Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

Two Aspergillus niger strains (GH1 and PSH) previously isolated from a semiarid region of Mexico were characterized for their effectiveness in converting pomegranate ellagitannins (ET) into ellagic acid (EA) in a solid state fermentation (SSF). Pomegranate seeds and husk were used as support for the SSF. Released EA was evaluated by liquid chromatography. Yields of 6.3 and 4.6 mg of EA per gram of dried pomegranate husk were obtained with A. niger GH1 and PSH, respectively. Total hydrolyzable polyphenols of pomegranate husk were degraded during the first 72 h of culture (71 and 61%, by GH1 and PSH strains, respectively). Tannin acyl hydrolase activity was not clearly associated with EA production. EA that accumulated in cultures of A. niger GH1 was remarkably pure after a simple extraction process. Pomegranate husk is a good support, and at the same time an excellent substrate in the production of high commercial interest metabolites like EA due the degradation of its ET content.

Although most pigs recover rapidly from stresses associated with the transition of weaning, a portion of the population lags behind their contemporaries in growth performance. The underlying biological and molecular mechanisms involved in postweaning differences in growth performance are poorly understood. The objective of this experiment was to use transcriptional profiling of skeletal muscle and adipose tissue to develop a better understanding of the metabolic basis for poor weaned-pig transition. A total of 1,054 pigs was reared in commercial conditions and weighed at birth, weaning, and 3 wk postweaning. Transition ADG (tADG) was calculated as the ADG for the 3-wk period postweaning. Nine pigs from both the lowest 10th percentile (low tADG) and the 60th to 70th percentile (high tADG) were harvested at 3 wk postweaning. Differential expression analysis was conducted in longissimus dorsi muscle (LM) and subcutaneous adipose tissue using RNA-Seq methodology. In LM, 768 transcripts were differentially expressed (DE), 327 with higher expression in low tADG and 441 with higher expression in high tADG pigs (q < 0.10). Expression patterns measured in LM by RNA-Seq were verified in 30 of 32 transcripts using quantitative PCR. No DE transcripts were identified in adipose tissue. To identify biological functions potentially underlying the effects of tADG on skeletal muscle metabolism and physiology, functional annotation analysis of the DE transcripts was conducted using DAVID and Pathway Studio analytic tools. The group of DE genes with lower expression in LM of low tADG pigs was enriched in genes with functions related to muscle contraction, glucose metabolism, cytoskeleton organization, muscle development, and response to hormone stimulus (enrichment score>> 1.3). The list of DE genes with higher expression in low tADG LM was enriched in genes with functions related to protein catabolism (enrichment score>> 1.3). Analysis of known gene-gene interactions identified possible regulators of these differences in gene expression in LM of high and low tADG pigs; these include forkhead box O1 (FOXO1), growth hormone (GH1), and the glucocorticoid receptor (NR3C1). Differences in gene expression between poor transitioning pigs and their contemporaries indicate a shift to decreased protein synthesis, increased protein degradation, and reduced glucose metabolism in the LM of low tADG pigs.

Idiopathic pituitary insufficiency (IPI) is diagnosed in 10% of all hypopituitary patients. There are several known and unknown aetiologies within the IPI group. The aim of this study was to investigate an adult IPI population for genetic cause according a screening schedule. From files of 373 GH deficient (GHD) patients on GH replacement 50 cases with IPI were identified. Of the 39 patients that approved to the study, 25 patients were selected for genetic investigation according to phenotype and 14 patients were not further tested, as sporadic isolated GHD (n = 9) and GHD with diabetes insipidus (n = 5) have low probability for a known genetic cause. Genotyping of all coding exons of HESX1, LHX4, PROP1, POU1F1 and GH1 genes were performed according to a diagnostic algorithm based on clinical, hormonal and neuroradiological phenotype. Among the 25 patients, an overall rate of 8% of mutations was found, and a 50% rate in familial cases. Among two sibling pairs, one pair that presented with complete anterior pituitary insufficiency, had a compound heterozygous PROP1 gene mutation (codons 117 and 120: exon 3 p Phe 117 Ile (c349 T>A) and p Arg 120 Cys (c358 C>T)) with a phenotype of very late onset ACTH-insufficiency. In the other sibling pair and in the sporadic cases no mutation was identified. This study suggests that currently known genetic causes are rare in sporadic adult IPI patients, and that systematic genetic screening is not needed in adult-onset sporadic cases of IPI. Conversely, familial cases are highly suspect for genetic causes.

