock Keratometer C-MES and Eyescan Model 55 separately. RESULTS: The corneal refraction of 3-year-old children, the anterior chamber depth and lens thickness after 5 years old, and the ocular axial length after 9 years old approach the adult magnitude. CONCLUSIONS: A 3-year-old child has been qualified with IOL implantation, the child younger than 9 years old should be implanted with a normal adult IOL and then corrected with glasses, and a child after 10 years old should be directly implanted with a proper dioptric IOL.

Adolescent↗
PubMed1996

[Experimental study of streptomycin on 45Ca intake of otolithic membranes].

45CaCO3 was injected into guinea pigs, and the radioactivity in otolithic membranes of utriculi and sacculi, auditory ossicles and femora was determined by liquid scintillation spectrometry, to study the dynamic effect of streptomycin on calcium content of otolithic membranes, as a means to explore the mechanism of streptomycin ototoxicity. The results showed that otoconium was a dynamic structure which took up 45Ca in a time course generally comparable to that of bone. Saccular otolithic membranes showed greater intake than utricular membranes. The calcium ion concentration of saccular endolymph was greater than that of the utriculus and the two maculas were not identical. Streptomycin may influence 45Ca uptake in the two macular otolithic membranes, causes calcium metabolic disorder and a fall of otolithic calcium content. It is possible that the fluid environments of the endolymph have changed, therefore dysfunction of otolithic organ may occur. This hypothesis may be one of the explanation of the mechanism of streptomycin ototoxicity.

Animals↗
PubMed1995

Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G0-G1 transition in unperturbed and drug-treated cell populations.

3T3 Cells↗
PubMed1995

Identification of the functionally relevant calmodulin binding site in smooth muscle caldesmon.

The C-terminal region of smooth muscle caldesmon (CaD) interacts with calmodulin (CaM) and reverses CaD's inhibitory effect on the actomyosin ATPase activity. We have previously shown that the major CaM-binding site (site A) in this region is within the segment from Met-658 to Ser-666 (Zhan, Q., Wong, S. S., and Wang, C.-L. A. (1991) J. Biol. Chem. 266, 21810-21814). Recently, another segment (site B), Asn-675 to Lys-695, was reported to bind CaM (Mezgueldi, M., Derancourt, J., Calas, B., Kassab, R., and Fattoum, A. (1994) J. Biol. Chem. 269, 12824-12832). To assess the functional relevance of these two putative CaM-binding sites, we have examined three synthetic peptides regarding their effects on CaM's ability to reverse CaD-induced inhibition of actomyosin ATPase activity: GS17C (Gly-651 to Ser-667), VG29C (Val-685 to Gly-713), each containing one CaM-binding site, and MG56C (Met-658 to Gly-713), which contains both sites. We found that although VG29C did bind CaM, its affinity was weakened by GS17C, and it failed to compete with CaD for CaM under the conditions where GS17C effectively displaced CaD from CaM. MG56C had an effect similar to that of GS17C. These experiments demonstrated that site A for CaM binding is involved in regulating the inhibitory property of CaD.

Amino Acid Sequence↗
PubMed1995

Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very low density lipoprotein secretion.

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.

Animals↗
PubMed1995

Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved.

Programmed cell death (apoptosis) is an active process by which cells initiate their own self-destruction. Growing evidence shows that this event is controlled by the activation of unique gene expression; some function as survival genes, such as bcl2, and others as killer genes, such as ced3 or interleukin converting enzyme. Likewise, external factors, such as the presence or absence of stimuli in the microenvironment of a cell, play a key role in ushering it towards survival or suicidal fate. Previously, I and others have reported that withdrawal of serum from culture medium can induce contact-inhibited quiescent mouse 3T3 fibroblasts to undergo rapid programmed cell death, as evidenced by the presence of massive DNA fragmentation within 24 h. I now report that, although the same process of serum withdrawal is capable of inducing apoptotic death in quiescent young human fibroblasts, the process takes as long as 2 weeks. Repeated attempts at the same serum withdrawal with cultures of senescent human fibroblasts show that phenotypic signs of apoptosis, such as DNA fragmentation and loss of cell viability, are not observed for up to 4 weeks; I suggest that in vitro aged human fibroblasts are resistant to undergoing programmed cell death. I have investigated the level of bcl2 presence as a possible protector of senescent human fibroblasts from apoptotic death; biochemical characterization shows that in mouse as well as human fibroblasts, bcl2 is present as an easily extractable (0.1% Triton) cytoplasmic protein. bcl2 level is in inverse relationship with the ease of induction of apoptotic death between young and senescent human fibroblasts. Immunofluorescence staining shows that, in senescent human fibroblasts, bcl2 is present not only in the cytoplasmic punctate spots seen in both mouse and young human fibroblasts but also in the nuclei as well as large granules surrounding the nuclei. Upon serum deprivation, the bcl2 level is reduced to undetectable in mouse 3T3 fibroblasts within 24 h and in young and intermediate aged human fibroblasts within 2 weeks; however, it remains unchanged in senescent human fibroblasts after the deprivation of serum for 2 weeks. These findings lead me to conclude that senescent fibroblasts are resistant to the induction of apoptotic death by serum deprivation. Furthermore, I suggest that repeated serial passaging during the in vitro aging process has inadvertently instituted a molecular mechanism whereby the bcl2 level cannot be repressed upon serum deprivation, which may subsequently allow senescent fibroblasts to be long-lived and protected from self-destruction.

