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Biomedical subjects

W Shi

Publications and source records attributed to W Shi.

At least 127 records · Page 7Linked to original sources
PubMed1999

The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor.

Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.

Animals↗
PubMed1999

Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-å crystal structure.

Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca(2+) ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k(cat) for the L. major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.

Amino Acid Sequence↗
PubMed1999

Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Abnormalities, Multiple↗
PubMed1999

Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues.

HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be approximately -70 mV in neonatal rat ventricle and -113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is approximately 5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for If activation, whereas the HCN2 isoform is associated with a more negative activation curve.

Animals↗
PubMed1999

Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol.

The mechanisms of action of the anticancer agent perillyl alcohol (POH), presently in Phase II clinical trials, were investigated in advanced rat mammary carcinomas. Gross and ultrastructural morphology of POH-mediated tumor regression indicated that apoptosis accounted for the marked reduction in the epithelial compartment. Characterization of cell growth and death indices revealed that apoptosis was induced within 48 h of chemotherapy, before the induction of cytostasis. RNA expression studies, based on a multiplexed-nuclease protection assay, demonstrated that cell cycle- and apoptosis-related genes were differentially expressed within 48 h of POH treatment; p21(Cip1/WAF1), bax, bad, and annexin I were induced; cyclin E and cyclin-dependent kinase 2 were repressed; and bcl-2 and p53 were unchanged. Next, a potential role for transforming growth factor beta (TGF-beta) signaling in POH-mediated carcinoma regression was explored. RNA expression studies, again based on a multiplexed-nuclease protection assay, showed that TGF-beta-related genes were induced and temporally regulated during POH treatment: (a) c-jun and c-fos were transiently induced within 12 h of chemotherapy; (b) TGF-beta1 was induced within 24 h of chemotherapy; (c) the mannose 6-phosphate/insulin-like growth factor II receptor and the TGF-beta type I and II receptors were induced within 48 h of chemotherapy; and (d) smad3 was induced during active carcinoma regression. In situ protein expression studies, based on fluorescence-immunohistochemistry in concert with confocal microscopy, confirmed up-regulation and demonstrated colocalization of TGF-beta1, the mannose 6-phosphate/insulin-like growth factor II receptor, the TGF-beta type I and II receptors, and Smad2/Smad3 in epithelial cells. Nuclear localization of Smad2/Smad3 indicated that the TGF-beta signaling pathway was activated in regressing carcinomas. Subpopulations of Smad2/Smad3-positive and apoptotic nuclei colocalized, indicating a role for Smads in apoptosis. Thus, Smads may serve as a potential biomarker for anticancer activity. Importantly, none of the POH-mediated anticancer activities were observed in normal mammary gland.

Animals↗
PubMed1999

Value of sublingual isosorbide dinitrate before isoproterenol tilt test for diagnosis of neurally mediated syncope.

Head-up tilt-table test is valuable in diagnosing type of syncope. Sensitivity varies in reports and is mostly poor. Isoproterenol or nitroglycerin is regularly used to increase head-up tilt-table test sensitivity. The aim of this study was to assess the value of sublingual isosorbide dinitrate administration before isoproterenol tilt test as a simple, practical test for diagnosis of neurally mediated syncope. Ninety-six patients (45 men and 51 women, mean age 42.5 years) with recurrent (median 4 times) loss of consciousness that remained unexplained after clinical and noninvasive assessment and 72 healthy volunteers (36 men and 36 women, mean age 40.1 years) were randomly divided into groups: isosorbide-isoproterenol test and isoproterenol test. During isosorbide-isoproterenol test, a positive response (syncope in association with sudden hypotension or bradycardia) occurred in 35 patients (72.9%) with unexplained syncope, an exaggerated response (minor symptoms in association with slowly increasing hypotension alone) occurred in 7 (14.6%), a negative response in 2 (4.2%), and drug intolerance in 4 (8.3%). During isoproterenol testing, these percentages were 52.1%, 16.7%, 25.0%, and 6.2%, respectively. Only 3 control volunteers (8.3%) had a positive response to isosorbide-isoproterenol test and 2 (5.6%) to isoproterenol test. The duration of the test and the time for syncope induction in the isosorbide-isoproterenol test were shorter than those in the isoproterenol test (24.84 +/- 5.15 vs 35.70 +/- 6.28 minutes [p <0.01]; 4.53 +/- 2.86 vs 6.27 +/- 4.11 minutes [p <0.05]). This study concluded that isosorbide-isoproterenol test could be valuable in diagnosing unexplained syncope for its high sensitivity, powerful specificity, and short duration.