Isolated growth hormone deficiency (IGHD) may be congenital, often due to genetic mutations, or acquired as a result of other factors such as cranial irradiation. The commonest genes implicated in its genetic etiology are those encoding growth hormone (GH1) and the receptor for GH-releasing hormone (GHRHR). Rarely, IGHD may be caused by mutations in transcription factors (HESX1, SOX3, OTX2) or be the first presentation before the development of other pituitary hormone deficiencies. IGHD has been classified in four genetic forms (type IA, IB, II and III). Despite the increasing number of genes implicated in the etiology of IGHD, mutations in known genes account only for a small percentage of cases; therefore, other as yet unidentified factors may be implicated in its etiology. Although there is no strict genotype/phenotype correlation in patients with IGHD, there are some emerging patterns that may guide us towards a genetic diagnosis of the condition. There is increasing understanding that the phenotype of patients with IGHD is highly variable and sometimes even evolving, dictating the need for long term follow-up in these cases.

OBJECTIVE

Five per cent to 30% of cases of idiopathic isolated GH deficiency (IGHD) have first-degree relatives with short stature, which is suggestive of a genetic aetiology. The HYPOPIT study aimed to obtain an overall picture of gene encoding pituitary GH (GH1) and gene encoding GH releasing hormone-receptor (GHRHR) defects in a Dutch IGHD cohort and to relate them with clinical parameters.

METHODS

Genetic analysis was performed of exons and exon-intron boundaries of GH1 and GHRHR in 89 Caucasian IGHD patients from 81 families, using denaturing high-performance liquid chromatography (dHPLC), DNA sequencing and multiplex ligation-dependent probe amplification. In addition, we performed functional studies on novel identified GH1 exonic variants.

RESULTS

Five different heterozygous GH1 mutations were present in 5 out of 81 participating families (6.1%), whereas no mutations in GHRHR were found. Patients with IGF-I SDS < -4.0 and peak GH levels < 5.7 mU/l had a mutation frequency of 40%, in contrast to 6.8% in patients with only one criterion, and 0.0% in patients with none of these criteria (P = 0.00007). Five new GH1 and two GHRHR variants were also identified; two of them (GH1 F92L and D153H) caused a marked reduction of GH secretion in vitro.

CONCLUSIONS

GH1 and GHRHR mutations are rare in Caucasian Dutch IGHD patients, which suggests the involvement of other genetic determinants in the aetiology of IGHD. IGF-I < -4.0 and peak GH levels < 5.7 mU/l are strong predictors of GH1 mutations in the studied population.

The somatotrophic axis consisting of pituitary-derived growth hormone and circulating insulin-like growth factor 1 has been well established as key regulators of animal health, metabolism, lactation, fertility, body composition and growth rate. The aim of this study was to simultaneously quantify the associations between SNPs in candidate genes of the somatotrophic axis (i.e., IGF-1, GH1 and GHR) with performance traits in Holstein-Friesian (HF) dairy cattle. Both novel SNPs identified previously by this group alongside other published SNPs within these genes were analysed for associations with performance in dairy cattle. Multiple regression analyses regressing genetic merit of up to 848 HF sires on novel SNPs (n = 76) and published SNPs (n = 33) were undertaken using weighted animal mixed linear models. Twenty-three SNPs were significantly associated with at least one of 18 traits analysed and involved in milk production, udder health, fertility and growth. Eight traits including milk fat composition, carcass conformation, stature, chest width, body depth, rump width, carcass and cull cow weight were independently associated with SNPs in two genes. Furthermore, for several traits including milk fat yield, somatic cell count, survival and carcass fat, SNPs in all three genes were independently associated with performance. Milk fat yield and carcass fat showed the highest number of independent associations across all three genes with five SNPs associated with both traits. The cumulative effects of the favourable alleles of all five SNPs across GH1, GHR and IGF-1 result in an increase of 5.9 kg and 28.6 units of milk fat and carcass fat, respectively. Cow survival was associated with a single SNP in each of the three genes with a cumulative allele effect of 1.5%. Independent effects of polymorphisms in GH1, GHR and IGF-1 reinforce the central role of the somatotrophic axis on animal development and performance.

