Biomedical subjects

Baohua Gu

Publications and source records attributed to Baohua Gu.

35 records · Page 2Linked to original sources

PubMed2004

Sequence requirements for the development of a chimeric HCV replicon system.

The hepatitis C virus (HCV) 3'nontranslated region (3'NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3'NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3'NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3'NTR supported stable replication suggesting that the interaction between the NS proteins and the 3'NTR may represent a critical determinant. Lastly, the type 1a 3'NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3'NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3'NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3'NTR sequences from relevant clinical samples.

3' Untranslated Regions↗

PubMed2004

Mapping cooperative activity of the hepatitis C virus RNA-dependent RNA polymerase using genotype 1a-1b chimeras.

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (RdRp) which is essential for viral replication. NS5B expression in bacteria generated 20- to 50-fold lower yield and 100-fold less product per mol of enzyme for gentoype 1a RdRp than type 1b. Further, unlike type 1b RdRp, type 1a enzyme failed to exhibit cooperative properties in the assays described herein. Differences in thermal stability may partially account for the inability to efficiently oligomerize. Superose gel filtration analyses confirm differences between these RdRp preparations, although affinity for the column rather than size may account for the differences in migration. To further address this complexity, a panel of RdRp type 1a-type 1b chimeras were evaluated and implicate a role for the thumb subdomain of genotype 1b RdRp as critical for cooperative function.

Amino Acid Sequence↗

PubMed2004

Kinetics of soil ozonation: an experimental and numerical investigation.

This study investigates the use of ozone for soil remediation. Batch experiments, in which ozone-containing gas was continuously recycled through a soil bed, were conducted to quantify the rate of ozone self-decomposition and the rates of ozone interaction with soil organic and inorganic matter. Column experiments were conducted to measure ozone breakthrough from a soil column. Parameters such as ozone flow rate, soil mass, and ozonation time were varied in these experiments. After ozone concentration had reached steady state, the total organic carbon concentration was measured for all soil samples. The ozonation efficiency, represented by the ratio of soil organic matter consumed to the total ozone input, was quantified for each experiment. Numerical simulations were conducted to simulate experimentally obtained column breakthrough curves. Experimentally obtained kinetic rate constants were used in these simulations, and the results were in good agreement with experimental data. In contrast to previous studies in which soil inorganic matter was completely ignored, our experiments indicate that soil inorganic matter may also promote depletion of ozone, thus reducing the overall ozonation efficiency. Three-dimensional numerical simulations were conducted to predict the efficacy of ozonation for soil remediation in the field. These simulations indicate that such ozonation can be very effective, provided that effective circulation of ozone is achieved through appropriately placed wells.

Bioreactors↗

PubMed2004

alpha-Galactosylceramide and novel synthetic glycolipids directly induce the innate host defense pathway and have direct activity against hepatitis B and C viruses.

alpha-Galactosylceramide is a glycolipid derived from marine sponges that is currently in human clinical trials as an anticancer agent. It has also been shown to be effective in reducing the amount of hepatitis B virus (HBV) DNA detected in mice that produce HBV constitutively from a transgene. It was assumed that all of the antiviral and antitumor activities associated with alpha-galactosylceramide were mediated through the activation of NK T cells. However, we report here an additional unpredicted activity of alpha-galactosylceramide as a direct antiviral agent and inducer of the innate host defense pathway. To exploit this activity, we have developed a new class of smaller, orally available glycolipids that also induce the innate host defense pathway and have direct activity against HBV and hepatitis C virus.

Animals↗

PubMed2004

Purification and characterization of the AAA+ domain of Sinorhizobium meliloti DctD, a sigma54-dependent transcriptional activator.

Activators of sigma54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3' end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD(Delta(1-142)), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD(Delta(1-142)) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD(Delta(1-142)) displayed multiple species. The DctD AAA+ domain, but not DctD(Delta(1-142)), formed a stable complex with sigma54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with sigma54 in addition to its role in recognition of upstream activation sequences.

Bacterial Proteins↗

PubMed2004

Raman spectroscopic detection for perchlorate at low concentrations.

Perchlorate (ClO4-) has recently emerged as a widespread environmental contaminant found in groundwater and surface water, and there is a great need for rapid detection and monitoring of this contaminant. In this study, we explore the use of surface-enhanced (SERS) and normal Raman spectroscopy for detecting ClO4- at low concentrations. We found that ClO4- is SERS active and, for the first time, were able to detect ClO4- at concentrations as low as 10(-6)-10(-7) M (or 10-100 microg/L) through the application of silver SERS substrates or selective sorbents such as bifunctional anion-exchange resins. The use of selective sorbents greatly enhanced the reproducibility and sensitivity of ClO4- detection by normal Raman spectroscopy. Further exploration and research may allow application of these techniques for in situ, real-time detection and monitoring of ClO4- in environmental samples at even lower concentrations.