Administration, Sublingual↗
PubMed1999

Expression of bvgAS of Bordetella pertussis represses flagellar biosynthesis of Escherichia coli.

BvgAS is a two-component system of Bordetella pertussis involved in the reciprocal regulation of the virulence genes and the flagellar biosynthesis. In this study, we found that expression of bvgAS in Escherichia coli also results in reduced motility. The repression was relieved by the addition of known chemical modulators of BvgAS such as MgSO4 and nicotinic acid, indicating that functional BvgAS proteins are required for the negative control of E. coli motility. In addition, BvgAS repressed the transcription of the flhDC master operon of E. coli, which consequently caused non-flagellation on the cell surface. However, expression of BvgAS had no effect on stress-resistant motile mutants of E. coli. These data suggest that E. coli may have BvgA-like protein(s) involved in the regulatory interactions between the stress response and the flagellar biosynthesis.

Bacterial Proteins↗
PubMed1999

Treatment of carotid-cavernous sinus fistulas retrograde via the superior ophthalmic vein (SOV).

BACKGROUND: Carotid-cavernous sinus fistula is an old difficulty in neurosurgery. At present, the main way to deal with it is by embolizing the fistula via an arterial route. But if the fistula is small or supplied by several branches, it is difficult to embolize via an arterial approach. METHODS: In this report, 21 patients with carotid-cavernous sinus fistulae were successfully cured by embolizing the fistula via the SOV approach. RESULTS: All patients were cured, and there were no serious postoperative complications. Follow-up has been carried out in 19 cases for 8 months to more than 4 years; no recurrence manifested. CONCLUSION: For carotid-cavernous sinus fistulae that cannot be embolized arterially, the superior ophthalmic vein route is an easy, safe, effective and promising approach.

Adolescent↗
PubMed1999

The 2.0 A structure of human hypoxanthine-guanine phosphoribosyltransferase in complex with a transition-state analog inhibitor.

The structure of human HGPRT bound to the transition-state analog immucillinGP and Mg2+-pyrophosphate has been determined to 2.0 A resolution. ImmucillinGP was designed as a stable analog with the stereoelectronic features of the transition state. Bound inhibitor at the catalytic site indicates that the oxocarbenium ion of the transition state is stabilized by neighboring-group participation from MgPPi and O5'. A short hydrogen bond forms between Asp 137 and the purine ring analog. Two Mg2+ ions sandwich the pyrophosphate and contact both hydroxyls of the ribosyl analog. The transition-state analog is shielded from bulk solvent by a catalytic loop that moves approximately 25 A to cover the active site and becomes an ordered antiparallel beta-sheet.

Binding Sites↗
PubMed1999

Regulation of pathways determining cholesterol availability in the baboon placenta with advancing gestation.

Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

Animals↗
PubMed1999

Human papillomavirus type 16 E7 DNA vaccine: mutation in the open reading frame of E7 enhances specific cytotoxic T-lymphocyte induction and antitumor activity.

A human papillomavirus type 16 E7 DNA vaccine with the open reading frame encoding mutations in two zinc-binding motifs expressed a rapidly degraded E7 protein. This vaccine induced a significantly stronger E7-specific cytotoxic T-lymphocyte response and better tumor protection in mice than did a wild-type E7 DNA vaccine expressing a stable E7 protein.

