Quantitative RT-PCR was used to determine the profiles of expression of 10 growth- or development-related genes in rainbow trout (Oncorhynchus mykiss) embryos prior to the formation of the somatotropic (ST) axis (pituitary somatotrops and liver); embryos were sampled immediately after fertilization and water-hardening (t(0)), 1-h post-fertilization, and 1-, 2-, 5-, 7-, 10- and 13-days post-fertilization (dpf); expression profiles were examined in embryos reared at two temperatures (6.0 and 8.5 degrees C), which had different developmental rates. Accumulation of mRNA encoding for GH1, GH2, IGF-1, IGF-2, two isoforms of GH receptors (GHR1, GHR2), two isoforms of IGF receptors (IGF-RIa, IGF-RIb) and two isoforms of thyroid receptor (TR), TRalpha and TRbeta, was measured. All of these genes were expressed in the t(0) samples, but the rates of expression of the different genes varied markedly. For most of the genes examined, the expression rates tended to fall within the first hour after fertilization, and remained at the lower level for between 2 and 7 days, after which there was a significant (P <0.05) and progressive increase in the number of accumulated copies of mRNA. This increase is probably associated with the commencement of embryonic genome transcription activity (EGTA), and it was generally, although not always, found later in embryos that were reared at 6.0 degrees C compared with the faster developing embryos reared at 8.5 degrees C. The study suggests that the EGTA begins between 2- and 5-dpf, with a staged increase in EGTA between 5- and 13-dpf.

β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.

The Gram-positive bacterium Cellulomonas fimi produces a large array of carbohydrate-active enzymes. Analysis of the collection of carbohydrate-active enzymes from the recent genome sequence of C. fimi ATCC 484 shows a large number of uncharacterized genes for glycoside hydrolase (GH) enzymes potentially involved in biomass utilization. To investigate the enzymatic activity of potential β-glucosidases in C. fimi, genes encoding several GH3 enzymes and one GH1 enzyme were cloned and recombinant proteins were expressed in Escherichia coli. Biochemical analysis of these proteins revealed that the enzymes exhibited different substrate specificities for para-nitrophenol-linked substrates (pNP), disaccharides, and oligosaccharides. Celf_2726 encoded a bifunctional enzyme with β-d-xylopyranosidase and α-l-arabinofuranosidase activities, based on pNP-linked substrates (CfXyl3A). Celf_0140 encoded a β-d-glucosidase with activity on β-1,3- and β-1,6-linked glucosyl disaccharides as well as pNP-β-Glc (CfBgl3A). Celf_0468 encoded a β-d-glucosidase with hydrolysis of pNP-β-Glc and hydrolysis/transglycosylation activities only on β-1,6-linked glucosyl disaccharide (CfBgl3B). Celf_3372 encoded a GH3 family member with broad aryl-β-d-glycosidase substrate specificity. Celf_2783 encoded the GH1 family member (CfBgl1), which was found to hydrolyze pNP-β-Glc/Fuc/Gal, as well as cellotetraose and cellopentaose. CfBgl1 also had good activity on β-1,2- and β-1,3-linked disaccharides but had only very weak activity on β-1,4/6-linked glucose.

The GH1 genes of probands of two families with familial isolated GH deficiency (IGHD) were sequenced. Double stranded sequencing of the polymerase chain reaction (PCR) amplification products from genomic DNA of two affected cousins in a consanguineous Turkish family revealed a G->>A transition in the 20th codon of the GH1 signal peptide. This substitution converts a TGG (Trp) to a TAG (stop) codon and generates a new AluI recognition site. PCR amplification of the GH1 alleles of family members, followed by AluI digestion, revealed that the G->>A transition segregated with the IGHD phenotype. In a Saudi Arabian family, a G->>C transversion was found that alters the first base of the donor splice site of intron IV. This substitution should perturb mRNA splicing, resulting in an altered protein product which should be unstable or bioinactive. This transversion also destroys an HphI site, which was used to assay samples from relatives. Digestion of PCR amplification products with HphI demonstrated cosegregation of the G->>C transversion with IGHD. These results demonstrate that familial IGHD is a heterogeneous disease that perturbs different steps in the expression of the GH1 gene.

