Biomedical subjects

L Cao

Publications and source records attributed to L Cao.

At least 145 records · Page 8Linked to original sources

PubMed1998

Cytokine gene transfer in cancer therapy.

New strategies based on gene transfer technology are employed in cancer therapy. Cytokines are polypeptides involved in immunity and inflammation, and essentially control the magnitude of the immune response. Genetically modified tumor cells releasing various cytokines have been shown to enhance tumor immunogenicity and to induce the regression of preexisting tumors. In some instances, immunological memory has been generated to resist the subsequent challenge with unmodified, parental tumor cells. Cytokine gene transfer into antitumor effector cells, as well as antigen presenting cells, is also being investigated to augment antitumor immune responses.

Animals↗

PubMed1998

The role of regulatory T cells in Lewis rats resistant to EAE.

Adult Lewis (LEW) rats are highly susceptible to experimental autoimmune encephalomyelitis (EAE), induced actively by immunization with guinea pig (GP) myelin basic protein (MBP) in complete Freund's adjuvant or adoptively transferred with activated T lymphocytes reactive to GP MBP peptide 68-88. Once LEW rats recover from active EAE or when given MBP in incomplete Freund's adjuvant (IFA), they become resistant to further attempts to induce active or passive EAE. In this study, we examined whether such EAE-resistant rats after MBP-IFA immunization have reduced frequencies of MBP-reactive T cells, whether these T cells are anergized, and whether the activity of regulatory T cells is increased to the event that they prevent activation of MBP-specific T cell subpopulations. By limiting dilution analyses (LDA) of unfractionated splenic T cells, the levels of MBP-reactive T cells in EAE-resistant rats appeared to be approximately 5% of the levels in EAE-susceptible rats. However, a subsequent analysis of CD4+ enriched T cell populations, depleted of the CD8 subset, showed similar frequencies of MBP-reactive cells in susceptible and resistant LEW rats. Not only were the frequencies on LDA altered by suppressor cells, but also LDA comparisons based on cell proliferation and cytokine production as indicators of MBP reactive cell frequencies gave markedly different results. We conclude that MBP-reactive T cells in this model of EAE-resistant LEW rats are hyporeactive to MBP as the result of an increased activity of a regulatory subset of CD8+ T cells. These results also demonstrate that the quantitation of MBP-reactive CD4+ T cells by LDA is strongly influenced by the presence of functionally antagonistic CD8+ T cells, which cause an underestimation of responder T cell frequencies, and by the method of detecting T cell reactivity.

Animals↗

PubMed1998

Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells.

Recent reports have implicated a possible role of reactive oxygen species (ROS) in the induction and mediation of apoptosis and DNA damage. Oxidative DNA base modification induced by cupric nitrilotriacetate (Cu-NTA) and the following apoptosis were observed in human promyelocytic leukemia HL-60 cells. We measured the level of ROS in the cells by using a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the amount of a modified DNA base, 8-hydroxydeoxyguanosine (8-OHdG) by HPLC-ECD. It was found that Cu-NTA exposure significantly enhanced ROS and 8-OHdG formation in the cells. Meanwhile, we observed both DNA fragmentation and morphological changes characteristic of apoptosis, which was also determined quantitatively by flow cytometry and showed dose- and time-dependent manners. Furthermore, several antioxidants such as dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and catalase were used to detect whether the apoptosis could be blocked. Only DMSO protected against this form of cell death. To elucidate molecular events in the apoptosis, expressions of Bcl-2 protein family members, such as Bcl-2, Bcl-X and Bax, and heat shock protein 70 (HSP-70) were measured by western blotting using specific antibodies. The levels of Bax and Bcl-Xs remained largely unchanged, but the Bcl-2 and Bcl-XL expression showed down-regulation. After 24 h incubation in the presence of copper, the levels of Bcl-2 and Bcl-XL reduced about 33.8% and 51.1% compared with untreated cells, respectively. Furthermore, after 16 h incubation, the level of HSP-70 expression was about 3.4-fold greater than that in untreated cells, suggesting that HSP-70 is important in increasing resistance to oxidative stress induced by Cu-NTA. But overexpression of HSP-70 failed to protect HL-60 cells from apoptosis induced by Cu-NTA. We inferred that Cu-NTA may induce oxidative DNA damage through free radical injuries, which may turn on the apoptosis in HL-60 cells.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen.

