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Biomedical subjects

L Cheng

Publications and source records attributed to L Cheng.

At least 289 records · Page 16Linked to original sources
PubMed1996

Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture.

In order to evaluate the characteristics and efficiency of gene transfer in primary cultures of oligodendrocytes, four different techniques including particle bombardment (Accell gene gun), cationic liposome-mediated transfection (lipofection), calcium phosphate co-precipitation and retroviral infection were compared using the LacZ and luciferase reporter genes. Highly purified postnatal adult rat oligodendrocytes were obtained by sequential immunopanning, plated in culture, and transfected using various reporter and promoter genes. The most efficient expression of LacZ and luciferase genes was found with particle mediated gene delivery. The transgene expression level obtained with gene gun delivery was at least two- to 100-fold greater than three other tested gene transfer methods. Comparison of the relative strength of four viral and two cellular promoters in these primary oligodendrocytes cultures demonstrated that the CMV promoter was the strongest. Using a human growth hormone (hGH) reporter gene, a long-term transgene expression pattern in primary oligodendrocytes was demonstrated to be sustained in culture for the entire experimental period (4 weeks) after particle-mediated gene transfer. These results demonstrate that expression of a foreign gene can be effectively achieved in primary cultures of adult oligodendrocytes, especially by using the particle bombardment method. The results also suggest that the current ex vivo gene transfer system may be used to manipulate oligodendrocytes for future application in gene therapy studies.

Adenoviridae↗
PubMed1996

Cultured Schwann cells constitutively express the myelin protein P0.

It is widely thought that mammalian Schwann cells do not express Po, the major glycoprotein in peripheral myelin, unless they are induced to do so by axonal signals that can be mimicked by agents that trigger cAMP signaling pathways. In contrast, we find that cultured Schwann cells make large amounts of Po without the addition of any axonal-like signal, provided they have not been exposed to serum during the culture process. We also report that glial growth factor/neuregulin inhibits this constitutive Po expression. Myelin basic protein is regulated in a similar way. We suggest that expression of Po by Schwann cells before the onset of myelination may be prevented by inhibitory signals within the nerve, rather than by the absence of a positive signal from axons.

Animals↗
PubMed1996

Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells.

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.

Animals↗
PubMed1996

Signal transduction and HIV transcriptional activation after exposure to ultraviolet light and other DNA-damaging agents.

Short wavelength (254 nm) ultraviolet light (UVC) radiation was much more potent in activating transcription of human immunodeficiency virus 1 (HIV) reporter genes stably integrated into the genomes of human and monkey cells than ionizing radiation (IR) from a 137Cs source at similarly cytotoxic doses. A similar differential was also observed when c-jun transcription levels were examined. However, these transcription levels do not correlate with activation of nuclear factor (NF)-kappa B and AP-1 measured by band-shift assays, i.e. both types of radiation produce similar increases in NF-kappa B and AP-1 activity, suggesting existence of additional levels of regulation during these responses. Because of the well-established involvement of cytoplasmic signaling pathways in the cellular response to tumor necrosis factor-alpha (TNF-alpha), UVC, and IR using other types of assays, the role of TNF-alpha in the UVC response of HIV and c-jun was investigated in our cell system. We demonstrate that UVC and TNF-alpha activate HIV gene expression in a synergistic fashion, suggesting that it is unlikely that TNF-alpha is involved in UVC activation of HIV transcription in stably transfected HeLa cells. Moreover, maximum TNF-alpha stimulation resulted in one order of magnitude lower levels of HIV expression than that observed after UVC exposure. We also observed an additive effect of UVC and TNF-alpha on c-jun steady-state mRNA levels, suggestive of a partial overlap in activation mechanism of c-jun by UVC and TNF-alpha; yet these responses are distinct to some extent. Our results indicate that the HIV, and to some extent also the c-jun, transcriptional responses to UVC are not the result of TNF-alpha stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. Additional levels of regulation that do not directly involve the NF-kappa B and AP-1 transcription factors, such as changes in chromatin structure associated with the UV repair process, may also be important for a full transcriptional response of HIV and c-jun to UVC. In addition to the new data, this report also summarizes our current views regarding UVC-induced activations of HIV gene expression in stably transfected cells.

Animals↗
PubMed1996

Fluorescence in situ hybridization method for measuring transfection efficiency.

