Cryptococcus fildesensis sp. nov., a psychrophilic basidiomycetous yeast isolated from Antarctic moss.

Two yeast strains isolated from the moss Chorisodontium aciphyllum from the Fildes Region, King George Island, maritime Antarctica, were classified as members of the genus Cryptococcus based on sequence analyses of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions. The rRNA gene sequence analyses indicated that the two strains represented a novel species of the genus Cryptococcus, for which the name Cryptococcus fildesensis sp. nov. is proposed (type strain: CPCC 300017(T) = DSM 26442(T) = CBS 12705(T)). The MycoBank number of the novel species is MB 805542.

Molecular analysis of fungal diversity associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica.

The fungal communities associated with three bryophytes species (the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata) in the Fildes Region, King George Island, maritime Antarctica, were studied using clone library analysis. Fungal communities showed low diversity; the 680 clones belonged to 93 OTUs. Of these, 78 belonged to the phylum Ascomycota, 13 to the phylum Basidiomycota, 1 to the phylum Zygomycota, and 1 to an unknown phylum. Among the OTUs, the most common orders in the Ascomycota were Helotiales (42 OTUs) and Chaetothyriales (14 OTUs) and the most common orders in the Basidiomycota were Sebacinales (3 OTUs) and Platygloeales (3 OTUs). Most OTUs clustered within clades that contained phylotypes identified from samples in Antarctic or Arctic ecosystems or from bryophytes in other ecosystems. In addition, we found that host-related factor may shape the fungal communities associated with bryophytes in this region. This is the first systematic study of the fungal community in Antarctic bryophytes to be performed using culture-independent method and the results may improve understanding of the endophytic fungal evolution and ecology in the Antarctic ecosystem.

Small molecular compounds inhibit HIV-1 replication through specifically stabilizing APOBEC3G.

APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(+) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD(50) of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.

[Establishment and drug susceptibility test of isoniazid resistant Mycobacterium smegmatis].

With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.

[Synthesis and antiviral activities of geldanamycin analog TC-GM in vitro].

In order to find antiviral compounds with novel structures, geldanamycin and lamivudine with different antiviral mechanisms were conjunctively synthesized to acquire a new compound TC-GM, and the antiviral activity of TC-GM was measured. The antiviral activity against HIV-1 was examined by p24 antigen ELISA kit. The activity against HBV was examined by dotblot. The activity against HSV and CoxB virus was examined by CPE. TC-GM exhibited broad-spectrum antiviral activities similarly like geldanamycin. TC-GM inhibited the replication of different viruses, including HIV-1, HBV, HSV 1 and 2, CoxB6. TC-GM showed more potent inhibitory activity against HIV-1 and HBV than other detected virus.

Heat stress cognate 70 host protein as a potential drug target against drug resistance in hepatitis B virus.

Heat stress cognate 70 (Hsc70) is a host protein associated with hepatitis B virus (HBV) replication. The goal of this study was to investigate whether Hsc70 could be an anti-HBV drug target. Our results showed that introducing Hsc70 increased HBV replication in HBV(+) human hepatocytes (HepG2.2.15 cells). The coiled-coil region on Hsc70 (nucleotides 1533 to 1608; amino acids 511 to 536) was the key sequence for HBV replication. Knockdown of Hsc70 expression by RNA interference (RNAi) largely inhibited HBV replication with no cytotoxicity to the host. Using an Hsc70 mRNA screening assay, the natural compound oxymatrine (OMTR) was found to be a selective inhibitor for Hsc70 expression. Then, OMTR was used to investigate the potential of Hsc70 as an anti-HBV drug target. OMTR inhibited Hsc70 mRNA expression by 80% and HBV DNA replication by over 60% without causing cytotoxicity. The anti-HBV effect of OMTR appeared to be mediated by destabilizing Hsc70 mRNA. The half-life (T(1/2)) of Hsc70 mRNA decreased by 50% in OMTR-treated hepatocytes. The Hsc70 mRNA 3'-untranslated-region (UTR) sequence was the element responsible for OMTR's destabilization activity. OMTR suppressed HBV de novo synthesis at the reverse transcription stage from pregenomic RNA (pgRNA) to DNA and was active against either wild-type HBV or strains resistant to lamivudine, adefovir, and entecavir. Therefore, host Hsc70 could be a novel drug target against HBV, and OMTR appears to inhibit HBV replication by destabilizing Hsc70 mRNA. As the target is not a viral protein, OMTR is active for either wild-type HBV or strains resistant to reverse transcriptase (RT) inhibitors.

In vivo antibacterial activity of nemonoxacin, a novel non-fluorinated quinolone.

