[Show abstract][Hide abstract] ABSTRACT: Bacteriophages, the viruses of eubacteria, have developed unique mechanisms to interact with their host bacteria. They have been viewed as potential antibacterial therapeutics. Mycobacteriophage-derived compounds may interact with Mycobacterium tuberculosis (MTB) and/or its components, such as the cord factor, trehalose-6,6'-dimycolate (TDM), which is the most abundant glycolipid produced on the surface of MTB. TDM emulsion injected intravenously into mice induces lung immunopathology that mimics many aspects of MTB infection. Thus, TDM is an important target for anti-MTB agent development. On the basis of genomics information of mycobacteriophages, 200 peptides were synthesized. Their effects on MTB, their interactions with TDM, and anti-inflammatory activities were tested. One of them (PK34) showed MTB-killing activity with a minimal inhibitory concentration of 50 μg/ml and TDM-binding ability. In a mouse model, PK34 showed comparable ability to clear MTB as rifampin did in vivo. It also exerted strong activity to inhibit MTB or TDM-induced inflammation in vivo. PK34 significantly inhibited inflammatory cytokines secretions by inactivating MAPK and PKB signals while it maintained certain proinflammatory cytokine production. It is possible to prospect for TDM-binding and/or anti-MTB peptides by mining the mycobacteriophages genome. In addition to its direct MTB-killing ability, PK34 might be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection or a template for developing antituberculosis (TB) agents because of its immunoregulative effects. As a TDM-binding peptide, PK34 may be a promising tool to study TDM's interactions with corresponding receptors and signal pathways.-Wei, L., Wu, J., Liu, H., Yang, H., Rong, M., Li, D., Zhang, P., Han, J., Lai, R.. A mycobacteriophage-derived trehalose-6,6'-dimycolate-binding peptide containing both antimycobacterial and anti-inflammatory abilities.
[Show abstract][Hide abstract] ABSTRACT: A series of novel bispyrazoles joined by arylmethylene at C-4 position were synthesized with aromatic aldehydes obtained from lignin and screened for their in vitro antioxidant activities by N,N-diphenyl-N′-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylenzothiazoline-sulphonic acid) diammonium salt (ABTS+) radical scavenging assays. All of these compounds exhibited good DPPH and ABST+ radical scavenging activities as compared to the standard, Trolox, which suggested their potential as promising agents for curing tumors or other free radical-related diseases.
No preview · Article · Mar 2012 · Chinese Journal of Chemistry
[Show abstract][Hide abstract] ABSTRACT: Triptolide, a diterpenoid epoxide, is one of the major active ingredients of Tripterygium wilfordii Hook F, a woody vine plant called lei gong teng in China, which is used in traditional Chinese Medicine (TCM) for treating many diseases. In this paper, we investigate the relation between inhibition of mitochondrial respiratory chain and liver injury induced by triptolide. Results indicate that the secondary β-oxidation impairment caused by inhibition of mitochondrial respiratory chain is involved in triptolide-induced liver injury, which featured by microvesicular steatosis, hyperlactacidemia and enhanced oxidant stress, although other mechanisms of triptolide-induced liver injury may also exist.
[Show abstract][Hide abstract] ABSTRACT: The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus.
Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased.
We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks.
In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA.
These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.
[Show abstract][Hide abstract] ABSTRACT: Metacavir (PNA) is a novel synthetic nucleoside analogue for the treatment of hepatitis B virus (HBV). Our recent studies showed that PNA, a prodrug of 2',3'-dideoxyguanosine (ddG), exhibited lower mitochondrial toxicity in long-term cultures of HepG2 cells. In the current study, we examined the long-term effects of PNA on mitochondrial toxicity in Marmota himalayana (Himalayan marmot). Himalayan marmots were treated daily with oral PNA (50 or 100 mg/kg), ziduvidine (AZT) (100 mg/kg), or water (control) for 90 days. PNA treatment did not alter the body weight or plasma lactate acid level. In livers from the animals treated with PNA at 100 mg/kg/day, histopathology showed mild steatosis or small focal liver cell necrosis. Electron microscopy also showed minor proliferation and partial mitochondrial swelling with crista reduction. Measurement of respiratory chain complex enzyme activity and mitochondrial DNA (mtDNA) content revealed no significant differences in skeletal muscle, liver, and kidney tissues between animals treated with PNA and controls. In contrast, in Himalayan marmots treated with AZT we observed delayed toxicity, including lactic acidosis, severe hepatic steatosis, obvious mitochondrial damage, and significant decreases in respiratory chain complex enzyme activity and mtDNA content. This is similar to the delayed toxicity syndrome observed previously in animals and humans. In summary, PNA treatment did not alter mitochondrial enzyme activity or mtDNA content. This suggests that PNA could pose a very low risk for adverse mitochondrion-related effects. However, long-term hepatotoxic effects of PNA were observed, and this indicates a need for continued monitoring of PNA-associated hepatotoxicity in clinical trials.