Perchlorates↗

PubMed2003

The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently.

The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3'-5' helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.

Adenosine Triphosphatases↗

PubMed2003

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase.

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.

Base Sequence↗

PubMed2003

The roles of natural organic matter in chemical and microbial reduction of ferric iron.

Although natural organic matter (NOM) is known to be redox reactive, the roles and effectiveness of specific functional groups of NOM in metal reduction are still a subject of intense investigation. This study entails the investigation of the Fe(III) reduction kinetics and capacity by three fractionated NOM subcomponents in the presence or absence of the dissimilatory metal reducing bacterium Shewanella putrefaciens CN32. Results indicate that NOM was able to reduce Fe(III) abiotically; the reduction was pH-dependent and varied greatly with different fractions of NOM. The polyphenolic-rich NOM-PP fraction exhibited the highest reactivity and oxidation capacity at a low pH (<4) as compared with the carbohydrate-rich NOM-CH fraction and a soil humic acid (soil HA) in reducing Fe(III). However, at a pH>4, soil HA showed a relatively high oxidation capacity, probably resulting from its conformational and solubility changes with an increased solution pH. In the presence of S. putrefaciens CN32, all NOM fractions were found to enhance the microbial reduction of Fe(III) under anaerobic, circumneutral pH conditions. Soil HA was found to be particularly effective in mediating the bioreduction of Fe(III) as compared with the NOM-PP or NOM-CH fractions. NOM-CH was the least effective because it was depleted in both aromatic and polyphenolic organic contents. However, because both soil HA and NOM-PP contain relatively high amounts of aromatic and phenolic compounds, results may indicate that low-molecular-weight polyphenolic organics in NOM-PP were less effective in mediating the bioreduction of Fe(III) at circumneutral pH than the high-molecular-weight polycondensed, conjugated aromatics present in soil HA. These research findings may shed additional light in understanding of the roles and underlying mechanisms of NOM reactions with contaminant metals, radionuclides, and other toxic chemicals in the natural environment.

Hydrogen-Ion Concentration↗

PubMed2003

Complete degradation of perchlorate in ferric chloride and hydrochloric acid under controlled temperature and pressure.

Despite favorable thermodynamics, the reduction of perchlorate (ClO4-) is kinetically limited in aqueous media because of its high activation energy. In this paper, a new methodology has been presented for degrading ClO4- in an FeCl3-HCl solution at an elevated temperature (< 200 degrees C) and/or pressure (approximately 20 atm). Results indicate that the rate constant for the pseudo-first-order reaction between ClO4- and ferrous Fe(II) (in excess) increased nearly 3 orders of magnitude when the temperature was increased from 110 to 195 degrees C, and a complete reduction of ClO4- occurred in < 1 h at 195 degrees C in the FeCl3-HCl solution. The activation energy of the reaction was calculated to be about 120 kJ/mol. Additionally, a flow-through reactor was constructed based on the batch kinetic measurements, and a nearly complete degradation of ClO4- was observed under continuous-flow mode. Because the FeCl3-HCl solution has been successfully used in regenerating selective anion-exchange resins sorbed with ClO4- during water treatment, this new methodology offers a cost-effective means to degrade ClO4- while not altering the chemical properties of the FeCl3-HCl regenerant solution so it can be reused, eliminating the production of secondary wastes.

Anion Exchange Resins↗

PubMed2003

Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.

Enzyme Inhibitors↗

PubMed2003

Fluorescence spectroscopic studies of natural organic matter fractions.

Because of the well-known molecular complexity and heterogeneity of natural organic matter (NOM), an aquatic bulk NOM was fractionated into well-defined polyphenolic-rich and carbohydrate-rich subfractions. These fractions were systematically characterized by fluorescence emission, three dimensional excitation-emission matrices, and synchronous-scan excitation spectroscopy in comparison with those of the reference International Humic Substances Society soil humic acid and Suwannee River fulvic acid. Results indicate that fluorescence spectroscopy can be useful to qualitatively differentiate not only NOM compounds from varying origins but also NOM subcomponents with varying compositions and functional properties. The polyphenolic-rich NOM-PP fraction exhibited a much more intense fluorescence and a red shift of peak position in comparison with the carbohydrate-rich NOM-CH fraction. Results also indicate that synchronous excitation spectra were able to provide improved peak resolution and structural signatures such as peak positioning, shift, and intensity among various NOM components as compared with those of the emission and excitation spectra. In particular, the synchronous spectral peak intensity and its red shift in the region of about 450-480 nm may be used to indicate the presence or absence of high molecular weight and polycondensed humic organic components, or the multicomponent nature of NOM or NOM subcomponents.

Chemical Fractionation↗

PubMed2003

Resistance profile of a hepatitis C virus RNA-dependent RNA polymerase benzothiadiazine inhibitor.

