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Biomedical subjects

W Shen

Publications and source records attributed to W Shen.

At least 91 records · Page 5Linked to original sources
PubMed2000

Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro.

OBJECTIVE: To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity. METHODS: PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC). Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15. The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and [3H]-TdR incorporation. RESULTS: Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells. With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC. CONCLUSIONS: Functional glycoprotein human B7-1 could be produced in bacterial cells. Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.

B7-1 Antigen↗
PubMed2000

Dysregulation of apoptosis: a possible mechanism leading to chronic progressive renal histological changes in lupus nephritis.

OBJECTIVE: To evaluate apoptosis in lupus nephritis and the relationship between the existence of apoptotic cells in renal tissue and histopathological or clinical changes. METHODS: Apoptosis was detected by in situ nick-end labeling techniques (TUNEL) in renal biopsies from 25 patients with type IV lupus nephritis (LN), 12 patients with IgA nephropathy IgAN, 4 patients with idiopathic mesangioproliferative glomerulonephritis (MsPGN) and 3 patients with acute poststreptococcal glomerulonephritis (APGN). Normal renal tissue obtained at nephrectomy for hypernephroma in 4 adults was used as control. Proliferating cells were identified by proliferating cell nuclear antigen (PCNA) in these patients. RESULTS: Compared to other proliferative glomerulonephritis and controls, the patients with lupus nephritis had less apoptotic cells, a higher ratio of PCNA + cells/TdT + cells (P/T) in renal tissues; and their P/T ratio in glomeruli and tubulointerstitium correlated with the chronicity index, r = 0.4983 (P = 0.0132), r = 0.8399 (P < 0.001), r = 0.6614 (P = 0.0033), respectively. P/T ratios in the glomerulus and tubule had a positive correlation with 24-hour urinary protein, r = 0.8554 (P < 0.001) and r = 0.7134 (P = 0.001); and a negative correlation with creatinine clearance (Ccr), r = -0.4880 (P = 0.0133) and r = -0.7229 (P = 0.001), which in tubules positively correlated with serum creatinine (Scr), r = 0.4107 (P = 0.0414). CONCLUSIONS: Apoptosis is reduced in proliferative lupus nephritis. Intense proliferation without a commensurate increase in apoptosis is a possible mechanism that leads to chronic progressive renal histopathological changes.

Adult↗
PubMed2000

[Analysis of influence of axial length and keratometric value on the curative effects of excimer laser photorefractive keratectomy for myopia].

OBJECTIVE: To evaluate the influence of axial length (AL) and keratometric value (KV) on the curative effects of excimer laser photorefractive keratectomy (PRK) for myopia. METHOD: A Keracor-17 excimer laser was used to treat 80 patients (121 eyes) with myopia. The cases were divided into three groups according to the AL and KV: The first group AL > 26 mm, KV 45 D, 36 eyes; the third group AL 0.05). The cases with uncorrected visual acuity >or= 0.5 had significant difference between the first and the second group and between the first and the third group (chi(2) = 6.30, P < 0.05). Multiple linear regression and correlative analysis showed that the increase in ocular axis was an unfavorable factor to visual acuity, while the increase in keratometric value was a favorable factor to visual acuity. CONCLUSION: PRK for treatment of myopia is an effective and safe refractive surgery. When the myopic degrees are identical, the postoperative outcome of myopia with increase in keratometric value as the main pathogeny is better than the myopia with increase in axial length as the main pathogeny.

Adolescent↗
PubMed2000

[Effects of 6 kinds of metal elements on meiotic maturation and in vitro fertilization on mouse oocyte].

To study the effects of Cd, Pb, Ni, Mn, Cr and Al on reproductive toxicity, the meiotic maturation and in vitro fertilization(IVF) of mouse oocytes were studied using in vitro culture and IVF of mouse oocytes. The results showed that except Al(NO3)2, the other groups [CdCl2, Pb(NO3)2, NiCl2, MnSO4 and CrCl3] could significantly reduce the number of superovulation oocyte. All of them could inhibit the meiotic maturation, the rates of IVF and extruding of the first polar body, but there was no impact on the germinal vesicle breakdown (GVBD) in vivo. The results also showed that except Al(NO3)3, other elements could obviously inhibit germinal vesicle breakdown of mouse oocytes. The results suggested that these elements might damage the process of meiotic maturation of oocyte of women at child-bearing age oocyte and reduce the fertility and reproductive capacity and result in sterile.

