Molecular and expression characterization of three gonadotropin subunits common alpha, FSH beta and LH beta in groupers

A SMART cDNA plasmid library was constructed from protogyous greasy grouper (Epinephelus coioides) pituitary, and the full-length cDNAs of three gonadotropin (GTH) subunits common alpha, FSH beta and LH beta were cloned and sequenced from the library. The nucleotide sequences of common alpha, FSH beta and LH beta subunit cDNAs are 647, 594 and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa, respectively. High homology was observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of common alpha, FSH beta and LH beta with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. Three polyclonal antibodies were prepared from the in vitro expressed common alpha, FSH beta and LH beta mature proteins, respectively. Western blot analysis and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three gonadotropin subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, common alpha was almost not detected in pituitaries, and FSH beta and LH beta expression levels were very low. While in the individuals with developing ovary, the expression of all three gonadotropin subunits reached to a high level. Immunofluoresence localization indicated that the grouper FSH beta cells mainly distributed in the middle area of PPD, while the LH beta cells distributed more widely, including in the area similar to the FSH beta cells and at the external periphery of pituitary near to the PI side. The common alpha might be expressed in both FSH beta and LH beta cells. Double immunofluoresence localization further demonstrated FSH beta and LH beta expression in distinct cells in the PPD area, although the FSH beta and LH beta cells were detected in the identical area of PPD. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Ovarian sertoli-leydig cell tumour with heterologous elements of gastrointestinal type associated with elevated serum alpha-fetoprotein level: an unusual case and literature review

Here we describe the case of a 19-year-old woman with a poorly differentiated ovarian Sertoli-Leydig cell tumor and an elevated serum alphafetoprotein level. The patient presented with diffuse abdominal pain and bloating. Physical examination, ultrasound, and magnetic resonance imaging revealed a right ovarian tumor that was histopathologically diagnosed as a poorly differentiated Sertoli-Leydig cell tumor with heterologous elements. Her alpha-fetoprotein serum level was undetectable after tumor resection. Sertoli-Leydig cell tumors are rare sex cord-stromal tumors that account for 0.5% of all ovarian neoplasms. Sertoli-Leydig cell tumors tend to be unilateral and occur in women under 30 years of age. Although they are the most common virilizing tumor of the ovary, about 60% are endocrineinactive tumors. Elevated serum levels of alpha-fetoprotein are rarely associated with Sertoli-Leydig cell tumors, with only approximately 30 such cases previously reported in the literature. The differential diagnosis should include common alpha-fetoprotein-producing ovarian entities such as germ cell tumors, as well as other non-germ cell tumors that have been rarely reported to produce this tumor marker.

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

The free alpha amino acid nitrogen contents of some common food fishes of Kakinada region, Andhra Pradesh

The moisture and free alpha amino nitrogen contents of some important food fishes and shell fishes of Kakinada region have been studied. Crustaceans and molluscs contain free alpha amino acids in quantities several times higher than all other aquatic animals examined in this study. Their probable role in the physiological activities of these animals has been discussed.

Identification and characterization of a novel splice variant of gonadotropin alpha subunit in the common carp Cyprinus carpio

In this study, an alternative splicing transcript GtH-alpha 291 was identified by RT-PCR, which is 291 nt and exists not only in the pituitary but also in the ovary in common carp Cyprinus carpio. The analysis of GtH-alpha 291 amino acid sequence by the SignalP server predicted that the 'missing segment' might characterize as a signal peptide. In the secretion experiment, GtH-alpha 357 subunit could be secreted out of HeLa cells while GtH-alpha 291 could not, which confirmed the prediction. Co-immunoprecipitation assay proved that GtH-alpha 291 subunit is able to interact with both FSH-beta and LH-beta as GtH-alpha 357 does. This is the first report concerning an alternative splicing transcript of a GtH alpha subunit. Further studies are necessary to elucidate the specific role of this variant in the regulation of gonadal development and sexual maturation. (c) 2007 The Authors.

