Molecular analysis of hprt mutants induced by 2-cyanoethylene oxide in human lymphoblastoid cells

The mutagenic epoxide metabolite of acrylonitrile, 2-cyanoethylene oxide (ANO), was used to treat human TK6 lymphoblasts (150 microM x 2 h ANO). A collection of hypoxanthine-phosphoribosyltransferase (hprt) mutants was isolated and characterized by dideoxy sequencing of cloned hprt cDNA. Base-pair substitution mutations in the hprt coding region were observed in 19/39 of hprt mutants: 11 occurred at AT base pairs and 8 at GC base pairs. Two -1 frameshift mutations involving GC bases were also observed. Approximately half (17/39) of the hprt mutants displayed the complete loss of single and multiple exons from hprt cDNA, as well as small deletions, some extending from exon/exon junctions. Southern blot analysis of 5 mutants with single exon losses revealed no visible alterations. Analysis of 1 mutant missing exons 3-6 in its hprt mRNA revealed a visible deletion in the corresponding region in its genomic DNA. The missing exon regions of 4 mutants (one each with exons 6, 7 and 8 loss and one mutant with a 17-base deletion of the 5' region of exon 9) were PCR amplified from genomic DNA and analyzed by Southern blot using exon-specific probes. The exons missing from the hprt mRNA were present in the genomic hprt sequence. DNA sequencing of the appropriate intron/exon regions of hprt genomic DNA from a mutant with exon 8 loss and a mutant exhibiting aberrant splicing in exon 9 revealed point mutations in the splice acceptor site of exon 8 (T----A) and exon 9 (A----G), respectively.     

Sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice

Several alphaviruses, including the Sindbis-group viruses, Ross River virus, O'nyong-nyong virus, and Chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. Mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. S.A.AR86 is a Sindbis-group alphavirus closely related to the arthralgia-associated Ockelbo and Girdwood S.A viruses. Following inoculation with S.A.AR86 derived from a molecular clone, infectious virus was isolated from bone and joint tissue 1 to 6 days postinfection. Studies using either in situ hybridization or S.A.AR86-derived double promoter viruses and replicons expressing green fluorescent protein localized sites of viral replication to the periosteum, tendons, and endosteum within the epiphyses of the long bones adjacent to articular joints. These results demonstrate that alphaviruses associated with arthralgia in humans replicate within bone-associated connective tissue adjacent to articular joints in an adult mouse model.     

Glutamate synthase, but not GABA shunt enzymes, contributes to nitrogen metabolism of the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

Glutamate synthase (E.C. 1.4.1.14) (GOGAT) activity was not detectable in L3 Haemonchus contortus, but was present in L3 Teladorsagia circumcincta and adult worms of both species. GOGAT activity was inhibited by 80% by azaserine. Activity (nmol min⁻¹ mg⁻¹ protein) was 33–59 in adult H. contortus, 51–91 in adult T. circumcincta and 24–41 in L3 T. circumcincta, probably depending on exposure to ammonia, as incubation with 1 mM NH₄Cl doubled GOGAT activity. The pH optimum was 7.5 in both species. Either NAD or NADP acted as co-factor. The mean apparent K_m for 2-oxoglutarate was 0.7 (0.5–0.9) mM and for glutamine was 1.0 (0.5–1.7) mM for different homogenates. There was no detectable activity in whole parasite homogenates of glutamate decarboxylase (E.C. 4.1.1.15) or succinic semialdehyde dehydrogenase (E.C. 1.2.1.24), the first and third enzymes of the GABA shunt, respectively, suggesting that the GABA shunt is not important in general metabolism in these species.     

Phylogeny of the Callandrena subgenus of Andrena (Hymenoptera: Andrenidae) based on mitochondrial and nuclear DNA data: polyphyly and convergent evolution

We propose a phylogenetic hypothesis of relationships within Callandrena, a North American subgenus of the bee genus Andrena, based on both mitochondrial and nuclear DNA sequences. Our data included 695 aligned base pairs comprising parts of the mitochondrial genes cytochrome oxidase subunits I and II and the intervening tRNA-leucine and 767 aligned base pairs of the F2 copy of the nuclear gene elongation factor-1alpha. We also suggest a preliminary hypothesis of relationships of the North American subgenera in the genus. Our analyses included 54 species of Callandrena, 42 species of Andrena representing 24 additional subgenera, and 11 outgroup species in the family Andrenidae. Parsimony analyses of each marker separately suggested that Callandrena was polyphyletic, with a combined analysis suggesting that there were at least two phylogenetically independent clades of bees with similar morphological features. Maximum likelihood and Bayesian analyses supported this conclusion, as did the non-parametric bootstrapping SOWH test. Convergence in morphological characters was likely due to their common use of members of Asteraceae as pollen hosts.     

Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer

The methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with the expression of a thermolabile enzyme with decreased activity that influences the pool of methyl-donor molecules. Several studies have reported an association between C677T polymorphism and susceptibility to colorectal cancer (CRC). Considering that methylation abnormalities appear to be important for the pathogenesis of CRC, we examined the correlation between the genotype of the MTHFR C677T polymorphism, hypermethylation of the promoter region of five relevant genes (DAPK, MGMT, hMLH1, p16(INK4a), and p14(ARF)), and microsatellite instability, in 106 patients with primary CRCs in Brazil. We did not find significant differences in the genotypic frequencies of the MTHFR C677T polymorphism when one or more loci were hypermethylated. However, we did find a significant excess of 677TT individuals among patients with CRC who had microsatellite instability. This strong association was independent of the methylation status of hMLH1 and of the biogeographical genomic ancestry of the patients. Although the mechanism responsible for the link between the C677T polymorphism and microsatellite instability was not apparent, this finding may provide a clue towards a better understanding of the pathogenesis of microsatellite instability in human colorectal cancer.     

Coxsackievirus-B3-induced myocarditis: virus receptor antibodies modulate myocarditis

Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.     

Granulocyte/macrophage colony-stimulating factor from mouse lung conditioned medium. Purification of multiple forms and radioiodination.

Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.