Cartilage is a hypocellular tissue in which a balance of matrix molecules, especially aggrecan and link protein, play a critical role in maintaining structural integrity. To study the role of aggrecan and link protein in mediating cell activities, we have stably expressed them in NIH/3T3 fibroblasts and observed the effect on cell-substratum interactions. Overexpression of either protein destabilized the cell-substratum interaction. However, when both were co-expressed, the interaction between cell and substratum was less impaired. Similar results were obtained on type II collagen-coated plates. The addition of exogenous gene products into fibroblast cell lines and chondrocyte culture had the same effect as expression of the genes. The addition of exogenous hyaluronan to the growth medium or treatment of cells with hyaluronidase also decreased cell adhesion, indicating that hyaluronan also plays a role in the cell-substratum adhesion. The presence of aggrecan seems to increase the amount of link protein on the cell surface. Chondrocytes expressing high concentrations of aggrecan and link protein were maintained within a matrix network and were able to survive in suspended culture. Imbalances in aggrecan or link protein concentrations, or degradation of hyaluronan, disrupted the network and caused the chondrocytes to aggregate or adhere to the plates.

3T3 Cells↗

PubMed1998

Expression of the G1 domain of aggrecan interferes with chondrocyte attachment and adhesion.

The proteoglycan aggrecan is a major component of cartilage, and degradation of aggrecan is associated with aging and a number of pathological conditions. To investigate the effects of the accumulation of G1 domain from degraded aggrecan, we overexpressed the G1 domain in cell culture. Exogenous addition of the expressed G1 gene product to chondrocyte cultures retarded the attachment of chondrocytes to tissue culture plates, type II collagen and hyaluronan. Expression of the G1 construct, aggrecan or link protein in chondrocytes led to decreased cell adhesion. Interestingly, aggrecan (and specifically G1) and link protein, when applied simultaneously, seem to be able to interact and inhibit the destabilization of chondrocyte adhesion observed when either is applied alone. The G3 domain of aggrecan has little effect on chondrocyte adhesion and did not change the effects of link protein and the G1 domain on chondrocyte adhesion. These effects probably involve the binding of G1 to cell surface molecules, since FACS (fluorescence-activated cell sorter) analysis demonstrated that the G1 domain binds to the chondrocyte surface with high affinity.

Aggrecans↗

PubMed1998

Expression of MDR1 mRNA and encoding P-glycoprotein in archival formalin-fixed paraffin-embedded gall bladder cancer tissues.

The aim of this study was to examine the expression of P-glycoprotein (Pgp) and MDR1 mRNA, in gall bladder carcinoma, a chemo-resistant tumour. 26 cases of gall bladder cancer and nine samples of normal gall bladder archival paraffin blocks were investigated for the presence of Pgp protein with immunohistochemistry (IHC) and MDR1 RNA by reverse transcription-polymerase chain reaction (RT-PCR). Monoclonal antibodies JSB-1 and UIC-2, recognising separate epitopes of Pgp, were used for IHC. For RT-PCR, total RNA was extracted from paraffin-embedded tissue. After RT, the samples were subjected to nested PCR (NPCR) using primers specific for the MDR1 gene, and evaluated by electrophoresis. In gall bladder carcinoma, the percentage of positive cases expressing Pgp (77% for JSB-1, 69% for UIC-2) and MDR1 mRNA (52%) was significantly higher than those in normal gall bladder. In earlier TNM stages Pgp and MDR1 mRNA were more frequently expressed (non-significant) than in advanced stages. The results of this study suggested that overexpression of MDR1 mRNA and Pgp in gall bladder carcinoma tissue probably is a very important reason why gall bladder cancer is generally not responsive to chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Telomerase activation in nasopharyngeal carcinomas.