Sex steroids play an important role in modulating pulsatile growth hormone (GH) release, acting at both hypothalamic and pituitary level in both humans and experimental animals. Selective estrogen receptor modulators (SERMs) act as either estrogen receptor agonists or antagonists in a tissue-selective manner. In postmenopausal women, serum GH levels correlate positively with endogenous estradiol levels and insulin-like grwoth factor-I (IGF-I) is positively related to bone mineral density (BMD) at the spine and hip. The aim of the present study was to evaluate, for the first time, the direct effect of LY117018, an analog of raloxifene, on GH secretion from both human and rodent pituitary cells in vitro. Our results demonstrated that pharmacological concentrations of the raloxifene analog LY117018 can stimulate GH secretion through a direct action on the pituitary. LY117018 also showed an estrogen-like activity, inducing the proliferation of rat pituitary GH-secreting adenomatous cells (GH1).

Using a gel mobility-shift assay, a nuclear protein factor was identified in cardiac myocyte which binds specifically to a DNA fragment from the 5' region of the alpha-myosin heavy chain gene shown previously to contain a thyroid hormone-sensitive element. Methylation interference experiments located the binding site within a 24-base pair sequence from positions -599 to -576. A double-stranded synthetic oligonucleotide containing this 24-base pair sequence bound to the factor and effectively competed with the natural binding site for factor binding. The factor was present in rat and human fibroblasts, and rat GH1 cells as well as L6E9 myoblasts and myotubes. The specificity with which this factor binds to DNA suggests that it could be involved in regulation of the alpha-myosin heavy chain gene.

Mutant N282T of a thermostable beta-glycosidase from GH1 family (TtbetaGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-beta-d-galactopyranoside (oNPGal) which competes with the condensation reaction and entails undesirable effects. In order to prevent this reaction, we rationally modified this enzyme at the [+1]/[+2] subsites so that oNPGal would bind less tightly. Molecular modeling (MM) suggested the mutation A221W, which decreased the affinity of the donor at these sites and moved it away from the bound galactose at the -1 subsite. A single (A221W) and a double mutant (A221W/N282T) were constructed, and they gave rise to a drastic decrease in self-condensation. The A221W mutant had no transglycosylation activity whereas the A221W/N282T mutant still displayed a condensation activity, comparable to that of the N282T mutant for the transfer on pNPGlcNAc. MM revealed that the double mutant A221W/N282T could induce the synthesis of a glycosidic bond between a donor and an acceptor displaying an equatorial 4-position. Moreover, it is suggested that mutation N282T could change the orientation of residue N219, leading to a stabilization of the acceptor with a new hydrogen bond. This finding opens the way to further improvements of evolved transglycosidases.

