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Biomedical subjects

P Wei

Publications and source records attributed to P Wei.

At least 37 records · Page 2Linked to original sources
PubMed1999

[Methodology of DNA fingerprinting and its application in identification of M. tuberculosis].

OBJECTIVE: To explore methodology of DNA fingerprinting technique and its application in identification of strains of M. tuberculosis. METHODS: This research was based on different copy numbers and location of chromosome DNA IS6110 in M. tuberculosis genome. After DNA of M. tuberculosis were cut by endonuclease PvuII, the products were transferred to nylon membrane, then detected and hybridized by enhanced chemiluminescent (ECL) labeling techniques. RESULTS: Fifty clinical isolates from different TB patients had unidentical DNA fingerprinting patterns. DNA fingerprinting patterns showed a great difference from sputum and pus specimens of the same patient. H(37) R(v) ofloxacin resistant and sensitive strains had an identical DNA fingerprinting pattern. CONCLUSIONS: Identification of M. tuberculosis strains is feasible by DNA fingerprinting technique.

DNA Fingerprinting↗
PubMed1998

The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein.

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.

Amino Acid Sequence↗
PubMed1998

Genes and regulatory sites of the "host-takeover module" in the terminal redundancy of Bacillus subtilis bacteriophage SPO1.

Early in infection of Bacillus subtilis by bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses. It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy. Here we analyze the nucleotide sequence of this 11.5-kb "host-takeover module," which appears to be designed for particularly efficient expression. Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function in B. subtilis. The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery. The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12. The genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified. Transcription-termination and RNase III cleavage sites were found at appropriate locations.

Amino Acid Sequence↗
PubMed1998

A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.

The HIV-1 Tat protein regulates transcription elongation through binding to the viral TAR RNA stem-loop structure. We have isolated a novel 87 kDa cyclin C-related protein (cyclin T) that interacts specifically with the transactivation domain of Tat. Cyclin T is a partner for CDK9, an RNAPII transcription elongation factor. Remarkably, the interaction of Tat with cyclin T strongly enhances the affinity and specificity of the Tat:TAR RNA interaction, and confers a requirement for sequences in the loop of TAR that are not recognized by Tat alone. Moreover, overexpression of human cyclin T rescues Tat activity in nonpermissive rodent cells. We propose that Tat directs cyclin T-CDK9 to RNAPII through cooperative binding to TAR RNA.

3T3 Cells↗
PubMed1998

Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury.

Neurotrophic factors have been proposed as a therapeutic treatment for traumatic brain and spinal cord injury. The present study determined whether exogenous administration of one such factor, brain-derived neurotrophic factor (BDNF), could effect behavioral recovery and/or histopathological changes after spinal cord injury. Adult rats received a mild or moderate contusion injury or complete transection of the mid-thoracic spinal cord. Immediately thereafter, they were infused intrathecally with vehicle or BDNF for 28 days. Behavioral recovery was evaluated for 6 weeks after injury, at which time the rats were sacrificed and the spinal cord tissue was examined histologically. The infusion of BDNF resulted in acute stimulation of hindlimb activity. These effects included activation of alternating airstepping in injured rats when the hindlimbs were unloaded as well as slight improvements in the rate of recovery in open field locomotion score. BDNF infusion was also associated with enhanced growth of cholinergic fibers at the injury epicenter, but did not affect white matter sparing or density of serotonergic axons at or below the injury site. Based on immunohistochemical detection of BDNF protein distribution, these described effects are likely to be mediated by the activation of cells and axons within the central injury region and the along the peripheral rim of the spinal cord. Together, these findings demonstrate that the exogenous infusion of BDNF after spinal trauma can influence postinjury outcome through mechanisms that include acute stimulation of hindlimb activity and neuritogenesis at the injury site.

Animals↗
PubMed1998

Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

Animals↗
PubMed1998

Tat-associated kinase, TAK, activity is regulated by distinct mechanisms in peripheral blood lymphocytes and promonocytic cell lines.

