In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.

BACKGROUND

Anaerobic fungi reside in the rumen and alimentary tract of herbivores where they play an important role in the digestion of ingested plant biomass. The anaerobic fungal isolate Orpinomyces sp. strain C1A is an efficient biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple types of lignocellulosic biomass. To understand the mechanistic and regulatory basis of biomass deconstruction in anaerobic fungi, we analyzed the transcriptomic profiles of C1A when grown on four different types of lignocellulosic biomass (alfalfa, energy cane, corn stover, and sorghum) versus a soluble sugar monomer (glucose).

RESULTS

A total of 468.2 million reads (70.2 Gb) were generated and assembled into 27,506 distinct transcripts. CAZyme transcripts identified included 385, 246, and 44 transcripts belonging to 44, 13, and 8 different glycoside hydrolases (GH), carbohydrate esterases, and polysaccharide lyases families, respectively. Examination of CAZyme transcriptional patterns indicates that strain C1A constitutively transcribes a high baseline level of CAZyme transcripts on glucose. Although growth on lignocellulosic biomass substrates was associated with a significant increase in transcriptional levels in few GH families, including the highly transcribed GH1 β-glucosidase, GH6 cellobiohydrolase, and GH9 endoglucanase, the transcriptional levels of the majority of CAZyme families and transcripts were not significantly altered in glucose-grown versus lignocellulosic biomass-grown cultures. Further, strain C1A co-transcribes multiple functionally redundant enzymes for cellulose and hemicellulose saccharification that are mechanistically and structurally distinct. Analysis of fungal dockerin domain-containing transcripts strongly suggests that anaerobic fungal cellulosomes represent distinct catalytic units capable of independently attacking and converting intact plant fibers to sugar monomers.

CONCLUSIONS

Collectively, these results demonstrate that strain C1A achieves fast, effective biomass degradation by the simultaneous employment of a wide array of constitutively-transcribed cellulosome-bound and free enzymes with considerable functional overlap. We argue that the utilization of this indiscriminate strategy could be justified by the evolutionary history of anaerobic fungi, as well as their functional role within their natural habitat in the herbivorous gut.

We have previously demonstrated that L-triiodothyronine (L-T3) induces an increase in growth hormone synthesis and messenger RNA in cultured GH1 cells, a rat pituitary cell line. In addition to regulating the growth hormone response, L-T3 elicits a time- and dose-dependent reduction in the level of its nuclear receptor, which is a direct function of the occupancy of the receptor binding site. In this study we have compared the relative affinity of L-T3, triiodothyroacetic acid, D-triiodothyronine (D-T3), and L-thyroxine (L-T4) for the receptor with the induction of the growth hormone synthesis and the ability of these compounds to elicit a reduction in thyroid hormone nuclear receptor levels. Triiodothyroacetic acid and D-T3 were specifically examined because the biologic effect of these compounds in the intact rat is significantly lower than predicted by their affinity for the receptor using isolated rat liver nuclei in vitro. In intact cells each compound demonstrated an excellent relationship between the relative receptor affinity, the induction of growth hormone production, and the concentration-dependent reduction in nuclear receptor levels. With the exception of D-T3, the relative affinity of iodothyronine was identical for the receptor using intact cells in serum-free media, or isolated GH1 cell nuclei in vitro. The apparent receptor affinity of D-T3 with intact cells was 5.5-fold lower than with isolated nuclei, which suggests a decrease in cell entry of D-T3 relative to the other iodothyronines. Quantitation of the [125I]iodothyronine associated with the receptor in GH1 cells after a 36-h incubation with L-125I-T4 was 90% L-T4 and 10% L-T3, which indicates that the major effect of L-T4 in GH1 cells is a result of intrinsic L-T4 activity. Studies with dispersed rat anterior pituitary cells demonstrated that L-T3 induces growth hormone synthesis and elicits a reduction in nuclear receptor levels in the same fashion as GH1 cells. The observation that thyroid hormone influences dispersed rat pituitary cells in a fashion qualitatively similar to GH1 cells may have implications for the growth hormone response of the somatotroph cell in vivo to different thyroidal states.