Nasopharyngeal carcinomas (NPC) are common in Hong Kong and southern China but rare in Western countries. Telomerase activation is common in human cancers but has not been reported previously in NPC. Telomerase activation in NPC was determined using the sensitive TRAP (telomerase rapid amplification protocol) assay in 45 nasopharyngeal biopsies (36 NPC, nine normal nasopharyngeal mucosae) in four xenografted NPC tumours established in nude mice and in five in vitro NPC cell lines. Telomerase activation is common in NPC and can be detected at high frequencies (85% in primary tumours and 100% in recurrent tumours). The frequency of telomerase activation was lowest in NPC biopsies without lymph node involvement (60%) compared with those with positive lymph node involvement (100%), and the difference is statistically significant (P < 0.05; Fisher exact test). All the xenografted NPC tumours and in vitro NPC cell lines were strongly positive for telomerase activity. Our results suggest that telomerase activation is common in NPC and it may be useful as a diagnostic marker in the detection of tumour cells in nasopharyngeal biopsies. The high frequency of telomerase activation in stage I NPC (80% positive) suggests that it is an early event in tumour progression.

Adolescent↗

PubMed1998

MP1 encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus Penicillium marneffei.

We cloned the MP1 gene, which encodes an abundant antigenic cell wall mannoprotein from the dimorphic pathogenic fungus Penicillium marneffei. MP1 is a unique gene without homologs in sequence databases. It codes for a protein, Mp1p, of 462 amino acid residues, with a few sequence features that are present in several cell wall proteins of Saccharomyces cerevisiae and Candida albicans. It contains two putative N glycosylation sites, a serine- and threonine-rich region for O glycosylation, a signal peptide, and a putative glycosylphosphatidylinositol attachment signal sequence. Specific anti-Mp1p antibody was generated with recombinant Mp1p protein purified from Escherichia coli to allow further characterization of Mp1p. Western blot analysis with anti-Mp1p antibody revealed that Mp1p has predominant bands with molecular masses of 58 and 90 kDa and that it belongs to a group of cell wall proteins that can be readily removed from yeast cell surfaces by glucanase digestion. In addition, Mp1p is an abundant yeast glycoprotein and has high affinity for concanavalin A, a characteristic indicative of a mannoprotein. Furthermore, ultrastructural analysis with immunogold staining indicated that Mp1p is present in the cell walls of the yeast, hyphae, and conidia of P. marneffei. Finally, it was observed that infected patients develop a specific antibody response against Mp1p, suggesting that this protein represents a good cell surface target for host humoral immunity.

Amino Acid Sequence↗

PubMed1998

Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis.

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.

AIDS-Related Opportunistic Infections↗

PubMed1998

Ligand-dependent regulation of NPR-A gene expression in inner medullary collecting duct cells.

Atrial natriuretic peptide (ANP) interacts with high-affinity, guanylyl cyclase-linked receptors in the inner medullary collecting duct (IMCD), where it exerts important regulatory control over sodium handling. We sought to determine whether receptor activity in these cells would be modulated (downregulated) by prolonged exposure to ligand. A number of natriuretic peptides (ANP, brain natriuretic peptide, and urodilatin) were found to decrease ligand-dependent natriuretic peptide receptor A (NPR-A) activity in IMCD cells. This inhibition was in direct proportion to their capacity to increase basal cGMP levels in this cell population. The reduction in receptor activity was accompanied by a dose- and time-dependent reduction in NPR-A mRNA levels in these cells. The decrease in transcript levels arose, in part, from a reduction in NPR-A gene transcription. ANP reduced NPR-A gene promoter activity in a transiently transfected IMCD cell population. 8-Bromo-cGMP was also effective in inhibiting NPR-A mRNA levels and NPR-A promoter activity, suggesting that the second messenger (i.e., cGMP) rather than ANP, itself, is responsible for downregulation of NPR-A gene expression.

Animals↗

PubMed1998

Comparison of two methods of staining apoptotic cells of leukemia cell lines. Terminal deoxynucleotidyl transferase and DNA polymerase I reactions.