Animals↗
PubMed2000

[A study on the inactivation of p16 genes and the expression of P16 protein in primary hepatocellular carcinomas].

The aim of the present study was to determine whether p16 gene is involved in the genesis of primary hepatocellular carcinoma (HCC). Twenty-five primary HCC tumor and corresponding non-tumor liver tissue specimens were examined for P16 gene alterations. The identification of deletion of p16 gene exon 1 and exon 2 was performed using comparative multiplex polymerase chain reaction (PCR) analysis. The point mutation of p16 gene exon 2 was investigated by single strand conformational polymorphism(SSCP) analysis, and the status of p16 gene methylation was screened using PCR-based methylation analysis. Moreover, 35 parafin specimens of primary HCCs with corresponding non-tumor liver tissues, including the 25 cases described above for screening of p16 gene alterations, were investigated also for P16 protein expression by using immunohistochemical analysis. The results of comparative multiplex PCR analysis showed that 12% (3/25) p16 gene deletions, including homozygous deletions in 2 tumors and hemizygous deletion in 1 tumor, were found in the primary HCCs samples. No point mutation was identified in the remaining 22 tumor samples without p16 gene deletions by using SSCP analysis. Hypermethylation was detected in 24% (6/25) of tumor samples by PCR-based analysis. However the corresponding non-tumor liver tissue specimens were always unmethylated at p16 locus. Loss of P16 protein expression, detected by immunohistochemistry, occurred in 16 of 35 (45.7%) tumor samples, whereas all the non-tumor liver tissue specimens showed positive p16 staining. These results indicate that inactivation of p16 gene is relevant to the genesis of HCC.

Carcinoma, Hepatocellular↗
PubMed2000

[Culture of endotheliocytes derived from guinea-pig common carotid artery and establishment of guinea-pig donor model].

Since endotheliocyte strains of guinea-pig and inbred strains of guinea-pig are not easily obtained, it is impossible to ensure the consistence of antigenicity between endotheliocytes and donor organs when endotheliocytes are used as an inductor in the research of immunological tolerance. So it is necessary to establish the guinea-pig endotheliocyte-donor model. One side of common carotid arteries was removed and reversed. After collagenase digestion, culture of endotheliocytes was performed. And the survival time of guinea-pig was observed. Cultured cells were confirmed as endotheliocytes by the study of morphology, immunohistochemistry and electron microscopy. The two-month-survival of guinea-pigs was 92% (103/112). The establishment of guinea-pig endotheliocyte-donor model can provide the foundation for the research of guinea-pig endotheliocyte and immunological tolerance.

Animals↗
PubMed2000

[A study on the preservation of rat kidney with HX-III solution].

This study was conducted to compare the effects of HX-III solution and UW solution on rat kidney transplantation. Inbred Sprague-Dawley rats weighing 200-280 g, were used in allotransplantation. The donors and recipients were of the same sex. The rats were randomly divided into control (UW solution) and experimental (HX-III solution) groups, and each group included 48- and 72-hour preservation subgroups. There were ten rats in each subgroup. Kidneys were flushed with UW solution or HX-III solution and stored at 4 degrees C for 48 or 72 hours. Then, kidney allotransplantations were performed. The results showed that the rates of survival were 100% and 90% in UW group for 48- and 72-hour cold storage, but those in HX-III were 100% and 60%, respectively. With 48-hour preservation, the maximum serum creatinine levels in UW group were similar to those in HX-III group (P > 0.05). With 72-hour preservation, the maximum serum creatinine levels in UW group were lower than those in HX-III group (P < 0.05). On the 14th day posttransplantation, the histological findings on Kidneys in the two groups for 48-hour cold storage were almost the same, but the grafted kidneys in HX-III group were damaged more seriously than those in UW group for 72 hour cold storage. On the 28th day posttransplantation, the histological findings on the grafted kidneys were basically normal in all groups. The authors concluded that HX-III solution could preserve rat kidney for 48 hours effectively.

