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Biomedical subjects

L P Zhu

Publications and source records attributed to L P Zhu.

At least 19 recordsLinked to original sources
PubMed2005

Inhibition of 6A8 alpha-mannosidase gene expression resulted in telomere length shortening in nasopharyngeal carcinoma cell CNE-2L2.

Telomere length shortening was observed in the nasopharyngeal carcinoma cell CNE-2L2 when 6A8 alpha-mannosidase expression was inhibited by antisense 6A8 DNA. Transduction with mock or an irrelevant DNA did not affect the telomere length in the carcinoma cells. Telomerase activity and mRNA transcription of TRF 1 and 2 were not changed in the cells treated with antisense 6A8. The Con A binding test showed an enhancement on the proteins isolated from the cells treated with antisense 6A8, but not on those from mock- or irrelevant DNA-treated cells. The data imply an association between glycosylation modification with telomere shortening in antisense 6A8-treated cells.

Cell Line, Tumor↗
PubMed2002

Modification of glycosylation reduces microvilli on rat liver epithelial cells.

Effect of glycosylation modification on the shape of microvillus was investigated on rat liver epithelial cell line WB-F344. Since rat ER alpha-mannosidase has 89% identity to human 6A8 alpha-mannosidase, we used an antisense 6A8 cDNA fragment to inhibit expression in WB-F344 cells. Cells were transfected with antisense 6A8, the relevant sense fragment or the mock plasmid. Genomic PCR for neo(R) demonstrated integration of the transfected gene into host DNA. Enzymatic activity assay on p -nitro-phenyl-alpha-D-mannopyranoside showed suppression of ER alpha-mannosidase expression in the cells transfected with antisense 6A8. Concanavalin A binding to these cells was enhanced, indicating a modification in glycosylation. Number reduction and blunting of microvillus on these cells was observed. Transduction with the sense fragment or the mock had no effect. Cells with suppressed ER alpha-mannosidase expression grew slower in culture. Our results indicate an obvious effect of glycosylation modification on microvilli, which might be related with malfunctioning of cells.

Animals↗
PubMed2001

Long-term expression of a transferred gene in Epstein-Barr virus transformed human B cells.

Delivering a gene into the Epstein-Barr virus (EBV)-transformed B cells is useful in studying effects of the gene on B-cell functions. However, although people have been able to efficiently transfer genes into and get them expressed in B-lympho blastoid cells for a time probably long enough to kill the cells using vectors harbouring oriP, the expression time of the delivered gene is not long enough in order to study the gene function in B cells. To solve this problem, we constructed an adeno-associated virus (AAV) plasmid pAGX(+) based on plasmids pSub201 and pRc/CMV. We developed and packaged recombinant AAV (rAAV) expression vectors containing an antisense or a sense DNA fragment of 6A8 cDNA encoding a human alpha-mannosidase, or an antisense fragment of 5D4 cDNA encoding a human cell membrane protein, or EYFP DNA. EBV-transformed B cell SKW6 and 3D5 were transduced with those rAAV or the mock. Transduction with the rAAV-EYFP showed an infection frequency of 64 +/- 3.5% and 58 +/- 6.2% for SKW6 and 3D5 cell, respectively. Genomic polymerase chain reaction (PCR) for neoR gene indicated an integration of the transferred gene into the host DNA. After being cultured and propagated for over 12 months, the cells were detected for the expression of the transferred gene. The RT-PCR, enzymatic assay and Con A binding test demonstrated an inhibition of 6A8 alpha-mannosidase in both SKW6 and 3D5 cells transduced with the antisense 6A8 DNA. Immunofluorescence staining with monoclonal antibodies (MoAb) 5D4 showed a reduction of the 5D4 protein expression on both the cells transduced with the antisense 5D4 DNA. The DNA fragmentation assay showed a resistance of the cells with 6A8 alpha-mannosidase inhibition to apoptosis induction by anti-Fas antibody. The data indicate that the AAV vector pAGX(+) can efficiently introduce genes into EBV-transformed B cells and the delivered gene can be expressed in the cells for more than 12 months which may be long enough for the study of gene functions in B cells.

Antibodies, Monoclonal↗
PubMed2001

[Growth inhibition of human nasopharyngeal carcinoma cell CNE-2L2 caused by suppression of 6A8 alpha-mannosidase expression].