We compared two methods to stain apoptotic cells, one using terminal deoxynucleotidyl transferase (TDT), the other DNA polymerase I, using leukemia cell lines treated with anti-Fas monoclonal antibody (MAb). Both TDT and polymerase I strongly reacted with fragmented nuclei of apoptotic MOLT-16 and Jurkat cells, but only polymerase I strongly reacted with nonfragmented nuclei of early apoptotic cells. Anti-Fas MAb-treated MOLT-4 cells showed morphological changes corresponding to early apoptosis and were strongly positive for polymerase I only. MOLT-16 and Jurkat cells treated with anti-Fas MAb and inhibitors of endonuclease and poly(ADP-ribose) polymerase showed the morphology of early apoptosis but were not strongly stained by TDT. Because DNA polymerase I has nick-translation activity, it is possible that DNA polymerase I reaction is positive in early apoptotic cells by detecting single-strand DNA cleavage, which occurs before extensive oligonucleosomal DNA cleavage and late morphological changes of apoptosis in leukemia cell lines. Although TDT is widely used to stain apoptotic cells, DNA polymerase I may be more applicable in special cases of apoptosis, in which cells undergo single-strand rather than double-strand DNA breaks. However, the procedure has limitations, such as the necessity to use cell smears for comparison with the TDT reaction. (J Histochem Cytochem 46:85-90, 1998)

Antibodies, Monoclonal↗

PubMed1998

[Expression and significance of tumor suppressor gene WAF1 in epithelial ovarian neoplasms].

The expression of P21WAF1 protein in epithelial ovarian carcinoma, borderline ovarian tumor, benign ovarian tumor and normal ovary were analyzed by immuno-histochemistry technique. Results indicated: 1. The positive expression for p21 was 45.7%(16/35) in epithelial carcinoma, 75%(9/12) in borderline tumor and benign tumor, 90.9%(10/11) in normal ovary. The difference for p21 protein between ovarian carcinoma and borderline tumor, benign tumor, normal ovary was obvious(P < 0.05). The differences in other groups were unimportant(P > 0.05). 2. The positive expression of p21 in stage I/II was 42.9%(6/14), in stage III/IV was 47.6%(10/21). The difference of them was insignificant(P > 0.05). 3. The positive expression for p21 in Grade I was 70%(7/10), in Grade II 50%(6/12), in Grade III 23.1%(3/13). The difference for p21 protein between Grade III and Grade I, Grade II was obvious(P < 0.05). The difference between Grade I and Grade II was unimportant(P > 0.05). 4. The positive expression for p21 in 35 ovarian carcinoma with lymphaden metastasis(17.6%) was lower than ovarian carcinoma without lymphaden metastasis(72.2%). The difference of them was obvious(P < 0.05). The conclusion is that deregulation of p21 protein is associate with tumoral differentiation, lymphaden metastasis and patient's prognosis, whereas it isn't associated with clinical stage. The loss for p21 protein may effect the onset and progress of ovarian carcinoma.

Carcinoma↗

PubMed1998

[Function analysis of regulative region of transforming gene Tx related to human nasopharyngeal carcinoma].

Tx gene is the DNA sequence with transforming activity which was cloned from the epithelial cell line (CNE2) of human nasopharyngeal carcinoma (NPC). In order to understand the role of Tx in carcinogenesis of NPC, the promoter function of potential promoter region in Tx2. 8 was detected using CAT reporter assay. The results showed that the region has no promoter function. It suggested that Tx2. 8 kb fragment homogenous to IGKC might be regulated by the specific mechanism of immunoglobulin gene.

Chloramphenicol O-Acetyltransferase↗

PubMed1998

[Detection of telomerase activity and its clinical significance in colonic cancers by PCR-ELISA].

Using the recently developed telomerase PCR-ELISA detection kit, the authors detected the telomerase activity in 12 paired colonic cancer biopsies. It was found that strong telomerase activity was detected in all of the 12 colonic cancer tissues but not in adjacent non-cancerous tissues. These results suggest that the detection of telomerase activity by the PCR-ELISA assay could be used as a supplementary means for the diagnosis of colonic cancer.

Biopsy↗

PubMed1998

[Construction of the internal standard RNA and DNA templates for MRP gene quantitative RT-PCR analysis].