[Show abstract][Hide abstract] ABSTRACT: Triptolide (CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), was reported to be sex-dependently metabolized mainly due to sex-related expression of CYP3A2. Sexual dimorphism in the expression of CYP isoforms is affected by sex difference in daily rhythm of growth hormone (GH) secretion. Neonatal administration of monosodium glutamate (MSG) can produce latent developmental defects in GH secretion and associated sex-dependent hepatic enzymes. In the present study, the triptolide metabolism, CYP3A2 expression and CYP3A-dependent activity were evaluated in Sprague-Dawley rats treated neonatally with MSG (4 mg/g) or saline (control) on postnatal days 1, 3, 5, 7 and 9. Treatment with MSG during the neonatal period in both sexes caused a number of disorders characterized by stunted body growth, notable obesity and suppression of GH secretion to barely detectable levels. In addition, neonatal treatment with MSG nearly eliminated the male-specific CYP3A2 expression and significantly reduced the microsomal erythromycin N-demethylation activity in males, while having no effects on CYP3A2 protein in females. Consistent with the P450 findings, the sexual dimorphism of triptolide metabolism completely disappeared in MSG-treated rats. This suggested that neonatal MSG treatment could eliminate the sex-dependent difference in metabolism of triptolide by suppressing CYP3A2 expression and activity in males to the same extent as females.
No preview · Article · Feb 2011 · Arzneimittel-Forschung
[Show abstract][Hide abstract] ABSTRACT: Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity.
Metacavir (PNA) is a novel synthetic nucleoside analog for oral administration with potent and specific antiviral activity
against hepatitis B virus (HBV). We assessed the potential for mitochondrial toxicity of PNA in long-term cultures of HepG2
hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and the
activities of respiratory-chain complexes I to IV. Exposure of HepG2 cells to PNA at concentrations up to 50 μM for 15 days
resulted in no deleterious effect on cell proliferation, levels of lactate or mtDNA, or enzyme activities of respiratory-chain
complexes I to IV. In contrast, dideoxycytosine at 10 μM and zidovudine at 50 μM have significant effects on cell proliferation,
levels of lactate and mtDNA, and enzyme activities of respiratory-chain complexes I to IV. However, PNA at a supratherapeutic
concentration of 250 μM could result in significant alterations in the levels of mtDNA and the activities of respiratory-chain
complex enzymes, revealing evidence of the potential mitochondrial toxicity of PNA. In summary, these in vitro results indicate that the potential for PNA to interfere with mitochondrial functions is low.
Full-text · Article · Nov 2010 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: In this study, we successfully expanded a full length gene encoding the monooxygenase eytochrome P450 2C9 gene from human liver of Chinese Han by RT-PCR. Our findings indicated that except G-->T mutation at the 190th nucleotide site, the other nucleotide sequences are completely consistent with CYP2C9 (NM017460) in GenBank. The SDS-PAGE and Western-Blot analysis showed that the CYP 2C9 gene was successfully expressed in the host cell E. coli BL21 (DE3). Our current study lays the foundation for the evaluation of pre-clinical drug metabolism and safety in the future.
No preview · Article · Apr 2010 · Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi
[Show abstract][Hide abstract] ABSTRACT: A number of H5N1 influenza outbreaks have occurred in aquatic birds in Asia. As aquatic birds are the natural reservoir of influenza A viruses and do not usually show clinical disease upon infection, the repeated H5N1 outbreaks have highlighted the importance of continuous surveillance on H5N1 viruses in aquatic birds. In the present study we characterized the biological properties of four H5N1 avian influenza viruses, which had been isolated from ducks, in different animal models. In specific pathogen free (SPF) chickens, all four isolates were highly pathogenic. In SPF mice, the S and Y isolates were moderately pathogenic. However, in mallard ducks, two isolates had low pathogenicity, while the other two were highly pathogenic and caused lethal infection. A representative isolate with high pathogenicity in ducks caused systemic infection and replicated effectively in all 10 organs tested in challenged ducks, whereas a representative isolate with low pathogenicity in ducks was only detected in some organs in a few challenged ducks. Comparison of complete genomic sequences from the four isolates showed that the same amino acid residues that have been reported to be associated with virulence and host adaption/restriction of influenza viruses were present in the PB2, HA, NA, M and NS genes, while the amino acid residues at the HA cleavage site were diverse. From these results it appeared that the virulence of H5N1 avian influenza viruses was increased for ducks and that amino acid substitutions at the HA cleavage site might have contributed to the differing pathogenicity of these isolates in mallards. A procedure for the intravenous pathogenicity index test in a mallard model for assessing the virulence of H5/H7 subtype avian influenza viruses in waterfowl is described.