Venom composition of parasitoid wasps attracts increasing interest - notably molecules ensuring parasitism success on arthropod pests - but its variation within and among taxa is not yet understood. We have identified here the main venom proteins of two braconid wasps, Psyttalia lounsburyi (two strains from South Africa and Kenya) and P. concolor, olive fruit fly parasitoids that differ in host range. Among the shared abundant proteins, we found a GH1 β-glucosidase and a family of leucine-rich repeat (LRR) proteins. Olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. Assuming that Psyttalia host larvae sequester ingested glycosides, the injected venom GH1 β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. Venom LRR proteins are similar to truncated Toll-like receptors and may possibly scavenge the host immunity. The abundance of one of these LRR proteins in the venom of only one of the two P. lounsburyi strains evidences intraspecific variation in venom composition. Altogether, venom intra- and inter-specific variation in Psyttalia spp. were much lower than previously reported in the Leptopilina genus (Figitidae), suggesting it might depend upon the parasitoid taxa.

Real time RT-PCR was used to measure the changes in the rates of synthesis of mRNA encoding for growth hormone-1 (GH1) and -2 (GH2) and insulin-like growth factor-1 (IGF-1) and -2 (IGF-2), and whole embryo GH content was measured in early stage rainbow trout (Oncorhynchus mykiss) embryos reared at two incubation temperatures (8.5 and 6.0 degrees C). Particular attention was paid to the phase of embryo development that preceded the appearance of the pituitary gland. GH was present in zygotes, and there were no significant changes in whole embryo GH content of the two temperature treatment groups from fertilization (t0) until the time at which GH was detectable in the pituitary gland by immunostaining. The expression of the two GH genes decreased during the first 24 h post-fertilization, and then increased significantly by 17 dpf in embryos reared at both temperatures. There was a subsequent steep increase in the number of copies of GH1 and GH2 mRNA associated with the formation of the pituitary gland evident at 23 and 34 dpf in the 8.5 and 6.0 degrees C groups, respectively. The number of copies of mRNA encoding for IGF-1 and IGF-2 did not change during the first 24 h post-fertilization; however, there was a significant increase in the numbers of transcripts for both genes evident by 13 dpf in embryos reared at the two incubation temperatures. The differences in the timing of the increases in GH and IGF mRNA may suggest that IGF gene expression is not GH-dependent at that stage. Moreover, the increased expression of the GH genes prior to the formation of the pituitary gland suggests that tissues other than the pituitary are expressing these genes in early embryos. The pattern of changes in GH content was similar to the pattern of GH gene expression in embryos reared at the two incubation temperatures when the age of embryos was plotted using degree-days. There were no apparent compensatory responses in GH1, GH2, IGF-1 or IGF-2 gene expression related to altered growth rates. The number of copies of IGF-2 mRNA was higher than that of IGF-1 mRNA during the early developmental period; this is consistent with the hypothesis that IGF-2 predominates during embryonic development. A differential expression of GH2 and GH1 was also observed with the overall copy numbers of GH2 mRNA being consistently higher than those of GH1.

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

New β-glucosidase activities were identified by screening metagenomic libraries constructed with DNA isolated from the topsoil of a winter wheat field. Two of the corresponding proteins, displaying an unusual preference for alkaline conditions, were selected for purification by Ni-NTA chromatography. AS-Esc6, a 762-amino-acid enzyme belonging to glycoside hydrolase family 3, proved to be a mesophilic aryl-β-glucosidase with maximal activity around pH 8 and 40 °C. A similar pH optimum was found for AS-Esc10, a 475-amino-acid GH1-family enzyme, but this enzyme remained significantly active across a wider pH range and was also markedly more stable than AS-Esc6 at pH greater than 10. AS-Esc10 was found to degrade cellobiose and diverse aryl glycosides, with an optimal temperature of 60 °C and good stability up to 50 °C. Unlike AS-Esc6, which showed a classically low inhibitory constant for glucose (14 mM), AS-Esc10 showed enhanced activity in the presence of molar concentrations of glucose. AS-Esc10 was highly tolerant to hydrogen peroxide and also to sodium dodecyl sulfate, this being indicative of kinetic stability. This unique combination of properties makes AS-Esc10 a particularly promising candidate whose potential in biotechnological applications is worth exploring further.