Association study of the calcitonin gene-related polypeptide-alpha (CALCA) and the receptor activity modifying 1 (RAMP1) genes with migraine

Migraine is a common neurovascular brain disorder characterised by recurrent attacks of severe headache that may be accompanied by various neurological symptoms. Migraine is thought to result from activation of the trigeminovascular system followed by vasodilation of pain-producing intracranial blood vessels and activation of second-order sensory neurons in the trigeminal nucleus caudalis. Calcitonin gene-related peptide (CGRP) is a mediator of neurogenic inflammation and the most powerful vasodilating neuropeptide, and has been implicated in migraine pathophysiology. Consequently, genes involved in CGRP synthesis or CGRP receptor genes may play a role in migraine and/or increase susceptibility. This study investigates whether variants in the gene that encodes CGRP, calcitonin-related polypeptide alpha (CALCA) or in the gene that encodes a component of its receptor, receptor activity modifying protein 1 (RAMP1), are associated with migraine pathogenesis and susceptibility. The single nucleotide polymorphisms (SNPs) rs3781719 and rs145837941 in the CALCA gene, and rs3754701 and rs7590387 at the RAMP1 locus, were analysed in an Australian Caucasian population of migraineurs and matched controls. Although we find no significant association of any of the SNPs tested with migraine overall, we detected a nominally significant association (p = 0.031) of the RAMP1 rs3754701 variant in male migraine subjects, although this is non-significant after Bonferroni correction for multiple testing.

Common variants in the regulative regions of GRIA1 and GRIA3 receptor genes are associated with migraine susceptibility

Background Glutamate is the principal excitatory neurotransmitter in the central nervous system which acts by the activation of either ionotropic (AMPA, NMDA and kainate receptors) or G-protein coupled metabotropic receptors. Glutamate is widely accepted to play a major role in the path physiology of migraine as implicated by data from animal and human studies. Genes involved in synthesis, metabolism and regulation of both glutamate and its receptors could be, therefore, considered as potential candidates for causing/predisposing to migraine when mutated. Methods The association of polymorphic variants of GRIA1-GRIA4 genes which encode for the four subunits (GluR1-GluR4) of the alpha-amino-3- hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor for glutamate was tested in migraineurs with and without aura (MA and MO) and healthy controls. Results Two variants in the regulative regions of GRIA1 (rs2195450) and GRIA3 (rs3761555) genes resulted strongly associated with MA (P = 0.00002 and P = 0.0001, respectively), but not associated with MO, suggesting their role in cortical spreading depression. Whereas the rs548294 variant in GRIA1 gene showed association primarily with MO phenotype, supporting the hypothesis that MA and MO phenotypes could be genetically related. These variants modify binding sites for transcription factors altering the expression of GRIA1 and GRIA3 genes in different conditions. Conclusions This study represents the first genetic evidence of a link between glutamate receptors and migraine.

Bayesian refinement of association signals for 14 loci in 3 common diseases

To further investigate susceptibility loci identified by genome-wide association studies, we genotyped 5,500 SNPs across 14 associated regions in 8,000 samples from a control group and 3 diseases: type 2 diabetes (T2D), coronary artery disease (CAD) and Graves' disease. We defined, using Bayes theorem, credible sets of SNPs that were 95% likely, based on posterior probability, to contain the causal disease-associated SNPs. In 3 of the 14 regions, TCF7L2 (T2D), CTLA4 (Graves' disease) and CDKN2A-CDKN2B (T2D), much of the posterior probability rested on a single SNP, and, in 4 other regions (CDKN2A-CDKN2B (CAD) and CDKAL1, FTO and HHEX (T2D)), the 95% sets were small, thereby excluding most SNPs as potentially causal. Very few SNPs in our credible sets had annotated functions, illustrating the limitations in understanding the mechanisms underlying susceptibility to common diseases. Our results also show the value of more detailed mapping to target sequences for functional studies. © 2012 Nature America, Inc. All rights reserved.

Common variants in the HLA-DRB1-HLA-DQA1 HLA class II region are associated with susceptibility to visceral leishmaniasis

To identify susceptibility loci for visceral leishmaniasis, we undertook genome-wide association studies in two populations: 989 cases and 1,089 controls from India and 357 cases in 308 Brazilian families (1,970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, and combined analysis across the three cohorts for rs9271858 at this locus showed P combined = 2.76 × 10 -17 and odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.30-1.52. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion, the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species. © 2013 Nature America, Inc. All rights reserved.