The growth hormone 1 (GH1)/insulin-like growth factor I (IGF-I) axis plays an important role in the development of breast cancer. By binding to its receptor, GH1 stimulates the production of IGF-I and its binding protein IGFBP3, resulting in the regulation of cell proliferation, differentiation and apoptosis. The GH1 gene expression is regulated by a highly polymorphic proximal promoter and a distal locus control region (LCR) 14.5 kb upstream of the gene. We investigated the effect of single nucleotide polymorphisms (SNPs) in the LCR and in the promoter region and an intron 4 SNP (IVS4+90 T/A) on breast cancer risk in a large cohort of Polish and German familial breast cancer cases and controls. SNPs in the LCR did not show an influence on breast cancer risk, either alone or in haplotypes. Three SNPs in the promoter region (G-340T, A-68G/C and A-63T/C) showed an increased and four SNPs (A-137G, G-119T, G-93delG and T-4G) a decreased allele frequency in the cases compared with the controls. Two of the SNPs (A-137G and G-93delG) lead to a decreased breast cancer risk among the minor allele carriers in the joint analysis of the two populations (odds ratio (OR) 0.62, 95% confidence interval (95% CI) 0.44-0.89, P = 0.01 and OR 0.65, 95% CI 0.47-0.90, P = 0.01, respectively). Haplotype analysis with these seven promoter SNPs revealed a protective association (OR 0.61, 95% CI 0.37-1.00, P = 0.04) for the haplotype GAGdAAT, containing the G-93delG variant allele, which in the single analysis already showed a protective effect. The effect was marginally stronger in combination with the LCR GC haplotype (OR 0.49, 95% CI 0.23-1.01, P = 0.04).

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (<em>GH1</em>) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (<em>GH1</em>0 and <em>GH1</em>1), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P<0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P<0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P<0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P<0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P<0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.

The interactions of two plant annexins, annexin 24(Ca32) from Capsicum annuum and annexin Gh1 from Gossypium hirsutum, with phospholipid membranes have been characterized using liposome-based assays and adsorption to monolayers. These two plant annexins show a preference for phosphatidylserine-containing membranes and display a membrane binding behavior with a half-maximum calcium concentration in the sub-millimolar range. Surprisingly, the two plant annexins also display calcium-independent membrane binding at levels of 10-20% at neutral pH. This binding is regulated by three conserved surface-exposed residues on the convex side of the proteins that play a pivotal role in membrane binding. Due to quantitative differences in the membrane binding behavior of N-terminally His-tagged and wild-type annexin 24(Ca32), we conclude that the N-terminal domain of plant annexins plays an important role, reminiscent of the findings in their mammalian counterparts. Experiments elucidating plant annexin-mediated membrane aggregation and fusion, as well as the effect of these proteins on membrane surface hydrophobicity, agree with findings from the membrane binding experiments. Results from electron microscopy reveal elongated rodlike assemblies of plant annexins in the membrane-bound state. It is possible that these structures consist of protein molecules directly interacting with the membrane surface and molecules that are membrane-associated but not in direct contact with the phospholipids. The rodlike structures would also agree with the complex data from intrinsic protein fluorescence. The tubular lipid extensions suggest a role in the membrane cytoskeleton scaffolding or exocytotic processes. Overall, this study demonstrates the importance of subtle changes in an otherwise conserved annexin fold where these two plant annexins possess distinct modalities compared to mammalian and other nonplant annexins.

The capacity of the globular domain of the chicken erythrocyte linker histone H5 (GH5) to self-associate in solution has been demonstrated by chemical cross-linking with dimethyl 3,3'-dithiobis-(propionimidate) (DTBP), dithiobis(succinimidyl propionate) (DSP), and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Several observations suggest that the GH5-GH5 interactions that mediate self-association are specific: (a) Incubation with each of the above reagents produces a discrete and characteristic pattern of cross-linked products; (b) GH1, the related peptide from chicken erythrocyte H1, is not cross-linked under the same conditions; (c) GH5 is not cross-linked with disuccinimidyl tartrate (DST), which has a shorter cross-linking span (6.4 A) than the other reagents (12 A); and (d) analysis of cross-linking as a function of peptide concentration provides an equilibrium constant for GH5 self-association of (4.8 +/- 1.3) x 10(3) M-1. The ability of GH5 to specifically self-associate is compatible with the proposal [Thoma, F., Koller, T., & Klug, A. (1979) J. Cell Biol. 83, 403-427] that linker histone globular domains occupy an axial position within the higher order chromatin fiber; the spatial juxtaposition of the GH5 domains at this location would be expected to promote their association and exert a stabilizing effect upon higher order chromatin structure.