The retinoic acid (RA) receptor-beta (RAR beta) gene is dramatically up-regulated by RA in F9 teratocarcinoma cells due to the presence of an RA-responsive element (RARE) in its promoter. Remarkably, however, RAR beta mRNA was essentially unaffected by RA in rat pituitary GH3 cells, even though the GH gene was induced by RA, and these cells express mRNAs specific for multiple RAR subtypes and for the potential RAR coactivators, retinoid X-receptors. The absence of RA induction of RAR beta was also observed in GH1 cells as well as in murine AtT-20 pituitary cells and rat H35 hepatocarcinoma cells. The RA unresponsiveness of the RAR beta gene in GH3 and AtT-20 cells was not due to a mutation in the RARE, which was identical to that in RA-responsive F9 cells. Furthermore, while AtT-20 and F9 cells both expressed multiple RAR beta isoforms, including RAR beta 2, which was profoundly induced by RA in F9 cells, none of these was highly regulated by RA in AtT-20 cells. The failure of RA to induce RAR beta mRNA in certain murine and rat pituitary and nonpituitary cell lines indicates that target gene responsiveness to RA is cell type specific in some cases.

A cluster of amino acid residues located in the AB-GH region of the alpha-chain are shown in intra-double strand axial interactions of the hemoglobin S (HbS) polymer. However, alphaLeu-113 (GH1) located in the periphery is not implicated in any interactions by either crystal structure or models of the fiber, and its role in HbS polymerization has not been explored by solution experiments. We have constructed HbS Twin Peaks (betaGlu-6->>Val, alphaLeu-113->>His) to ascertain the hitherto unknown role of the alpha113 site in the polymerization process. The structural and functional behavior of HbS Twin Peaks was comparable with HbS. HbS Twin Peaks polymerized with a slower rate compared with HbS, and its polymer solubility (C(sat)) was found to be about 1.8-fold higher than HbS. To further authenticate the participation of the alpha113 site in the polymerization process as well as to evaluate its relative inhibitory strength, we constructed HbS tetramers in which the alpha113 mutation was coupled individually with two established fiber contact sites (alpha16 and alpha23) located in the AB region of the alpha-chain: HbS(alphaLys-16->>Gln, alphaLeu-113->>His), HbS(alphaGlu-23->>Gln, alphaLeu-113->>His). The single mutants at alpha16/alpha23 sites were also engineered as controls. The C(sat) values of the HbS point mutants involving sites alpha16 or alpha23 were higher than HbS but markedly lower as compared with HbS Twin Peaks. In contrast, C(sat) values of both double mutants were comparable with or higher than that of HbS Twin Peaks. The demonstration of the inhibitory effect of alpha113 mutation alone or in combination with other sites, in quantitative terms, unequivocally establishes a role for this site in HbS gelation. These results have implications for development of a more accurate model of the fiber that could serve as a blueprint for therapeutic intervention.

