Biomedical subjects

C Du

Publications and source records attributed to C Du.

At least 73 records · Page 4Linked to original sources

PubMed1998

Sustained gene expression in retrovirally transduced, engrafting human hematopoietic stem cells and their lympho-myeloid progeny.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.

Animals↗

PubMed1998

[Construction of a competitor for bcr-abl cDNA by recombinant PCR].

OBJECTIVE: Competitive PCR is a powerful method for quantification of mRNA. This study sought to construct an internal standard which can be used as a competitor for bcr-abl fusion cDNA. METHODS: Using recombinant PCR for site-directed mutagenesis. RESULTS A 240bp mimic of a 276bp fragment in b3a2 type of bcr-abl cDNA was obtained. The difference in mimic,which was verified by DNA sequencing, was a 55bp deletion of the target sequence and a 19bp insertion of exogenous unrelated sequence with a XbaI restriction site. In it, except for the inner 36bp, the mimic contained the same sequence and shared the same primer recognition sites as the target b3a2 cDNA(276bp) or as the target b2a2 cDNA(201bp). The obtained recombinant vector, named pGEM-mimic, can be used as a common competitor for either b3a2 or b2a2 type of bcr-abl cDNA. CONCLUSION: This provided a simple and reliable method for constructing an internal standard used in competitive PCR.

DNA, Complementary↗

PubMed1998

[p16INK4/CDKN2 gene deletions and mutations in non-small cell lung cancers].

OBJECTIVE: To investigate the genomic status of cyclindependent kinase-4 inhibitor, p16 INK4/CDKN2 gene in primary non-small cell lung cancers. METHODS: p16 gene was analysed by duplex PCR, PCR-SSCP, and sequencing in 31 primary non-small cell lung cancer (NSCLC) tissues. RESULTS: Gene deletions were found in three samples (3/31), one case had whole p16 gene deletion and the other two had deletion in exon 1 and in exon 2 respectively. Two cases of point mutations (2/30) in the gene had been found, one in exon 1 and another one in exon 2. In the 5 cases of p16 gene abnormal samples, 4 cases (two cases of deletions and two cases of mutations) were non-small cell lung cancers with lymphnode metastasis (4/20). CONCLUSION: These results indicate that inactivation of p16 gene is associated with some NSCLC (5/31), and probably be a late event in NSCLC carcinogenesis.

Adult↗

PubMed1998

Mechanism of inhibition of LPS-induced IL-12p40 production by IL-10 and TGF-beta in ANA-1 cells.

IL-12, a macrophage-derived proinflammatory cytokine, consists of two polypeptide subunits (p40 and p35) encoded by two separate genes. The p35 subunit is constitutively expressed, whereas the p40 subunit is induced after activation. The bioactive interleukin-12 (IL-12; p70) influences the development of Th1 responses and is a potent activator of natural killer (NK) and T cells. In contrast to IL-12, transforming growth factor beta (TGF-beta) and IL-10 inhibit production of proinflammatory cytokines, including IL-12, and attenuate Th1-mediated immune response. We have examined the molecular mechanisms by which TGF-beta and IL-10 inhibit production of the IL-12p40 subunit in LPS-stimulated murine macrophage cell line. We show that both IL-10 and TGF-beta suppress IL-12p40 production by inhibiting the transcription of IL-12p40 gene. At equal concentrations, IL-10 was more potent than TGF-beta in inhibiting IL-12p40 gene transcription. TGF-beta also reduces the stability of IL-12p40 mRNA, accounting thereby to an additional mechanism of inhibition of IL-12 production.

Animals↗

PubMed1998

Protein phosphorylation plays an essential role in the regulation of alternative splicing and sex determination in Drosophila.

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. We show that mutations in the Drosophila LAMMER kinase, Doa, alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated similarly to dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing.

Alternative Splicing↗

PubMed1998

Improved folding yields of a model protein using protein disulfide isomerase.

PURPOSE: To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2). METHODS: C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH. The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI. The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC. RESULTS: The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1. The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3. The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C. CONCLUSIONS: These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2. In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange.

Chromatography, High Pressure Liquid↗

PubMed1998

Conformational and topological requirements of cell-permeable peptide function.