[Show abstract][Hide abstract] ABSTRACT: Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.
Full-text · Article · Jul 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: In the current study, we characterized H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation during a 5 year period (1998-2002). Phylogenetic analysis of the 8 genes of 11 representative viruses showed that they all shared high similarity to that of the first isolate, A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages. Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of the neuraminidase gene. These genetic characteristics strongly suggest that these viruses are descendants of the first isolate. In addition, our study also showed that the H9N2 viruses circulating in the operation during this 5 year period were evolving, as shown by antigenic variations between viruses manifested by reactivity with polyclonal antisera and monoclonal antibodies, by haemagglutination with erythrocytes from different animals, by amino acid differences in haemagglutinin and neuraminidase proteins, and by variation in their ability to replicate in the respiratory and intestinal tract and to be transmitted by aerosol. Phylogenetic analysis revealed that the internal genes from some H5N1 viruses of duck origin clustered together with those from H9N2 virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more recent reassortment events between these two subtypes of viruses. Continuous surveillance of influenza virus in poultry and waterfowl is critical for monitoring the genesis and emergence of potentially pandemic strains in this region.
Full-text · Article · Jan 2009 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Recently H5N1 subtype avian influenza virus (AIV) is capable of mortality to aquatic bird. The molecular basis for the virulence of this virus is still poorly understood.
We characterized two H5N1 subtype viruses, A/mallard/Huadong/Y/2003 (Y) is nonpathogenic to mallard whereas A/mallard/Huadong/S/2005 (S) is highly pathogenic to mallard. Using reverse genetics, we constructed a series of single-gene or multiple-gene reassortants from these two viruses.
Substitution of single-gene for PB2, PB1, PA (3P), HA and of combination for 3P gene resulted in complete attenuation of S virus in mallard. However, these corresponding substitutions only slightly increased virulence of Y virus in mallard. Other gene segments had little contribution to the virulence of both viruses.
These results indicate that the pathogenicity of H5N1 AIV to mallard was regulated by multiple gene segments, and these regulations had more sensitive effect on highly pathogenic virus backbone than on low pathogenic virus backbone.
[Show abstract][Hide abstract] ABSTRACT: Infection in chicken flocks on a farm, where AI outbreak took place in 1998, was monitored in a four-year consecutive period and 22 H9N2 subtype viruses were isolated. The HA gene and antigenicity of 9 strains among isolates were compared with that of isolate of 1998. The results indicated that mutations of HA genes of H9 subtype influenza A viruses were observed during five years. However these mutations of HA genes didn't lead to sufficient antigenic variation to change the cross-protective pattern of these strains. These findings may be fruitful for making further study of genetic mutations of AIVs in the field and improving the control strategies.
No preview · Article · Jan 2004 · ACTA MICROBIOLOGICA SINICA
[Show abstract][Hide abstract] ABSTRACT: Eight full-length genes of an H9N2 subtype avian influenza virus A/Chicken/Shanghai/F/ 98(H9N2), were amplified by RT-PCR and rapid amplification of cDNA ends (RACE) and sequenced. The results of phylogenetic analysis showed that A/Chicken/Shanghai/F/98 was entirely different from Quail/Hong Kong/G1/97 which was directly related to human infection with avian influenza virus in Hong Kong, whereas its HA, NA, M and NS genes belonged to A/Chicken/Beijing/ 1/94-like sublineage with the nucleotide homology of 96.7%, 96.4%, 97.5% and 98.0% respectively. The homology of NA gene between A/Chicken/Shanghai/F/98 and A/Duck/Hong Kong/Y280/ 97 was 97.4% and they both lost 9 nucleotides between nt 205 to 206. However, the PB2, PB1, PA, NP genes did not show any close relationship with those of the three known sublineages and the later three didn't share immediate ancestors with available isolates whose sequences had been reported. Therefore, A/Chicken/Shanghai/F/98 is a product of natural reassortment between H9N2 avian influenza viruses from different sublineages.