Three monoclonal antibodies (M318T, M56S and M815) against an external envelope glycoprotein (gp110) of simian immunodeficiency virus (SIV) mac251 were obtained by immunizing BALB/c mice with recombinant gp110 (rgp110). All three monoclonal antibodies reacted with the surface of cells infected with SIVmac251 but not with that of uninfected counterparts. The binding activity of these monoclonal antibodies against native gp110 was confirmed by means of Western blotting. One of them, M318T neutralized SIVmac251 infection both by cell-free and cell-associated viruses. M318T cross-reacted with human immunodeficiency virus type 2 strains (HIV-2 GH1 and ROD isolates) and SIVmac239 isolates. However, the antibody did not cross-neutralize these viral strains. Epitope mapping revealed that the neutralizing epitope recognized by M318T was localized at 8 residues between amino acids 178 and 185 (KRDKTKEY) in gp110, corresponding to the V2 region of human immunodeficiency virus type 1 (HIV-1). Because some antibodies against the V2 region of HIV-1 reportedly neutralize virus infection by interfering with CD4-gp120 interaction, we tested the activity of M318T against the binding of CD4-gp110. However, M318T did not inhibit CD4-gp110 interaction, suggesting the involvement of another unknown mechanism of M318T-mediated neutralization. In analogy with the V2 region of HIV-1, the V2 region of SIV contains a type specific neutralizing epitope recognized by M318T. Although some amino acid sequence in the epitope was conserved for the isolates of SIV and HIV-2 and there was cross-reactivity of the antibody against the strains, neutralization by M318T was associated with a single amino acid (182 T) in the epitope.

Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides.

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) β-glucosidase, hydrolyzes β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the β-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a (4)C(1) chair conformation, while that of the noncovalently bound DNP2FG had a (1)S(3) skew boat, consistent with hydrolysis via a (4)H(3) half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 β-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.

Silver-Russell syndrome (SRS) is a syndrome of severe pre- and postnatal growth retardation and typical dysmorphic features. Rare chromosomal aberrations have been reported in SRS; among these are two balanced translocations involving 17q24-q25. Recently, we described a patient with a paternally inherited heterozygous deletion of the chorionic somatomammotropin hormone 1 (CSH1) gene. The CSH1 gene is member of the growth hormone (GH) gene cluster on 17q, which consists of two growth hormone genes and three CSH genes. Genomic alterations in the GH cluster are well known, causing different phenotypes depending on the size of the deletion and the genes involved. By screening 63 SRS cases with marker D17S254, we have detected 2 further patients with a heterozygous deletion in the GH cluster. Quantitative analysis using restriction assays confirmed these findings. Additionally, in a cohort of 17 patients with isolated intrauterine and postnatal growth retardation, we detected a further patient to be carrier of a CSH1 deletion. Screening of 141 unrelated controls revealed hemizygosity in one person for which data on growth were not available. We additionally analyzed our cohort of SRS patients for mutations in CSH1 and its 3' neighbour GH2. However, analyses failed to reveal any pathogenic mutation. While the central role of GH1 in human growth is well established, the physiological roles of CSH1 and other components of the cluster are unclear. The increased prevalence of hemizygosity of CSH1 in our population in comparison to controls indicates a role for CSH1 haploinsufficiency in the etiology of growth retardation. Investigation of CSH1 deletions in further SRS and growth retarded patients will enable us to establish under which circumstances haploinsufficiency of CSH1 is likely to result in clinical changes.

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.

Originally native to the eastern United States, American cranberry ( Vaccinium macrocarpon Aiton, family Ericaceae) cultivation of native and hybrid varieties has spread across North America. Herein is reported the phytochemical diversity of five cranberry cultivars (Stevens, Ben Lear, Bergman, Pilgrim, and GH1) collected in the Greater Vancouver Regional District, by anthocyanin content and UPLC-TOF-MS metabolomic profiling. The anthocyanin content for biological replicates (n = 5) was determined as 7.98 ± 5.83, Ben Lear; 7.02 ± 1.75, Bergman; 6.05 ± 2.51, GH1; 3.28 ± 1.88, Pilgrim; and 2.81 ± 0.81, Stevens. Using subtractive metabonomic algorithms 6481 compounds were found conserved across all varietals, with 136 (Ben Lear), 84 (Bergman), 91 (GH1), 128 (Pilgrim), and 165 (Stevens) unique compounds observed. Principal component analysis (PCA) did not differentiate varieties, whereas partial least-squares discriminate analysis (PLS-DA) exhibited clustering patterns. Univariate statistical approaches were applied to the data set, establishing significance of values and assessing quality of the models. Metabolomic profiling with chemometric analysis proved to be useful for characterizing metabonomic changes across cranberry varieties.