Animals↗
PubMed1999

TGF-beta3-null mutation does not abrogate fetal lung maturation in vivo by glucocorticoids.

Newborn transforming growth factor (TGF)-beta3-null mutant mice exhibit defects of palatogenesis and pulmonary development. Glucocorticoids, which play a central role in fetal lung maturation, have been postulated to mediate their stimulatory effects on tropoelastin mRNA expression through TGF-beta3 in cultured lung fibroblasts. In the present study, we analyzed the abnormally developed lungs in TGF-beta3-null mutant mice and compared the effects of glucocorticoids on gene expression and lung morphology between TGF-beta3 knockout and wild-type mice. Lungs of TGF-beta3-null mutant mice on embryonic day 18.5 did not form normal saccular structures and had a thick mesenchyme between terminal air spaces. Moreover, the number of surfactant protein C-positive cells was decreased in TGF-beta3-null mutant lungs. Interestingly, glucocorticoids were able to promote lung maturation and increased expression of both tropoelastin and fibronectin but decreased the relative number of surfactant protein C-positive cells in fetal lungs of both genotypes. This finding provides direct evidence that glucocorticoid signaling in the lung can use alternative pathways and can exert its effect without the presence of TGF-beta3.

Animals↗
PubMed1999

Outcome for cancer patients requiring mechanical ventilation.

PURPOSE: To describe hospital survival for cancer patients who require mechanical ventilation. MATERIALS AND METHODS: A prospective, multicenter observational study was performed at five academic tertiary care hospitals. Demographic and clinical variables were obtained on consecutive cancer patients at initiation of mechanical ventilation, and information on vital status at hospital discharge was acquired. RESULTS: Our analysis was based on 782 adult cancer patients who met predetermined inclusion criteria. The overall observed hospital mortality was 76%, with no statistically significant differences among the five study centers. Seven variables (intubation after 24 hours, leukemia, progression or recurrence of cancer, allogeneic bone marrow transplantation, cardiac arrhythmias, presence of disseminated intravascular coagulation, and need for vasopressor therapy) were associated with an increased risk of death, whereas prior surgery with curative intent was protective. The predictive model based on these variables had an area under the receiver operating characteristic curve of 0.736, with Hosmer-Lemeshow goodness-of-fit statistics of 7.19; P = .52. CONCLUSION: This model can be used to estimate the probability of hospital survival for classes of adult cancer patients who require mechanical ventilation and can help to guide physicians, patients, and families in deciding goals and direction of treatment. Prospective independent validation in different medical settings is warranted.

Critical Care↗
PubMed1999

Serum leptin concentrations and expression of leptin transcripts in placental trophoblast with advancing baboon pregnancy.

Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. Recently, however, both leptin messenger ribonucleic acid (mRNA) and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in primate pregnancy. In the present study, venous blood samples were collected at 5-day intervals during gestation from baboons (Papio sp), an established model for the study of human pregnancy, as well as from nonpregnant baboons, and leptin concentrations were determined by RIA. Additionally, placental villous tissue was collected upon cesarean delivery at early (days 60-62; n = 5), mid (days 98-102; n = 5), and late (days 159-167; n = 5) gestation (term = approximately 184 days), and leptin mRNA was quantitated by competitive RT-PCR. Finally, in situ hybridization was employed to localize transcripts to specific placental cell types. Results determined that maternal leptin levels (mean +/- SEM), which were dramatically greater (P<0.01) than those in nonpregnant cycling baboons (1.4+/-0.1 ng/mL), increased (P<0.005) with gestational age from 63.6+/-10.4 ng/mL on day 60 of gestation to 157.8+/-16.1 near term. Levels declined to those found in cycling baboons by 15 days postdelivery. In contrast to maternal leptin concentrations, placental leptin mRNA decreased (P<0.02) with advancing pregnancy, as transcript abundance declined approximately 8-fold from early to late gestation. Maternal peripheral leptin concentrations were positively correlated (r = 0.66; P<0.001) whereas placental leptin mRNA levels were negatively correlated (r = -0.64; P<0.01) with gestational age. Expression of leptin mRNA transcripts, as evidenced by RT-PCR in villous tissue, was localized principally within syncytiotrophoblast by in situ hybridization. In summary, changes in maternal peripheral leptin concentrations and placental leptin mRNA abundance that occur commensurate with advancing gestational age may imply evolving roles for the polypeptide with advancing primate pregnancy. In this capacity, localization of leptin transcripts within the baboon syncytiotrophoblast suggests the potential for autocrine or paracrine interactions within this endocrinologically active tissue. Finally, both the similarities in leptin ontogeny in baboon and human pregnancy and the singular enhancement of maternal leptin levels inherent throughout baboon gestation emphasize the potential of this nonhuman primate model for the study of leptin action in the maternal-fetoplacental unit.