3T3 Cells↗
PubMed1995

Modelling the effects of age-related mtDNA mutation accumulation; complex I deficiency, superoxide and cell death.

Deleterious mitochondrial mutations accumulate during normal human aging in postmitotic tissues. How these mutations affect aging cells is currently unknown. This issue has been addressed in two ways. The first is to determine the likeliest effect of random mutations in the mitochondrial genome, and of the 4977 bp deletion and MELAS point mutation that rise in frequency with age. The results indicate that Complex I is statistically much more likely to be affected than any other product of the mitochondrial genome. We have also attempted to model Complex I deficiency in animals with the drug MPTP, a specific inhibitor of Complex I. We find that MPTP causes massive damage in brains of mice with a genetic deficiency in the mitochondrial superoxide dismutase, MnSOD, but less in mice that overexpress the enzyme. We conclude from these data that MPTP-induced cell death must be mediated through an increase in the steady-state concentration of superoxide anion in mitochondria. Since the likeliest target of mitochondrial mutation is Complex I, deficiency of which causes MnSOD-inhibitable lethality, we propose that rising mtDNA mutations with age will cause an increase in superoxide-mediated cell death. Such a mechanism for age-related cell death has the potential to explain several age-related phenotypes.

Aging↗
PubMed1995

Phenotypic and cell cycle properties of human oligodendrocytes in vitro.

The remyelination, albeit limited, which occurs at the lesion sites in the central nervous in multiple sclerosis has been attributed to both myelin production by previously myelinating cells and to precursor cells which mature into myelin-producing cells. Oligodendrocyte (OL) number may be increased at the periphery of the lesions. In this study, we assessed the state of maturation and cell cycle-dependent properties of OLs derived from surgically resected adult human cerebral cortex specimens. In 6-day-old OL cultures, a small proportion of cells (14.1 +/- 3.5%: range 4-24%) expressed an immature phenotype, defined as A007+:myelin basic protein (MBP)-negative. Using lack of statin expression as an index of cells exiting the G0 phase of the cell cycle, we observed that 4.6 +/- 1.6% of A007+ cells, but only rare MBP+ cells (0.4 +/- 1.8%) were non-reactive with the anti-statin antibody, S44. The proportion of non-statin-reactive cells was not affected by treatment with basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF). The oligodendrocytes did not incorporate BrdU during a 48-h pulse and did not immunoreact with Ki-67 antibody. In 4-week-old cultures, we found that all A007+ cells were also MBP+ and that 99.5 +/- 0.7% were statin-positive. Exposing 4-week-old OLs to conditions of serum deprivation or to 1,000 units/ml of recombinant human TNF-beta for 4 days induced nuclear fragmentation in a high proportion (> 70%) of cells, as measured by a TUNEL technique; in these cultures, a similarly high proportion of cells were non-immunoreactive with anti-statin antibody. Our results suggest that a small number of phenotypic 'pre-oligodendrocytes' can be derived from the adult human CNS and that a proportion of these cells have exited the G0 phase of the cell cycle. Attempt at cell cycling, however, could reflect abortive mitosis and activation of programmed cell death.

Cell Cycle↗
PubMed1995

Optical detection of macromolecular heparin via selective coextraction into thin polymeric films.

Thin plasticized polymer films, poly(vinyl chloride) doped with a specific ion pairing quaternary ammonium compound, tridodecylmethylammonium chloride, and a lipophilic pH indicator, 3-hydroxy-4-(4-nitrophenylazo)phenyl octadeconate, are shown to exhibit significant and analytically useful optical response toward macromolecular heparin. The response mechanism is based on favorable extraction of heparin into the bulk organic film, owing to the specific ion-pairing complexation reaction between the quaternary ammonium species and the polyanion. A simultaneous coextraction of hydrogen ions results in protonation of the pH chromophore and hence a change in the optical absorbance of the polymeric film. When used in a limited volume/fixed exposure (10 min) detection mode, film absorbances change as a function of the initial heparin concentration in the range of 0.2-3.0 units/mL (1.2-18 micrograms/mL). The practical measurement response time is controlled by heparin diffusion through the stagnant diffusion layer adjacent to the surface of the film as well as within the bulk of the polymer film and is shown to increase with the molecular weight of the heparin species tested. No optical response to heparin is observed when a strong heparin complexing agent (e.g., protamine) is present in the test solution, suggesting that the polymer film can be used to conveniently monitor heparin-protamine (or other antagonist) titrations. The theory relating to the operation of the sensing film in either the equilibrium or the kinetic mode and the selectivity of the optimized film to heparin relative to small anions are presented.

