MALDI-TOF MS analysis of ribosomal proteins coded in S10 and spc operons rapidly classified the Sphingomonadaceae as alkylphenol polyethoxylate-degrading bacteria from the environment

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using ribosomal subunit proteins coded in the S10-spc-alpha operon as biomarkers was applied for the classification of the Sphingomonadaceae from the environment. To construct a ribosomal protein database, S10-spc-alpha operon of type strains of the Sphingomonadaceae and their related alkylphenol polyethoxylate (APEO(n) )-degrading bacteria were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains. The observed MALDI mass spectra of intact cells were compared with the theoretical mass of the constructed ribosomal protein database. The nine selected biomarkers coded in the S10-spc-alpha operon, L18, L22, L24, L29, L30, S08, S14, S17, and S19, could successfully distinguish the Sphingopyxis terrae NBRC 15098(T) and APEO(n) -degrading bacteria strain BSN20, despite only one base difference in the 16S rRNA gene sequence. This method, named the S10-GERMS (S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum) method, is a significantly useful tool for bacterial discrimination of the Sphingomonadaceae at the strain level and can detect and monitor the main APEO(n) -degrading bacteria in the environment.     

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

The evolutionary history of maternal plant-manipulation and larval feeding behaviours in attelabid weevils (Coleoptera; Curculionoidea)

Attelabid weevils manipulate specific structures of their host plants in a species-specific manner, e.g., cutting a shoot, cutting a leaf, rolling a leaf, or constructing sophisticated wrapped leaf rolls, presumably to secure the survivorship of eggs or larvae. To depict the evolutionary history of maternal plant-manipulation behaviours and larval feeding strategies of the family Attelabidae, molecular phylogenetic analyses were conducted by sequencing the nuclear 18S and 28S ribosomal DNA and the mitochondrial cytochrome oxidase subunit I genes. Our analyses indicated that the attelabid weevils form a monophyletic group, and that maternal plant-cutting behaviour originated in a common ancestor of Attelabidae, but was subsequently lost in several lineages. Monophyly of the subfamily Attelabinae was also recovered with high support, but the subfamily Rhynchitinae was not recovered as monophyletic. By employing maximum-likelihood-based ancestral state reconstructions, larval leaf-blade feeding was inferred to have evolved from boring of cut shoots/petioles. Moreover, maternal leaf-rolling behaviours likely originated independently in the Attelabinae and Byctiscini lineages, and in several Deporaini lineages. As the sophisticated behaviours constructing wrapped leaf rolls of Attelabinae originated only once and has not been lost from the lineage, these complex and innovative behaviours may have contributed to the success and diversification of the lineage.     

Polymorphism rs7895833 in the SIRT1 gene and its association with dyslipidaemia in the elderly

ObjectivesSirtuin 1 is a human protein involved in gene silencing and in inducing the deacetylation of proteins involved in the metabolic and adaptive response mechanisms. Polymorphisms in the SIRT1 gene have been studied with respect to aging. This study aims to determine the allelic and genotypic frequencies of the rs7895833 A/G polymorphism in the SIRT1 gene, and to identify the association between this polymorphism and the co-morbidities prevalent in the elderly population.Material and methodsA total of 216 patients were evaluated in an outpatient clinic in Central Brazil. The individuals underwent validated tests for cognitive impairment and falls risk, serum biochemistry analysis, as well as polymer chain reaction (PCR) with confronting two-pair primers for polymorphism genotyping.Resultsrs7895833 polymorphism in SIRT1 gene was observed in these patients as follows: AA (56/216), AG (138/216), and GG (22/216). The frequency of allele A was 0.58, and that of allele G was 0.42. In the multivariate analysis of the exploratory variables, glucose, high density lipoprotein (HDL) cholesterol, systemic arterial hypertension, dyslipidaemia, and depression, which were associated in the univariate analysis with the polymorphism rs7895833, only dyslipidaemia showed a statistically significant difference in a greater number of individuals with this polymorphism.ConclusionThe variant allele G of the SIRT1 gene polymorphism was found in 42% of these Brazilian geriatric patients, and was associated with dyslipidaemia. Further studies should be performed to confirm this result and to elucidate the role of SIRT1 in lipid metabolism.     

Bis-pyrene-labeled molecular beacon: a monomer-excimer switching probe for the detection of DNA base alteration

A new bis-pyrene-labeled oligonucleotide probe (BP-probe) has been designed for the detection of a single base mismatch in single strand (ss) DNA as a target. The sequence of BP-probe was chosen to form stem-loop structure similar to a molecular beacon (MB-probe), yielding bis-pyrene-labeled molecular beacon (BP-MB-probe). Partially double stranded (ds) BP-MB-probes were prepared by complexation with oligonucleotides whose sequences are complementary to the loop segment but not to the stem and exchangeable with the target DNA. The partially ds BP-MB-probes were shown to exhibit monomer fluorescence as major fluorescence, while the ss BP-MB-probe in the stem-loop form displays strong excimer fluorescence. The strand exchange reactions between partially ds BP-MB-probe and target ss DNA in the presence of cationic comb-type copolymer as a catalyst were monitored by the excimer fluorescence changes. The existence of a mismatched base can be determined by the slower PASE rates compared with fully matched DNA.     

Isolation of bacteria whose growth is dependent on high levels of CO2 and implications of their potential diversity

Although some bacteria require an atmosphere with high CO(2) levels for their growth, CO(2) is not generally supplied to conventional screening cultures. Here, we isolated 84 bacterial strains exhibiting high-CO(2) dependence. Their phylogenetic affiliations imply that high-CO(2) culture has potential as an effective method to isolate unknown microorganisms.     

Effects of low-intensity resistance exercise with short interset rest period on muscular function in middle-aged women

We investigated the effect of low-intensity resistance exercise training on muscular size and strength where the interset rest period was shortened so as to reduce the metabolite clearance. Female subjects (aged 45.4 +/- 9.5 years, n = 10) performed bilateral knee extension exercises in a seated position on an isotonic leg extension machine. The exercise sessions consisted of 3 sets of exercise at a mean intensity of approximately 50% 1RM with an interset rest period of 30 seconds and was performed twice a week for a period of 12 weeks. The strength and the cross-sectional area (CSA) of the knee extensors and flexors were examined with an isokinetic dynamometer and magnetic resonance imaging (MRI), respectively. The CSAs of the knee extensors and flexors increased by 7.1 +/- 1.6% (p < 0.01, Wilcoxon signed rank test) and 2.5 +/- 1.4% (not significant), respectively. Isometric and isokinetic strengths increased significantly (p < 0.01) at all velocities examined, whereas no significant change was observed in those of knee flexors. These results indicate that a low-intensity resistance exercise with a short interset rest period is substantially effective in inducing muscular hypertrophy and concomitant increase in strength.     

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.