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Identification of Lactobacillus plantarum Genes That Are Induced in the Gastrointestinal Tract of Mice. Peter A. Bron, 2004.Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of environmental niches, including the human gastrointestinal (GI) tract . Moreover, this lactic acid bacterium can survive passage through the human or mouse stomach in an active form . To investigate the genetic background of this persistence, resolvase-based in vivo expression technology (R-IVET) was performed in L . plantarum WCFS1 by using the mouse GI tract as a model system . This approach identified 72 L . plantarum genes whose expression was induced during passage through the GI tract as compared to laboratory media . Nine of these genes encode sugar-related functions, including ribose, cellobiose, sucrose, and sorbitol transporter genes . Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract . Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L . plantarum encounters in the GI tract . The four extracellular protein encoding genes identified could potentially be involved in interaction with host specific factors . The rest of the genes are part of several functionally unrelated pathways or encode (conserved) hypothetical proteins . Remarkably, a large number of the functions or pathways identified here have previously been identified in pathogens as being important in vivo during infection, strongly suggesting that survival rather than virulence is the explanation for the importance of these genes during host residence . N-Substituted Pyrrole Derivatives as Novel Human Immunodeficiency Virus Type 1 Entry Inhibitors That Interfere with the gp41 Six-Helix Bundle Formation and Block Virus Fusion. Shibo Jiang, 2004.A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs . However, T-20 must be injected twice daily and is too expensive . Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors . By screening a chemical library consisting of "drug-like" compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range . The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity . NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the  -helical conformation . They blocked a D-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain . Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge . These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry . Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41 .
New Strategies for Cultivation and Detection of Previously Uncultured Microbes. Bradley S. Stevenson, 2004.An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites . Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media . The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O2 [vol/vol]) and anoxic atmospheres . Some bacteria were incubated with elevated concentrations of CO2 (5% [vol/vol]) . Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO2 . A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed . This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates . Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture . The Chloramphenicol-Inducible catB Gene in Agrobacterium tumefaciens Is Regulated by Translation Attenuation. Elizabeth J. Rogers, 2002.Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991) . The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible . Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp . Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis. Kimiko Yabe, 2003.The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus . When (1'S,5'S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed . When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed . However, (1'R,5'R)-AVR did not serve as the substrate . In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later . Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed . When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos . The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE . Separate feeding experiments performed with A . parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins . Remarkably, we found that VONE and VOROL had ring-opened structures . Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported . These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1'S,5'S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA . A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered .
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