BACKGROUND

Growth hormone (GH) is an essential regulator of intrahepatic lipid metabolism by activating multiple complex hepatic signaling cascades. Here, we examined whether chronic exogenous GH administration (via gene therapy) could ameliorate liver steatosis in animal models of alcoholic fatty liver disease (AFLD) and explored the underlying molecular mechanisms.

METHODS

Male C57BL/6J mice were fed either an alcohol or a control liquid diet with or without GH therapy for 6 weeks. Biochemical parameters, liver histology, oxidative stress markers, and serum high molecular weight (HMW) adiponectin were measured. Quantitative real-time PCR and western blotting were also conducted to determine the underlying molecular mechanism.

RESULTS

Serum HMW adiponectin levels were significantly higher in the GH1-treated control group than in the control group (3.98 ± 0.71 μg/mL vs. 3.07 ± 0.55 μg/mL; P < 0.001). GH1 therapy reversed HMW adiponectin levels to the normal levels in the alcohol-fed group. Alcohol feeding significantly reduced hepatic adipoR2 mRNA expression compared with that in the control group (0.71 ± 0.17 vs. 1.03 ± 0.19; P < 0.001), which was reversed by GH therapy. GH1 therapy also significantly increased the relative mRNA (1.98 ± 0.15 vs. 0.98 ± 0.15) and protein levels of SIRT1 (2.18 ± 0.37 vs. 0.99 ± 0.17) in the alcohol-fed group compared with those in the control group (both, P < 0.001). The alcohol diet decreased the phosphorylated and total protein levels of hepatic AMP-activated kinase-α (AMPKα) (phosphorylated protein: 0.40 ± 0.14 vs. 1.00 ± 0.12; total protein: 0.32 ± 0.12 vs. 1.00 ± 0.14; both, P < 0.001) and peroxisome proliferator activated receptor-α (PPARα) (phosphorylated protein: 0.30 ± 0.09 vs. 1.00 ± 0.09; total protein: 0.27 ± 0.10 vs. 1.00 ± 0.13; both, P < 0.001), which were restored by GH1 therapy. GH therapy also decreased the severity of fatty liver in alcohol-fed mice.

CONCLUSIONS

GH therapy had positive effects on AFLD and may offer a promising approach to prevent or treat AFLD. These beneficial effects of GH on AFLD were achieved through the activation of the hepatic adiponectin-SIRT1-AMPK and PPARα-AMPK signaling systems.

We have studied the effect of iopanoic acid (IOP), a radiographic contrast agent which inhibits T4 to T3 conversion, on thyroid hormone nuclear receptors, GH response to T4 and T3, and T4 5'-monodeiodination in GH1 cells, a rat pituitary cell line. IOP at concentrations higher than 10 microM inhibits iodothyronine binding to the nuclear receptor without changing the dissociation constant (Kd) (0.1 nM for T3 and 1 nM for T4), and reduces the GH response to 50 nM T4, 5 nM T3, or the combined effect of T4 and glucocorticoids. These results could be explained by an inhibition of protein synthesis which was reduced by more than 50% by 50 microM IOP. By contrast, nontoxic concentrations of IOP did not change the GH response to different doses of T4 ranging from 1 nM to 50 nM. We also examined T3 generation from T4 and found that the intracellular T3 levels of cells incubated with 50 nM T4 were almost as high as those of cells incubated with 5 nM T3 which induces a full GH response. Intracellular T3 levels were markedly reduced in the cells incubated with T4 and IOP, but GH production was not reduced despite these differences in T3 levels. Additionally, more than 40% of the nuclear receptor was occupied by T3 in cells incubated with T4, whereas more than 90% was occupied by T4 in cells receiving the same amount of T4 with 5 microM IOP. Our results suggest that the effect of T4 on GH production by GH1 cells could be attributed to an important extent to the T3 generated from it, whereas when T4 monodeiodination is strongly inhibited, most of the biological activity is a result of intrinsic T4 activity.