Genome mapping in the dog is in its early stages. Here we illustrate an approach to combined physical and linkage mapping of type 1 anchor (gene) loci in the dog using information on syntenic homology from human and mouse, an interbreed cross/backcross, and a strategy for isolation of dog genomic clones containing both gene-specific sequences and simple sequence repeat polymorphisms. Eleven gene loci from human chromosome 17q (HSA17q) were mapped to the centromeric two-thirds of dog chromosome 9 (CFA9), an acrocentric chromosome of medium size: P4HB, GALK1, TK1, GH1, MYL4, BRCA1, RARA, THRA1, MPO, NF1, and CRYBA1. Eight of these were also positioned on a linkage map spanning 38.6 cM. Based on combined fluorescence in situ hybridization and linkage mapping, the gene order on CFA9 is similar to that of the homologous genes on HSA17q and mouse chromosome 11 (MMU11), but in the dog the gene order is inverted with respect to the centromere. Canine loci, GALK1, TK1, GH1, MYL4, THRA1, and RARA constitute a closely linked group near the centromeric end of CFA9, spanning a genetic distance of only 4.7 cM. Canine NF1 and CRYBA1 lie distally, near the lower border of the Giemsa band adjacent to the distal one-third of CFA9. NF1 and CRYBA1 are loosely linked to the more centromeric group (31.2 cM). No HSA17 genes were found on the telomeric one-third of CFA9. Painting of dog chromosomes with a human whole chromosome 17 probe showed hybridization with only the proximal two-thirds of CFA9, consistent with the conclusion that the distal one-third corresponds to a segment or segments of other human chromosomes. Two loci, GLUT4 and PMP22, located on HSA17p, were mapped by FISH to dog chromosome 5 in a region also identified by the whole human chromosome 17 paint, indicating disruption of HSA17 syntenic homology at the centromere.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.

BACKGROUND

The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the optimization of cellobiose consumption and ethanol productivity by simultaneously engineering both proteins of the pathway, the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1), in an example of pathway engineering through directed evolution.

RESULTS

The improved pathway was assessed based on the strain specific growth rate on cellobiose, with the final mutant exhibiting a 47% increase over the wild-type pathway. Metabolite analysis of the engineered pathway identified a 49% increase in cellobiose consumption (1.78 to 2.65 g cellobiose/(L · h)) and a 64% increase in ethanol productivity (0.611 to 1.00 g ethanol/(L · h)).

CONCLUSIONS

By simultaneously engineering multiple proteins in the pathway, cellobiose utilization in S. cerevisiae was improved. This optimization can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. The improved in vivo cellobiose utilization demonstrated here could help to decrease the in vitro enzyme load in biomass pretreatment, ultimately contributing to a reduction in the high cost of biofuel production.

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A-chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB-ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti-CD3 IT, anti-CD5 IT and anti-CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti-CD4 IT inhibited poorly and anti-CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine-labelled antibodies. Anti-CD7 showed the fastest internalization rate, followed by anti-CD3 and anti-CD5. Anti-CD4 and anti-CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.

Isolated growth hormone deficiency (IGHD) represents conditions of GH deficiency that are not necessarily associated with other pituitary hormone deficiencies or with an organic lesion. Three sub-categories of IGHD have been clinically identified (IGHD types 1-3), and IGHD type 1 has been further separated into IGHD types 1a and b. However, this clinical sub-categorization of IGHD may need reconsideration due to the recent identification of molecular heterogeneity within each sub-type of IGHD. In a small number of children with IGHD, defects in the GH, GH-releasing hormone receptor (GHRH-R), and GH1 genes have been identified. In most cases, no cause for IGHD can be identified; however, the proportion of idiopathic IGHD cases may be decreasing due to identification of causative factors. The phenotype of IGHD is variable depending in part on the underlying genetic disorders in the affected individuals. Several studies have focused on the usefulness of MRI findings in patients with GHD but anatomic abnormalities of the pituitary gland are variable. We review current studies and the clinical, biochemical, and molecular features described for different groups of affected individuals with IGHD.

The white-rot fungus Phanerochaete chrysosporium has two intracellular beta-glucosidases (BGL1A and BGL1B) belonging to glycoside hydrolase (GH) family 1. BGL1B effectively hydrolyzes cellobiose and cellobionolactone, but BGL1A does not. We have determined the crystal structure of BGL1A in substrate-free and gluconolactone complexed forms. The overall structure and the characteristic of subsite -1 (glycone site) were similar to those of other known GH1 enzymes. The loop regions covering on the (beta/alpha)(8) barrel was significantly deviated, and they form a unique subsite +1 (aglycone site) of BGL1A.

