BACKGROUND

Pretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose is typically recovered through precipitation with an anti-solvent. An industrially feasible pretreatment and hydrolysis process requires robust cellulases that are stable and active in the presence of either small amounts of ILs co-precipitated with recovered cellulose or for saccharifications in the presence of IL. β-glucosidase (BG) hydrolyzes cellobiose into two molecules of glucose (Glc) and is the last step of biomass hydrolysis. These enzymes are prone not only to product inhibition by glucose but also to inactivation by ILs. With increasing interest in IL-based pretreatment methods, there is increasing focus toward a search for Glc-tolerant and IL-tolerant BG.

RESULTS

We identified a BG belonging to the GH1 family, H0HC94, encoded in Agrobacterium tumefaciens 5A, and cloned and overexpressed the protein in Escherichia coli. H0HC94 exhibited high enzymatic activity with β-glycosidic substrates (248 µmol/min/mg on pNPGlc and 262 µmol/min/mg on cellobiose) and tolerant to Glc (apparent K i = 686 mM). Further evidence of Glc-based stabilization came from the increase in melting temperature of H0HC94, with increasing Glc concentrations. The half-life of H0HC94 also increased between 2- and 20-fold in the presence of increasing concentrations of Glc. In the presence of 0.9 M of different [C2mim]-based ionic liquids, the specific activity of H0HC94 decreased by around 20-30 %. However, the addition of 100 mM glucose to the IL-enzyme mix resulted in a more stable enzyme as evidenced by the slight recovery of H0HC94 melting temperature and up to tenfold increase in half-life. This higher stability came at a cost of 2-10 % decrease in specific activity. The steady-state kinetic analyses for a subset of the ionic liquids tested indicate that the enzyme undergoes uncompetitive inhibition by glucose and ionic liquid, indicating the possibility of binding of the ionic liquid and glucose to the enzyme-substrate complex.

CONCLUSIONS

H0HC94 is a Glc-stabilized BG that is also tolerant up to 0.9 M concentrations of different IL's and indicates the possibilities of using an IL-Glc-based cellulose solvent that displays enzyme-compatibility.

Human cytomegalovirus (CMV) is a major cause of morbidity in fetuses following intrauterine infection. The glycoprotein (g) envelope trimeric gH/gL/gO and pentameric gH/gL/pUL128/pUL130/pUL131A complexes are required for CMV entry into fibroblasts and endothelial/epithelial cells, respectively, and both are targets for neutralizing antibodies. The role of sequence variability among viral strains in the outcome of congenital CMV infection is controversial. Variation in the CMV UL75 gene encoding glycoprotein H (gH), the UL115 (gL), the UL74 (gO), and the UL128 locus (UL128L) encoding three structural proteins (pUL128, pUL130, and pUL131A) was determined in 82 newborns with congenital CMV infection and 113 infants with postnatal or unproven congenital CMV infection. Genotyping was performed by sequencing analysis of PCR-amplified fragments and the PCR-restriction fragment length polymorphism (RFLP) method, and the viral load was measured by quantitative real-time PCR. The obtained results demonstrated that (1) different CMV variants and mixed CMV infections can be detected in newborns infected congenitally; (2) the gH1 genotype, UL130 variant 6, and UL131A variant 1 were associated with some signs/symptoms within cohort of pediatric patients, mainly consisting of infants with symptomatic CMV infection. The results revealed that pUL130, pUL131A, and gH polymorphisms seemed to be associated with the outcome of CMV infection in infants.

