A novel 62‐bp indel mutation in the promoter region of transforming growth factor‐beta 2 (TGFB2) gene is associated with body weight in chickens

Transforming growth factor‐beta 2 (encoded by TGFB2) is a growth factor that regulates a plethora of cellular functions. In this study, we sequenced the promoter and full‐length exon region of the chicken TGFB2 and found two mutations (g.‐640C>T and g.‐851_‐790del) within the promoter. The two polymorphisms were genotyped in 1030 pedigreed hens recorded for body weight at 7 (BW7), 9 (BW9), 11 (BW11), 13 (BW13), 17 (BW17) weeks old, egg weight at 36 weeks of age (EW36) and egg numbers from the age at first egg (AFE) to 40 weeks of age (EN40). Despite the fact that no mutations were found to have statistically significant genetic effects on egg production, the association results of growth traits showed that both g.‐640C>T and g.‐851_‐790del had significant effects on body weights and that both genotype g.‐640TT and g.‐851_‐790wt/wt were positive for body weight performance. Therefore, the polymorphisms of TGFB2, especially the g.‐851_‐790del mutation associated with body weight at almost all periods, could be potential useful genetic markers to improve the growth of Beijing You chickens.     

Two mutations in the 5′‐flanking region of the KITLG gene are associated with litter size of dairy goats

In this study, Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the 5′‐flanking region of the KITLG gene by DNA sequencing and primer‐introduced restriction analysis–polymerase chain reaction. Two novel SNPs (g.13090G>T and g.13664C>A) were identified (GenBank Accession no. KM658964). Furthermore, g.13090G>T and g.13664C>A loci were closely linked in SN and GZ breeds (r² > 0.33). Association analysis results showed that g.13090G>T and g.13664C>A SNPs significantly affected litter size (P < 0.05). The litter size of individuals with the combined genotype GG/CC from both dairy goat breeds was greater than that of individuals with TT/AA in average parity (P < 0.05). Known biochemical and physiological functions, along with our results, indicated that GG/CC could be used in marker‐assisted selection to choose individuals with greater litter size from both breeds. These results extend the spectrum of genetic variation in the caprine KITLG gene and may contribute to genetic resources and breeding of goats.     

Allelic variation in the serotonin transporter (5HTT) gene contributes to idiopathic pulmonary hypertension in children

Pulmonary hypertension is a potentially lethal condition, which affects adults and children alike. Genetic factors are implicated in the causation of primary pulmonary hypertension. We investigate the role of polymorphism in the 5HTT gene in the etiology of pulmonary hypertension in children aged 1-18.8 years. We have tested the hypothesis that the 5HTT gene does contribute to the pathogenesis of this disease in children by comparing the allelic frequencies of both the long and short variants between children with idiopathic pulmonary hypertension and pulmonary hypertension secondary to underlying pulmonary disease. We found that homozygosity for the long variant of 5HTT was highly associated with idiopathic pulmonary hypertension in children, suggesting perhaps a more important role for 5HTT gene function in the pathogenesis of early onset disease.     

Sequences from the 5′ flanking region of the ϵ‐globin gene support the relationship of Callicebus with the pitheciins

The purpose of this study was to determine nucleotide sequences from the 5′ flanking region of the ϵ‐globin gene of selected platyrrhine primates and to analyze the data for phylogenetic information and estimated times of divergence. We report new sequence data for two species of New World monkeys, Callicebus torquatus and Pithecia irrorata. We analyzed these data in conjunction with homologous sequences from other primate species. The data support the hypothesis that the titi monkeys (Callicebus) and seed predators (Tribe Pitheciini) form a clade (Subfamily Pitheciinae), and also provide limited support for that subfamily being allied with the atelines. We also present estimated dates of divergence for the Callicebus and pitheciin lineages. Am. J. Primatol. 48:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

White matter microstructure in bipolar disorder is influenced by the serotonin transporter gene polymorphism 5‐HTTLPR