Animals↗
PubMed1999

Utilization of two seven-transmembrane, G protein-coupled receptors, formyl peptide receptor-like 1 and formyl peptide receptor, by the synthetic hexapeptide WKYMVm for human phagocyte activation.

Trp-Lys-Tyr-Val-D-Met (WKYMVm) is a synthetic leukocyte-activating peptide postulated to use seven-transmembrane, G protein-coupled receptor(s). In the study to characterize the receptor(s) for WKYMVm, we found that this peptide induced marked chemotaxis and calcium flux in human phagocytes. The signaling induced by WKYMVm in phagocytes was attenuated by high concentrations of the bacterial chemotactic peptide fMLP, suggesting that WKYMVm might use receptor(s) for fMLP. This hypothesis was tested by using cells over expressing genes encoding two seven-transmembrane receptors, formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1), which are with high and low affinity for fMLP, respectively. Both FPR- and FPRL1-expressing cells mobilized calcium in response to picomolar concentrations of WKYMVm. While FPRL1-expressing cells migrated to picomolar concentrations of WKYMVm, nanomolar concentrations of the peptide were required to induce migration of FPR-expressing cells. In contrast, fMLP elicited both calcium flux and chemotaxis only in FPR-expressing cells with an efficacy comparable with WKYMVm. Thus, WKYMVm uses both FPR and FPRL1 to stimulate phagocytes with a markedly higher efficacy for FPRL1. Our study suggests that FPR and FPRL1 in phagocytes react to a broad spectrum of agonists and WKYMVm as a remarkably potent agonist provides a valuable tool for studying leukocyte signaling via these receptors.

Cell Migration Inhibition↗
PubMed1999

Progressive loss of myocardial ATP due to a loss of total purines during the development of heart failure in dogs: a compensatory role for the parallel loss of creatine.

BACKGROUND: Whether myocardial ATP content falls in heart failure is a long-standing and controversial issue. The mechanism(s) to explain any decrease in ATP content during heart failure have not been identified. METHODS AND RESULTS: Cardiac dysfunction, heart failure, and a prolonged steady state of heart failure were induced by chronic right ventricular pacing for 1 to 2 weeks, 3 to 4 weeks, and 7 to 9 weeks in dogs. Cardiac function and myocardial O(2) consumption (Mf1.gif" BORDER="0">O(2)) were measured with the dogs in the conscious state. ATP, total purine, and creatine were measured in biopsy specimens obtained at each stage. ATP and the total purine pool progressively fell at rates of 0.12 and 0.15 nmol. mg protein(-1). d(-1), despite an increase in Mf1.gif" BORDER="0">O(2). The rate of loss of creatine was 1.06 nmol. mg protein(-1). d(-1), 7 times faster than the depletion of total purine. CONCLUSIONS: (1) ATP contents progressively decreased during heart failure as a result of a loss of the total purine pool. The loss of purines may be due to inhibition of de novo purine synthesis. (2) Loss of creatine is an early marker of heart failure and may serve as a compensatory mechanism minimizing the reduction of the total purine pool in the failing heart.

Adenosine Triphosphate↗
PubMed1999

Zinc-dependent structural stability of human Sonic hedgehog.

