Extremely high conservation in the untranslated Region as well as the coding region of CNP mRNAs throughout elasmobranch species

C-type natriuretic peptide (CNP) is a crucial osmoregulatory hormone in elasmobranchs, participating in salt secretion and drinking. In contrast to teleosts and tetrapods in which the NP family is composed of a group of structurally related peptides, we have shown that CNP is the sole NP in sharks. In the present study, CNP cDNAs were cloned from four species of batoids, another group of elasmobranchs. The cloned batoid CNP precursors contained a plausible mature peptide of 22 amino acid residues that is identical to most shark CNP-22s, but five successive amino acids were consistently deleted in the prosegment compared with shark precursors, supporting the diphyletic classification of sharks and rays. In addition, molecular phylogenetic trees of CNP precursors were consistent with a diphyletic interpretation. Except for the deletion, the nucleotide and deduced amino acid sequences of the CNP cDNAs are extremely well-conserved among all elasmobranch species, even between sharks and rays. Surprisingly, high conservation is evident not only for the coding region, but also for the untranslated regions. It is most likely that the high conservation is due to the low nucleotide substitution rate in the elasmobranch genome, and high selection pressure. The 3′-untranslated region of the elasmobranch CNP cDNAs contained three to six repeats of the ATTTA motif that is associated with the regulation of mRNA stability and translation efficiency. Alternative polyadenylation sites were also found; the long 3′-untranslated region contains a core of ATTTA motifs while the short form has only one or no ATTTA motif, indicating that the post-transcriptional modification of mRNA is important for regulation of CNP synthesis. These characteristics in the 3′-untranslated region were conserved among all elasmobranch CNP cDNAs. Since CNP has been implicated as a fast-acting hormone to facilitate salt secretion from the rectal gland, the conserved 3′-untranslated region most likely contributes to rapid regulation of CNP synthesis in elasmobranchs in response to acute changes in internal and external environments.

Non-quantized minimum free energy in untranslated region exons

In an attempt to improve automated gene prediction in the untranslated region of a gene, we completed an in-depth analysis of the minimum free energy for 8,689 sub-genetic DNA sequences. We expanded Zhang's classification model and classified each sub-genetic sequence into one of 27 possible motifs. We calculated the minimum free energy for each motif to explore statistical features that correlate to biologically relevant sub-genetic sequences. If biologically relevant sub-genetic sequences fall into distinct free energy quanta it may be possible to characterize a motif based on its minimum free energy. Proper characterization of motifs can lead to greater understanding in automated genefinding, gene variability and the role DNA structure plays in gene network regulation.

Our analysis determined: (1) the average free energy value for exons, introns and other biologically relevant sub-genetic sequences, (2) that these subsequences do not exist in distinct energy quanta, (3) that introns exist however in a tightly coupled average minimum free energy quantum compared to all other biologically relevant sub-genetic sequence types, (4) that single exon genes demonstrate a higher stability than exons which span the entire coding sequence as part of a multi-exon gene and (5) that all motif types contain a free energy global minimum at approximately nucleotide position 1,000 before reaching a plateau. These results should be relevant to the biochemist and bioinformatician seeking to understand the relationship between sub-genetic sequences and the information behind them.

Repair and tolerance of DNA damage in higher plants

DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana.

Exercise, skeletal muscle and circulating microRNAs

Regular exercise stimulates numerous structural, metabolic, and morphological adaptations in skeletal muscle. These adaptations are vital to maintain human health over the life span. Exercise is therefore seen as a primary intervention to reduce the risk of chronic disease. Advances in molecular biology, biochemistry, and bioinformatics, combined with exercise physiology, have identified many key signaling pathways as well as transcriptional and translational processes responsible for exercise-induced adaptations. Noncoding RNAs, and specifically microRNAs (miRNAs), constitute a new regulatory component that may play a role in these adaptations. The short single-stranded miRNA sequences bind to the 3' untranslated region of specific messenger RNAs (mRNAs) on the basis of sequence homology. This results in the degradation of the target mRNA or the inhibition of protein translation causing repression of the corresponding protein. While tissue specificity or enrichment of certain miRNAs makes them ideal targets to manipulate and understand tissue development, function, health, and disease, other miRNAs are ubiquitously expressed; however, it is uncertain whether their mRNA/protein targets are conserved across different tissues. miRNAs are stable in plasma and serum and their altered circulating expression levels in disease conditions may provide important biomarker information. The emerging research into the role that miRNAs play in exercise-induced adaptations has predominantly focused on the miRNA species that are regulated in skeletal muscle or in circulation. This chapter provides an overview of these current research findings, highlights the strengths and weaknesses identified to date, and suggests where the exercise-miRNA field may move into the future.