We describe here the use of fluorescence in situ hybridization (FISH) to measure the transfection efficiency of the transient expression vector pCMVcat in lymphoblasts and fibroblasts. By using a pCMVcat probe, we can visualize the location of the plasmid after transfection and thus determine transfection efficiency. In this report, we show that, for transfection of pCMVcat by the diethylaminoethyl-dextran method, the transfection efficiency was about 15 and 70 times greater in fibroblasts and lymphoblasts, respectively, when measured by the FISH method as compared to the efficiency measured by cotransfection with pCMV beta gal. Based on these results, we conclude that the FISH method is a highly sensitive, specific and direct measure of transfection efficiency of a transient expression vector and that it may be useful for evaluating laboratory assays in which the quantitative aspects of transfection and the effect of plasmid DNA damage on transfection efficiency are important.

Cell Line↗
PubMed1996

Primary and secondary infections with Cryptosporidium parvum in immunosuppressed adult mice.

The present study was undertaken to determine if infection of immunocompetent adult C57BL/6N mice with Cryptosporidium parvum would render them more resistant to a challenge infection following immunosuppression with dexamethasone (DEX). Fecal oocyst shedding and parasite colonization of the terminal ilea were greater in immunosuppressed mice than in nonimmunosuppressed mice. Secondary infections with C. parvum resulted in decreased oocyst shedding and reduced parasite colonization compared with primary infections. Flow cytometry revealed fewer splenic B cells but more splenic total T, CD4+ T, CD8+ T cells and macrophages in immunosuppressed mice than in nonimmunosuppressed mice. The CD4+ to CD8+ T cell ratios and blastogenic responses to lipopolysaccharide (LPS), but not to concanavalin A, were decreased in immunosuppressed mice compared with nonimmunosuppressed mice. Blastogenic responses to LPS and percentages of splenic total B cells and macrophages were increased in secondary infections compared to primary infections. Enhanced susceptibility to C. parvum infection in immunosuppressed mice revealed DEX-mediated effects on both cell-mediated and humoral immunity. Our results suggest that increased resistance in immunosuppressed mice to secondary infections with C. parvum may involve increases in B cells and macrophages.

Animals↗
PubMed1996

Increased expression of 72-kd type IV collagenase (MMP-2) in human aortic atherosclerotic lesions.

MMP-2, a secreted 72-kd metalloproteinase that specifically degrades type IV collagen as well as denatured collagens, has been implicated in smooth muscle cell migration. To evaluate the possible contribution of this enzyme to the formation and progression of the atherosclerotic lesion, the expression of MMP-2 was studied in human aortic tissue. MMP-2 was visualized in frozen sections of the aortic wall by an immunofluorescent technique with a polyclonal antibody. Expression of MMP-2 in the aortic extracts was also studied by zymography and Western blotting. Our results reveal that a greater amount of MMP-2 is present in fatty streaks and atherosclerotic plaques as compared with normal regions of the aorta. Immunoblotting analysis showed that MMP-2 was expressed in atherosclerotic plaque > fatty streak > normal aortic wall in a ratio of approximately 4:2:1. Zymograms show that both forms (activated and latent) of MMP-2 increased in the atherosclerotic plaques. The presence of macrophages, detected by an immunohistochemical technique in some areas of higher MMP-2 expression suggests that these cells are a possible source of MMP-2. We conclude that MMP-2 collagenase may have a role in the formation and progression of the atherosclerotic lesion and may be involved in clinical complications of atherosclerosis, such as fissure and rupture, leading to thrombosis.

Adolescent↗
PubMed1996

Expression of E-cadherin in primary and metastatic prostate cancer.

Immunohistochemical studies have suggested that E-cadherin may be a useful prognostic marker in prostate cancer. Previous studies have depended on cryostat sections of tissues selected grossly. Many prostate cancers, even when extensive, are not visible grossly; many others cannot be demarcated sharply grossly. The wide applicability of prognostic markers after total prostatectomy will depend upon methods that can be applied to tissue selected based upon the histopathological examination of the entire prostate. Our purpose was to investigate the possibility that E-cadherin could be demonstrated in paraffin-embedded whole prostates and metastatic prostate cancer. Microwaving in citrate buffer was the best of five methods tested for the demonstration of E-cadherin in paraffin-embedded prostate and was used to investigate 53 primary prostate cancers from 44 patients and lymph node metastases from 14 patients. Metastases of prostate cancer to lymph nodes expressed less (P = 0.008) E-cadherin than primary prostate cancers. The expression of E-cadherin correlated with the histopathological differentiation (Gleason grade) of primary prostate cancers (P = 0.03, Ptrend = 0.003). The use of monoclonal anti-human E-cadherin (HECD-1) with microwaving in citrate buffer followed by immunoperoxidase staining with heavy metal enhancement for the demonstration of E-cadherin in paraffin-embedded tissue will, for the first time, allow the use of archival tissue for prognostic studies of E-cadherin in prostate cancer and other tissue. Our results are consistent with the hypothesis that aggressive prostate cancers exhibit decreased expression of E-cadherin and demonstrate the feasibility of long-term prognostic studies of this molecule in the usually multiple prostate cancers found in whole, formalin-fixed, paraffin-embedded resected prostates.

