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Results for “reverse engineering”

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PubMed2000

Alleviation of murine leukemia virus repression in embryonic carcinoma cells by genetically engineered primer binding sites and artificial tRNA primers.

The primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNA(B2) to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNA(B2) to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNA(Lys1.2) suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS.

3T3 Cells↗

PubMed2001

Generation of a humanised single chain Fv (Scfv) derived from the monoclonal Eric-1 recognising the human neural cell adhesion molecule.

BACKGROUND: Murine monoclonal antibody (MoAb) ERIC-1 recognises an epitope on the neural cell adhesion molecule (NCAM) whose expression in paediatric and adult tissues is confined mainly to the brain, peripheral nerve, and adrenal medulla. Anti-NCAM antibodies have been used for the treatment and diagnosis of a number of tumours, including neuroblastoma. However, whole antibody exhibits poor penetration into solid tumour deposits and rapid systemic clearance upon repeated administration due to development of a human antimouse antibody (HAMA) response. PROCEDURE: To overcome these problems, recombinant DNA techniques have been used to humanise and assemble the ERIC-1 immunoglobulin variable heavy (VH) and light (VL) chains into a single chain Fv (scFv). RESULTS: Three humanised scFv clones were identified which differ from the predicted humanised sequence by occasional amino acid changes, but these maintain the same specificity as the original ERIC-1 MoAb. CONCLUSIONS: The humanised scFv may prove to be a useful reagents in the treatment and diagnosis of a variety of neuroectodermal tumours and can clearly form a suitable template for the generation of a fully humanised ERIC-1 MoAb.

Amino Acid Sequence↗

PubMed2000

Development of a plant-derived subunit vaccine candidate against hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 "mimotopes" previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines.

Administration, Intranasal↗

PubMed2025

Mapping early PRC2 nucleation sites upon Suz12 reintroduction reveals features of de novo Polycomb recruitment.

Polycomb domains safeguard cell identity by maintaining lineage-specific chromatin states enriched in repressive histone modifications, preserving the epigenetic memory of cell lineages. While Polycomb Repressive Complex 2 (PRC2) can re-establish its occupancy after perturbation, the mechanisms that guide de novo Polycomb recruitment remain unclear. To address this, we engineered an auxin-inducible degradation system to reversibly deplete and reintroduce the endogenous PRC2 core subunit Suz12 in mouse embryonic stem cells (mESCs). Genome-wide profiling at an early recovery time point revealed ~1,100 PRC2 nucleation sites, characterized by rapid Suz12 and histone H3K27me3 re-accumulation with strong signal, with minimal impact on gene expression. These sites were significantly enriched at bivalent promoters, coinciding with unmethylated CpG islands and chromatin states associated with developmental regulation, and were largely conserved in differentiated cells. Motif analysis identified G/C-rich DNA sequences associated with E2F and zinc-finger proteins, alongside strong co-occupancy with MTF2 and JARID2, two PRC2 cofactors previously implicated in Polycomb targeting. Notably, a subset of nucleation sites overlapped with long-range chromatin interaction anchors in histone H3K27me3 HiChIP datasets. These findings reveal that PRC2 de novo nucleation sites are associated with a combination of chromatin states, DNA sequence features, cofactor co-occupancy and spatial genome organization, suggesting that epigenetic memory can be re-established through defined genomic and chromatin features.

Epigenetic memory↗

PubMed1992

Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I.

The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.

Amino Acid Sequence↗

PubMed1995

Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus.

Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.

Animals↗

PubMed1998

Conformational changes necessary for gene regulation by Tet repressor assayed by reversible disulfide bond formation.