BACKGROUND

Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures.

RESULTS

Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14-16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10-14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends.

CONCLUSIONS

Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results.

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

The fast technological development, particularly single nucleotide polymorphism array, array-comparative genomic hybridization, and whole exome sequencing, has led to the discovery of many novel genetic causes of growth failure. In this review we discuss a selection of these, according to a diagnostic classification centred on the epiphyseal growth plate. We successively discuss disorders in hormone signalling, paracrine factors, matrix molecules, intracellular pathways, and fundamental cellular processes, followed by chromosomal aberrations including copy number variants (CNVs) and imprinting disorders associated with short stature. Many novel causes of GH deficiency (GHD) as part of combined pituitary hormone deficiency have been uncovered. The most frequent genetic causes of isolated GHD are GH1 and GHRHR defects, but several novel causes have recently been found, such as GHSR, RNPC3, and IFT172 mutations. Besides well-defined causes of GH insensitivity (GHR, STAT5B, IGFALS, IGF1 defects), disorders of NFκB signalling, STAT3 and IGF2 have recently been discovered. Heterozygous IGF1R defects are a relatively frequent cause of prenatal and postnatal growth retardation. TRHA mutations cause a syndromic form of short stature with elevated T3/T4 ratio. Disorders of signalling of various paracrine factors (FGFs, BMPs, WNTs, PTHrP/IHH, and CNP/NPR2) or genetic defects affecting cartilage extracellular matrix usually cause disproportionate short stature. Heterozygous NPR2 or SHOX defects may be found in ∼3% of short children, and also rasopathies (e.g., Noonan syndrome) can be found in children without clear syndromic appearance. Numerous other syndromes associated with short stature are caused by genetic defects in fundamental cellular processes, chromosomal abnormalities, CNVs, and imprinting disorders.

Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH5, consistent with the role of E176 as the catalytic acid-base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90Å resolution, respectively. The Q176Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.

Specific receptors for insulin and the somatomedin peptides insulin-like growth factors I and II (IGF-I and IGF-II) have been characterized on three separate cloned strains of rat pituitary tumor cells (GH3, GH1, and GC). Binding of 125I-labeled peptides was time, temperature, and pH dependent for all three cell lines. Specific binding of [125I]insulin, which was extremely low in normal rat adenohypophyseal cells, was demonstrable for all three lines, with the Kd for the high affinity receptor ranging from 10(-10) to 4 X 10(-10) M/liter. A specific high affinity IGF-I receptor was also identified, with a Kd of approximately 10(-9) M/liter. IGF-II and insulin were, respectively, 10% and 1% as potent as IGF-I in competing for this receptor. When [125I]insulin and [125I]IGF-I were cross-linked to GH3 cells with disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both receptors were found to have an apparent mol wt greater than 300,000 in the unreduced state, with subunits of apparent mol wt 125,000 after reduction. A third discrete receptor, which bound [125I]IGF-II, was also identified on all three cell lines. IGF-I was only 10% as potent as IGF-II at displacing [125I]IGF-II, and insulin was virtually unreactive. When [125I]IGF-II was cross-linked to GH3 cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two receptors were identified. One had an apparent mol wt of 205,000 unreduced and 250,000 upon reduction, and presumably represents the type II receptor. Additionally, a band was observed at an apparent mol wt greater than 300,000 unreduced and 125,000 upon reduction, probably representing IGF-II binding to the IGF-I or insulin receptor. The presence of specific high affinity receptors for insulin, IGF-I, and IGF-II in these transformed cell lines is consistent with previous observations in normal rat and human pituitary cells and suggests a role for these peptides in the modulation of pituitary function.