Bipolar disorder (BD) is associated with signs of widespread disruption of white matter (WM) integrity. A polymorphism in the promoter of the serotonin transporter (5‐HTTLPR) influenced functional cortico‐limbic connectivity in healthy subjects and course of illness in BD, with the short (s) allele being associated with lower functional connectivity, and with earlier onset of illness and poor response to treatment. We tested the effects of 5‐HTTLPR on diffusion tensor imaging (DTI) measures of WM microstructure in 140 inpatients, affected by a major depressive episode in course of BD, of Italian descent. We used whole brain tract‐based spatial statistics in the WM skeleton with threshold‐free cluster enhancement of DTI measures of WM microstructure: axial, radial and mean diffusivity and fractional anisotropy. Compared with l/l homozygotes, 5‐HTTLPR*s carriers showed significantly increased radial and mean diffusivity in several brain WM tracts, including corpus callosum, cingulum bundle, uncinate fasciculus, corona radiata, thalamic radiation, inferior and superior longitudinal fasciculus and inferior fronto‐occipital fasciculus. An increase of mean and radial diffusivity, perpendicular to the main axis of the WM tract, is thought to signify increased space between fibers, thus suggesting demyelination or dysmyelination, or loss of bundle coherence. The effects of 5‐HTTLPR on the anomalous emotional processing in BD might be mediated by changes of WM microstructure in key WM tracts contributing to the functional integrity of the brain.     

Differential promoter activities of functional haplotypes in the 5′‐flanking region of human Sulfotransferase 1A1

Previously, we reported five common single nucleotide polymorphisms (SNPs), ?624G>C, ?396G>A, ?358A>C, ?341C>G, and ?294T>C, and six common haplotypes (CGACT, GAACT, GGAGC, GGACC, CAACT, and GAACC) in the 5′‐flanking region of the SULT1A1 gene that were associated with altered enzymatic activity. In the present study, we performed in vitro assays to determine the functional impact of these genetic variations on the promoter activity. Dual luciferase reporter assays revealed that these SNPs are located in a negative regulatory fragment of the SULT1A1 gene. Further experiments demonstrated that these SNPs and haplotypes affected promoter activities of SULT1A1. Electrophoretic mobility shift assays showed distinctive binding patterns for the SNPs ‐396G>A and ‐294T>C, due to differential binding affinities of the G/A alleles and the T/C alleles to nuclear proteins extracted from the liver carcinoma cell lines, HepG2 and Huh7. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:422–428, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21437     

A functional variant in ST2 gene is associated with risk of hypertension via interfering MiR‐202‐3p

Recent studies have suggested that interleukin 1 receptor‐like 1 (ST2) plays a critical role in pathogenesis of several cardiovascular disease conditions. In this study, we examined association of 13 single nucleotide polymorphisms (SNPs) of ST2 gene with essential hypertension (EH) risk in 1151 patients with EH and 1135 controls. Our study showed that variants rs11685424, rs12999364 and rs3821204 are highly associated with an increase in risk of EH, while rs6543116 is associated with a decrease risk of EH. Notably, in silico analyses suggested the G>C change of rs3821204, which located within the 3′UTR of soluble ST2 mRNA, disrupted a putative binding site for miR202‐3p. Functional analyses suggested that miR‐202‐3p significantly decreased soluble ST2‐G mRNA stability and inhibited its endogenous expression. Furthermore, we found increased plasma‐soluble ST2 (sST2) level was highly associated with CC genotype of rs3821204 in vivo. Taken together, our findings provide the first evidence that genetic variants in ST2 gene are associated with EH risk and variant rs3821204 may influence the development of EH by controlling sST2 expression.     

N‐β‐alanyldopamine metabolism,locomotor activity and sleep in Drosophila melanogaster ebony and tan mutants

Drosophila melanogaster Meigen mutants for N‐β‐alanyldopamine (NBAD) metabolism have altered levels of NBAD, dopamine and other neurotransmitters. The ebony¹ mutant strain has very low levels of NBAD and higher levels of dopamine, whereas the opposite situation is observed in the tan¹ mutant. Dopamine is implicated in the control of movement, memory and arousal, as well as in the regulation of sleep and wakefulness in D. melanogaster. N‐β‐alanyldopamine, which is best known as a cuticle cross‐linking agent, is also present in nervous tissue and has been proposed to promote locomotor activity in this fly. The daily locomotor activity and the sleep patterns of ebony¹ and tan¹ mutants are analyzed, and are compared with wild‐type flies. The tan¹ mutant shows reduced locomotor activity, whereas ebony¹ shows higher levels of activity than wild‐type flies, suggesting that NBAD does not promote locomotor activity. Both mutants spend less time asleep than wild‐type flies during night‐time; ebony shows more consolidated activity during night‐time and increased sleep latency, whereas tan is unable to consolidate locomotor activity and sleep in either phase of the day. The daily level of NBAD‐synthase activity is measured in vitro using wild‐type and tan¹ protein extracts, and the lowest NBAD synthesis is observed at the time of higher locomotor activity. The abnormalities in several parameters of the waking/sleep cycle indicate some dysfunction in the processes that regulates these behaviours in both mutants.     