We constructed and characterized four Tet repressor (TetR) variants with engineered cysteine residues which can form disulfide bonds and are located in regions where conformational changes during induction by tetracycline (tc) might occur. All TetR mutants show nearly wild-type activities in vivo, and the reduced proteins also show wild-type activities in vitro. Complete and reversible disulfide bond formation was achieved in vitro for all four mutants. The disulfide bond in NC18RC94 immobilizes the DNA reading head with respect to the protein core and prevents operator binding. Formation of this disulfide bond is possible only in the tc-bound, but not in the operator-bound conformation. Thus, these residues must have different conformations when bound to these ligands. The disulfide bonds in DC106PC159' and EC107NC165' immobilize the variable loop between alpha-helices 8 and 9 located near the tc-binding pocket. A faster rate of disulfide formation in the operator-bound conformation and a lack of induction after disulfide formation show that the variable loop is located closer to the protein core in the operator-bound conformation and that a movement is necessary for induction. The disulfide bond in RC195VC199' connects alpha-helices 10 and 10' of the two subunits in the dimer and is only formed in the tc-bound conformation. The oxidized protein shows reduced operator binding. Thus, this bond prevents formation of the operator-bound conformation. The detection of conformational changes in three different regions is the first biochemical evidence for induction-associated global internal movements in TetR.

Cysteine↗

PubMed1985

Reversible dissociation of dimeric tyrosyl-tRNA synthetase by mutagenesis at the subunit interface.

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus exhibits half-of-the-sites reactivity and negative cooperativity in binding of tyrosine. Protein engineering has been applied to the enzyme to determine whether it can be reversibly dissociated into monomers and if the monomers are active. The target for mutation is the residue Phe-164. The side chain of Phe-164 in one subunit interacts with its symmetry-related partner in the other. Mutation of Phe-164----Asp-164 gives a mutant [TyrTS(Asp-164)] that undergoes dissociation at high pH when the aspartate residues are ionized. The monomer is inactive and does not bind tyrosine. Dissociation is enhanced at low concentrations of enzyme by a mass action effect. Kinetic and binding measurements on TyrTS(Asp-164) with tyrosine and tyrosyl adenylate show that the monomer has very weak affinity for these ligands. Accordingly, dimerization is favored by high concentrations of tyrosine and ATP since the dimeric form has a high affinity for the ligands. The presence of tRNA does not encourage dimer formation, and so it must bind to the monomer. TyrTS(Asp-164) is fully active at pH 6 where dimerization is favored but has low activity at pH 7.8 where dissociation is favored. It should now prove possible to engineer heterodimers that may be used to investigate the subunit interactions further.

Amino Acyl-tRNA Synthetases↗

PubMed2000

Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Amino Acid Substitution↗

PubMed2001

A unified theory of enzyme kinetics based upon the systematic analysis of the variations of k(cat), K(M), and k(cat)/K(M) and the relevant DeltaG(0 not equal) values-possible implications in chemotherapy and biotechnology.

To elucidate the kinetic properties of critical enzymatic situations that have previously escaped classification, we performed a systematic analysis of all the possible variations of the kinetic constants k(cat,) K(M,) and k(sp) = k(cat)/K(M,) encompassing all aspects of enzymology. The equation gives a total of thirteen theoretically possible cases, comprising the reference case plus 12 different sets of variations, which can be divided into six principal cases and six specular ones. The six relevant cases are examined individually in the context of each of the main chapters of enzymology, i.e. as regards mechanism of action, specificity of substrate and isoenzyme, reversible and irreversible inhibition, and mutation of residues (enzyme evolution and enzyme engineering). Some critical cases where k(sp) does not hold as a specificity index are classified for the first time. Interestingly, the six possible cases correspond to the five known cases of reversible inhibition (competitive, non-competitive, incompetitive, mixed competitive/non-competitive, and mixed incompetitive/non-competitive) plus an additional case of biphasic nature (activation-inhibition), which is crucial for a full understanding of specificity and which leads us to propose some modification to the definition of enzyme specificity. The systematic approach to enzymology outlined herein could find practical applications in various sectors of biotechnology, including chemotherapy.

Binding, Competitive↗

PubMed2001

Genetically engineered macrophages expressing IFN-gamma restore alveolar immune function in scid mice.

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cells in vivo. Many options exist for successfully transfecting cells in vitro, but they are not easily adapted to the in vivo condition. To overcome this limitation, we transduced macrophages in vitro with the murine IFN-gamma (mIFN-gamma) gene and intratracheally delivered the macrophages to express mIFN-gamma in vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-gamma and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 +/- 4.2 microg/ml mIFN-gamma at 24 h in vitro. The mIFN-gamma-producing macrophages were intratracheally instilled into mechanically ventilated scid mice. mIFN-gamma levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 +/- 5.1 pg/ml at 48 h (P < 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces of scid mice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P < 0.05). Tumor necrosis factor alpha was increased from undetectable at baseline to 103.5 +/- 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-gamma levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-gamma gene can partially restore significant immune activity in the lungs of immunodeficient mice.