POEM, A 3-dimensional exon taxonomy and patterns in untranslated exons

Background: The existence of exons and introns has been known for thirty years. Despite this knowledge, there is a lack of formal research into the categorization of exons. Exon taxonomies used by researchers tend to be selected ad hoc or based on an information poor de-facto standard. Exons have been shown to have specific properties and functions based on among other things their location and order. These factors should play a role in the naming to increase specificity about which exon type(s) are in question.

Results: POEM (Protein Oriented Exon Monikers) is a new taxonomy focused on protein proximal exons. It integrates three dimensions of information (Global Position, Regional Position and Region), thus its exon categories are based on known statistical exon features. POEM is applied to two congruent untranslated exon datasets resulting in the following statistical properties. Using the POEM taxonomy previous wide ranging estimates of initial 5' untranslated region exons are resolved. According to our datasets, 29–36% of genes have wholly untranslated first exons. Untranslated exon containing sequences are shown to have consistently up to 6 times more 5' untranslated exons than 3' untranslated exons. Finally, three exon patterns are determined which account for 70% of untranslated exon genes.

Conclusion: We describe a thorough three-dimensional exon taxonomy called POEM, which is biologically and statistically relevant. No previous taxonomy provides such fine grained information and yet still includes all valid information dimensions. The use of POEM will improve the accuracy of genefinder comparisons and analysis by means of a common taxonomy. It will also facilitate unambiguous communication due to its fine granularity

Molecular cloning of natriuretic peptides from the heart of reptiles : loss of ANP in diapsid reptiles and birds

Atrial natriuretic peptide (ANP) and B-type NP (BNP) are hormones involved in homeostatic control of body fluid and cardiovascular regulation. Both ANP and BNP have been cloned from the heart of mammals, amphibians, and teleost fishes, while an additional cardiac peptide, ventricular NP, has been found in selected species of teleost fish. However, in chicken, BNP is the primary cardiac peptide identified thus far. In contrast, the types of NP/s present in the reptilian heart are unknown, representing a considerable gap in our understanding of NP evolution. In the present study, we cloned and sequenced a BNP cDNA from the atria of representative species of reptile, including crocodile, lizard, snake, and tortoise. In addition, we cloned BNP from the pigeon atria. The reptilian and pigeon BNP cDNAs had ATTTA repeats in the 3′ untranslated region, as observed in all vertebrate BNP mRNAs. A high sequence homology was evident when comparing reptile and pigeon preproBNP with the previously identified chicken preproBNP. In particular, the predicted mature BNP-29 was identical between crocodile, tortoise, and chicken, with pigeon having a single amino acid substitution; lizard and snake BNP had seven and nine substitutions, respectively. Furthermore, an ANP cDNA could only be cloned from the tortoise atria. Since ANP was not isolated from the heart of any non-chelonian reptile and appears to be absent in birds, we propose that the ANP gene has been lost after branching of the turtles in the amniote line. This data provides new avenues for research on NP function in reptiles.

Dopamine transporter (DAT1) VNTR polymorphism and alcoholism in two culturally different populations of South India

It is well established that the central dopaminergic reward pathway is likely involved in alcohol intake and the progression of alcohol dependence. Dopamine transporter (DAT1) mediates the active re-uptake of DA from the synapse and is a principal regulator of dopaminergic neurotransmission. The gene for the human DAT1 displays several polymorphisms, including a 40-bp variable number of tandem repeats (VNTR) ranging from 3 to 16 copies in the 3′-untranslated region (UTR) of the gene. To assess the role of this gene in alcoholism, we genotyped the VNTR of DAT1 gene in a sample of 206 subjects from the Kota population (111 alcohol dependence cases and 95 controls) and 142 subjects from Badaga population (81 alcohol dependence cases and 61 controls). Both populations inhabit a similar environmental zone, but have different ethnic histories. Phenotype was defined based on the DSM-IV criteria. Genotyping was performed using PCR and electrophoresis. The association of DAT1 with alcoholism was tested by using the Clump v1.9 program which uses the Monte Carlo method. In both Kota and Badaga populations, the allele A10 was the most frequent allele followed by allele A9. The genotypic distribution is in Hardy–Weinberg equilibrium in both cases and control groups of Kota and Badaga populations. The DAT1 VNTR was significantly associated with alcoholism in Badaga population but not in Kota population. Our results suggest that the A9 allele of the DAT gene is involved in vulnerability to alcoholism, but that these associations are population specific.