A 66‐bp deletion in growth hormone releasing hormone gene 5′‐flanking region with largemouth bass recessive embryonic lethal

Growth hormone releasing hormone (GHRH) regulates the secretion of growth hormone (GH) in the pituitary gland. A 66‐bp deletion (c.‐923_‐858del) was detected in the 5′‐flanking sequence of the largemouth bass (Micropterus salmoides) GHRH gene. In two cultured random populations of adult individuals (A: n = 170 and B: n = 150), the genotype ratios of +/+:+/? were 2.5:1 and 2.8:1 respectively. Only one ?/? fish was detected. A Largemouth bass family was constructed with two heterozygous individuals (+/?) as parents. The genotype ratio of +/+:+/?:?/? in the filial generation embryos was 1:1.6:0.1 at the neurula and 1:2:0 at hatched larvae stages. This indicated that the 66‐bp deletion was a recessive lethal site and that homozygous individuals (?/?) died off in embryonic development. The growth traits (body weight, body length and body depth) were measured, and the GHRH mRNA expression levels in brain tissue were detected using real‐time PCR. The effects of genotype (+/?) on growth traits and GHRH mRNA expression were not significant. Although the cause of death was not clear, the results hint that the 66‐bp deletion site in GHRH 5′‐flanking sequence significantly affects the livability in largemouth bass embryonic development.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Kinetic studies of the effect of a 17‐nucleotide difference in the 0.3‐kb region containing the R‐U5‐5′ leader sequence of Friend murine leukemia virus on viral gene expression

In addition to the env gene, a 0.3‐kb fragment containing the R‐U5‐5′ leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr‐MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3‐kb fragment, and Rec5, carrying the 0.3‐kb fragment of the non‐neuropathogenic Fr‐MLV clone 57. These analyses suggested that the 0.3‐kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env‐mRNA rather than the amount of total viral mRNA or viral production.     

Comparative genome analysis with the human genome reveals chicken genes associated with fatness and body weight

The selection of meat-type chickens (broilers) for rapid growth has been accompanied by excessive fat deposition. In this study, we analysed 53 candidate genes that are associated with obesity and obesity-related traits in humans, for which we found chicken orthologues by BLAST searches. We have identified single nucleotide polymorphisms (SNPs) with significant differences in allele frequencies between broilers and layers in each of the following six candidate genes: adrenergic, beta-2-, receptor, surface (ADRB2); melanocortin 5 receptor (MC5R); leptin receptor (LEPR), McKusick-Kaufman syndrome (MKKS), milk fat globule-EGF factor 8 protein (MFGE8) and adenylate kinase 1 (AK1). To examine associations with fatness and/or body weight, we used birds of extreme phenotypes in F(2) and backcross populations with varying levels of abdominal fat weight per cent (%AFW) and body weight. We then assessed the level of gene expression by real-time PCR. In two genes, ADRB2 and MFGE8, we found significant association with %AFW. The ADRB2 gene was found to have a significantly higher expression in the liver of lean chickens compared with those of the fat individuals. We believe that this approach can be applied for the identification of other quantitative genes.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Hyperthermia‐induced seizures alter adenosine A1 and A2A receptors and 5′‐nucleotidase activity in rat cerebral cortex

Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia‐induced seizures on adenosine A₁ and A_2A receptors and 5′‐nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real‐time PCR, and 5′‐nucleotidase activity assays. Hyperthermic seizures were induced in 13‐day‐old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A₁ receptor density was observed using [³H]DPCPX as radioligand, and mRNA coding A₁ was observed 48 h after hyperthermia‐induced seizures. In contrast, a significant decrease in A_2A receptor density was detected, using [³H]ZM241385 as radioligand, 48 h after hyperthermia‐evoked convulsions. These short‐term changes in A₁ and A_2A receptors were also accompanied by a loss of 5′‐nucleotidase activity. No significant variations either in A₁ or A_2A receptor density or 5′‐nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A₁ and A_2A receptors and loss of 5′‐nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures.

     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.