Animals↗

PubMed1997

Protein engineering of the HMG-CoA reductase of Pseudomonas mevalonii. Construction of mutant enzymes whose activity is regulated by phosphorylation and dephosphorylation.

The activity of Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) is not regulated by phosphorylation, presumably due to the absence of a suitable target serine and protein kinase recognition motif. We have engineered P. mevalonii HMG-CoA reductase to a form whose activity, like that of mammalian HMG-CoA reductases, is regulated by phosphorylation/dephosphorylation. We substituted serine for arginine 387, the residue that corresponds to the regulatory serine of the HMG-CoA reductases of higher eukaryotes. A recognition motif for cAMP-dependent protein kinase was added by replacing leucine 384 by histidine (enzyme L384H/R387S) and also valine 391 by leucine (enzyme L384H/R387S/V391L). The activity of P. mevalonii HMG-CoA reductase mutant enzymes L384H/R387S and L384H/R387S/V391L was attenuated by phosphorylation. Restoration of activity accompanied subsequent dephosphorylation catalyzed by lambda protein phosphatase. Incorporation and subsequent release of phosphate paralleled the attenuation and restoration of catalytic activity. Incorporation of 0.5 mol of phosphate per subunit was accompanied by an approximately 50% decrease in initial activity. As in the analogous Syrian hamster mutant enzyme S871D, P. mevalonii mutant enzyme R387D exhibited 10% wild-type activity, suggesting that the attenuation of activity that accompanies phosphorylation results at least in part from the introduction of negative charge. Engineering of P. mevalonii HMG-CoA reductase to forms whose activity is reversibly regulated by phosphorylation/dephosphorylation provides an attractive model for future structure-based mechanistic studies. Solution of the X-ray structure of phosphorylated and dephosphorylated forms of engineered P. mevalonii HMG-CoA reductase should then reveal interactions of the active site phosphoseryl residue that result in attenuation of catalytic activity.

Animals↗

PubMed2026

Virus-induced gene silencing as a tool for functional genomics in weeds: Challenges and future directions.

Virus-induced gene silencing (VIGS) has evolved from a conceptual demonstration of antiviral defense into a pivotal reverse-genetics platform for plant functional genomics. By exploiting engineered DNA- or RNA-based viral vectors, VIGS enables rapid, sequence-specific transcript knockdown through RNA-mediated degradation of target transcripts. Recent refinements in vector design, inoculation strategies, and viral species selection, such as TRV, BSMV, and FoMV, have expanded its application to previously recalcitrant plants, including major crops and emerging weed models. In weeds, functional genomics remains particularly challenging due to high genetic variability, limited genomic resources, and incompatibility with conventional viral vectors and transformation systems. In this context, VIGS provides a tractable approach to investigate genes associated with herbicide resistance, metabolic adaptation, and stress tolerance. Beyond weed biology, its application to studies of immune signaling, hormonal crosstalk, and secondary metabolism highlights VIGS as a versatile biotechnology for elucidating gene function and supporting next-generation strategies in plant improvement and integrated pest management.

Journal Article↗

PubMed1995

Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli.

Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.

Amino Acid Sequence↗

PubMed2001

In vitro characterization of FIV-pPPR, a pathogenic molecular clone of feline immunodeficiency virus, and two drug-resistant pol gene mutants.

