Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.     

Novel protein genes in animal mtDNA: a new sex determination system in freshwater mussels (Bivalvia: Unionoida)?

Mitochondrial (mt) function depends critically on optimal interactions between components encoded by mt and nuclear DNAs. mitochondrial DNA (mtDNA) inheritance (SMI) is thought to have evolved in animal species to maintain mito-nuclear complementarity by preventing the spread of selfish mt elements thus typically rendering mtDNA heteroplasmy evolutionarily ephemeral. Here, we show that mtDNA intraorganismal heteroplasmy can have deterministic underpinnings and persist for hundreds of millions of years. We demonstrate that the only exception to SMI in the animal kingdom, that is, the doubly uniparental mtDNA inheritance system in bivalves, with its three-way interactions among egg mt-, sperm mt- and nucleus-encoded gene products, is tightly associated with the maintenance of separate male and female sexes (dioecy) in freshwater mussels. Specifically, this mother-through-daughter and father-through-son mtDNA inheritance system, containing highly differentiated mt genomes, is found in all dioecious freshwater mussel species. Conversely, all hermaphroditic species lack the paternally transmitted mtDNA (=possess SMI) and have heterogeneous macromutations in the recently discovered, novel protein-coding gene (F-orf) in their maternally transmitted mt genomes. Using immunoelectron microscopy, we have localized the F-open reading frame (ORF) protein, likely involved in specifying separate sexes, in mitochondria and in the nucleus. Our results support the hypothesis that proteins coded by the highly divergent maternally and paternally transmitted mt genomes could be directly involved in sex determination in freshwater mussels. Concomitantly, our study demonstrates novel features for animal mt genomes: the existence of additional, lineage-specific, mtDNA-encoded proteins with functional significance and the involvement of mtDNA-encoded proteins in extra-mt functions. Our results open new avenues for the identification, characterization, and functional analyses of ORFs in the intergenic regions, previously defined as "noncoding," found in a large proportion of animal mt genomes.     

In vitro bioactivation of bazedoxifene and 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol in human liver microsomes

Bazedoxifene is a selective estrogen receptor modulator (SERM) that has been developed for use in post-menopausal osteoporosis. However, it contains a potentially toxic 5-hydroxy-3-methylindole moiety. Previous studies on the 5-hydroxyindole and the 3-alkylindole-containing drugs indometacine, zafirlukast and MK-0524 structural analogs have shown that they are bioactivated by cytochrome P450s through a dehydrogenation process to form quinoneimine or 3-methyleneindolenine electrophilic species. In the present study, bazedoxifene was synthesized and then evaluated, together with raloxifene and 2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol (13), a 3-methyl-5-hydroxyindole-based structural fragment of bazedoxifene, for its ability to form reactive electrophilic species when incubated with human liver microsomes (HLMs) or recombinant CYP isozymes. We showed that bazedoxifene was bioactivated only in trace amounts with recombinant CYP isozymes. In contrast, the N-dealkylated fragment of bazedoxifene (2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol) was bioactivated in considerable amounts to an electrophilic intermediate, which was trapped with glutathione and identified by LC-MS/MS. This suggests that bazedoxifene would require initial N-dealkylation, which could subsequently lead to the formation of the reactive intermediate. However, such an N-dealkylated metabolite of bazedoxifene was not detected after the incubation of bazedoxifene in HLM or recombinant CYP isozymes.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.     

Population dynamics of banded thrips (Aeolothrips intermedius Bagnall, Thysanoptera, Aeolothripidae) and its potential prey Thysanoptera species on white clover

In 2002, the occurrence of banded thrips (Aeolothrips intermedius Bagnall) and some other Thysanoptera species on white clover (Trifolium repens L.) was monitored at two locations in the continental part of Slovenia. White clover presents in many countries important intercrop in integrated vegetable production. Light blue sticky boards were placed on grasslands (one parcel on each location) with high percentage of white clover. Sticky boards were changed in about 10-days intervals from the end of April till the beginning of October. Number of caught individuals on the boards was counted. They were classified in three different groups: 1. Aeolothrips intermedius, 2. representatives of Haplothrips, Odontothrips and Frankliniella genera, 3. representatives of Thrips genus. We stated that, compared with the other Thysanoptera species in the open, predatory thrips occurs in lower number. Predatory species Aeolothrips intermedius was the most numerous during the flowering of white clover. It was established that other Thysanoptera species (the most of them are facultative phytophagous species) were more numerous also in the periods of less favourable weather conditions and during the non-flowering growing stages of white clover. Based on the results of present research we concluded that A. intermedius has a potential to control onion thrips (Thrips tabaci Lindeman), especially in July and August, when in the open both species occur in high numbers.     

High frequency hearing loss correlated with mutations in the GJB2 gene

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.     

Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA

Within the tribe Stenodermatini the systematics of the complex of species allied with the genus Artibeus has generated several alternative phylogenetic hypotheses. The most recent treatment recognized four genera (Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the most recent common ancestor of these four genera would include the common ancestor of all other currently recognized Stenodermatini genera except Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear satellite DNA repeat and 402 bp of DNA sequence variation from the mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern blot analyses, in situ hybridization, and mitochondrial DNA sequence data indicate that Enchisthenes is not closely related to Dermanura, Artibeus, or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common ancestor after diverging from the remainder of the Stenodermatini. If our conclusions are correct, then justification for recognizing Dermanura and Koopmania as generically distinct from Artibeus must be based on the magnitude of difference that distinguishes each rather than on the conclusion that to place them as congeneric with Artibeus creates a paraphyletic taxon.