Cell-permeable peptide import recently was developed to deliver synthetic peptides into living cells for studying intracellular protein functions. This import process is mediated by an N-terminal carrier sequence which is the hydrophobic region of a signal peptide. In this study, the conformational consequence of the interaction of cell-permeable peptides with different mimetic membrane environments was investigated by circular dichroism analysis. We showed that cell-permeable peptides adopted alpha-helical structures in sodium dodecyl sulfate (SDS) micelles or aqueous trifluoroethanol (TFE). The potency of these peptides in forming helical structures is higher in an amphiphilic environment (SDS) than in a hydrophobic environment (TFE), suggesting that some hydrophilic molecules associated with the cell membrane may be involved in peptide import. We also studied topological requirements of cell-permeable peptide function. We demonstrated that peptides containing the carrier sequence in their C-termini can also be imported into cells efficiently. This important discovery can avoid repetitious synthesis of the membrane-translocating sequence for peptides with different functional cargoes and is potentially useful for developing a cell-permeable peptide library. Finally, we showed that, when a retro version of the carrier sequence was used, the peptide lost its translocating ability despite retaining a high content of alpha-helical structure in mimetic membrane environments. This suggests that the propensity of peptides to adopt a helical conformation is required but not sufficient for cellular import and that other structural factors such as the side-chain topology of the carrier sequence are also important. Our studies together contribute to the more rational design of useful cell-permeable peptides.

3T3 Cells↗

PubMed1998

Modulation of nicotinic AChR channels by prostaglandin E2 in chick sympathetic ganglion neurons.

The effects of prostaglandin E2 (PGE2), an important metabolite of arachidonic acid, were studied on the activity of nicotinic AChR channels in cultured chick sympathetic ganglion neurons. In whole cell recordings, PGE2 (25 nM) inhibited significantly the ACh-evoked macroscopic current. In cell-attached patch recordings, PGE2 significantly inhibited single AChR channel currents as a result of a decrease in the frequency of channel opening, with no change in open time and conductance. PGE2 did not alter the extent or rate of agonist-induced desensitization of the AChR channels. These effects are specific since the related compound PGD2 had no effect on AChR channel function. Because there is an abundant endogenous production of PGE2 within sympathetic ganglia in response to certain stimuli, the inhibition of AChR channel function by PGE2 could serve an important role to modulate synaptic transmission in the sympathetic nervous system.

Animals↗

PubMed1998

[Factors analysis on ceramic crack of metal-ceramic restorations].

This article enumerated many possible causes of ceramic crack of metal-ceramic restorations and analysed the possible causes of ceramic crack of metal-ceramic restorations. And authors also discussed the effective ways to eliminate the improper factors to keep the good bond strength of ceramics to metal alloy and to avoid ceramic crack of metal-ceramic restorations.

Dental Restoration Failure↗

PubMed1998

[SEM study of machinable vita cerec mark II porcelain surface morphology after HF acid etching].

After HF acid etching under 5 different HF concentrations and 6 exposing times, the surface morphology of machinable porcelain was examined by scanning electron microscope, and the porsosity percentage and ething depth were determined. The results showed that the etching depth increased with the rising of HF concentration and disposing time until 5%-7.5 min. The porosity percentage of ething surface was positively interrelated with HF concentration and disposing time. It was the first time to classify the etching patterns of machinable porcelain into four groups.

Acid Etching, Dental↗

PubMed1998

[Shear bond test of HF acid etching machinable porcelain bonded to enamel with different concentration and disposing time].

After HF acid etching under 5 different HF concentrations and 6 exposing times, shear bonding strength of porcelain to enamel were tested. The results showed that HF acid etching technique had a positive influence on the bonding strength of porcelain to enamel. The most favorable concentration-time groups were 2.5%-5.0 min, 5.0%-5.0 min, 7.5%-2.5 min, 10.0%-1.0 min, 15.0%-0.5 min, and the debonding type of porcelain-resin-enamel composite body was mixed debonding.

Acid Etching, Dental↗

PubMed1997

Carboxyfullerenes as neuroprotective agents.

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 microM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Abeta1-42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Animals↗

PubMed1997

Neuronal and glial apoptosis after traumatic spinal cord injury.

Cell death was examined by studying the spinal cords of rats subjected to traumatic insults of mild to moderate severity. Within minutes after mild weight drop impact (a 10 gm weight falling 6.25 mm), neurons in the immediate impact area showed a loss of cytoplasmic Nissl substances. Over the next 7 d, this lesion area expanded and cavitated. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive neurons were noted primarily restricted to the gross lesion area 4-24 hr after injury, with a maximum presence at 8 hr after injury. TUNEL-positive glia were present at all stages studied between 4 hr and 14 d, with a maximum presence within the lesion area 24 hr after injury. However 7 d after injury, a second wave of TUNEL-positive glial cells was noted in the white matter peripheral to the lesion and extending at least several millimeters away from the lesion center. The suggestion of apoptosis was supported by electron microscopy, as well as by nuclear staining with Hoechst 33342 dye, and by examination of DNA prepared from the lesion site. Furthermore, repeated intraperitoneal injections of cycloheximide, beginning immediately after a 12.5 mm weight drop insult, produced a substantial reduction in histological evidence of cord damage and in motor dysfunction assessed 4 weeks later. Present data support the hypothesis that apoptosis dependent on active protein synthesis contributes to the neuronal and glial cell death, as well as to the neurological dysfunction, induced by mild-to-moderate severity traumatic insults to the rat spinal cord.