The role of the zinc site in the N-terminal fragment of human Sonic hedgehog (ShhN) was explored by comparing the biophysical and functional properties of wild-type ShhN with those of mutants in which the zinc-coordinating residues H140, D147, and H182, or E176 which interacts with the metal ion via a bridging water molecule, were mutated to alanine. The wild-type and E176A mutant proteins retained 1 mol of zinc/mol of protein after extensive dialysis, whereas the H140A and D147A mutants retained only 0.03 and 0.05 mol of zinc/mol of protein, respectively. Assay of the wild-type and mutant proteins in two activity assays indicated that the wild-type and E176A mutant proteins had similar activity, whereas the H140A and D147A mutants were significantly less active. These assays also indicated that the H140A and D147A mutants were susceptible to proteolysis. CD, fluorescence, and (1)H NMR spectra of the H140A, D147A, and E176A mutants measured at 20 or 25 degrees C were very similar to those observed for wild-type ShhN. However, CD measurements at 37 degrees C showed evidence of some structural differences in the H140A and D147A mutants. Guanidine hydrochloride (GuHCl) denaturation studies revealed that the loss of zinc from the H140A and D147A mutants destabilized the folded proteins by approximately 3.5 kcal/mol, comparable to the effect of removing zinc from wild-type ShhN by treatment with EDTA. Thermal melting curves of wild-type ShhN gave a single unfolding transition with a midpoint T(m) of approximately 59 degrees C, whereas both the H140A and D147A mutants displayed two distinct transitions with T(m) values of 37-38 and 52-54 degrees C, similar to that observed for EDTA-treated wild-type ShhN. Addition of zinc to the H140A and D147A mutants resulted in a partial restoration of stability against thermal and GuHCl denaturation. The ability of these mutants to bind zinc was confirmed using a fluorescence-based binding assay that indicated that they bound zinc with K(d) values of approximately 1.6 and approximately 15 nM, respectively, as compared to a value of </=100 pM for wild-type ShhN. The properties of the E176A mutant were indistinguishable from those of wild-type ShhN in all biophysical and functional assays, indicating that this residue does not contribute significantly to stabilization of the zinc-binding site and that ShhN does not require hydrolase activity for in vitro biological function.

Alkaline Phosphatase↗
PubMed1999

Substrate turnover by transporters curtails synaptic glutamate transients.

Although inhibitors of glutamate transport prolong synaptic currents at many glutamate synapses, the cause of the current prolongation is unclear. Transport inhibitors may prolong synaptic currents by simply interfering with synaptic glutamate binding to transporters, by inhibiting substrate translocation, or by promoting accumulation of ambient glutamate, which may act cooperatively at receptors with synaptic glutamate. We show that reversal of the membrane potential of astrocytes surrounding the synapse prolongs synaptic currents but does not decrease the apparent affinity of transporters or significantly alter glutamate-dependent kinetics of macroscopic transporter currents in excised membrane patches. Positive membrane potentials do not affect binding of a nontransported glutamate analog, nor do positive membrane potentials alter the number of transporters available to bind analog. We also test the hypothesis that glutamate accumulation during uptake inhibition by transporter substrates is the direct cause of synaptic current prolongations. Transporter substrates elevate ambient glutamate near synapses by fostering reverse transport of endogenous glutamate. However, increases in ambient glutamate cannot account for the prolongations of synaptic currents, because a nonsubstrate transport inhibitor does not foster reverse uptake yet it prolongs synaptic currents. Moreover, exogenous glutamate does not mimic synaptic current prolongations induced by substrate inhibitors. These results provide strong support for a major role of substrate translocation in determining the time course of the glutamate concentration transient at excitatory synapses.

2-Amino-5-phosphonovalerate↗
PubMed1999

Conditions providing enhanced transfection efficiency in rat pheochromocytoma PC12 cells permit analysis of the activity of the far-upstream and proximal promoter of the brain creatine kinase gene.

While brain creatine kinase (CKB) is expressed at highest levels in the brain, where it functions in regenerating ATP, the gene elements and protein factors regulating CKB transcription in neuronal and glial cells have not been identified. To investigate the regulation of CKB in neuronal cells, we examined the expression of the promoter proximal and 5' far-upstream regions of the rat CKB gene transiently transfected into rat PC12 pheochromocytoma cells. Initially, these experiments were hampered by the extremely low transfection efficiency of PC12 cells. We increased efficiency by greater than 200-fold by employing CaPO4-precipitated DNA transfection into PC12 cells which were optimized for transient transfection by: (i) culturing cells in polylysine-coated dishes to insure attachment throughout transfection; (ii) exposing cells to transfected DNA for an optimal time and employing a glycerol shock; and, most importantly, (iii) dissociating the characteristic self-adhesive clumps of PC12 into mostly single cells. Use of the plasmid expressing green fluorescent protein allowed identification of the transfected cells that averaged 10-20% of the total. Analyses of CKB promoter-CAT gene constructs showed that in PC12 cells expression of the proximal (0.2 kb) CKB promoter was low while expression of the 1.4 kb promoter was three fold higher and the 2.9 kb promoter was ten fold higher, suggesting the presence of at least two upstream cis-acting, positive regulatory elements. In agreement, the steady-state CKB mRNA level was higher in PC12 than in other neuronal cell lines examined, possibly reflecting the effects of positive upstream factors. The results are discussed in relation to how this economical and straightforward transfection procedure may be useful in identify factors regulating the transcription of CKB and other genes expressed in neuronal cells.