Two bacterial strains were isolated from greenhouse soils of Daejeon and Yangpyeong regions in Korea. The strains, designated <em>GH1</em>-9T and <em>GH1</em>9-3T, were Gram-negative and aerobic, with rod-shaped cells. Their DNA G+C contents were 61.7 and 67.3 mol%, respectively. The major fatty acids of strain <em>GH1</em>-9T were iso-C16 : 0, iso-C15 : 0, iso-C14 : 0, iso-C17 : 1omega9c and iso-C11 : 0 3-OH and the major components of strain <em>GH1</em>9-3T were iso-C16 : 0, iso-C15 : 0, C16 : 1omega7c alcohol, iso-C17 : 1omega9c and iso-C11 : 0 3-OH. None of the species of the genus Lysobacter with validly published names showed 16S rRNA gene sequence similarity values of more than 97 % with respect to the novel isolates. The closest sequence similarity of strain <em>GH1</em>-9T was with Lysobacter concretion is DSM 16239T (96.4 %), whereas strain <em>GH1</em>9-3T showed the highest sequence similarity with Lysobacter enzymogenes DSM 2043T (96.6 %). Polyphasic taxonomic studies indicated that the two strains should be classified as representing novel members of the genus Lysobacter. The names Lysobacter daejeonensis sp. nov. and Lysobacter yangpyeongensis sp. nov. are proposed, with strains <em>GH1</em>-9T (=KACC 11406T=DSM 17634T) and <em>GH1</em>9-3T (=KACC 11407T=DSM 17635T), respectively, as the type strains.

We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) from Neurospora crassa. Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.

The human growth hormone receptor (GHR) mediates the effects of growth hormone (GH1), starting a signalling cascade that is involved in the regulation of proliferation, differentiation and apoptosis. Recently, an isoform of the GHR gene lacking exon 3 (GHRd3) was associated with accelerated responsiveness to growth hormone. In this study, we investigated the association of the GHRd3 polymorphism with breast cancer risk and performed a haplotype analysis with 3 additional single nucleotide polymorphisms (SNPs) (Gly186Gly, Cys440Phe and Ile544Leu) in the GHR coding region in a Polish cohort. We did not observe any effect of the 4 polymorphisms on breast cancer risk. Neither did the 3 most common haplotypes influence breast cancer risk. However, a rare haplotype (dGGC), containing the GHRd3 allele, was associated with a decreased breast cancer risk (OR 0.30, 95% CI 0.11-0.80).

The Ligand Epitope Antigen Presentation System (L.E.A.P.S.) approach to vaccine development allowed construction of immunogens from defined T cell epitopes from herpes simplex virus (HSV) proteins that conferred protection against lethal challenge by the virus. This technology utilizes specific peptides which bind to CD4, CD8 or other proteins on the surface of T cells (T cell binding ligand (TCBL)), macrophage and dendritic cells (immune cell binding ligand (ICBL)) to promote the immunogenicity of an epitope, activate T cell and other protective responses, and direct the immune response to either a Th1 or a Th2 type of response. The J TCBL/ICBL is a peptide from beta-2-microglobulin which binds to the CD8 protein and promotes Th1 responses and the G TCBL/ICBL is a peptide from the beta chain of MHC II molecules that binds to the CD4 protein and promotes Th2 responses. Epitopes from the ICP27 (H1, H2), glycoprotein B (gB) and glycoprotein D (gD) proteins of HSV-1 were attached to either the J TCBL/ICBL or the G TCBL/ICBL. The JH1, JH2, JgB and JgD vaccines elicited DTH responses without antibody but conferred protection upon lethal challenge. Th1 related antibody was produced after challenge of the JgB and JgD immunized mice. Immunization with the GH1, GgB or GgD vaccines did not yield protection. The GgB and GgD produced Th2 related antibodies upon virus challenge. Initiation of the immune response by the JgD vaccine was dependent on functional CD4, CD8 expressing cells and interferon gamma and delivery of protection was dependent upon CD4 and interferon gamma. The L.E.A.P.S. HSV vaccines appear to elicit the appropriate immune responses for protection and further work is being performed to develop the JgD vaccine for human use.

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular β-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g).