As a master hormone controlling growth and metabolism, GH is also known to regulate reproduction. Studies in mammals have shown that mutations in GH or its receptor (GHR) not only result in retardation in body growth but also reproductive dysfunctions in both sexes. However, the roles of GH in reproduction of other vertebrates are poorly defined. In this study, we created two zebrafish GH (gh1) mutant lines using CRISPR/Cas9. The mutant developed normally up to 14 days postfertilization (dpf); however, a high rate of mortality was observed afterward in both lines, and only a small number of mutant fish could survive to adult stage. The body growth of the mutants was significantly retarded in both sexes in a gene dose-dependent manner compared with their wild-type siblings. A severe dysfunction of gonadal development was observed in survived mutant females, with ovarian folliculogenesis being arrested completely at primary growth stage until 100 dpf. Interestingly, the folliculogenesis in the mutant resumed after months of delay with a certain number of follicles entering vitellogenic growth. As for male reproduction, although the spermatogenesis in mutant males seemed normal in adults, the GH-insufficient heterozygote showed an obvious delay of spermatogenesis (puberty onset) at early developmental stages. The adult mutant males could not breed with wild-type females through natural spawning; however, the sperm isolated from the mutant testes could fertilize eggs through artificial fertilization. This study provides further genetic evidence for the dependence of puberty onset on somatic growth, but not age, in fish.

β-Glucosidase is the rate-limiting component of a cellulase-hydrolyzing reaction. Thermostability and glucose-tolerance are two critical criteria of the enzyme, which practically determine its performance in industrial applications. In this study, a thermostable and glucose-tolerant β-glucosidase (named Bgl1317) belonging to the glycoside hydrolase family 1 was acquired from a metagenomic library of Turpan soil through functional screening. Bgl1317 showed excellent thermostability and glucose-tolerance and its crystal structure was subsequently determined at a high resolution. Rational design based on the structure was conducted, producing three beneficial mutations A397R, L188A and A262S. While A397R improved the cellobiose activity by 80%, L188A and A262S increased the IC₅₀ value of glucose from 0.8 to 1.5 M. The residues that may play a role in glucose-tolerance of GH1 β-glucosidases were summarized and the performances of glucose-tolerant β-glucosidases reported in recent years were discussed and compared. This study provides insights into enzymatic properties of Bgl1317 for engineering it into a powerful catalyst and β-glucosidases in general.

BACKGROUND

Growth hormone (GH) has been used to treat children with GH deficiency (GHD) since 1966.

OBJECTIVE

Using a combined retrospective and cross-sectional approach, we explored the long-term outcomes of patients with GHD, analysed factors influencing therapeutic response, determined persistence into adulthood, investigated pituitary morphology, and screened for mutations in causative genes.

METHODS

The files of 96 GH-deficient children were reviewed. In a subset of 50 patients, re-assessment in adulthood was performed, including GHRH-arginine testing, pituitary magnetic resonance imaging (MRI), and mutational screening for the growth hormone-1 gene (GH1) and the GHRH receptor gene (GHRHR) in isolated GHD (IGHD), and HESX1, PROP1, POU1F1, LHX3, LHX4, and GLI2 in multiple pituitary hormone deficiency (MPHD) patients.

RESULTS

GH was started at a height SDS of -3.2 ± 1.4 in IGHD patients and of -4.1 ± 2.1 in MPHD patients. Relative height gain was 0.3 SDS/year, absolute gain 1.6 SDS, and 1.2/2.6 SDS in IGHD/MPHD, respectively. Mid-parental target height was reached in 77%. Initial height SDS, bone age retardation and duration of GH replacement were correlated with height SDS gain. GHD persisted into adulthood in 19 and 89% of subjects with IGHD and MPHD, respectively. In 1/42 IGHD patients a GH1 mutation was detected; PROP1 mutations were found in 3/7 MPHD subjects. Anterior pituitary hypoplasia, combined with posterior pituitary ectopy and pituitary stalk invisibility on MRI, was an exclusive finding in MPHD patients.

CONCLUSIONS

GH replacement successfully corrects the growth deficit in children with GHD. While the genetic aetiology remains undefined in most cases of IGHD, PROP1 mutations constitute a major cause for MPHD. Persistence of GHD into adulthood is related to abnormal pituitary morphology.