Animals↗
PubMed1999

[Biomechanical experimental study on effective fraction of radix salviae miltiorrhizae on healing of bone fracture].

OBJECTIVE: To assess the effect of Danshen-9403 (DS-9403, an effective fraction of Radix Salviae Miltiorrhizae) on healing of bone fracture. METHODS: Standardized radial fracture was performed in 120 Wistar rats. The model animals were randomized into four groups: group A was fed with DS-9403, group B injected with Staphylococcus Aureus, group C with normal saline, and group D with Salviae injection. The treatment began at the first day of fracture. The rats were sacrificed on the 25th, 39th and 50th day separately in batches and their intact radii were removed by dissection for detecting load and stress of three-point bending test with autograph universal material testing machine (Shimazu, Japan). RESULTS: The parameters of load in DS-9403 treated groups on the 39th day and that of stress at 25th, 39th and 50th day were (6.20 +/- 1.32) N, (5.71 +/- 3.58) N/mm2, (8.27 +/- 2.42) N/mm2 and (66.25 +/- 26.21) N/mm2 respectively, which were significantly higher than those in other groups, P < 0.05. CONCLUSION: DS-9403 has the action of increasing the strength of fracture healing site.

Animals↗
PubMed1999

[IL-12 gene treatment of hepatocellular carcinoma: experimental study].

OBJECTIVE: To investigate the inhibitory effects of retrovirus vector containing IL-12 gene on hepatoma growth in vivo and to explore a new approach of gene therapy to hepatocellular carcinoma (HCC). METHODS: Retrovirus vector containing IL-12 gene was constructed and transfected into packaged cell PA317. Positive PA317 was injected into the rat which suffered from experimental HCC and its anti-tumor effects and immunity changes were recorded. RESULTS: The packaged cell PA317 containing IL-12 gene could inhibit the proliferation of hepatocellular cell line CBRH3. The rats injected at day 1 or 3 can survive permanently, while those injected at day 5 or 7 can survive longer than those not injected. However, tumor generated in rats injected with blank control or package cells containing retroviral vector without IL-12 gene (P < 0.01). CONCLUSIONS: Package cells transfected with retroviral vector containing IL-12 either injected to the hepatoma tissue locally or given splenic exercises anti-hepatoma effects efficiently. The direct intrasplenic injection route is new, safe and effective.

Animals↗
PubMed1999

[Clinical diagnosis of fungal keratitis by confocal microscopy].

OBJECTIVE: To illustrate the value of confocal microscope in the diagnosis of fungal keratitis. METHODS: In vivo confocal microscopy was performed on 43 patients of suspected fungal keratitis. The results were compared to that of smears and biopsies. RESULTS: Fungal filaments were imaged in 31 of 43 patients' corneas. Fungal keratitis was diagnosed in 32 patients based on clinical signs and all the methods of examinations. The positive rate of confocal microscopy is 96.9%. CONCLUSION: Confocal microscopy can be a rapid, effective and noninvasive imaging technique, and it is helpful in the diagnosis, treatment and study of fungal keratitis.

Adolescent↗