TAK, a multisubunit cellular protein kinase that specifically associates with the human immunodeficiency virus Tat proteins and hyperphosphorylates the carboxyl-terminal domain of RNA polymerase II, is a cofactor for Tat and mediates its transactivation function. The catalytic subunit of TAK has been identified as cyclin-dependent kinase Cdk9, and its regulatory partner has been identified as cyclin T1; these proteins are also components of positive transcription elongation factor P-TEFb. TAK activity is up-regulated upon activation of peripheral blood lymphocytes and following macrophage differentiation of promonocytic cell lines. We have found that activation of peripheral blood lymphocytes results in increased mRNA and protein levels of both Cdk9 and cyclin T1. Cdk9 and cyclin T1 induction occurred in purified CD4(+) primary T cells activated by a variety of stimuli. In contrast, phorbol ester-induced differentiation of promonocytic cell lines into macrophage-like cells produced a large induction of cyclin T1 protein expression from nearly undetectable levels, while Cdk9 protein levels remained at a constant high level. Measurements of cyclin T1 mRNA levels in a promonocytic cell line suggested that regulation of cyclin T1 occurs at a posttranscriptional level. These results suggest that cyclin T1 and TAK function may be required in differentiated monocytes and further show that TAK activity can be regulated by distinct mechanisms in different cell types.

CD4-Positive T-Lymphocytes↗
PubMed1998

Transrepression of c-jun gene expression by the glucocorticoid receptor requires both AP-1 sites in the c-jun promoter.

The c-jun protooncogene encodes a nuclear protein, cJun, which is a major component of the AP-1 transcription factor. AP-1 regulates various aspects of cell proliferation and differentiation. As an immediate early response gene, the expression of the c-jun gene is affected by various extracellular stimuli, such as serum, phorbol esters, and glucocorticoids. In mouse L929 fibroblasts, dexamethasone (DEX) treatment caused a 60% reduction of c-jun mRNA levels. Previous studies indicated that this reduction is due to the alteration of the transcription rate of the c-jun gene. To further investigate the molecular mechanisms of transcriptional repression of c-jun by DEX, a full-length human c-jun promoter, from -1780 to +731, was amplified from genomic DNA using PCR and then linked to the luciferase reporter gene. To identify the regulatory elements responsible for the down-regulation, nested deletions spanning the promoter were generated, and the promoter/luciferase constructs were transiently transfected into L929 cells. Upon hormone treatment, basal activity of the full-length c-jun promoter was reduced by approximately 40%, which accounts for two-thirds of the overall down-regulation observed at the mRNA level. This reduction of c-jun promoter activity was abolished after deletion of the region between -1780 to -63, where two AP-1 sites (-182 and -64) are located. Site-directed deletion of these AP-1 sites reduced the basal activity of the c-jun promoter and prevented repression by DEX. Repression of the c-jun gene is due to the transrepression activity of the glucocorticoid receptor (GR), as determined using GR mutants lacking this activity. Overexpression of cJun overcame the negative effect of DEX, suggesting that down-regulation of the c-jun gene by hormone is mediated by the interaction between the GR and the cJun protein. These studies are the first to show that glucocorticoids can repress c-jun promoter activity through the AP-1 sites in the c-jun promoter in mouse fibroblast cells. They also suggest that inhibition of cell proliferation by glucocorticoids may be due not only to the interference with AP-1 activity on other cellular genes, but also because of a direct transcriptional suppression of c-jun gene expression by the GR.

Animals↗
PubMed1998

[Radioimmunological image in tumor bearing nude mice with monoclonal antibody against human laryngeal carcinoma].

OBJECTIVE: To investigate the specific autoradiographic diagnosis of laryngeal carcinoma and its metastasis. METHODS: Three strains of the mixed monoclonal antibodies against human laryngeal carcinoma (McAbLC9, 11, 12) labelled with 131I were injected(intraperitoneally) into the nude mice bearing squamous cell carcinoma. The tumor autoradiography was done by single photo-emission computed tomography (ECT). The percent injected dose/g of tumor were measured, and the radioactivity ratio of tumor to non-tumor tissues(T/NT) were calculated. RESULTS: The 131I-McAbLC 9, 11, 12 can accumulate in the area of the laryngeal carcinoma within limited time, and demonstrate a satisfactory tumor imaging at 96 h. In experimental groups: The %ID/g of tumor tissues (mean +/- s, n = 6) was 18.8 +/- 0.61; The %ID/g of tumor tissues in the control group was 4.46 +/- 0.82 (mean +/- s, n = 6) at 96 h. The difference was statistically significant (P < 0.01, t = 34.97). CONCLUSION: The results suggest that 131I McAbLC9, 11, 12 is specific and sensitive in recognising human laryngeal carcinoma cell, and the area and size of tumor can be easily detected by ECT. It is feasible for localization of tumor and its metastasis.

Animals↗
PubMed1997

Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats.

The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7-28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions.

Animals↗
PubMed1997

Regulation of the glucocorticoid receptor gene by the AP-1 transcription factor.