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species.

The secretion of prolactin is increased by treatment of prolactin producing rat pituitary cells with the hypothalamic tripeptide thyroliberin. To investigate the underlying mechanisms we used three closely related rat pituitary tumor cell strains (GH1 2C1, GH3 and GH4C1), which synthesize and spontaneously secrete prolactin and/or growth hormone. Growth hormone and prolactin released into the culture medium over a period of 24 h were measured by radioimmunoassay. Initial rates of synthesis were measured by immunoprecipitation of intracellular growth hormone and prolactin after incubation of cell cultures with 3H-leucine. The observed increase in prolactin synthesis and release was correlated with morphological effects of thyroliberin treatment. The volume density of Golgi complexes and the volume and surface densities of rough endoplasmic reticulum were compared in untreated cells and thyroliberin treated cells. As normal distribution could not be assumed the non-parametric rank test of Wilcoxon was used whereby the densities calculated for each cell section were ranked. All three morphological parameters increased after thyroliberin treatment in cells secreting prolactin only (GH4C1), implying that the increase of prolactin secretion, at least in part, is due to increased prolactin synthesis.

Considerable information regarding the regulation of GH and prolactin (PRL) release has been generated using pituitary cell lines as model systems. Inasmuch as these cultures have been derived from single cells by clonal selection it has frequently been assumed that they are composed of homogeneous populations of hormone secretors. However, experience with GH3 cells clearly demonstrates that such is not the case, since this GH- and PRL-producing line is comprised of a mixture of cells that are bihormonal, secrete only GH, or secrete neither hormone. Interestingly, the relative amount of these phenotypic subpopulations is not fixed, but can be altered by treatment with established regulators of GH and PRL secretion. This potential for secretory heterogeneity and phenotypic plasticity prompted us to examine the cellular composition of other commonly used GH- and/or PRL-secreting cell lines under control and treatment conditions. First, GH4C1, GH1, GC, MMQ and P0 cells were maintained according to established media and culture protocols and the relative abundance of GH and PRL secretors was assessed by reverse haemolytic plaque assays. As shown previously for GH3 cells, two cell lines were found to be functionally heterogeneous. Specifically, GH4C1 and GH1 were comprised of mixed populations of GH (25.9 +/- 1.1% and 51.3 +/- 6.5% (S.E.M.) respectively) and PRL (44.8 +/- 3.7 and 66.1 +/- 4.1% respectively) secretors. However, MMQ and GC cell cultures were relatively homogeneous with respect to hormone secretion in that the MMQ cells released PRL (72.2 +/- 4.9%) but not GH, while GC cells released GH (93.6 +/- 1.4%) but not PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

The aim of this work is to evaluate levels of placental growth hormone (PGH), pituitary growth hormone (GH1), insulin-like growth factor (IGF-I) and ghrelin in pregnant women's blood serum before, during and after delivery. Furthermore, the aim is to search for links and interdependence of GH1, PGH and IGF-I concentrations.

METHODS

Seventy nine blood samples were taken one to two hours before, during and half an hour after expulsion of placenta. All proteins studied were determined by ELISA method, using ELISA Kit.

RESULTS

The highest PGH concentration and IGF-I concentration in pregnant women's blood serum was observed before delivery while GH1 concentration was lowest. During and after delivery PGH and IGF-I concentration decreased proportionately and pituitary growth hormone concentration increased accordingly. About half an hour after delivery of the placenta, GH1 concentration was highest.

CONCLUSIONS

In pregnant women's blood there is a metabolic interdependence between PGH and IGF-I. Their concentration increases proportionately during pregnancy and decreases after delivery. It appears that labor and delivery releases GH1 blockade, which level rises three-fold during delivery. After parturition its role and concentration returns to levels before pregnancy.

