Mineralization of sulfonamides from wastewater using ozone-based systems.

Ozone, UV/ozone, ozone/persulfate (PS) and UV/ozone/PS systems were used to mineralize sulfonamides. Sulfadiazine (SDZ), sulfamerazine (SMR) and sulfamethazine (SMZ) were the target compounds. The novel contribution of this study is its determination of the effects of PS addition, sulfonamide structure, pH and salinity on sulfonamide mineralization in ozone-based systems. The mineralization rate of sulfonamides satisfied pseudo-first-order kinetics. The SMZ mineralization rate constant in ozone, UV/ozone, ozone/PS and UV/ozone/PS systems at pH 5 were 0.0058; 0.0101; 0.0069 and 0.0802 min-1, respectively, and those at pH 7 were 0.0075; 0.0116; 0.0083 and 0.0873 min-1, respectively. The increase in the number of methyl substituents in the heterocyclic group of SMZ and the corresponding increase in the steric hindrance of radical addition, reduced mineralization rates below those of SMR and SDZ. The addition of PS promoted sulfonamide mineralization in the ozone-based systems; conversely, salinity inhibited sulfonamide mineralization.

Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.

Enterohaemorrhagic Escherichia coli (EHEC) causes life-threatening infections in humans as a consequence of the production of Shiga-like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen-activated protein kinase (MAPK) pathway, an evolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Our results validate the EHEC-C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.

Cytotoxic and novel compounds from Solanum indicum.

Solavetivone (1), cytotoxic to OVCAR-3 cells with an IC(50) value of 0.1 mM, has been isolated from Solanum indicum. In addition, a novel solafuranone (2) and three known compounds, scopoletin, N-(p-trans-coumaroyl)tyramine, and N-trans-feruloyltyramine, were isolated for the first time from this plant. The structures of the above compounds were established by means of spectroscopic and X-ray analyses.

Construction of a tagging system for subcellular localization of proteins encoded by open reading frames.

We have previously characterized a monoclonal antibody (SC1D7) that is directed to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses. To better map the epitope, we took advantage of an available plasmid, pMAL-c2, that encodes the E. coli MBP-coding sequence and constructed plasmids to express MBP fragments. A construct containing the N-terminal portion of MBP does not react with SC1D7, whereas a second construct expressing glutathione S-transferase fused with the C-terminal half of MBP does react with SC1D7. To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7. Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced. This sequence matches MBP with an amino acid stretch of KDPRIAA. To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulting recombinant protein did react with SC1D7. Thereafter, this epitope was incorporated into a eucaryotic expression plasmid containing a previously defined hepatitis delta virus epitope for protein tagging. This two-epitope-tagging vector is useful in various molecular analyses. We demonstrate its usage for localization of a bacterial virulence factor in host cells. This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing.

The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication.

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.

Immunohistochemical differentiation of hepatitis D virus genotypes.

Determination of hepatitis D virus (HDV) genotypes is epidemiologically and clinically important. Phylogenic analysis based on sequencing analysis of multiple HDV strains isolated from sera of patients is not convenient for mass screening in routine laboratories. This study was designed to develop genotype-specific antibodies against hepatitis delta antigen (HDAg) and to apply these antibodies for immunohistochemical differentiation of HDV genotypes in formalin-fixed, paraffin-embedded liver biopsies of patients. Divergence in the carboxyl-terminal 19 amino acids of the large HDAg between genotypes I and II is more than 70%. Peptides covering these residues were conjugated to keyhole limpet hemocyanin and were used for immunization. The generated antibodies were confirmed for their specificity by binding to type-specific HDAgs expressed in DNA-transfected Huh-7 hepatoma cells. Liver biopsies from 6 patients who had dominant genotype I HDV and 33 patients who had dominant genotype II HDV in sera were stained with these antibodies. The accuracy for these antibodies was 94.9%, and the agreement between dominant HDV genotypes in serum and dominant hepatic HDV genotypes based on HDAg staining was nearly perfect (kappa = 0.83). In summary, the carboxyl-terminal 19 amino acids of the large HDAg can be used as immunogens to generate genotype-specific antibodies. These antibodies were proven to be useful in immunohistochemical differentiation of HDV genotypes in liver biopsies.

Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7.

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.

DNA-Based immunization produces Th1 immune responses to hepatitis delta virus in a mouse model.

Hepatitis delta virus (HDV) superinfection is one of the major causes of fulminant hepatitis in endemic areas of hepatitis B virus (HBV) infection. Currently, there is no effective treatment or vaccine against HDV superinfection. DNA-based immunization is a promising antiviral strategy to prevent or treat persistent viral infections. In this study, we investigated the immunological effects of DNA vaccines against HDV in BALB/c mice. Plasmid (pD) encoding large hepatitis D antigen (L-HDAg), or plasmid (pS/pD) coexpressing hepatitis B surface antigen (HBsAg) and L-HDAg, were injected into mice intramuscularly. The seroconversion rate, anti-HBs levels, anti-HDV titers, T-cell proliferation responses, and T-helper (Th)-release cytokine profiles were analyzed. Mice immunized with plasmids, pS/pD or pD, produced low, but significant, titers of anti-HDV antibodies. In contrast, pS/pD induced much stronger anti-HBs titers in the immunized animals. Interestingly, splenic lymphocytes derived from pS/pD-inoculated mice demonstrated significant proliferation responses to recombinant HBsAg and HDAg, and resulted in a Th1-like immune response as suggested by the production of interferon gamma (INF-gamma) and interleukin-2 (IL-2), but not IL-4. The splenic lymphocyte derived from the pD-inoculated mice showed a similar Th1 response to the stimulation of HDAg, but not to HBsAg. In conclusion, our results suggest that DNA vaccines against HDV can induce significant cellular immune responses with a Th1 preference. HBV and HDV coimmunization can be performed by DNA vaccines. These results are promising for the future development of prophylactic and therapeutic HDV vaccines.

Characterization of a strain-specific monoclonal antibody to hepatitis delta virus antigen.

Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates. A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity. HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II. The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides. A consensus peptide deduced has the best match with that of residues 4-10 of HDAg (isolate 25). To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed. The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1-10. When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1. It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25). Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored. The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system.

Recombination of hepatitis D virus RNA sequences and its implications.

Recombination between RNA sequences plays a role in the fast evolution of a few viruses. There has been no report on hepatitis D virus (HDV) recombination. In this study, we analyzed genetic recombination of HDV and its possible impact on evolution and clinical course. The aligned HDV sequences allowed us to construct a phylogenetic tree which supported the notion of distinct lineages of HDV. The tree was also used in the analysis of recombination using partial likelihoods assessed through optimization. Nine segments of the HDV genome with significant levels of genetic recombination were detected. Five segments were in the hypervariable region, and four were in the delta-antigen- coding region. None could be found in the well-conserved autocleavage region that is essential for replication. Recombination occurred both between and within types. The results of this study indicated that the remarkable variation in HDV genomic sequences, particularly in the hypervariable region, among different genotypes may at least partly result from multiple episodes of genetic recombination during evolution. Genetic recombination may play a significant role in increasing genetic diversity. Importantly, a genetic recombination (nt 1082-1093) occurred in one of the immunogenic domains of hepatitis delta virus antigen recognized by human and woodchuck antibodies (amino acids 174-195). Genetic recombination also occurred at another segment between nt 1517 and 1535, which was close to one of the predicted T-cell epitopes (amino acids 26-41). In longitudinal analysis of HDV genomes at different time points during chronic infection, novel dominant HDV strains with amino acid changes at these epitopes usually emerged after severe hepatitis attacks. In the comparison of HDV clones during or shortly after flare-up of liver disease, Ka/Ks ratios of > 1 were frequently found, suggesting Darwinian positive selection. Therefore, recombination in these two segments may play an important role for HDV in the evasion of immunity.