Animals↗
PubMed1999

[Effect of aluminium on meiotic maturation of mouse oocyte in vitro].

The effect of aluminium nitrate on the meiotic maturation of mouse oocyte was studied in the culture of mouse oocyte. The results showed that aluminium nitrate could inhibit the extruding of the first polar body and the dose of 30 mg/L and 60 mg/L could affect the viability of mouse oocyte, but little impact on the germinal vesicle breakdown (GVBD) of oocyte. The results indicated that aluminium nitrate might be hazardous to the meiotic maturation of mouse oocyte.

Aluminum Compounds↗
PubMed1999

Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons.

1. Whole-cell voltage clamp experiments were performed on isolated spiking retinal neurons from the salamander retina. Calcium channel currents were studied using barium as the charge carrier while potassium and sodium currents were suppressed with TEA and TTX, respectively. 2. Baclofen, a metabotropic GABA receptor agonist, both enhanced and suppressed high-voltage-activated calcium channel current. Baclofen facilitated an L-type channel current, and this effect was not voltage dependent. As reported previously, baclofen inhibited an N-type channel current and this action was voltage dependent. 3. While the suppressive effect was mediated by a fast-acting, direct G-protein action, the facilitatory effect was slower and was blocked by inhibitors of protein kinase C (PKC), either GF-109203x or the PKC (19-36) sequence fragment. 4. The pharmacology of the inhibitory and facilitatory responses differed. Commonly used antagonists of metabotropic GABA receptors, CGP35348 and CGP55845, were more potent antagonists of the inhibitory response. Similarly, a selective agonist at the metabotropic GABA receptor, APMPA, was also more effective in eliciting the inhibitory response. 5. These observations indicate that there may be two baclofen-sensitive metabotropic GABA receptors with opposing effects on calcium channel current. This is the first description of a facilitatory action of GABAB receptors and indicates that GABA may not function exclusively as an inhibitory transmitter.

Ambystoma↗
PubMed1999

HOX11 interacts with CTF1 and mediates hematopoietic precursor cell immortalization.

HOX11 is a homeodomain-containing oncogenic transcription factor that immortalizes hematopoietic precursor cells. The mechanism by which HOX11 facilitates this initial step of leukemogenesis is, however, not well understood. We have used a DNA binding site selection assay to investigate cooperative DNA binding by HOX11 with other transcription factors. A consensus sequence was derived and identified as the binding site for the CCAAT-box-binding transcription factors (CTF). HOX11 was shown to interact in vitro and in vivo with CTF1. Retrovirus-mediated transduction of an antisense CTF1 cDNA dramatically reduced the proliferative capacity of HOX11-immortalized hematopoietic precursor cells. CTF1 is, therefore, the first HOX11 protein partner identified that plays an important role in hematopoietic precursor cell immortalization.

Binding Sites↗
PubMed1999

Potent inhibitors of protein farnesyltransferase: heteroarenes as cysteine replacements.

Synthesis and biological evaluation of heteroarenes as reduced cysteine replacements are described. Of the heteroaryl groups examined with respect to FT inhibitor FTI-276 (1), pyridyl was the replacement found to be most effective. Substitutions at C4 of the pyridyl moiety did not affect the in vitro activity. Compound 9a was found to have moderate in vivo bioavailability.

3T3 Cells↗