The ability of avian-specific linker histone H5, and the globular domains of H5 (GH5) and H1(0) (GH1(0), to self-associate either free in solution or when bound to DNA was investigated. All three proteins underwent a salt-dependent increase in turbidity that may be indicative of nonspecific interactions. Dithiobis(succinimidyl propionate) cross-linking was used to measure specific contacts for both H5 and GH5 free in solution and bound to DNA. H5 and GH5 each became cross-linked in solution, with GH5 displaying divalent polymerization interactions, which suggests that two specific surfaces were involved in the assembly process. For GH5-DNA complexes, cross-linking appeared to be largely the consequence of aggregation, but under low concentrations of DSP, cross-linking GH5 was observed to assemble preferentially onto DNA before oligomerizing to form massive aggregates. Both linear and supercoiled DNA facilitated GH5 interactions compared to assembly in solution; differences in the distribution of cross-linked polymer sizes indicates that assembly is dependent on both the presence of DNA and the morphology of the DNA. Finally, on the basis of a technique referred to as quantitative proteolysis, GH5 assembly on DNA appears to involve specific protein-protein contacts involving the C terminus of one partner. Overall, the cumulative results reported here support the premise that linker histones assemble specifically both in solution and on DNA.

Human GH has two disulfide bridges linking Cys-53 to Cys-165 and Cys-182 to Cys-189. Although absence of the first disulfide bridge has been shown to affect the bioactivity of GH in transgenic mice, little is known of the importance of this bridge in mediating the GH/GH-receptor (GHR) interaction in humans. However, we have identified a missense mutation (G705C) in the GH1 gene of a Serbian patient. This mutation was found in the homozygous state and leads to the absence of the disulfide bridge Cys-53 to Cys-165. To study the impact of this mutation in vitro, GHR binding and Janus kinase (Jak)2/signal transducer and activator of transcription (Stat)5 activation experiments were performed, in which it was observed that at physiological concentrations (3-50 ng/ml) both GHR binding and Jak2/Stat5 signaling pathway activation were significantly reduced in the mutant GH-C53S, compared with wild-type (wt)-GH. Higher concentrations (400 ng/ml) were required for this mutant to elicit responses similar to wt-GH. These results demonstrate that the absence of the disulfide bridge Cys-53 to Cys-165 affects the binding affinity of GH for the GHR and subsequently the potency of GH to activate the Jak2/Stat5 signaling pathway. In conclusion, we have demonstrated that GH-C53S is a bioinactive GH at the physiological range and that the disulfide bridge Cys-53 to Cys-163 is required for mediating the biological effects of GH.

Insulin-like growth factor-I (IGF-I) stimulates cell proliferation and can enhance the development of tumors in different organs. Epidemiologic studies have shown that an elevated level of circulating IGF-I is associated to increased risk of breast cancer as well as other cancers. Genetic variants affecting the release or biological action of growth hormone (GH), the main stimulator of IGF-I production, may predict circulating levels of IGF-I and have an effect on cancer risk. We tested this hypothesis with a large case-control study of 807 breast cancer patients and 1,588 matched control subjects nested within the European Prospective Investigation into Cancer and Nutrition. We genotyped 22 common single nucleotide polymorphisms in 10 genes involved in GH production and action (GHRH, GHRHR, SST, SSTR1-SSTR5, POU1F1, and GH1), and in parallel, we measured serum levels of IGF-I and IGFBP-3, its major binding protein, in samples of cases and controls. SST and SSTR2 polymorphisms showed weak but statistically significant associations with breast cancer risk. SSTR5 polymorphisms were associated with IGF-I levels, whereas one polymorphism in GHRHR and one in POU1F1 were associated with IGFBP-3 levels. Our conclusion is that common genetic variation in the GH synthesis pathway, as measured by single nucleotide polymorphisms selected in the present study, is not a major determinant of IGF-I and IGFBP-3 circulating levels, and it does not play a major role in altering breast cancer risk.

Linkage studies were performed in six European families with hyperkalaemic periodic paralysis (PPII) with myotonia, an autosomal dominantly inherited disorder characterised by episodic weakness. The weakness is caused by non-inactivating sodium channels of reduced single channel conductance of the muscle fibre membrane. Recently, portions of the gene coding for the alpha subunit of the sodium channel of the adult human skeletal muscle (h-Na2) have been cloned and localised on chromosome 17q with no recombinants to the human growth hormone locus (GH1). Linkage between these two chromosome 17 markers and the disease was shown in our families (Z = 7.14, 0 = 0.00). These results, combined with the linkage data of a single large American family, suggest that the disease is caused by dominant mutations of the adult sodium channel, and that it is probably a genetically homogeneous disorder. Hyperkalaemic periodic paralysis is the first non-progressive myotonic disorder to be localised on the human genome.