Using cultured GH1 cells, a growth hormone and prolactin-producing rat pituitary cell line, we have shown that n-butyrate and other short chain carboxylic acids stimulate histone acetylation and elicit a reduction of thyroid hormone nuclear receptor which is inversely related to the extent of acetylation (Samuels, H. H., Stanley, F., Casanova, J., and Shao, T. C. (1980) J. Biol. Chem. 255, 2499-2508). In this study, we compared the n-butyrate and propionate modulation of receptor levels to regulation of the growth hormone and prolactin response by 3,5,3'-triiodo-L-thyronine (L-T3). n-Butyrate (0.1-10 mM) did not stimulate growth hormone production. L-T3 stimulated the growth hormone response 4- to 5-fold and n-butyrate (0.5-1 mM) increased L-T3 stimulation of growth hormone production 1.5- to 2-fold compared to L-T3 alone. L-T3 stimulation of growth hormone production at higher n-butyrate concentrations decreased in parallel with the n-butyrate-mediated reduction of receptor levels. In contrast with the growth hormone response, n-butyrate (0.5 mM) increased basal prolactin production about 5-fold. Prolactin production, which is inhibited 25 to 50% by L-T3, was stimulated between 20- and 70-fold by L-T3 + n-butyrate (0.5-1 mM) and this decreased at higher n-butyrate levels. Prolactin mRNA and growth hormone mRNA levels paralleled the changes in prolactin and growth hormone production rates. These effects of L-T3, n-butyrate, or L-T3 + n-butyrate appeared unrelated to changes in cAMP levels or global changes in DNA methylation of the growth hormone or prolactin genes. Propionate elicited the same effects as n-butyrate but at a 5- to 10-fold higher concentration consistent with their relative effect on stimulating acetylation of chromatin proteins. These results suggest that prolactin gene expression is under partial regulatory repression which is reversed by a carboxylic acid-mediated postsynthetic modification event which allows for stimulation of the prolactin gene by thyroid hormone.

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-β-1,4-glucosidase and exo-β-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.

OBJECTIVE

Identification of signal transduction genes related to drug resistance in pediatric acute lymphoblastic leukemia (ALL).

METHODS

Ex vivo drug resistance in 107 children, divided into study and validation groups, was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) drug resistance assay. The gene expression profile was identified by microarray analysis and validated by quantitative reverse transcription polymerase chain reaction.

RESULTS

A set of five genes involved in signal transduction, present in each resistance profile, was identified. The expression of four genes was up-regulated: Gardner-Rasheed feline sarcoma viral oncogene homolog, v-Fgr (FGR), S100 calcium binding protein A11 (S100A11), formyl peptide receptor 1 (FPR1), ArfGAP with RhoGAP domain, ankyrin repeat and PH1 domain (ARAP1), while the expression of growth hormone 1 (GH1) was found to be down-regulated in resistant leukemia blasts.

CONCLUSIONS

Ex vivo exposure of leukemia cells to anticancer drugs induces changes in the expression of genes involved in cell signaling pathways. These genes play an important role in the mechanism of cellular drug resistance.

BACKGROUND

The pituitary is a critical neuroendocrine gland that is comprised of five hormone-secreting cell types, which develops in tandem during the embryonic stage. Some essential genes have been identified in the early stage of adenohypophysial development, such as PITX1, FGF8, BMP4 and SF-1. However, it is likely that a large number of signaling molecules and transcription factors essential for determination and terminal differentiation of specific cell types remain unidentified. High-throughput methods such as microarray analysis may facilitate the measurement of gene transcriptional levels, while Expressed sequence tag (EST) sequencing, an efficient method for gene discovery and expression level analysis, may no-redundantly help to understand gene expression patterns during development.

