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A L Wang

Publications and source records attributed to A L Wang.

At least 19 recordsLinked to original sources
PubMed2001

Structural analysis of the -1 ribosomal frameshift elements in giardiavirus mRNA.

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that required a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implicated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a -1 ribosomal frameshift in the GLV transcript.

Giardiavirus↗
PubMed2000

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia.

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Effect of pH and Zn(2+) on subcultured muscle cells from Macrobrachium nipponense.

A cell culture system was devised for muscle cell of Macrobrachium nipponense in the study. The juvenile and adult shrimps were held in laboratory aquaria with penicillin 1000 IU/ml and streptomycin 1000 microg/ml for 12-24 hours. Cell cultures were established in medium 199 supplemented with 20% fetal bovine serum, 1 g/L glucose, 5.2 g/L NaCl, 1.43 g/L CaCl(2), 0.05 g/L MgCl(2), 100 IU/mL penicillin and 100 microg/ml streptomycin. Fibroblast-like cells were passaged up to three times and survived for 54 days. The results showed the optimum for subculture in vitro was in medium 199 with pH 7.6. Moreover, basal medium supplemented with Zn(2+) 60 microg/L could enhance the growth of the muscle cells. It was found that better results for cell culture would be obtained more easily with juvenile shrimps caught in spring than adults in summer or autumn; and shrimps caught within 12 hours after ecdysis could grow much better than the intermoult shrimps.

Animals↗
PubMed2000

Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme.

Giardia lamblia is a primitive eukaryotic microorganism that derives its metabolic energy primarily from anaerobic glycolysis. In trophozoites, pyruvate-ferredoxin oxidoreductase (PFOR) converts pyruvate to acetyl-CoA with the transfer of a pair of electrons to ferredoxin, which can then reduce metronidazole and activate it into a potent antigiardiasis agent. It is unclear, however, whether this anaerobic disposal of electrons is essential for the energy metabolism in Giardia. In the present study, cDNAs encoding hammerhead ribozyme flanked with various lengths of antisense PFOR RNA were cloned into a viral vector pC631pac derived from the genome of giardiavirus (GLV). RNA transcripts of the plasmids showed high cleavage activities on PFOR mRNA in vitro. They were introduced into GLV-infected G. lamblia trophozoites by electroporation and stablized in the transfected cells via serial passages under puromycin selection. PFOR mRNA and enzyme activity in the transfected cells were decreased by 46-60% with the ribozyme PRzS flanked with 20 nt PFOR antisense RNA on each arm and by 69-80% with the ribozyme PRzL flanked with 600 and 1500 nt PFOR antisense RNA. PRzS without the inserted ribozyme or ribozyme flanked with alcohol dehydrogenase E antisense RNA showed no effect on PFOR mRNA and activity. The ribozyme-transfected cells demonstrated significantly enhanced resistance to metronidazole and grew equally well under anaerobic and aerobic conditions. In contrast, the wild-type cells grew slightly better anaerobically than the transfectants but did not grow at all in aerobic conditions. Thus, the reduced PFOR expression enables Giardia to grow under molecular oxygen and the presence of PFOR enhances the anaerobic growth of Giardia with an increased susceptibility towards metronidazole. In addition, this study demonstrated for the first time the feasibility of using a viral RNA vector to express a ribozyme targeted at a specific mRNA in G. lamblia to reduce the expression of a specific gene.

Animals↗
PubMed1998

Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA.

Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.

5' Untranslated Regions↗
PubMed1997

Long-term protection from myocardial ischemic events in a randomized trial of brief integrin beta3 blockade with percutaneous coronary intervention. EPIC Investigator Group. Evaluation of Platelet IIb/IIIa Inhibition for Prevention of Ischemic Complication.

CONTEXT: Abciximab, a monoclonal antibody fragment against the platelet receptor alphaIIb beta3 integrin, prevents platelet aggregation. A randomized, placebo-controlled study showed that abciximab improves outcomes for patients undergoing percutaneous coronary angioplasty at 30 days and at 6 months. OBJECTIVE: To determine whether abciximab improves outcomes 3 years after coronary angioplasty. DESIGN: Double-blind, placebo-controlled, randomized trial. SETTING: A total of 56 academic and community hospitals in the United States. PATIENTS: A total of 2099 high-risk patients undergoing coronary angioplasty were randomized. Sufficient time elapsed for 2.5 years of follow-up among 2001 patients and for 3 years of follow-up among 1599 patients. INTERVENTIONS: Abciximab bolus of 0.25 mg/kg followed by infusion at 10 microg/min for 12 hours; abciximab bolus of 0.25 mg/kg followed by placebo infusion; or placebo bolus followed by placebo infusion. MAIN OUTCOMES MEASURES: The primary outcome was the composite of death, myocardial infarction, or coronary revascularization. Secondary outcomes were death, myocardial infarction, or coronary revascularization individually. Subgroups having refractory unstable angina or evolving myocardial infarction and having different elevations of creatine kinase during initial angioplasty were analyzed. RESULTS: At 3 years, composite end points occurred in 41.1% of those receiving abciximab bolus plus infusion; 47.4% of those receiving abciximab bolus only; and 47.2% of those receiving placebo only (for abciximab bolus plus infusion vs placebo, P=.009). Death occurred in 6.8%, 8.0%, and 8.6%, respectively (for abciximab bolus plus infusion vs placebo, P=.20); myocardial infarction in 10.7%, 12.2%, and 13.6%, respectively (for abciximab bolus plus infusion vs placebo, P=.08); and revascularization in 34.8%, 38.6%, and 40.1%, respectively (for abciximab bolus plus infusion vs placebo, P=.02). Among those with refractory unstable angina or evolving myocardial infarction, death occurred in 5.1%, 9.2%, and 12.7%, respectively (for abciximab bolus plus infusion vs placebo, P=.01). Death rates increased as periprocedural creatine kinase levels increased. CONCLUSIONS: Abciximab bolus with infusion given at the time of coronary angioplasty improves outcomes as long as 3 years after the procedure.