OBJECTIVE: To study the relationship between 6A8 alpha-mannosidase expression and growth of CNE-2L2 cells, a human nasopharyngeal carcinoma cell line. METHODS: Recombinant adeno-associated virus vector (rAAV) was used as a mediator to transfer an antisense or a sense fragment of 6A8 cDNA into CNE-2L2 cells. 6A8 alpha-mannosidase expression was detected by means of mAb 6A8a staining, alpha-mannosidase activity assay and ConA binding test. Cell growth was examined by means of MTT and colony formation. Tumor growth at the inoculated site of the cells in nude mice was detected after 8 weeks. RESULTS: Transduction of antisense 6A8 could reduce the expression of 6A8 alpha-mannosidase. In comparison to those in controls, the wild type, the mock-transduced and the sense 6A8-transduced cells, the MTT value, the colony number formed and the tumor weight grown at the inoculated site of cells were significantly decreased in the antisense 6A8-transduced cells (P < 0.001). CONCLUSION: Decreased expression of 6A8 alpha-mannosidase caused an inhibition of CNE-2L2 cell growth.

Animals↗
PubMed2001

[6A8 alpha-mannosidase catalyzes p-nitrophenyl-alpha-D-mannopyranoside].

OBJECTIVE: To confirm the alpha-mannosidase nature of the protein encoded by 6A8 cDNA. METHODS: 1) To construct a full-length 6A8 cDNA based on the three cloned DNA fragments by means of gene recombinant technique; 2) To insert the 6A8 cDNA into eukaryotic expression vector pCDI; 3) To transfect the recombinant pCDI-6A8 into COS-7 cells; 4) To characterize the nature of the protein encoded by 6A8 cDNA by means of enzymic activity assay and Western blotting assay. RESULTS: The constructed 6A8 cDNA was the right cDNA in sequence. The enzymetic activity of the homogenate of COS-7 cells transfected with pCDI-6A8 was 3-4 times higher than that of the cells transfected with the mock or the wild cells. The enzymetic reaction could not be inhibited by swainsonine. Western blot showed a band of 120,000 recognized by mAb 6A8. The band in the cells transfected with pCDI-6A8 cDNA was much darker than that in the cells transfected with the mock or in the wild cells. CONCLUSION: The protein encoded by 6A8 cDNA is a kind of alpha-mannosidase, which belongs to type II alpha-mannosidase.

Animals↗
PubMed2000

Cloning, expression and characterization of a cDNA (6A8) encoding a novel human alpha-mannosidase.

A 3300-bp cDNA (6A8) has been isolated from a human tonsil cell lambdagt11 cDNA library (GenBank accession number: AF044414). The 6A8 gene is localized on human chromosome 13q31-32. Its cDNA has an open reading frame from position 57 bp to 3243 bp, encoding a 1062 amino-acid polypeptide. The sequence of the polypeptide has 89% identity to rat liver ER alpha-mannosidase. Homogenates of COS-7 cells transfected with 6A8 cDNA showed an enhanced enzymatic activity with p-nitro-phenyl-alpha-D-mannopyranoside, which was not inhibited by swainsonine. These data suggest that 6A8 alpha-mannosidase belongs to the class II alpha-mannosidase. Western blot analysis showed a band for 6A8 cDNA encoded protein of approximately 120 kDa. Northern blot analysis revealed two 6A8 mRNA transcripts with different tissue distribution. Enhanced concanavalin A (ConA) binding to CNE-2L2 cells transfected with a reverse 6A8 DNA was observed, indicating that the 6A8 protein is an important cellular alpha-mannosidase.

Amino Acid Sequence↗
PubMed2000

Mitochondrial Ca(2+)homeostasis in the regulation of apoptotic and necrotic cell deaths.

Using distinct models of apoptosis and necrosis, we have investigated the effect of mitochondrial Ca(2+)(Ca(m)) homeostasis in the regulation of cell death in neuroblastoma cells as well as cardiac myocytes. The steady state level of Ca(m)was determined as the FCCP-releasable Ca(2+). Culturing cells with low concentration of extracellular Ca(2+)(Ca(o)) or with EGTA triggered an early reduction in both the Ca(m)store and the membrane potential (DeltaPsi(m)). This was followed by the detection of cytochrome c release, caspase activation, and apoptosis. Inhibitors of the mitochondrial permeability transition pore such as cyclosporin A and Bcl-2 blocked the release of Ca(m)and inhibited apoptosis. In contrast, mitochondrial Ca(2+)overload resulted in necrotic cell death. Culturing cells in the presence of excess Ca(o)led to increased Ca(m)load together with a decrease of DeltaPsi(m)that reached maximum at 1 h, with necrosis occurring at 2 h. While the decline of Ca(m)and DeltaPsi(m)was a coupled reaction for apoptosis, this relationship was uncoupled during necrosis. Clonazepam, a relatively specific inhibitor of the mitochondrial Na/Ca exchanger, was able to protect the cells from necrosis by reducing Ca(m)overload. Importantly, combination of clonazepam and cyclosporin showed a cooperative effect in further reducing the Ca(m)overload and abolished cell death. The data imply the participation of Ca(m)homeostasis in the regulation of apoptosis and necrosis.