We have recently demonstrated substantial stereospecific nuclear/cytosolic free triiodothyronine (T3) gradients within T3 responsive rat tissues in situ. These studies have now been extended to examine T3 transport in a rat pituitary tumor cell line, GH1. L-T3 had a 7.6-fold higher affinity for the nuclear receptor when assayed in whole cell incubations in comparison to isolated nuclei, though D-T3 affinity was not altered under these conditions. An apparently higher number of receptors for D-T3 was explained by racemic contamination of the isotopes used. Measurement of free hormone concentration ratios for both enantiomers revealed a small step up from medium to cytosol for L-T3 (1.65) but a reverse ratio for D-T3 (0.46). The nuclei were able to concentrate both enantiomers, though stereospecificity was maintained (nucleus/cytosol, L-T3, 4.5, D-T3 1.7). Transport of L-T3 at both boundaries could be inhibited by monodansylcadaverine. Thus, stereospecific transport functions are found within GH1 cells, though the magnitude of the free nucleus/cytosol gradient is reduced from those seen in rat tissues in situ.

A somatic cell hybrid panel was constructed consisting of seven hybrids with translocation breakpoints spanning the region 17q23->>q25. Hybrid clones carrying the longarm derivative of chromosome 17 in the absence of the normal chromosome 17 and of the derivative 17 were initially identified by PCR typing for a proximal and distal 17q marker. The translocation breakpoints of the hybrids were then mapped in more detail by PCR analysis for a number of microsatellite markers from chromosome 17q as well as for five gene loci (CACNLG, GH1, SOX9, TIMP2, TK1) previously mapped to the region 17q23->>q25. In addition, the locus for GDIA1 was mapped by FISH to 17q25.3 and fine mapped with the help of the hybrid panel. These seven new hybrids complement the existing somatic cell hybrid panel for the long arm of chromosome 17q.

In this study, whole genome sequencing and comparative genomic analyses were performed for Mucilaginibacter polytrichastri RG4-7 and its carboxymethyl cellulose degradation potential was assessed. The results showed that the genome of strain RG4-7 was 5.84 Mb and contained 5019 predicted genes, in which a high proportion of strain-specific genes were related to carbohydrate metabolism. The carboxymethyl cellulose (CMC) degradation and cellulase activity tests revealed the strong cellulose degradation ability, CMCase and β-glucosidase activity in strain RG4-7. Real-time RT-PCR testing of most cellulose degradation related glycoside hydrolase (GH) families showed that GH9 (OKS85969), GH1 (OKS85832), GH3 (OKS89331 and OKS85615) were significantly up-regulated when strain RG4-7 was inoculated with CMC-Na, which suggested that GH9, GH1 and GH3 might determine its cellulose degradation ability. Certainly, further research need to be done to elucidate cellulose degradation mechanisms in strain RG4-7 in order to develop its industrial application value in lignocellulosic biomass degradation and waste management.

YxaL is conserved within the Bacillus subtilis species complex associated with plants and soil. The mature YxaL protein contains a repeated beta-propeller domain, but the subcellular location and function of YxaL has not been determined. The gene encoding the mature YxaL protein was PCR amplified from genomic DNA of B. velezensis strain GH1-13 and used for recombinant protein production. A rabbit polyclonal antibody against the purified YxaL was generated and used for western blotting to determine the constitutive expression and secretion of YxaL. During normal culture growth of strain GH1-13, levels of the constitutively secreted YxaL were slowly rising to 100 μg L-1, and degraded with a half-life of 1.6 h in the culture medium. When the effects of YxaL on plant seed germination and seedling growth were examined, it was shown that seed treatment of Arabidopsis thaliana and rice (Oryza sativa L.) with purified YxaL at the optimal concentration of 1 mg L-1 was effective at improving the root growth of plants. Seedlings from the treated Arabidopsis seeds markedly increased transcription of a 1-aminocyclopropane-1-carboxylate synthetase marker gene (ACS11) but reduced expression of auxin- and abscisic acid-responsive marker genes (IAA1, GH3.3, and ABF4), especially when provided with exogenous auxin. Horticulture experiments showed that pepper (Capsicum annuum) seeds treated with 1 mg L-1 YxaL in a soaking solution increased shoot growth and improved tolerance to drought stress. We hypothesize that YxaL secreted from plant growth-promoting Bacillus cells has a significant impact on plant roots, with the potential to improve plant growth and stress tolerance.