OBJECTIVES: To evaluate the in vivo antibacterial efficacy of nemonoxacin, a novel C8-methoxy non-fluorinated quinolone in murine systemic and local infection models. METHODS: The efficacy of nemonoxacin in systemic infections was evaluated in mouse peritonitis models using isolates of methicillin-susceptible Staphylococcus aureus (MSSA, n=1), methicillin-resistant S. aureus (MRSA, n=1), methicillin- and levofloxacin-resistant Staphylococcus capitis (levofloxacin-resistant MRSC, n=1), penicillin-intermediate Streptococcus pneumoniae (PISP, n=1), penicillin-resistant S. pneumoniae (PRSP, n=2), Enterococcus faecalis (n=2, including 1 vancomycin-resistant Enterococcus, VRE) and Escherichia coli (n=3). The local infections included mouse pulmonary infections caused by PRSP (n=1), Klebsiella pneumoniae (n=1) and mouse ascending urinary tract infection caused by E. coli (n=1). RESULTS: In the mouse systemic infection model, nemonoxacin demonstrated potent activity against MSSA (ED(50) =2.08 mg/kg), MRSA (ED(50) =2.59 mg/kg), levofloxacin-resistant MRSC (ED(50) =2.52 mg/kg), PISP (ED(50) =5.47 mg/kg), PRSP (ED(50) =3.68-5.28 mg/kg) and E. coli (ED(50) =3.13-5.28 mg/kg), and moderate activity towards E. faecalis infection (ED(50) =8.48-15.16 mg/kg). The therapeutic efficacy of nemonoxacin was significantly higher (P<0.01) than that of levofloxacin in infections caused by Gram-positive isolates (MSSA, MRSA, levofloxacin-resistant MRSC, PISP, PRSP and E. faecalis), but less potent than that of levofloxacin against E. coli infection (P<0.01). Nemonoxacin in vivo efficacy results with Gram-positive isolates (2- to 5-fold ED(50) advantage over levofloxacin) are consistent with the MIC data (4- to 16-fold MIC advantage of nemonoxacin over levofloxacin). In the mouse pulmonary infection model, nemonoxacin showed potent activity towards PRSP (higher than levofloxacin) and K. pneumoniae (lower than levofloxacin) infections. In the mouse ascending urinary tract infection model, nemonoxacin exhibited potent activity against E. coli infection (lower than levofloxacin). CONCLUSIONS: The results validated the potent efficacy of nemonoxacin in vivo. The higher efficacy of nemonoxacin than of levofloxacin towards infections caused by Gram-positive cocci (especially MRSA, levofloxacin-resistant MRSC, PRSP and VRE) warrants investigation of its clinical use.

[Establishment and application of a high-throughput screening assay for premature activation of HIV-1 precursors].

Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).

In vivo antibacterial activity of vertilmicin, a new aminoglycoside antibiotic.

Vertilmicin is a novel aminoglycoside antibiotic with potent activity against gram-negative and -positive bacteria in vitro. In this study, we further evaluated the efficacy of vertilmicin in vivo in systemic and local infection animal models. We demonstrated that vertilmicin had relatively high and broad-spectrum activities against mouse systemic infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. The 50% effective doses of subcutaneously administered vertilmicin were 0.63 to 0.82 mg/kg, 0.18 to 0.29 mg/kg, 0.25 to 0.99 mg/kg, and 4.35 to 7.11 mg/kg against E. coli, K. pneumoniae, S. aureus, and E. faecalis infections, respectively. The therapeutic efficacy of vertilmicin was generally similar to that of netimicin, better than that of gentamicin in all the isolates tested, and better than that of verdamicin against E. coli 9612 and E. faecalis HH22 infections. The therapeutic efficacy of vertilmicin was further confirmed in local infection models of rabbit skin burn infection and mouse ascending urinary tract infection.

In vitro antibacterial activity of vertilmicin and its susceptibility to modifications by the recombinant AAC6'-APH2'' enzyme.