Corymbia leaf oils, latitude, hybrids and herbivory: a test using common-garden field trials

Foliar oils, particularly monoterpenes, can influence the susceptibility of plants to herbivory. In plants, including eucalypts, monoterpenes are often associated with plant defence. A recent analysis revealed an increase in foliar oil content with increasing latitudinal endemism, and we tested this pattern using three eucalypt taxa comprising a latitudinal replacement cline. We also examined the relative concentrations of two monoterpenes (alpha-pinene and 1,8-cineole), for which meta-analyses also showed latitudinal variation, using hybrids of these three taxa with Corymbia torelliana. These, and pure C. torelliana, were then assessed in common-garden field plots for the abundance and distribution of herbivory by four distinct herbivore taxa. Differing feeding strategies among these herbivores allowed us to test hypotheses regarding heritability of susceptibility and relationships to alpha-pinene and 1,8-cineole. We found no support for an increase in foliar oil content with increasing latitude, nor did our analysis support predictions for consistent variation in alpha-pinene and 1,8-cineole contents with latitude. However, herbivore species showed differential responses to different taxa and monoterpene contents. For example, eriophyid mites, the most monophagous of our censused herbivores, avoided the pure species, but fed on hybrid taxa, supporting hypotheses on hybrid susceptibility. The most polyphagous herbivore (leaf blister sawfly Phylacteophaga froggatti) showed no evidence of response to plant secondary metabolites, while the distribution and abundance patterns of Paropsis atomaria showed some relationship to monoterpene yields.

A Case Study of the Conformation of Poly(alpha-aminoisobutyric acid): alpha- or 310-Helix

The relative stabilities of a- and Blo-helical structures for polymers of a-aminoisobutyric acid (Aib) have been worked out, using the classical potential energy functions. To make a comparative study, we have used Buckingham "6-exp" and Kitaigorodsky's potential functions. Conformational analysis of the dipeptide segment with Aib residue indicates the necessity for nonplanar distortion of the peptide unit, which is a common feature in the observed crystal structures with Aib residues. In the range of Aw -10 to +loo studied, a-helical conformations are preferred in the region -3" < Aw < +loo, and Blo-helical conformations are preferred in the region -3" > Aw > -10'. Minimum energy conformations for right-handed structures are found in the +ue region of Aw and correspondingly for left-handed structures in the -ue region of Aw. For Aw - 6", a-helical structures have four- or near fourfold symmetry with h - 1.5 A. Such a helix with n = 4 and h = 1.5 A is termed an a'-helix. This structure is found to be consistent with the electron diffraction data of Malcolm3 and energetically more favorable than the standard 310-helix.

Recognition of Interaction Interface Residues in Low-Resolution Structures of Protein Assemblies Solely from the Positions of C alpha Atoms

Background: The number of available structures of large multi-protein assemblies is quite small. Such structures provide phenomenal insights on the organization, mechanism of formation and functional properties of the assembly. Hence detailed analysis of such structures is highly rewarding. However, the common problem in such analyses is the low resolution of these structures. In the recent times a number of attempts that combine low resolution cryo-EM data with higher resolution structures determined using X-ray analysis or NMR or generated using comparative modeling have been reported. Even in such attempts the best result one arrives at is the very course idea about the assembly structure in terms of trace of the C alpha atoms which are modeled with modest accuracy. Methodology/Principal Findings: In this paper first we present an objective approach to identify potentially solvent exposed and buried residues solely from the position of C alpha atoms and amino acid sequence using residue type-dependent thresholds for accessible surface areas of C alpha. We extend the method further to recognize potential protein-protein interface residues. Conclusion/Significance: Our approach to identify buried and exposed residues solely from the positions of C alpha atoms resulted in an accuracy of 84%, sensitivity of 83-89% and specificity of 67-94% while recognition of interfacial residues corresponded to an accuracy of 94%, sensitivity of 70-96% and specificity of 58-94%. Interestingly, detailed analysis of cases of mismatch between recognition of interface residues from C alpha positions and all-atom models suggested that, recognition of interfacial residues using C alpha atoms only correspond better with intuitive notion of what is an interfacial residue. Our method should be useful in the objective analysis of structures of protein assemblies when positions of only C alpha positions are available as, for example, in the cases of integration of cryo-EM data and high resolution structures of the components of the assembly.

Common and rare variants of the renin-angiotensin system and their relation to antihypertensive drug responses

Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.

Proteomic Identification of Haptoglobin alpha 2 as a Glioblastoma Serum Biomarker: Implications in Cancer Cell Migration and Tumor Growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin alpha 2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = < 0.0001 and < 0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.