Regulation of miRNAs in human skeletal muscle following acute endurance exercise and short-term endurance training

The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r=-0.71; P= 0.04), miR-31 and HDAC4 protein (r =-0.87; P= 0.026) and miR-31 and NRF1 protein (r =-0.77; P= 0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3′ untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health

The role of jak2a in zebrafish hematopoiesis

Janus kinase 2 (Jak2) transduces signals from hematopoietic cytokines, and a gain-of-function mutation (Jak2617V>F) is associated with myeloproliferative diseases, particularly polycythemia vera. In this study, we examined the role of jak2a in zebrafish embryos in knock-down and overexpression studies using morpholinos (MOs) targeting the 5' untranslated region (UTR) (jak2aUTR-MO) and splice-site junction (jak2aSS-MO) of jak2a, a Jak inhibitor AG490 and a constitutive-active form of jak2a (jak2aca). At 18 and 24 hours after fertilization (hpf), jak2a is expressed predominantly in the intermediate cell mass (ICM; site of primitive hematopoiesis) of wild-type and chordin morphant embryos (characterized by expansion of ICM). Both jak2a MOs and AG490 reduced gata1+ (erythroid) cells in Tg(gata1:GFP) embryos, signal transducer and activation of transcription 5 (stat5) phosphorylation, and gene expression associated with early progenitors (scl and lmo2) and erythroid (gata1, he1 and ßhe1) and myeloid (spi1 [early] and mpo [late]) lineages. The chordin morphant is associated with increased stat5 phosphorylation, and both jak2a MOs and treatment with AG490 significantly ameliorated ICM expansion and hematopoietic gene up-regulation in these embryos. Injection of plasmid encoding jak2aca significantly increased erythropoiesis and expression of gata1, he1 and ßhe1, spi1, mpo, and l-plastin. In conclusion, zebrafish jak2a is involved in primitive hematopoiesis under normal and deregulated conditions.

Altered expression of two zinc transporters, SLC30A5 and SLC30A6, underlies a mammary gland disorder of reduced zinc secretion into milk

Two cases of zinc deficiency in breastfed neonates were investigated where zinc levels in the mothers' milk were reduced by more than 75 % compared to normal. The objective of this study was to find the molecular basis of the maternal zinc deficiency condition. Significant reductions in mRNA expression and protein levels of the zinc transporters SLC30A5 and SLC30A6 were found in maternal tissue, suggesting a causal link to the zinc-deficient milk. Novel splice variants of the SLC30A6 transcript were detected. No modifications were found in coding regions, or in transcription binding sites of promoter regions or in 5' and 3' untranslated regions of both transporters in lymphoblasts and fibroblasts isolated from both mothers. Altered DNA methylation in SLC30A5 at two CpG sites was detected and may account for the reduced levels of SLC30A5 mRNA and protein in lymphoblasts. Reduced SLC30A6 mRNA and protein levels in lymphoblasts may be secondary to reduced SLC30A5 expression, as they function as a heterodimer in zinc transport. In conclusion, two cases of zinc deficiency are linked to low levels of the SLC30A5 and SLC30A6 zinc transporters. These two zinc transporters have not been previously associated with zinc deficiency in milk.

The baby makes 3 plus project in the Great South Coast region: final report from the external evaluator, January 2016

This report presents the evaluation of the Baby Makes 3 Plus project in the Great South Coast region of Victoria. Baby Makes 3 Plus was one of 12 projects funded by the Department of Justice and Regulation in Victoria under initiative to support primary prevention and early intervention-focused partnership projects to address violence against women and children. The project provided the Baby Makes 3 relationship education program to new parents across the region, and conducted a variety of training to increase the skills of Great South Coast early years practitioners (the Plus component of the project). The three key objectives of the project were:• To increase the capacity of first time parents to build equal and respectful relationships in response to the lifestyle and relationship changes that follow the birth of a child.• To increase the capacity of health professionals and organisations to promote equal and respectful relationships during the transition to parenthood.• To building capacity to identify women at risk of experiencing family violence through a gender equity component of in-service training.

2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human A1 adenosine receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5- dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro 4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino- 3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA¹AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist- A₁AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH₂)_n- bridge, n ) 3 < n ) 4 < n ) 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA₁AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.

There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region–specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3µmol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion–induced molecular changes.

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two coppertransporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNKand WNDand their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles forMNKandWND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.

An exploration of sense of community, part 3: dimensions and predictors of psychological sense of community in geographical communities

Within the discipline of community psychology there remains considerable debate as to the latent structure of psychological sense of community (PSOC). One of the few theoretical discussions is that of McMillan and Chavis (1986), who hypothesized four dimensions: Belonging; Fulfillment of Needs; Influence; and Shared Connections. Discussion has also emerged in the literature regarding the role of identification within PSOC. However few studies have empirically investigated the role of identification in PSOC. The current study explored PSOC in a sample of residents of rural, regional and urban geographical communities (N = 669). In an endeavor to clarify the underlying dimensions of PSOC, a test battery included several measures of PSOC as well as measures of identification with the community. The study also examined the role of demographic factors in predicting PSOC. Results provided support for McMillan and Chavis' (1986) four dimensions of PSOC. Further, a fifth dimension emerged, that of Conscious Identification, suggesting that identification is separate to existing dimensions of PSOC. The demographic factors significantly associated with PSOC were type of region, with rural participants displaying higher PSOC than their urban counterparts; participation in local organizations; having children; and a vision of one’s neighborhood as broader than just a street or block. These results, and the implications for PSOC research, are discussed.