OBJECTIVE: To construct the internal standard RNA and DNA templates for quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitatively measure the mRNA expression of MRP gene. METHODS AND RESULTS: A 292 bp fragment was amplified by PCR from plasmid pRC/RSV-MRP containing full-length MRP cDNA and inserted into the Sma I site of PUC19 vector, constructing a recombinant plasmid-pUMRP292. A 238 bp HGV fragment was amplified by PCR from plasmid pUHGV and inserted into the Cla I site of pUMRP292, constructing a new recombinant plasmid-pUMRP 292/HGV as the internal DNA competitive template for quantitative PCR of MRP. A 530 fragment containing the 292 bp of MRP and the 238 bp of HGV was cut down by EcoR I and Xba I pUMRP292/HGV and cloned into the transcriptional vector pSP72 by the same enzyme sites, constructing a recombinant plasmid-pSMRP292/HGV, and then pSMRP292/HGV was cleaved with EcoR V and transcripted in vitro by SP6 RNA polymerase, obtaining a 530 bp mutant MRP-RNA positive strand as the internal RNA competitive template for quantitative RT-PCR. CONCLUSION: The established quantitative RT-PCR assay is simple, rapid and sensitive, and the DNA competitive template is more simple, economic and reliable than the RNA one.

ATP-Binding Cassette Transporters↗

PubMed1998

[Study on the pharmacognosy of traditional Chinese medicine tubeimu].

In this article, we have studied the traditional Chinese medicine Tubeimu on the pharmacognosy. This paper reports the thickening condition on the epidermis cell walls of the bulbuls of Tubeimu [Bolbostemma paniculatum (Maxim) Franquet] for the first time and corrects the wrong record in before literature.

Chromatography, Thin Layer↗

PubMed1997

Quantitative assessment of myelin basic protein-reactive T cell entry to the central nervous system by using oligonucleotide probes complementary to VDJ junctional sequences of rat TCR beta-chain.

The VDJ junctional region represents the most diverse part of the antigenic TCR. We have previously reported that of 200 sequenced TCR beta-chains of rat MBP-reactive T cells, rarely did two share sequence homology over the entire CDR3 region. In this study, we demonstrate that sequences of the TCR CDR3 region are excellent clonotypic markers for rat MBP-reactive T cell clones and oligonucleotide probes complementary to the CDR3 region of three T cell clones specifically recognized the TCR from which they were derived, but failed to recognize syngeneic T cells that express a similar TCR beta-chain or T cells that share both V beta and J beta usage. To explore this observation, we determined the ability of MBP-reactive T cell clones to enter the CNS. We were able to show that some MBP-reactive T cell clones have an augmented ability to enter the CNS and that fully-activated T cells have a higher penetrating activity than their less-activated T cell counterparts.

Amino Acid Sequence↗

PubMed1997

Immunological effects of an arginine side chain contaminating synthetically prepared peptides.

The side chain, 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), is a protective group coupled to arginine to mask the omega-nitrogen, in order to protect the guanidino function during peptide synthesis by the 9-fluorenylmethoxycarbonyl (Fmoc) procedure (Walker, 1994). This group is removed at the completion of peptide synthesis; however, the cleavage process can be incomplete. We have found that animals injected with a mixed population of pure, i.e. unmodified, and Mtr-containing MBP peptides have an immunodominant humoral response to the Mtr-bearing peptide. This response is dependent on the characteristics of the MBP peptide involved. For two MBP peptides, the Mtr-containing peptide had increased binding to antibody over pure peptide. For two other peptides, only the Mtr-containing peptide bound antibody while the unmodified peptide did not. In a separate system involving a polyclonal response to an unrelated peptide from beta2-microglobulin (beta2 m), the dominance of the Mtr group was also evident. These results provide further evidence that a small side chain on a single amino acid in a peptide can markedly alter the immunogenicity and antigenicity of that peptide for antibody reactivity. This evidence emphasizes the need for a critical awareness of each component of peptide synthesis and its potential to alter the immunoreactivity of the final product.

Amino Acid Sequence↗