OBJECTIVE: To compare in vitro replication kinetics and nucleoside analog susceptibilities of a natural feline immunodeficiency virus (FIV) isolate (FIV-Maxam), a molecular clone of FIV (FIV-pPPR), and two (-)-beta-L-2',3'-dideoxy-3'-thiacytidine- (3TC-) resistant mutants of FIV-pPPR. SAMPLE POPULATION: Peripheral blood mononuclear cells (PBMC) from 4 specific-pathogenfree cats. PROCEDURE: Two point mutations corresponding to mutations of human immunodeficiency virus type 1 (HIV-1) were engineered into the highly conserved YMDD motif of the reverse transcriptase- (RT-) encoding region of the FIV-pPPR pol gene. Replication kinetics and nucleoside analog susceptibilities of FIV-Maxam, FIV-pPPR, and the 2 mutant viruses were measured in vitro, using feline PBMC. RESULTS: Replication kinetics and nucleoside analog susceptibilities were similar between FIV-Maxam and FIV-pPPR. However, FIV-Maxam was significantly more susceptible to 3TC. A methionine-to-valine mutation at codon 183 (M183V) of the RT-encoding region of the pol gene of FIV-pPPR conferred high-level phenotypic resistance to 3TC and cross-resistance to the related compound (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine. CONCLUSIONS AND CLINICAL RELEVANCE: Similarities between FIV-Maxam and FIV-pPPR suggest that results of studies performed using FIV-pPPR will have relevance to natural FIV infection in cats. In vitro evaluation of nucleoside analog susceptibilities of FIV-Maxam may help determine concentrations of nucleoside analogs required for effective treatment of FIV-infected cats. IMPACT FOR HUMAN MEDICINE: 3TC resistance of FIV-pPPR M183V was similar in magnitude to that of HIV-1 M184V, a mutant described in infected humans treated with 3TC. Thus, FIV-pPPR M183V may be a useful model for studying the in vivo effects of 3TC resistance on lentivirus pathogenesis.

Animals↗

PubMed1995

Tetracycline-reversible silencing of eukaryotic promoters.

A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments. This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site. We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein. Upon addition of tetracycline, luciferase expression was induced more than 50-fold above the baseline level, with half-maximal induction by 2 days. Furthermore, a protein of around 110 kDa was found to coimmunoprecipitate with the TetR-KRAB fusion protein. This protein might play a role as an adaptor protein mediating the silencing exerted by the TetR-KRAB protein. The TetR-KRAB silencing system should be useful as a genetic switch for regulating the expression of chromosomally integrated heterologous and endogenous genes present in mammalian genomes.

Amino Acid Sequence↗

PubMed2001

Regulation of interleukin-8 expression by nitric oxide in human pancreatic adenocarcinoma.

The regulation of interleukin-8 (IL-8) expression by nitric oxide (NO) was determined in human pancreatic cancer cell lines. CaPan-2 and FG human pancreatic adenocarcinoma cells were incubated for 24 h in medium alone or medium containing a cytokine mixture in the presence or absence of an NO synthase (NOS) inhibitor, N(G)-monomethyl-L-arginine (NMA). The NOS activity and level of IL-8 expression were determined. IL-8 expression was induced in the two cell lines. A low level of NOS activity was detectable only in CaPan-2 cells. Moreover, the presence of NMA did not reverse the induction of IL-8. The FG cells were then engineered to produce a physiologic level of NO and incubated in medium alone or medium containing 1 mM NMA. No significant IL-8 expression was induced in those producing a low level of NO, whereas IL-8 expression was induced in those producing a high level of NO. Inhibition of NO production by NMA reversed this effect. Incubation of FG cells with an NO donor, S-nitroso-D,L.-acetyl-penicillamine (SNAP), led to a concentration-dependent and time-dependent induction of IL-8 expression. This NO-mediated upregulation of IL-8 expression correlated with an increase in IL-8 gene transcription and mRNA stability. Our results indicate that NO is involved in the regulation of IL-8 expression in and contributes to the progression of human pancreatic cancer.

Adenocarcinoma↗

PubMed2000

[Development of influenza vaccines against newly emerging A/H5N1 virus].

Emergence of highly virulent influenza A/H5N1 viruses in Hong Kong in 1997 posed a threat of pandemic and brought an urgent need to develop a suitable seed virus for vaccine production. The virulence of the H5N1 viruses to chicken embryos should hamper the efficient production of the vaccine. In addition, potential virulence to humans raised safety issue in manufacturing vaccine. Toward vaccine development, one approach is to use an avirulent avian influenza virus antigenically similar to the virulent ones as a surrogate vaccine strain. The other approach is based on the attenuation of pathogenicity of virulent H5N1 virus by genetic engineering of the hemagglutinin gene and selection of a gene constellation. The reverse genetics technique can make the latter approach possible. Candidate strains suitable for vaccine production could be prepared by using either approach.

Animals↗