The glucocorticoid receptor (GR) is a ligand-activated nuclear transcription factor, and AP- 1 (Fos/Jun or Jun/Jun) is a transcription factor whose components are nuclear proteins encoded by c-fos and c-jun protooncogenes. Serum stimulation of serum-starved NIH 3T3 cells resulted in an approx 188-fold induction of c-fos mRNA at 30 min and an approximately ninefold induction of c-jun mRNA at 1 h, followed by an increase in GR mRNA levels at 3-12 hour (twofold). Sequential induction of cFos, cJun, and GR protein levels also occurred. Overexpression of the cFos protein in NIH 3T3 cells (NIH 3T3 [cFos 3] and NIH 3T3 [cFos 10]) caused an increase in the endogenous GR protein. Previous and present studies showed that a putative AP-1 site within the GR promoter binds AP-1 proteins (both Jun and Fos family members). To address the molecular mechanism involved in transcriptional activation of the GR gene, we investigated the relevance of AP-1 binding complexes in this activation and in overall regulation of GR gene transcription. Transient transfection with a full length GR promoter linked to a luciferase gene into both NIH 3T3 (cFos 3) and NIH 3T3 (cFos 10) cells gave rise to an induction of luciferase activity. This induction was abolished following mutation or deletion of the GR AP-1 site from the promoter. These findings suggest that cFos is responsible for the induction of GR expression in serum-stimulated NIH 3T3 cells, and serum growth factors may stimulate GR transcription by a cFos-dependent mechanism at the putative AP-1 site. These studies support a role for the AP-1 transcription factor in regulating GR gene expression.

3T3 Cells↗
PubMed1997

[Choice of treatment for fracture of distal tibia with ankle joint involvement].

It is difficult to treat the intra-articular fracture of distal tibia or Pilion's fracture. From 1987 to 1995, 43 cases of Pilon's fracture were admitted and treated with different methods. After treatment, they had been followed up for 1 to 8 years. According to Riiedi's Classification, there were type I 12 cases, type II 21 cases and type III 10 cases. The methods used on these patients included manual reduction and plaster of paris immobilization, calcaneous tubercle traction and plaster of paris immobilization, open reduction and internal fixation with Kirschner wires, and open reduction and internal fixation with AO plate. The outcome was evaluated according to Ovadia's criteria, for type I fracture, 8 cases were treated with conservative method with a satisfactory rate of 79.17% and 4 cases were treated with operation with a satisfactory rate of 91.67%; for type II, conservative method for 12 cases and the satisfactory rate was 33.33%, and operation for 9 cases with a satisfactory rate of 70.37%; for type III, conservative method for 2 cases, with poor result, and operation for 8 cases with satisfactory rate of 79.17%. The indication for conservative treatment was type I fracture. It showed that for Pilon's fracture, the outcome of open reduction and internal fixation was superior to that of the conservative treatment, especially in those having internal fixation with AO plate.

Adolescent↗
PubMed1997

[The effect of vascular endothelial cell growth factor on survival of skin flap in rats].

In order to study the effect of vascular endothelial cell growth factor (VEGF) on the survival of skin flap 30 SD rats were used. A randomized flap measuring 7.5 cm x 3.0 cm was created on the back of each SD rat. The treatment group (n = 10) received VEGF 40 ng/flap by subcutaneous injection with microinjector during and 24 hours after operation. The control groups received heparin 16 U/flap (n = 10) or normal saline 800 microliters/flap (n = 10). After operation, on the 3rd and 11th day, the survival rate of the skin flaps and the dermovascular density of each flap were investigated by histological and histo-morphometrical examination. The results showed that there was no significant difference in the survival rate between the treatment group and the controls on the 3rd day after operation, while on the 11th day, there was a significant difference between them, and the survival rate was much higher in the treatment group. Besides, dermovascular density was much more increased in the treatment group than that in the controls, especially in the distal 1/3 of the flap (P < 0.02). The conclusion was that VEGF could accelerate the vascularization of the flap and enhance the survival rate of the flap.

Animals↗
PubMed1995

Genes that protect against the host-killing activity of the E3 protein of Bacillus subtilis bacteriophage SPO1.

A cloned rpoB gene, specifying an apparently mutant RNA polymerase beta subunit, protected Escherichia coli against the cytocidal effects of the E3 protein of bacteriophage SPO1, suggesting that RNA polymerase is the primary cellular target of the E3 protein. Two segments of the wild-type E. coli genome, one of which specifies a suppressor of dnaK mutations, and thus, possibly, a molecular chaperone, also provided protection when overexpressed, but wild-type rpoB did not.

Bacillus Phages↗