Animals↗

PubMed1997

A GFP reporter system to assess gene transfer and expression in human hematopoietic progenitor cells.

Hematopoietic stem cells are widely recognized as attractive targets for gene therapy but current protocols to transduce these cells using recombinant retroviral vectors are inefficient. To evaluate optimization of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (GFP) was explored as a reporter. We first improved sensitivity of detection > 100-fold over that achieved previously by using a novel retroviral vector (termed MGIN) expressing a high level of an enhanced GFP gene. Primitive human hematopoietic cells bearing the CD34 surface antigen and lacking lineage differentiation markers (CD34+ Lin-) were transduced with the MGIN vector using a clinically applicable supernatant procedure. Under the conditions employed, > 75% of the target cells retained the CD34+ Lin- primitive phenotype after 4-5 days in culture, of those > or = 25% expressed a high level of GFP detectable by both flow cytometric analysis and fluorescence microscopy. When transduced cells were cultured in clonogenic progenitor assays, GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and not detrimental to the differentiative potential of the transduced CD34+ Lin- cells. We conclude that GFP is effective as a vital marker to quantity retrovirus-mediated gene transfer into human hematopoietic and perhaps other types of stem/progenitor cells, and monitor gene expression during their subsequent cell lineage determinations.

Antigens, CD34↗

PubMed1997

[The correlation between lung cancer lymph node metastasis and nm23-H1 gene mutation, mRNA expression].

OBJECTIVE: To examine the genomic status and the mRNA expression of the nm23-H1 gene, and to analyse the relationship between metastasis of non-small cell lung carcinomas and the gene abnormalities. METHOD: By using PCR-SSCP and semiquantitative RT-PCR, the nm23-H1 gene mutation and mRNA expression were studied in 31 cases of non-small cell lung cancer. The normal tissue adjacent to carcinoma and normal lung tissue were used as control. RESULT: None of the nm23-H1 gene mutation was found in all the lung cancer samples. Decreased expressions of this gene were found in 14 of 20 lung cancer cases with lymph node metastasis, the rate (14/20) of this was significantly higher than that (3/11) of the non-metastatic cancers (P < 0.05). CONCLUSION: These data indicate that nm23-H1 may be a putative metastasis suppressor gene which had shown reverse regulating activation in the metastatic progression of lung cancers, and the level of nm23-H1 mRNA may be considered as one of the pathological indicators in predicting metastatic potential of lung cancers.

Adult↗

PubMed1997

[Study on the permeability of the infiltration glass of GI- I slip casting aluminous ceramic core].

In order to explore the infiltration condition of GI- I slip casting aluminous ceramic core glass material to aluminous ceramic substratum and provide reference for clinical use, the linear change relationship between the minimal infiltration depth and infiltration time was determined by measuring the minimal depth of glass material infiltrated to aluminous ceramic substratum from one to six hours. The result showed: there is a high linear relationship between the minimal infiltration depth for GI- I infiltration glass and the infiltration time. For clinical use, the minimal infiltration time for GI- I slip casting aluminous ceramic core at least 2.5 hrs and 5 hrs for bridge framework.

Crowns↗

PubMed1997

[Preliminary determination of the properties of HX-I refractory die material].

The setting time, the setting expansion ratio and the compatibility with impression materials of the HX-I refractory die material were determined. The results showed: the setting time was 11 +/- 3 min: the setting expansion ratio was 0.23%; the compatibility with impression materials was excellent between HX-I die material and agar or silicone rubber respectively, but the compatibility was bad between HX-I die material and alginate. These properties of HX-I die material were close to the ones of Lamina die material.

Dental Materials↗

PubMed1997

[Identification of G6PD gene mutations in several nationalities from Yunnan province].

OBJECTIVE: To analyze the G6PD gene mutations in several nationalities from Yunnan province. METHODS: G6PD gene mutation of 29 samples from G6PD deficient patients were analyzed by using common and mis-matched primering PCR followed by restriction enzyme digestion. The samples were collected from Yunnan province among several nationalities. RESULTS: In these 29 cases, 20 were of two known mutations, including 19 of 1388 (G-->A) and 1 of 1376 (G-->T) mutation. The 7 cases of national minorities, including 3 of Yi ([symbol: see text]), 2 of Bai ([symbol: see text]), 1 of Dai ([symbol: see text]), and 1 of Naxi ([symbol: see text]), were all 1388 mutations. No report of the G6PD gene 1388 mutation in these ethnic groups was published excepting for Dai. CONCLUSION: The mutation probably had been prevalent before the emergence of these national minorities. The approximate rate of G6PD gene mutations provided in Yunnan province may, in some respects, help to interpret the molecular evolution.

Asian People↗