Defining boundaries of chromosomal rearrangements at the molecular level would benefit from landmarks that link the cytogenetic map to physical, genetic, and transcript maps, as well as from large-insert FISH probes for such loci to detect numerical and structural rearrangements in metaphase or interphase cells. Here, we determined the locations of 24 genetically mapped CEPH-Mega YACs along the FLpter scale (fractional length from p-telomere) by quantitative fluorescence in situ hybridization analysis. This generated a set of cytogenetically mapped probes for chromosome 17 with an average spacing of about 5 cM. We then developed large-insert YAC, BAC, PAC, or P1 clones to the following 24 known genes, and determined refined map locations along the same FLpter scale: pter-TP53-TOP3-cen-TNFAIP1-ERBB2-TOP2A- BRCA1-TCF11-NME1-HLF-ZNF147/CL N80-BCL5/MPO/SFRS1-TBX2-PECAM1-DDX5/ PRKCA-ICAM2-GH1/PRKAR1A-GRB2-CDK3 /FKHL13-qter. Taken together, these 48 cytogenetically mapped large-insert probes provide tools for the molecular analysis of chromosome 17 rearrangements, such as mapping amplification, deletion, and translocation breakpoints in this chromosome, in cancer and other diseases.

OBJECTIVE

Expression of the human growth hormone (GH) gene (GH1) is regulated by a locus control region (LCR) and the highly polymorphic GH1 promoter. We analyzed GH1 LCR/promoter single nucleotide polymorphisms (SNPs) in patients with isolated growth hormone deficiency (IGHD) in relation to clinical data.

METHODS

We directly sequenced the GH1 LCR/promoter of 62 Dutch IGHD patients without mutations or deletions in GH1 or GHRHR and of 72 controls with normal height. We related GH1 LCR/promoter SNPs to height, serum insulin-like growth factor 1 (IGF-I) levels and response to GH treatment.

RESULTS

In IGHD patients, promoter SNPs 6, 8 and 9 were associated with height and IGF-1 levels. In controls, SNPs 6 and 11 were associated with height. Homozygosity for the minor allele of SNP 9, associated with lower IGF-I levels in patients, was significantly more frequent among patients than among controls. Genotypes based on SNPs 6, 8, 9 and 11 explained 10.8% of IGF-I SDS variation in IGHD patients and 15.9% of height SDS variation in controls.

CONCLUSIONS

GH1 Promoter SNPs 6, 8 and 9 were associated with height and IGF-1 levels among patients, and SNPs 6 and 11 with height in controls.

A novel marine Gram-negative bacterium, designated strain GH1-23T, was isolated from a tidal mudflat sample collected at Dongmak seashore on Gangwha Island, Republic of Korea and its taxonomic position was determined through a polyphasic investigation. The bacterium was strictly aerobic, chemoheterotrophic, catalase- and oxidase-positive, consisted of non-motile rods and grew optimally at 30 °C, pH 7 and 1 % NaCl. The predominant cellular fatty acids were C18 : 1ω7c and cyclo-C19 : 0ω8c. The major isoprenoid quinone was Q-10. The major polar lipids were phosphatidylglycerol, a phosphoglycolipid and an aminolipid. Comparative 16S rRNA gene sequence analysis revealed that the isolate was closely related to members of the genus Maribius. The highest 16S rRNA gene sequence similarity was found to be 97.5 % to Maribius salinus followed by 97.4 % to Maribius pelagius; levels of 16S rRNA gene sequence similarity between the novel strain and other representatives of family Rhodobacteraceae were <95.5 %. The DNA G+C content was 66.7 mol % and DNA-DNA relatedness values with the type strains of species of the genus Maribius were 33-39 %. Based on combined data from a polyphasic investigation, strain GH1-23T (=KCTC 52957T=DSM 104950T) represents a novel species of the genus Maribius, for which the name Maribius pontilimi sp. nov. is proposed.