Vertilmicin is a new semisynthetic aminoglycoside with a structure similar to that of netilmicin except for a methyl group at the C-6' position. In the present study, the in vitro antibacterial activity of vertilmicin was studied, and its susceptibility to modifications by the recombinant aminoglycoside bifunctional modifying enzyme AAC(6')-APH(2'') was compared with those of verdamicin and netilmicin. A total of 1,185 clinical isolates collected from hospitals in Beijing between 2000 and 2001 were subjected to the in vitro antibacterial activity evaluations, including MIC, minimum bactericidal concentration (MBC), and time-kill curve tests. The MICs were evaluated in non-gentamicin-resistant (gentamicin-susceptible and gentamicin-intermediate) strains and gentamicin-resistant strains, respectively. For most of the non-gentamicin-resistant bacteria (except for the isolates of Pseudomonas spp.), the MIC(90)s of vertilmicin were in the range of 0.5 to 8 microg/ml, comparable to those of the reference aminoglycosides. For the gentamicin-resistant isolates, the three semisynthetic aminoglycosides (vertilmicin, netilmicin, and amikacin) demonstrated low MIC(50)s and/or MIC(90)s, as well as high percent susceptibility values. Among the study drugs, vertilmicin showed the lowest MIC(90)s, 16 microg/ml, for the gram-positive gentamicin-resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis. Meanwhile, vertilmicin was a potent bactericidal agent, with MBC/MIC ratios in the range of 1 to 2 for Escherichia coli, Klebsiella pneumoniae, and S. aureus and 1 to 4 for S. epidermidis. The time-kill curve determination further demonstrated that this effect was rapid and concentration dependent. In evaluations of susceptibility to modifications by the recombinant AAC(6')-APH(2'') with maximum rate of metabolism/K(m) measurements, vertilmicin exhibited susceptibilities to both acetylation and phosphorylation lower than those of netilmicin and verdamicin.

Chemomicin A, a new angucyclinone antibiotic produced by Nocardia mediterranei subsp. kanglensis 1747-64.

A new angucyclinone antibiotic, chemomicin A was isolated from cultured broth of Nocardia mediterranei subsp. kanglensis 1747-64. Its chemical structure was determined to be 1,2,3,4a,5,6,6a,12a,12b-nonahydro-1,2,3,8,12,12b-hexahydroxy-3-methyl-6a,12a-epoxybenz[a]anthracen-4,7(12H)-dione by a detailed spectroscopic analysis. Chemomicin A had antimicrobial activity against Bacillus subtilis and Enterococcus faecium with MIC values of 10.2 and 20.4 microM, respectively, and showed cytotoxicity against human colorectal cancer HCT116 cells and human esophageal carcinoma YES-2 cells with IC50 values of 127 and 153 microM, respectively.

Antiviral therapeutic efficacy of foscarnet in hepatitis B virus infection.

Foscarnet (PFA), a viral DNA polymerase inhibitor, is a clinical agent for herpes viruses. The goal of the study was to evaluate the therapeutic efficacy of PFA in hepatitis B virus (HBV) infection. Intravenous infusion of PFA (1 g/day) for 4 weeks significantly reduced serum HBeAg (p<0.01) and HBV DNA copies (p<0.05) in 31 patients who were diagnosed with active chronic HBV infection (CHB) and had not received antiviral treatment previously. Alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and gamma glutamyl transpeptidase (gamma-GT) of the patients declined (p<0.001, 0.001 and 0.01, respectively). Kidney function (blood creatinine and urea nitrogen) remained unchanged. Another 21 lamivudine-resistant CHB patients with mutations at the tyrosine-methionine-aspartate-aspartate motif (YMDD) displayed a response to PFA similar to that mentioned above, with reductions in HBeAg (p<0.05), HBV DNA (p<0.01) and liver enzymes (ALT and AST, p<0.001; gamma-GT, p<0.05). Moreover, PFA reduced serum HBeAg (p<0.01), HBV DNA (P<0.05), AST (p<0.05) and ALT (p<0.02) in a cohort of 13 severe CHB patients with advanced liver damage. PFA was also evaluated in vitro and in vivo. PFA inhibited HBV DNA replication in HBV-transfected human HepG2 cells (2.2.15 cells) with reduced amount of HBV RC-DNA and DS-DNA. In the duck HBV-infected ducklings, PFA reduced viral DNA and duck HBsAg in the serum (p<0.01 for both).

Diversity and cold adaptation of culturable endophytic fungi from bryophytes in the Fildes Region, King George Island, maritime Antarctica.

Endophytic fungi associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica, that is, the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata, were studied by culture-dependent method. A total of 128 endophytic fungi were isolated from 1329 tissue segments of 14 samples. The colonization rate of endophytic fungi in three bryophytes species were 12.3%, 12.1%, and 8.7%, respectively. These isolates were identified to 21 taxa, with 15 Ascomycota, 5 Basidiomycota, and 1 unidentified fungus, based on morphological characteristics and sequence analyses of ITS region and D1/D2 domain. The dominant fungal endophyte was Hyaloscyphaceae sp. in B. hatcheri, Rhizoscyphus sp. in C. aciphyllum, and one unidentified fungus in S. uncinata; and their relative frequencies were 33.3%, 32.1%, and 80.0%, respectively. Furthermore, different Shannon-Weiner diversity indices (0.91-1.99) for endophytic fungi and low endophytic fungal composition similarities (0.19-0.40) were found in three bryophyte species. Growth temperature tests indicated that 21 taxa belong to psychrophiles (9), psychrotrophs (11), and mesophile (1). The results herein demonstrate that the Antarctic bryophytes are an interesting source of fungal endophytes and the endophytic fungal composition is different among the bryophyte species, and suggest that these fungal endophytes are adapted to cold stress in Antarctica.

