Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional 1H NMR.

A series of two-dimensional (2D) correlated 1H NMR spectra of reduced and oxidized Escherichia coli thioredoxin have been used to probe the effects of pH in the vicinity of the active site, -Cys32-Gly-Pro-Cys35-, using the complete proton resonance assignments available for thioredoxin. In either oxidation state, the majority of residues of the thioredoxin molecule remain unchanged between pH 5.7 and pH 10, as indicated by the identical chemical shifts of the C alpha H, C beta H, and other protons. In reduced thioredoxin, a fairly widespread region around the active-site dithiol is affected by the titration of a group or groups with pKa approximately 7.1-7.4 in 2H2O. Another titration, with pKa approximately 8.4, affects a smaller region of the protein. Oxidized thioredoxin contains a disulfide and no free thiol groups; nevertheless, the proton resonances of many groups in the active-site region were observed to titrate with a pKa of 7.5, probably as a result of an abnormally high pKa value for the carboxyl group of the buried Asp-26 residue. For reduced thioredoxin, the results indicate that Asp-26 is titrating in this pH range, as well as both thiol groups. The new results are strongly suggestive that the mechanism of thioredoxin-catalyzed protein disulfide reduction may be critically dependent on proton transfer as well as electron transfer within the active site.     

Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.     

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.     

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.     

Molecular Cloning and Analysis of the Endogenous Retrovirus Chemically Induced from RFM/Un Mouse Cell Cultures

We molecularly cloned and analyzed an N-tropic ecotropic retrovirus induced with iododeoxyuridine from RFM/Un mouse cell cultures. Based on the restriction map, the RFM/Un virus appears to be indistinguishable from other induced N-tropic retroviruses. A nucleotide sequence analysis of the long terminal repeat of an infectious clone revealed structural features characteristic of murine type C retrovirus long terminal repeats. The U3 region of the RFM/Un virus long terminal repeat, however, contained no short sequence duplication or insertion found in other murine leukemia virus isolates.     

Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program

This paper is an extension of compilations published previously in this journal. (Ashby and Tennant, 1988; Ashby et al., 1989). A summary of the rodent carcinogenicity bioassay data on a further 39 chemicals tested by the U.S. National Toxicology Program (NTP) is presented. An evaluation of each chemical for structural alerts to DNA-reactivity is also provided, together with a summary of its mutagenicity to Salmonella. Chemicals with an aliphatic nitro group (-C-NO2) have been added to the composite structure of DNA-reactive sub-groups. The 39 chemicals were numbered and evaluated as an extension of the earlier analysis of 264 NTP chemicals. The activity patterns and conclusions derived from the earlier studies are followed by these 39 chemicals, albeit a detailed analysis of the total database of 301 chemicals is reserved for the succeeding paper.