Anions↗
PubMed1995

Cells en route to apoptosis are characterized by the upregulation of c-fos, c-myc, c-jun, cdc2, and RB phosphorylation, resembling events of early cell-cycle traverse.

Density-arrested quiescent murine Balb/c-3T3 cells are dependent upon growth factors for their survival. Withdrawal of serum from their medium induces rapid cell death, the mechanism of which is not yet fully understood. We have studied the effect of serum deprivation on density-inhibited quiescent Swiss 3T3 cells and found that they undergo rapid cell death upon total withdrawal of serum. The nature of this cell death is similar to apoptosis, as shown by cellular and nuclear morphology and DNA fragmentation into oligonucleosomal fragments. Investigating the regulation of early cell-cycle genes during this process, we found that c-myc, c-jun, c-fos, and cdc2 protein presence is induced after serum deprivation, when the phosphorylated form of the RB protein also appears. The upregulation of these genes' protein products is coupled with the appearance of PCNA, a proliferation-specific nuclear antigen, as well as significant incorporation of BrdU, which may reflect DNA repair activity; in situ analysis shows that BrdU-positive cells are also positive for DNA fragmentation. These results suggest that en route to apoptosis, cells undergo events typical of early cell-cycle traverse by expressing early G1 genes and may even experience the late G1/S phase boundary, as shown by the presence of PCNA. However, the demonstrated ability of these cells to traverse the G1 phase of the cell cycle seems to be an abortive event, since they die shortly afterwards.

3T3 Cells↗
PubMed1995

Down-regulation of statin, a nonproliferation-specific nuclear protein, and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts.

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G1 phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G1 phase in the apoptotic process is an abortive one, with the end result of cell demise.

3T3 Cells↗
PubMed1995

In situ detection of Epstein-Barr virus in non-small cell lung carcinomas.

Epstein-Barr virus (EBV) is strongly associated with nasopharyngeal carcinoma and lymphoepithelioma-like carcinomas (LELC) of foregut-derived organs. Recently this group of EBV-associated carcinomas has been expanded by the identification of the virus in conventional adenocarcinomas of the stomach. In situ hybridization (ISH) using a sensitive digoxigenin-labelled EBER RNA probe was performed on 167 consecutive unselected primary non-small cell lung carcinomas, to determine the frequency of EBV association in these tumours. Nine cases (5.4 per cent) showed strong EBER signals in the tumour cell nuclei. By immunohistochemistry, four of the EBER-positive tumours showed patchy expression of the viral latent membrane protein (LMP-1) and none showed any expression of the EBV nuclear antigen 2 (EBNA2). Morphologically, all the positive tumours were LELC, whereas no conventional type of non-small cell lung carcinoma showed EBV association. The LELC presented a morphological spectrum from undifferentiated to squamoid or glandular differentiation. The patients showed a male to female ratio of 8:1. The mean age at presentation was 48 years. Smoking was not a risk factor. All patients were alive at follow-up periods of 23-52 months. Southern blot analysis performed on eight of the nine positive tumours showed a clonal episomal form of EBV, suggesting the clonal expansion of an infected tumour cell early in oncogenesis. These characteristics of the EBV-associated lung tumours justify their consideration as a distinct clinicopathological entity.

Adult↗
PubMed1995

Terminal differentiation-dependent alteration in the expression of translation elongation factor-1 alpha and its sister gene, S1, in neurons.

Elongation factor-1 alpha (EF-1 alpha) is a highly conserved, abundantly expressed protein that functions in peptide elongation during mRNA translation. Mammalian species contain a second EF-1 alpha gene, S1, whose expression is limited to brain, heart, and skeletal muscle. Such tissue specificity in S1 gene expression led us to hypothesize that this specialized member of the EF-1 alpha family is possibly essential to terminally differentiated, long-lived cells such as neurons, cardiomyocytes, and myocytes. We report here that during development, EF-1 alpha mRNA levels remain unchanged in S1-negative tissues, but sharply decrease in S1-positive tissues. RNase protection as well as tissue distribution analyses of the S1 message suggest that down-regulation of EF-1 alpha expression coincides well with that of S1 up-regulation during postnatal development. Analysis of primary cultures of the rat neonatal cerebral cortex cells has shown that S1 gene expression is indeed restricted to neurons only, whereas nonneuronal cell types such as astrocytes and microglia are S1-negative. EF-1 alpha mRNA on the other hand was detected in all three cell types, namely, neurons, astrocytes, and microglia. This report confirms the terminal differentiation-dependent expression of the S1 gene in neurons of the cerebral cortex.

Animals↗