Signal transducer and activator of transcription 5b (STAT5b) has been identified as a key downstream mediator of growth hormone (GH) signaling in somatic growth of mammalian. However, the corresponding homologue gene of Stat5b is unknown in fish species. In this study, we generated loss-of-function mutants in stat5.1 and stat5.2, two stat5 homologues existing in zebrafish. In stat5.1-deficient zebrafish, a significant reduction of body length and body weight was detected in the embryos/larvae and adults compared with the wild-type control fish, and sexual size dimorphism in adult zebrafish was also eliminated. However, the stat5.2-deficient zebrafish displayed a normal developmental phenotype during all lifespan. Chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) method was adopted to further investigate the potential transcriptional targets of Stat5 protein and cast much light upon the biological function of Stat5. We identified more than 800 genes as transcriptional targets of Stat5 during zebrafish embryogenesis. KEGG analysis indicated that the Stat5 target gene network is predominantly linked to the metabolic pathways, neuroactive ligand-receptor interaction and JAK-STAT signaling pathways. Further validation studies suggested that Stat5.1 protein could directly regulate the expression of gh1, and stat5.1-mutated zebrafish showed a reduction of gh1 mRNA level. In the present study, stat5.1 was revealed as the corresponding homologue gene of Stat5b in fish species. Additionally, we found a novel molecular interaction between Stat5.1/Stat5b and GH, and unraveled a positive feedback loop Stat5.1-GH-Stat5.1 which is necessary for somatic growth and body development in zebrafish.

The aims of this study were to estimate the genetic variation of traditional milk coagulation properties (MCPs), milk acidity, curd firmness (CF) modeled on time t (CF(t) ; comprising: RCT(eq), rennet coagulation time estimated from the equation; CF(P), the asymptotic potential curd firmness; k(CF), the curd firming instant rate constant; and k(SR), the syneresis instant rate constant) and maximum CF traits (MCF; comprising CF(max), the maximum CF value; and tmax, the time of attainment). Furthermore, we investigated 96 single nucleotide polymorphisms (SNPs) from 54 candidate genes, testing their associations with the above-listed traits. Milk and blood samples were collected from 1271 cows (each sampled once) from 85 herds. Genotyping was performed using a custom Illumina VeraCode GoldenGate approach. A Bayesian linear animal model (including the effects of herd, days in milk, parity and additive polygenic effects) was used to estimate the genetic parameters of the studied traits. The same model with the addition of the SNP genotype effect was used for our association analysis. The heritability estimates of CF t and the MCF traits (RCT(eq)=0.258; k(CF)=0.230; CF(max)=0.191; t(max)=0.278) were similar to those obtained using traditional MCPs (0.187 to 0.267), except for the lower estimates for CF(P) (0.064) and k(SR) (0.077). A total of 13 of the 51 tested SNPs had relevant additive effects on at least one trait. We observed associations between MCPs and SNPs in the genes encoding ATP-binding cassette sub-family G member 2 (ABCG2), chemokine ligand 2 (CCL2), growth hormone 1 (GH1), prolactin (PRL) and toll-like receptor 2 (TLR2). Whereas, CF(t) and the MCF traits were associated with polymorphisms in the α-s1-casein (CSN1S1), β-casein (CSN2), GH1, oxidized low-density lipoprotein receptor 1 (OLR1), phospholipase C β1 (PLCB1), PRL and signal transducer and activator of transcription 5A (STAT5A) genes.

