Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) from Landfill Sites

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.     

High-affinity uptake of [3H]serotonin in cultured neurones of the cockroach Periplaneta americana

Cultured central neurons from the American cockroach, Periplaneta americana, have been used to investigate the uptake of [³H]serotonin. The neurones accumulate [³H]serotonin from the extracellular medium by both a high-and a low-affinity system. The activity of the high-affinity mechanism is decreased by low temperature and metabolic poisons, and is dependent on sodium and chloride ions. Both depolarising levels of external potassium ions and veratridine decrease the high-affinity uptake system, suggesting it is influenced by the transmembrane potential. The pyrethroid insecticides, deltamethrin and permethrin, enhance the inhibitory effect of veratridine. Pyrethroid enhancement is completely blocked by tetrodotoxin, and neither pyrethroid affects the uptake system in the absence of veratridine. Avermectin B_1A is a powerful inhibitor of the high-affinity uptake system, and its effect is blocked by picrotoxin. High-affinity uptake of [³H]serotonin is inhibited by imipramine and amitriptyline; desipramine has no significant effect on this uptake. The activity of the high-affinity system is also reduced by 8-hydroxy-dipropylaminotetralin, α-methyl-serotonin, and 1-(3-chlorophenyl)piperazine. Dopamine, noradrenaline, octopamine, and the formamidine insecticides, chlordimeform and demethylchlordimerform, are moderate inhibitors of the high-affinity uptake system. The formamidine effect is not blocked by tetrodotoxin or picrotoxin.     

Cold-induced photoinhibition affects establishment of Eucalyptus nitens (Deane and Maiden) Maiden and Eucalyptus globulus Labill

The effects of cold-induced photoinhibition on Eucalyptus globulus and Eucalyptus nitens seedlings were assessed between planting and age 23 weeks. The seedlings were subjected to four treatments before planting: non-hardened (NH) E. globulus and E. nitens, cold-hardened (CH) E. nitens, and nutrient-starved (NS) E. nitens. Seedlings were planted alongside established 1-year-old E. nitens saplings. The experimental site was at 350 m above sea level, which is considered marginal for the establishment of E. globulus plantations due to low mean minimum temperatures. Cold and sunny conditions after frost increased photoinhibition in the order: NH E. globulus > NH and CH E. nitens > NS and established E. nitens. As a result there was 20% mortality of NH E. globulus seedlings. NS E. nitens seedlings were severely photoinhibited and had high anthocyanin levels at planting; levels of photoinhibition decreased after planting, anthocyanin levels remained high and there was no mortality. Carotenoid levels were low in E. globulus compared to E. nitens treatments. It was concluded that cold-induced photoinhibition is a factor determining the range of environments where E. globulus can be successfully planted, and not frost tolerance alone. Anthocyanin was synthesised in response to increased photoinhibition. Anthocyanin levels were correlated to the severity of the photoinhibition. Thus, E. nitens seedlings nutrient starved in the nursery are pre-conditioned to photoinhibitory conditions experienced soon after planting. This treatment is a useful risk-management tool where cold-induced photoinhibition is likely to occur during seedling establishment.     

The R-spondin protein family

The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/β-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.     

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.      

Assessment of changes in the brca2 and p53 genes in breast invasive ductal carcinoma in northeast Brazil

Background

BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models.

Results

BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant.

Conclusions

There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.     

The localisation of alkaline phosphatase in the midgut epithelium of Carausius morosus

Summary Alkaline phosphatase was found to occur only in those cells of the midgut epithelium of C. morosus that had a high lipid content. As it was localised specifically in the microvilli of the cell border it is suggested that in this insect the enzyme might be concerned with lipid absorption.