RESULTS

A total of 9,271 ESTs were generated from both fetal and adult pituitaries, and assigned into 961 gene/EST clusters in fetal and 2,747 in adult pituitary by homology analysis. The transcription maps derived from these data indicated that developmentally relevant genes, such as Sox4, ST13 and ZNF185, were dominant in the cDNA library of fetal pituitary, while hormones and hormone-associated genes, such as GH1, GH2, POMC, LHbeta, CHGA and CHGB, were dominant in adult pituitary. Furthermore, by using RT-PCR and in situ hybridization, Sox4 was found to be one of the main transcription factors expressed in fetal pituitary for the first time. It was expressed at least at E12.5, but decreased after E17.5. In addition, 40 novel ESTs were identified specifically in this tissue.

CONCLUSIONS

The significant changes in gene expression in both tissues suggest a distinct and dynamic switch between embryonic and adult pituitaries. All these data along with Sox4 should be confirmed to further understand the community of multiple signaling pathways that act as a cooperative network that regulates maturation of the pituitary. It was also suggested that EST sequencing is an efficient means of gene discovery.

BACKGROUND

Causal mutations have been detected only in a minority of isolated GH deficiency (IGHD) patients. Idiopathic IGHD might be the result of the interaction between several low-penetrance genetic factors and the environment.

OBJECTIVE

The aim of this study was to test the contribution to IGHD of genetic variations in the GH1 gene regulatory regions.

METHODS

A case-control association study was performed including 118 sporadic IGHD patients with a nonsevere phenotype (height -4/-1 sd score and partial GH deficiency) and two control groups, normal stature (n=200) and short-stature individuals with normal GH secretion (n=113). Seven single-nucleotide polymorphisms in the GH1 promoter, one in the IVS4 region, and two in the locus control region were analyzed.

RESULTS

The -57T allele within the vitamin D-responsive element showed a positive significant association when comparing patients with normal (P=0.006) or short stature (P=0.0011) controls. The genotype -57TT showed an odds ratio of 2.93 (1.44-5.99) and 2.99 (1.42-6.31), respectively. The functional relevance of the -57 variation was demonstrated by the luciferase assay in the presence of vitamin D. The vitamin D-induced inhibition of luciferase activity was significantly (P=0.012) stronger for the promoter haplotype carrying the associated variation -57T [haplotype #1 (hp#1)] with respect to hp#2, bearing -57G. Replacement of the T with a G at -57 on hp#1 abolished the repression, demonstrating that the T at position -57 is necessary to determine the greater vitamin D-induced inhibitory effect of hp#1. EMSA experiments showed a different band-shift pattern of the T and G sequences.

CONCLUSIONS

The common -57G->>T polymorphism contributes to IGHD susceptibility, indicating that it may have a multifactorial etiology.

Human Growth Hormone gene ( GH1 ) resides on chromosome 17q22-24 and it is expressed in somatotropic cells of the anterior pituitary gland. While there are multiple causes of GH Deficiency (GHD) a significant proportion have a genetic basis. The most severe Mendelian form of IGHD, called IGHD IA, has an autosomal recessive mode of inheritance. While affected individuals can have short lengths at birth and hypoglycemia in infancy, all develop severe dwarfism by six months of age. Although short stature, delayed growth velocity, and delayed skeletal maturation all occur with IGHD, none are specific for IGHD 1A. GH1 gene deletions, frameshifts, or nonsense mutations cause complete absence of GH in IGHD 1A. Thus IGHD 1A is best described as being complete GHD caused by severe loss of function GH1 gene mutations rather than being limited to only those having GH1 gene deletions. Interestingly, GH1 gene deletions are recurring mutations that can arise through unequal recombination in meiosis rather than by allele sharing through common descent. Individuals with IGHD 1A develop severe dwarfism in early infancy and often develop anti-GH antibodies after receiving exogenous GH. These antibodies can prevent the growth response expected from exogenous GH therapy. Individuals who are heterozygous for a GH1 gene deletion but whose other GH1 allele is not deleted and produces a non truncated product are usually immune tolerant.