Abciximab↗
PubMed1997

[Clinical observation on effect of xiaoyu zhixue tablet on 104 patients with idiopathic multifocal bleeding and platelet aggregation defect].

OBJECTIVE: To explore the effect and pharmacological mechanism of Xiaoyu Zhixue Tablet (XZT). METHODS: The XZT mainly composed of Chinese medicinal herbs to replenishing Qi and removing stasis was used to treat patients with idiopathic multifocal bleeding and platelet aggregation defect. Patients were divided into 2 groups. The TCM group (104 cases) treated with XZT, and the control group (84 cases) treated with vitamins C, K and P, adrenosin and (or) artificial menstruation, for at least 4 months. RESULTS: The hemostatic effective rate was 92.3% in TCM group and 27.4% in control group (P < 0.005), the recovery rate of platelet aggregation after 6 months therapy was 69.2% and only 1.8% respectively. The difference between two groups was very significant (P < 0.005). CONCLUSION: XZT could regulate the physiological functions of blood-vessel and platelet to realize the hemostatic action and platelet aggregation.

Adolescent↗
PubMed1996

Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia.

Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.

Amebicides↗
PubMed1996

Vagal bronchopulmonary C-fibers and acute ventilatory response to inhaled irritants.

Experiments were carried out in anesthetized Sprague-Dawley rats to determine the role of vagal bronchopulmonary C-fiber afferents in regulating the respiratory responses to inhaled irritants. Spontaneous inhalation of 2 tidal breaths of a known airway irritant (sulfur dioxide, 0.5%; ammonia, 1%; cigarette smoke, 50%) into the lower airways invariably elicited an immediate and transient inhibitory effect on breathing, characterized by apnea or bradypnea and accompanied by bradycardia, which lasted for 3-8 breaths. A delayed hyperpnea was also induced by inhalation of cigarette smoke, but not by sulfur dioxide or ammonia. After perineural capsaicin treatment of both cervical vagi to selectively block the conduction of capsaicin-sensitive C-fibers, these inhaled irritants no longer evoked any inhibitory effect on breathing; conversely, an augmented inspiration was triggered within the first 3 breaths from the onset of cigarette smoke inhalation in > 85% of the rats studied, but after a delay of several breaths following inhalation of ammonia or sulfur dioxide in only 30% of the rats. The augmented breaths were completely abolished when both cervical vagi were cooled to 6-7 degrees C. Bilateral vagotomy eliminated all the immediate responses to these irritants. These results suggest that both vagal C-fiber endings and irritant receptors in the airways are activated by these inhaled irritants, but the more dominant and consistent inhibitory effect on breathing is elicited primarily by stimulation of C-fiber afferents.

Administration, Inhalation↗
PubMed1996

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

Animals↗
PubMed1995

Sequences at four termini of the giardiavirus double-stranded RNA.

The linear double-stranded RNA (dsRNA) genome of giardiavirus (GLV) was estimated to be 6100 nucleotides (nt) [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 8585-8599]. As the 5'- and 3'-untranslated regions (UTR) of viral genomes are known to contain critical information for viral replication, we reexamine the sequences at all four termini of GLV dsRNA by (i) direct RNA sequencing with RT, (ii) tailing GLV dsRNA with UTP or CTP in addition to ATP in 3' rapid amplification of cDNA ends (RACE) and (iii) adding poly(dG) to the products of primer extension in 5'-RACE. The results confirmed the reported sequence for the 5'-terminus of the GLV sense strand RNA, but uncovered an additional 177 nt at the 3'-terminus. The new study also showed conclusively that there are no protruding overhangs at either terminus of GLV dsRNA.

Base Sequence↗
PubMed1995

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors.

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.

Abciximab↗
PubMed1995

Inhibition of hepatitis B viral gene expression by antisense phosphorothioate oligodeoxynucleotides.

The inhibitory effect of antisense phosphorothioate oligodeoxyribonucleotides (S-oligos) on the production of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) was studied using Hep G2 cells transfected with hepatitis B virus (HBV). Synthetic antisense oligos (15-mers) directed against the cap site of mRNA transcribed from the SP II promoter and regions of the translational initiation site of the S gene showed a sequence-specific, dose-dependent inhibitory effect on HBV gene expression between concentrations of 1.0 microM-5.0 microM. Oligos directed against the middle of the S gene had little effect on HBsAg and HBeAg expression, as did non-complementary random-sequence control. The cells remained viable throughout the experiments and no morphological abnormalities were observed with antisense S-oligos at concentrations below 20.0 microM. These results suggest a therapeutic potential for antisense oligonucleotides in the treatment of patients who are chronically infected with HBV.

Cell Division↗
PubMed1995

Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease.

The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.

Amino Acid Sequence↗
PubMed1995

Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

Animals↗