Antibodies, Monoclonal↗
PubMed1996

DNA repair capacity correlates with mutagen sensitivity in lymphoblastoid cell lines.

This study describes a correlation between cellular DNA repair capacity and the frequency of mutagen-induced in vitro chromosomal breaks in selected lymphoblastoid cell lines. Two assays, host cell reactivation (HCR) assay for measuring cellular DNA repair capacity and in vitro mutagen sensitivity assay, have recently been shown to be useful biomarkers for such susceptibility. Increased in vitro mutagen sensitivity, measured by the number of induced chromatid breaks, has been postulated to reflect decreased capacity of DNA repair, as measured by the HCR assay. However, these two assays have not been examined in parallel to test this hypothesis. In this study, we performed both assays in 16 established lymphoblastoid cell lines derived from patients with xeroderma pigmentosum (n = 3), ataxia telangiectasia (n = 2), head and neck cancer (n = 3), and melanoma (n = 2), and from normal human subjects (n = 6) using UV light, 4-nitroquinoline-1-oxide (4-NQO; an UV-mimetic agent), and gamma-irradiation as the test agents. The measurements from the HCR assay correlated significantly with the frequency of chromatid breaks induced by either UV irradiation (r = -0.69; P < 0.01) or 4-NQO (r = -0.70; P < 0.01). Although published data suggest that damage induced by UV and 4-NQO may be repaired by different pathways, the two agents induced similar frequencies of chromatid breaks (r = 0.68; P < 0.01) in the tested cell lines. Our results also indicated that the HCR assay is not suitable to test agents that cause DNA strand breaks, such as gamma-irradiation, whereas the mutagen sensitivity assay is. Although reduced cellular DNA repair capacity correlated with increased frequency of mutagen-induced chromatid breaks in these cell lines, these two assays have different sensitivities in measuring the repair of damage induced by different carcinogens; therefore, the use of both assays is recommended for future molecular epidemiological studies of cancer susceptibility.

4-Nitroquinoline-1-oxide↗
PubMed1996

[Analysis of survival in 240 patients with unresectable primary hepatocellular carcinoma treated with hepatic artery chemo-embolization].

The effect of transcatheter hepatic artery chemoembolization (TAE) on hepatocellular carcinoma (HCC) was evaluated. The survival time of 240 patients with unresectable primary HCC who were treated by TAE during the period from Jan. 1987 to Jan. 1995 were followed up. The results showed that in this series the overall 1-, 2-, 3-, 5- and 7-year survival rate was 61.0%, 39.2%, 30.7%, 18.9%, 16.0%, respectively. The survival rate of patients treated with TAE plus hepatectomy at 1, 2, 3, 5 and 7 years was 95.2%, 90.4%, 85.0%, 59.0% and 59.0% respectively, while that in patients treated with TAE in 1, 2, 3, 5, 7 years was 58.1%, 34.0%, 24.4%, 13.7%, 9.1%, respectively. This results indicate that TAE therapy is a treatment of choice for patients with advanced hepatocellular carcinoma.

Adolescent↗
PubMed1996

[Percutaneous cervical discectomy: a report of twenty-five cases].

Twenty-five patients with herniations of the cervical intervertebral disks were treated with percutaneous cervical discectomy. The surgical instruments and technique were improved based on those for percutaneous lumbar discectomy. All the patients well recovered with an average follow-up period of 15.6 months (from 4 to 23 months) and no major complications was encountered. The method is safe, easy and effective. The anatomy, principles and maneuvers related to the operation are discussed in detail.