Recently, a benzo-1,2,4-thiadiazine antiviral agent (C(21)H(21)N(3)O(4)S; compound 4) was shown to be a potent, highly specific inhibitor of the primary catalytic enzyme of the hepatitis C virus (HCV) replicase complex. In this study, we selected for resistance to confirm the mechanism of action for compound 4 in HCV replicon cells. As expected, spontaneous mutations or fluidity in the HCV polymerase (NS5B) coding sequence occurred upon routine passage of the HCV replicon cells in the absence of compound 4. After 1 month of culture in the presence of 10 microM compound 4, or 20 times the 50% inhibitory concentration of the replicon, replicon cells were almost 20-fold less susceptible to compound 4. Twenty-one NS5B cDNA clones were generated from the resistant replicon cells. Five mutations in the 21 NS5B clones were present at frequencies higher than that of control replicon cells, and no clone contained more than a single mutation within the polymerase gene. RNA-dependent RNA polymerase studies using purified recombinant NS5B containing these single point mutations allowed the identification of residue 414 as sufficient for biochemical resistance to compound 4. Further, the contribution of this residue to confer cell-based resistance to compound 4 was validated using a stable recombinant mutant replicon cell line which harbors a methionine-to-threonine change at residue 414. The potential for additional mutations in other nonstructural genes of HCV to contribute to the resistance profile of compound 4 is discussed.

Benzothiadiazines↗

PubMed2003

Replication studies using genotype 1a subgenomic hepatitis C virus replicons.

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.

Adaptation, Physiological↗

PubMed2002

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.

Cell Line↗

PubMed2002

Spectroscopic characterization of the structural and functional properties of natural organic matter fractions.

Natural organic matter (NOM) is known to be complex in nature with varying structural and functional characteristics. In this study, an aquatic NOM was fractionated into the polyphenolic-rich (NOM-PP) and the carbohydrate-rich (NOM-CH) fractions in an attempt to better characterize their chemical and structural properties along with a reference soil humic acid (SHA). Various spectroscopic techniques were employed for the study, including ultraviolet-visible (UV/Vis). 13C-nuclear magnetic resonance, Fourier-transform infrared, fluorescence, and electron paramagnetic resonance spectroscopies. Results indicate that the relative abundance of aromatic C=C and methoxyl (-OCH3) functional groups are in the order of SHA > NOM-PP > NOM-CH. However, the aquatic NOM-PP and NOM-CH fractions are characterized by high contents of carboxylic and alcoholic functional groups relative to the SHA. In particular, the NOM-PP fraction appears to contain more phenolic and ketonic functional groups than the NOM-CH and SHA fractions, and it gives a strong fluorescence and high paramagnetic spin count. On the other hand, the NOM-CH fraction possesses a relatively low amount of carbon but a high amount of oxygen or oxygen-containing structural features, such as carbohydrate-OH and carboxylic groups, and shows the least fluorescence intensity and paramagnetic spin counts. Results of these spectroscopic studies confirm the heterogeneous nature of NOM, and point out the importance of isolation and improved characterization of various NOM subcomponents in order to better understand the behavior and roles of NOM in the natural environment.

Humic Substances↗

PubMed2002

Microbiological characteristics in a zero-valent iron reactive barrier.

Zero-valent iron (Fe0)-based permeable reactive barrier treatment has been generating great interest for passive groundwater remediation, yet few studies have paid particular attention to the microbial activity and characteristics within and in the vicinity of the Fe0-barrier matrix. The present study was undertaken to evaluate the microbial population and community composition in the reducing zone of influence by Fe0 corrosion in the barrier at the Oak Ridge Y-12 Plant site. Both phospholipid fatty acids and DNA analyses were used to determine the total microbial population and microbial functional groups, including sulfate-reducing bacteria, denitrifying bacteria, and methanogens, in groundwater and soil/iron core samples. A diverse microbial community was identified in the strongly reducing Fe0 environment despite a relatively high pH condition within the Fe0 barrier (up to pH approximately 10). In comparison with those found in the background soil/groundwater samples, the enhanced microbial population ranged from approximately 1 to 3 orders of magnitude and appeared to increase from upgradient of the barrier to downgradient soil. In addition, microbial community composition appeared to change over time, and the bacterial types of microorganisms increased consistently as the barrier aged. DNA analysis indicated the presence of sulfate-reducing and denitrifying bacteria in the barrier and its surrounding soil. However, the activity of methanogens was found to be relatively low, presumably as a result of the competition by sulfate/metal-reducing bacteria and denitrifying bacteria because of the unlimited availability of sulfate and nitrate in the site groundwater. Results of this study provide evidence of a diverse microbial population within and in the vicinity of the iron barrier, although the important roles of microbial activity, either beneficially or detrimentally, on the longevity and enduring efficiency of the Fe0 barriers are yet to be evaluated.

Adsorption↗