Evidence that methylation of the FMR-I locus is responsible for variable phenotypic expression of the fragile X syndrome.

DNA at the FMR-1 locus was analyzed by Southern blot using probe StB12.3 in an unusual fragile X family with six brothers, three of whom are affected with fragile X to varying degrees, two of whom are nonpenetrant carriers, and one of whom is unaffected. Fragile X chromosome studies, detailed physical examinations, and psychological testing were completed on all six. Two of the affected brothers and the two nonpenetrant brothers were found to be methylation mosaics. The three affected males spanned the phenotypic and cognitive spectrum of the fragile X syndrome. A correlation was seen between the degree of methylation and the phenotypic expression identified in the three affected males. The two males initially classified as nonpenetrant were found to have mild phenotypic expression which consisted of minor cognitive deficits and a partial physical phenotype. These two, who were negative on fragile X chromosome studies, were found on DNA analysis to have large broad smears, with approximately 97% of the DNA unmethylated. The results described here indicate that some "nonpenetrant" carrier males may have varying amounts of methylation of the FMR-1 region, which can result in mild expression of the fragile X syndrome. The apparently mild phenotypic and cognitive expression of the fragile X syndrome in the two males, initially classified as nonpenetrant, who are mosaic for hypermethylation of an expansion of the CGG repeat in the premutation range, indicates that expression of the syndrome is not confined to males with large, hypermethylated expansions (full mutation) but has instead a gradient effect with a threshold for the full expression of the phenotype.     

Isolation and characterization of ilvA, ilvBN, and ilvD mutants of Caulobacter crescentus.

Caulobacter crescentus strains requiring isoleucine and valine (ilv) for growth were shown by transduction and pulsed-field gel electrophoresis to contain mutations at one of two unlinked loci, ilvB and ilvD. Other C. crescentus strains containing mutations at a third locus, ilvA, required either isoleucine or methionine for growth. Biochemical assays for threonine deaminase, acetohydroxyacid synthase, and dihydroxyacid dehydratase demonstrated that the ilvA locus encodes threonine deaminase, the ilvB locus encodes acetohydroxyacid synthase, and the ilvD locus encodes dihydroxyacid dehydratase. C. crescentus strains resistant to the herbicide sulfometuron methyl, which is known to inhibit the action of certain acetohydroxyacid synthases in a variety of bacteria and plants, were shown to contain mutations at the ilvB locus, further suggesting that an acetohydroxyacid synthase gene resides at this locus. Two recombinant plasmids isolated in our laboratory, pPLG389 and pJCT200, were capable of complementing strains containing the ilvB and ilvD mutations, respectively. The DNA in these plasmids hybridized to the corresponding genes of Escherichia coli and Serratia marcescens, confirming the presence of ilvB-like and ilvD-like DNA sequences at the ilvB and ilvD loci, respectively. However, no hybridization was observed between any of the other enteric ilv genes and C. crescentus DNA. These results suggest that C. crescentus contains an isoleucine-valine biosynthetic pathway which is similar to the corresponding pathway in enteric bacteria but that only the ilvB and ilvD genes contain sequences which are highly conserved at the DNA level.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Antigen-specific Th1 but not Th2 cells provide protection from lethal Trypanosoma cruzi infection in mice

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.     

Trypanoside,anti-tuberculosis,leishmanicidal, and cytotoxic activities of tetrahydrobenzothienopyrimidines

The synthesis of 2-(5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-yl)hydrazone-derivatives (BTPs) and their in vitro evaluation against Trypanosoma cruzi trypomastigotes, Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, and six human cancer cell lines is described. The in vivo activity of the most active and least toxic compounds against T. cruzi and L. amazonensis was also studied. BTPs constitute a new family of drug leads with potential activity against infectious diseases. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents in developing countries.