Animals↗
PubMed1995

Activity changes of ATP synthase and adenylate kinase in rat-liver mitochondria during the early phase after burn injury.

Using succinate as a substrate, the activities of ATP synthase and adenylate kinase in liver mitochondria of male Sprague-Dawley rats 30 min after 20 per cent full skin thickness burns were increased in comparison with the sham burn rats, suggesting that these two enzymes might contribute to the increased rates of ATP formation and oxidative phosphorylation coupling during the early phase after burn injury.

Adenylate Kinase↗
PubMed1990

Establishment of a human B cell line that responds specifically to B cell growth factor.

A human B cell line (3D5) that responds specifically to B cell growth factor (BCGF) has been developed by a sequence of Staphylococcus aureus Cowen I activation, EB virus immortalization, and cloning. Proliferative response to PHA-stimulated T cell supernatant (PHA-T-Sup) and nonresponsiveness to rIL-2 stimulation were factors used to screen positive cells. Phenotype analysis with a flow cytometer indicated that: 1) 3D5 is a B cell line: 100% of the cells were positive for B1 marker and 59% were positive for sIg, while T3 and Mo 1 were negative; 2) 3D5 is an activated B cell line: both Tac and 4F2 markers of activated (but not of resting) B cells were 100% positive; 3) 3D5 expresses high molecular weight BCGF (HMW-BCGF) receptor-associated epitope BA5. 3D5 cells proliferated in response to cpBCGF stimulation in a dose-dependent manner. HMW-BCGF also induced 3D5 cells to proliferate. Interestingly, no proliferation could be detected in the presence of rIL-2, rIL-4, or rIFN-r. The data show that 3D5 cells are specifically BCGF-responsive B cells. Using 3D5 cells as target, BCGF activity was detected in crude BCGF preparation sedimented by 85% (NH4)2SO4 and chromatographed in a DEAE-Sephadex A-25 column from PHA-T-Sup. T24 cell supernatant with B cell differentiation factor (BCDF) activity could not induce 3D5 cells to differentiate into immunoglobulin-secreting cells.

B-Lymphocytes↗
PubMed1989

[Phenotype analysis of peripheral blood T lymphocytes from SLE patients by flow cytometry].

Phenotype of OKT3, OKT4 and OKT8 markers on surface of peripheral blood T lymphocytes from 31 SLE patients were analyzed with a flow cytometer and the results were compared with those of T cells from 36 normal controls. The results showed that the rates of OKT3+ and OKT4+ cells were within the normal range, while that of OKT8+ cells (44.52 +/- 12.07%) was significantly higher than normal (30.39 +/- 6.06%, P less than 0.01). Besides, the OKT4+/OKT8+ ratio in patients (0.69 +/- 0.24) was lower than that of normal controls (1.06 +/- 0.28, P less than 0.01), 5/13 (38.5%) cases with kidney lesion showing obvious decrease of OKT4+/OKT8+ ratio, while 1/18 cases (5.56%) without kidney lesion also showing such decrease.

Adult↗
PubMed1987

Selective effects of cyclophosphamide therapy on activation, proliferation, and differentiation of human B cells.

The immune function of B lymphocytes from 12 patients with nonneoplastic immune-mediated diseases receiving chronic low-dose (2 mg/kg per d) cyclophosphamide (CY) was evaluated. There was a selective and differential suppressive effect of CY therapy on the various stages of the B cell cycle including activation, proliferation, and differentiation. The proliferative responses to Staphylococcus aureus Cowan strain I (SAC) and mitogenic concentrations of anti-mu were suppressed. In contrast, B cells that have been presumably activated in vivo proliferated with a normal pattern when exposed to B cell growth factor in vitro. Chronic low-dose CY therapy also suppressed B cell differentiation. Secretion of immunoglobulin by B cells following in vitro triggering with SAC and a T cell supernatant was suppressed in CY-treated patients. Moreover, differentiation of the large in vivo-activated B cells (which do not require an in vitro activation signal) in the presence of appropriate T lymphocyte supernatant was also suppressed. This selective suppression of B cell function at multiple points in the B cell cycle may be responsible for the efficacy of CY therapy in certain antibody and immune complex-mediated diseases.

Adolescent↗