Adult↗
PubMed1995

Simultaneous amplification of four DNA repair genes and beta-actin in human lymphocytes by multiplex reverse transcriptase-PCR.

We describe here the development, optimization, and use of a non-radioactive, quantitative, multiplex reverse transcriptase-PCR technique to measure, in a single reaction, the relative levels of the transcripts of four DNA repair genes (XPCC, hMSH2, XRCC1, and ERCC1) and the beta-actin gene in lymphoblastoid cell lines and frozen peripheral blood lymphocytes. Expression of defective DNA repair genes was not detected in DNA repair-deficient human cell lines, whereas the intact genes were detected in repair-proficient cell lines and in lymphocytes from a normal donor. The assay was reproducible, and repeated determinations of the same samples generated highly consistent results for each target gene. This approach should facilitate molecular epidemiological studies that incorporate screening for germline alterations that may affect gene expression and for changes in the levels of gene expression.

Actins↗
PubMed1995

Biological characterization and molecular cloning of murine C-type retroviruses derived from the TSZ complex from mainland China.

Characterization of the SRS murine retrovirus complex, derived from the TSZ system of murine leukemia developed in China, was carried out. The initial stock contained XC+, NB-tropic virus (and possibly other viruses), and induced several neoplastic diseases in neonatally inoculated NIH Swiss mice: erythroid leukemia, myeloid leukemia (acute myeloblastic leukemia), and lymphoblastic lymphoma (both B- and T-lymphoid). In addition, approximately 30% of inoculated animals developed central nervous system disease--hindlimb paralysis or semilateral paralysis. Rescue of virus from the spleen of an animal with combined erythroid/myeloid leukemia, followed by endpoint dilution gave two stocks: 19-6 (XC+, NB-tropic) and 19-7 (XC-, NB-tropic). Both stocks induced erythroid and myeloid leukemia, and 19-6 induced CNS symptoms as well. Southern blot analysis indicated that the predominant viruses from the 19-6 and the 19-7 cultures were related, but different in the env region. An infectious virus molecular clone of provirus from 19-6 cells was obtained. The resulting cloned virus [SRS 19-6 murine leukemia virus (MuLV)] induced four kinds of leukemia: erythroid, myeloid, B-lymphoma, and T-lymphoma; in many cases, more than one tumor type was identified in the same animal. Such a broad spectrum of leukemias induced by a cloned MuLV is unusual. Flaccid hindlimb paralysis induced by SRS 19-6 MuLV could be attributed to meningeal B-lymphoma. Immunofluorescent staining with a panel of env-specific monoclonal antibodies confirmed that the 19-6 and 19-7 viral stocks contained different viruses, which differed from previously characterized MuLVs. The viruses of the SRS complex may provide interesting reagents for investigations of MuLV-induced disease.

Animals↗
PubMed1995

Effect of cations on the elution of pectic polysaccharides from anion-exchange resins.

Conditions for quantitatively eluting six plant pectic polysaccharides from diethylaminoethyl (DEAE)-columns were established. Cations, surprisingly, greatly affected the elution of pectic polysaccharides from these anion-exchange columns. Quantitative elution of apple pectic acid was achieved when columns of DEAE-Sephadex A-25 and DEAE-Trisacryl Plus-M were developed with 0.5 M NH4Cl, 0.5 M LiCl, or 0.5 M CsCl in buffer. In contrast, up to 1 M NaCl and KCl in buffer only eluted 6.3 to 54% of the apple pectic acid from the two types of columns. In each case the retained apple pectic acid was eluted basically quantitatively by 0.5 M NH4Cl in buffer. The elution of a pectic polysaccharide fraction from Apium graveolens (celery), commercial citrus polygalacturonic acid, and two polysaccharides isolated from purified cell walls of Lemna minor (duckweed) from columns of DEAE-Trisacryl was incomplete when the columns were developed wit 0.5 M NaCl or 0.5 M KCl in buffer, ranging from 0 to 89%; however, again, the retained portion of each sample was eluted quantitatively or almost so from the columns with 0.5 M NH4Cl in buffer. One pectic polysaccharide, commercial citrus pectin, was eluted equally well and almost quantitatively by 0.5 M NaCl, KCl and NH4Cl in buffer. When a pectic polysaccharide fraction from purified cell walls of L. minor was applied to a preparative column of DEAE-Trisacryl Plus-M, 99% of the sample was eluted by a gradient of 0 to 0.5 M NH4Cl in buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