BACKGROUND

Dominant-negative GH1 mutations cause familial isolated growth hormone deficiency type II (IGHD II), which is characterized by GH deficiency, occasional multiple anterior pituitary hormone deficiencies, and anterior pituitary hypoplasia. The basis of the variable expression and progression of IGHD II among relatives who share the same GH1 mutation is poorly understood.

OBJECTIVE

We hypothesized that the cellular ratios of mutant/normal GH1 transcripts would correlate with the severity of the IGHD II phenotype. We determined the relative amounts of mutant and normal GH1 transcripts in cell lines and correlated transcript ratios with severity.

METHODS

Members of the same IGHD II kindred were genotyped for the GH1 E3+1 G/A mutation by DNA sequencing. Ratios of their 17.5-kDa (mutant)/22-kDa (normal) GH1 transcripts were determined in cultured lymphocytes (CLs), and these ratios were correlated with height sd scores obtained before GH replacement therapy.

RESULTS

Ratios of 17.5-/22-kDa GH1 transcripts in CLs from family members with the same IGHD II mutation correlated with differences in their height SD scores.

CONCLUSIONS

Our findings suggest that expression levels of both the mutant and normal GH1 allele are important in the pathogenesis of IGHD II, that the ratio of mutant/normal transcripts may be a predictive marker of the penetrance and severity of IGHD II, and that CLs may be useful as surrogates to study GH1 transcript expression of subjects whose anterior pituitary cells are not available.

Growth hormone deficiency (GHD) results from variations affecting the production and release of growth hormone (GH) and is of 2 types: isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD). IGHD results from mutations in GH1 and GHRHR while CPHD is associated with defects in transcription factor genes PROP1, POU1F1, and HESX1. The present study reports on screening of POU1F1, PROP1, and HESX1 in CPHD patients and the novel variations identified. Fifty-one CPHD patients from 49 unrelated families clinically diagnosed on the basis of biochemical and imaging investigations along with 100 controls were enrolled. Detailed family history was noted from all participants and 5 ml blood samples drawn were processed for DNA isolation followed by direct sequencing of POU1F1, PROP1, and HESX1genes. Of the 51 patients, 8 were females and 43 were males. Mean height standard deviation score (SDS) and weight SDS were -5.50 and -2.76, respectively. Thirty-six of the 51 patients underwent MRI of which 9 (25%) had normal pituitary structure and morphology while 27 (75%) showed abnormalities. Molecular analysis revealed 10 (20%) patients to have POU1F1 and PROP1 mutations/variations of which 5 were novel and 2 previously reported. No mutations were identified in HESX1. The novel variations identified were absent in the 100 healthy individuals screened and the control database Exome Aggregation Consortium (ExAC). Reported POU1F1 and PROP1 mutation hotspots were absent in our patients. Instead, novel POU1F1 changes were identified suggesting existence of a distinct mutation spectrum in our population.

BACKGROUND

The human cytomegalovirus (HCMV) is a common pathogen responsible for asymptomatic and persistent infections in healthy individuals. However, cytomegalovirus infections are a major cause of morbidity and mortality in immunocompromised patients, especially in recipients of solid-organ transplants and AIDS patients.

METHODS

HCMV DNA from 42 patients who received kidney transplants between 2004 and 2008 were subjected to polymerase chain reaction and restriction fragment length polymorphism to identify HCMV gB and gH genotypes.

RESULTS

HCMV gB1 and gH1 genotypes were the most the predominant HCMV genotypes (P < .05, P < .05, respectively). In addition, both HCMV gB1 and gH1 genotype were significantly more often associated with the development of fever with leukopenia and severe HCMV disease than other gB or gH2 genotypes. No significant differences were observed among viral loads between the HCMV genotypes among infected individuals.

CONCLUSIONS

This study demonstrated the prevalence and role of HCMV genotypes in infection and disease in renal transplant patients in Kuwait.