Retinoic acid produced a time and dose-dependent depletion of thyroid hormone receptors in GH1 cells without modifying their affinity for triiodothyronine (T3). A maximal decrease (50-70%) was obtained after 24-48 h incubation with 5-10 microM retinoic acid. Treatment with 0.8 nM T3 for 24 h caused a similar reduction and did not potentiate the decrease produced by these concentrations of retinoic acid. However, the combination of sub-maximally effective doses of both ligands had an additive effect on receptor levels. The reduction of receptor caused by retinoic acid is accompanied by a decreased expression of c-erbA alpha 1 and alpha 2 mRNAs, but the retinoid did not reduce the abundance of c-erbA beta mRNA. In contrast, T3 decreased the levels of both alpha and beta transcripts.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

Growth hormone 1 (GH1) gene deletions occur in approximately 10-15% of patients with severe isolated, GH deficiency (GHD). The standard treatment for GHD is GH replacement. Individuals with GH gene defects, however, may form GH antibodies that interfere with the efficacy of exogenous recombinant GH (rhGH) therapy.

OBJECTIVE

We describe the growth measures and metabolic studies of two Hispanic sisters with the same 7.6-kb GH1 gene deletion who presented with short stature and increased body fat, and who developed neutralizing GH antibodies secondary to rhGH exposure.

METHODS

The younger sister has now been treated with recombinant human insulin-like growth factor-I (rhIGF-I) for 4 years, and is continuing treatment. The older sister was not given rhIGF-I based on her normal height velocity and age. After the first 4 years of rhIGF-I treatment of the younger sister, we summarized the longitudinal anthropometric measures and serial laboratory studies, including GH surrogates, fasting lipid studies, oral glucose tolerance tests, and HbA1c, of both sisters. Body composition was quantified using DEXA analysis.

RESULTS

The older sister achieved an adult stature at the low end of her mid-parental target height range, having been treated only with rhGH for ~2.5 years (between 11 months and 3.5 years of age). Treatment of the younger sister with rhIGF-I for 4 years has led to persistent improvement in height velocity, but was associated with adverse short-term effects on all lipids. Her BMI increased modestly (+4.1 kg/m(2)) during rhIGF-I treatment, though her change in percent body fat was negligible by DEXA (Δ -0.7%).

CONCLUSIONS

In individuals with a GH gene deletion, rhIGF-I promotes increased height velocity, but may be associated with adverse effects on lipids and BMI. It is clear that the long-term effects of rhIGF-I on lipid metabolism and body composition require further monitoring and assessment with continued treatment.

The level and hierarchical distribution of genetic variation in complete sequences of the Atlantic salmon (Salmo salar) growth hormone (GH1) gene were investigated in populations from Europe and North America with a view to inferring the major evolutionary forces affecting genetic variation at this locus. Seventeen polymorphic sites were identified in complete sequences from nine populations, with levels of noncoding (intron and untranslated region sequences) nucleotide diversity being similar to those observed in other species. No variation, however, was observed in exonic sequences, indicating that nucleotide diversity in the Atlantic salmon GH1 gene is three and 25 times less than that estimated for human and Drosophila coding sequences, respectively. This suggests that purifying selection is the predominant contemporary force controlling the molecular evolution of GH1 coding sequences. Comparison of haplotype relationships within and between populations indicated that differentiation between populations from Europe and North America was greater than within-continent comparisons. However, several haplotypes observed in the northernmost European populations were more similar to those observed in North American than to any other haplotypes observed in Europe. This is most likely to be a result of historical, rather than contemporary, gene flow. Neutrality test statistics, such as Tajima's D, were significantly positive in the European populations in which North American-like haplotypes were observed. Although a positive Tajima's D is commonly interpreted as the signal of balancing selection, a more likely explanation in this case is that either historical migration or ascertainment bias, rather than within population local adaptation, has given rise to an excess of intermediate frequency alleles.

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.