Transition-state mimicry is increasingly important both to understand enzyme mechanism and to direct the synthesis of putative therapeutic agents. X-ray crystallography is able to provide vital information on the interactions between an enzyme and the potential inhibitor. Here we report the structures, at approximately 2 A resolution, of a family GH1 beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus, in complex with both covalently (derived from 2-fluoro-glycosides) and noncovalently (hydroximolactam) bound inhibitors. The enzyme has broad specificity, accommodating both gluco- and galacto-configured substrates, and the crystallographic data demonstrate that the only difference in the way these ligands bind lies in the interactions between Gln18, Glu432, and Trp433, and the hydroxyl group at the O3 and O4 positions. Inhibition by the differently configured ligands was also shown to be extremely similar, with K(i) values of 1.04 and 1.08 microM for the gluco and galacto epimers, respectively. The noncovalently bound inhibitors have a trigonal anomeric carbon, adopt a (4)H(3) (half-chair) conformation, and an interaction is formed between O2 and the catalytic nucleophile, all of which contribute to (partial) mimicry of the oxocarbenium-ion-like transition state. The inhibition of the beta-glycosidase from S. solfataricus by hydroximolactams is discussed in light of the emerging work on family GH1 glycosidase inhibition by a spectrum of putative transition-state mimics.

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.

Reduced birth weight and slow neonatal growth are risks correlated with the development of common diseases in adulthood. The Human Growth Hormone/Chorionic Somatomammotropin (hGH/CSH) gene cluster (48 kb) at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placental genes (GH2, CSH1, CSH2, and CSHL1) may contribute to common variation in intrauterine and infant growth, and also to the regulation of feto-maternal and adult glucose metabolism. In contrast to GH1, there are limited genetic data on the hGH/CSH genes expressed in utero. We report the first survey of sequence variation encompassing all five hGH/CSH genes. Resequencing identified 113 SNPs/indels (ss86217675-ss86217787 in dbSNP) including 66 novel variants, and revealed remarkable differences in diversity patterns among the homologous duplicated genes as well as between the study populations of European (Estonians), Asian (Han Chinese), and African (Mandenkalu) ancestries. A dominant feature of the hGH/CSH region is hyperactive gene conversion, with the rate exceeding tens to hundreds of times the rate of reciprocal crossing-over and resulting in near absence of linkage disequilibrium. The initiation of gene conversion seems to be uniformly distributed because the data do not predict any recombination hotspots. Signatures of different selective constraints acting on each gene indicate functional specification of the hGH/CSH genes. Most strikingly, the GH2 coding for placental growth hormone shows strong intercontinental diversification (F(ST)=0.41-0.91; p<10(-6)) indicative of balancing selection, whereas the flanking CSH1 exhibits low population differentiation (F(ST)=0.03-0.09), low diversity (non-Africans, pi=8-9 x 10(-5); Africans, pi=8.2 x 10(-4)), and one dominant haplotype worldwide, consistent with purifying selection. The results imply that the success of an association study targeted to duplicated genes may be enhanced by prior resequencing of the study population in order to determine polymorphism distribution and relevant tag-SNPs.

In anthropoid primates, growth hormone (GH) genes have undergone at least 2 independent locus expansions, one in platyrrhines (New World monkeys) and another in catarrhines (Old World monkeys and apes). In catarrhines, the GH cluster has a pituitary-expressed gene called GH1; the remaining GH genes include placental GHs and placental lactogens. Here, we provide cDNA sequence evidence that the platyrrhine GH cluster also includes at least 3 placenta expressed genes and phylogenetic evidence that placenta expressed anthropoid GH genes have undergone strong adaptive evolution, whereas pituitary-expressed GH genes have faced strict functional constraint. Our phylogenetic evidence also points to lineage-specific gene gain and loss in early placental mammalian evolution, with at least three copies of the GH gene present at the time of the last common ancestor (LCA) of primates, rodents, and laurasiatherians. Anthropoid primates and laurasiatherians share gene descendants of one of these three copies, whereas rodents and strepsirrhine primates each maintain a separate copy. Eight of the amino-acid replacements that occurred on the lineage leading to the LCA of extant anthropoids have been implicated in GH signaling at the maternal-fetal interface. Thus, placental expression of GH may have preceded the separate series of GH gene duplications that occurred in catarrhines and platyrrhines (i.e., the roles played by placenta-expressed GHs in human pregnancy may have a longer evolutionary history than previously appreciated).