HCV IRES-mediated core expression in zebrafish.

The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-ß, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism.

High performance liquid chromatographic separation of thirteen drugs collected in Chinese Pharmacopoeia 2010(Ch.P2010) on cellulose ramification chiral stationary phase.

The enantiomers separation of thirteen drugs collected in Ch.P2010 was performed on chiral stationary phase of cellulose ramification (chiralpak OD and chiralpak OJ) by high performance liquid chromatographic (HPLC) methods, which included ibuprofen (C1), ketoprofen (C2), nitrendipine (C3), nimodipine (C4), felodipine (C5), omeprazole (C6), praziquantel (C7), propranolol hydrochloride (C8), atenolol (C9), sulpiride (C10), clenbuterol hydrochloride (C11), verapamil hydrochloride (C12), and chlorphenamine maleate (C13). The mobile phase consisted of isopropanol and n-hexane. The detection wavelength was set at 254 nm and the flow rate was 0.7 mL/min. The enantiomers separation of these thirteen racemates on chiralpak OD column and chiralpak OJ column was studied, while the effects of proportion of organic additives, alcohol displacer and temperature on the separation were studied. And the mechanism of some of racemates was discussed. The results indicated that thirteen chiral drugs could be separated on chiral stationary phase of cellulose ramification in normal phase chromatographic system. The chromatographic retention and resolution of enantiomers could be adjusted by factors including column temperature and the concentration of alcohol displacer and organic alkaline modifier in mobile phase. It was shown that the resolution was improved with reducing concentration of alcohol displacer. When concentration of organic alkaline modifier was 0.2% (v/v), the resolution and the peak shape were fairly good. Most racemates mentioned above had better resolution at column temperature of 25 °C. When racemates were separated, the temperature should be kept so as to obtain stable separation results.

Berberine reduces insulin resistance through protein kinase C-dependent up-regulation of insulin receptor expression.

Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

Combination of simvastatin with berberine improves the lipid-lowering efficacy.

We have identified berberine (BBR) as a novel cholesterol-lowering drug acting through stabilization of the low-density lipoprotein receptor (LDLR) messenger RNA. Because the mechanism differs from that of statins, it is of great interest to examine the lipid-lowering activity of BBR in combination with statins. Our results showed that combination of BBR with simvastatin (SIMVA) increased the LDLR gene expression to a level significantly higher than that in monotherapies. In the treatment of food-induced hyperlipidemic rats, combination of BBR (90 mg/[kg d], oral) with SIMVA (6 mg/[kg d], oral) reduced serum LDL cholesterol by 46.2%, which was more effective than that of the SIMVA (28.3%) or BBR (26.8%) monotherapy (P < .01 for both) and similar to that of SIMVA at 12 mg/(kg d) (43.4%). More effective reduction of serum triglyceride was also achieved with the combination as compared with either monotherapy. Combination of BBR with SIMVA up-regulated the LDLR messenger RNA in rat livers to a level about 1.6-fold higher than the monotherapies did. Significant reduction of liver fat storage and improved liver histology were found after the combination therapy. The therapeutic efficacy of the combination was then evaluated in 63 hypercholesterolemic patients. As compared with monotherapies, the combination showed an improved lipid-lowering effect with 31.8% reduction of serum LDL cholesterol (P < .05 vs BBR alone, P < .01 vs SIMVA alone). Similar efficacies were observed in the reduction of total cholesterol as well as triglyceride in the patients. Our results display the rationale, effectiveness, and safety of the combination therapy for hyperlipidemia using BBR and SIMVA. It could be a new regimen for hypercholesterolemia.

Synthesis and anti-influenza activities of carboxyl alkoxyalkyl esters of 4-guanidino-Neu5Ac2en (zanamivir).

Three alkoxyalkyl 2-carboxylate ester derivatives related to zanamivir were synthesized. All of the analogs of zanamivir modified at carboxylic moiety with alkoxyalkyl esters 1a-c showed higher activities than ribavirin on influenza A and B virus in the MDCK cells. Oral treatment or intraperitoneal administration of compound 1c showed significantly protective effects in mice infected with influenza A virus with low toxicities.