Risk alleles for chronic hepatitis B are associated with decreased mRNA expression of HLA-DPA1 and HLA-DPB1 in normal human liver.

A genome-wide association study identified single nucleotide polymorphisms (SNPs) rs3077 and rs9277535 located in the 3' untranslated regions of human leukocyte antigen (HLA) class II genes HLA-DPA1 and HLA-DPB1, respectively, as the independent variants most strongly associated with chronic hepatitis B. We examined whether these SNPs are associated with mRNA expression of HLA-DPA1 and HLA-DPB1. We identified gene expression-associated SNPs (eSNPs) in normal liver samples obtained from 651 individuals of European ancestry by integrating genotype (~650 000 SNPs) and gene expression (>39 000 transcripts) data from each sample. We used the Kruskal-Wallis test to determine associations between gene expression and genotype. To confirm findings, we measured allelic expression imbalance (AEI) of complementary DNA compared with DNA in liver specimens from subjects who were heterozygous for rs3077 and rs9277535. On a genome-wide basis, rs3077 was the SNP most strongly associated with HLA-DPA1 expression (p=10(-48)), and rs9277535 was strongly associated with HLA-DPB1 expression (p=10(-15)). Consistent with these gene expression associations, we observed AEI for both rs3077 (p=3.0 × 10(-7); 17 samples) and rs9277535 (p=0.001; 17 samples). We conclude that the variants previously associated with chronic hepatitis B are also strongly associated with mRNA expression of HLA-DPA1 and HLA-DPB1, suggesting that expression of these genes is important in control of HBV.

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori.

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

Multi-subunit assembly of the Pyrococcus furiosus small heat shock protein is essential for cellular protection at high temperature.

The hyperthermophilic archaeon, Pyrococcus furiosus, expresses a small, alpha-crystallin-like protein in response to exposure to extreme temperatures, above 103 degrees C. The P. furiosus small heat shock protein (Pfu-sHSP) forms large oligomeric complexes. Based on the available crystal structures of the Methanocaldococcus jannaschii and wheat sHSPs, the protruding carboxy terminal domain is probably involved in subunit interactions. We constructed Pfu-sHSP mutants to analyze chaperone function and to study multi-subunit assembly. The results confirmed that the carboxy terminus of Pfu-sHSP is involved in inter-dimer interactions, whereas the amino terminal deletion mutant still exhibited the wild-type assembly characteristics. The ability to form oligomeric complexes via the carboxy terminal domain was shown to be necessary for thermotolerance of Escherichia coli overexpressing Pfu-sHSP. The amino terminal domain was not required for inter-species thermotolerance.

Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus.

The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105 degrees C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105 degrees C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56 degrees C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50 degrees C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.

Evidence of recent lateral gene transfer among hyperthermophilic archaea.

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.

Achieving department safety: a team approach to development of a department safety plan template.

Hospitals develop safety plans to teach employees to work safely with hazards, to maintain a safe patient care environment, and to enable appropriate response to emergencies affecting the healthcare facility. This article explains the process used to create the Department Specific Safety and Infection Control Plan at United Hospital, St. Paul, MN.

Structure determination of the glutamate dehydrogenase from the hyperthermophile Thermococcus litoralis and its comparison with that from Pyrococcus furiosus.

Glutamate dehydrogenase catalyses the oxidative deamination of glutamate to 2-oxoglutarate with concomitant reduction of NAD(P)(+), and has been shown to be widely distributed in nature across species ranging from psychrophiles to hyperthermophiles. Extensive characterisation of this enzyme isolated from hyperthermophilic organisms has led to its adoption as a model system for analysing the determinants of thermal stability. The crystal structure of the extremely thermostable glutamate dehydrogenase from Thermococcus litoralis has been determined at 2.5 A resolution, and has been compared to that from the hyperthermophile Pyrococcus furiosus. The two enzymes are 87 % identical in sequence, yet differ 16-fold in their half-lives at 104 degrees C. This is the first reported comparative analysis of the structures of a multisubunit enzyme from two closely related yet distinct hyperthermophilies. The less stable T. litoralis enzyme has a decreased number of ion pair interactions; modified patterns of hydrogen bonding resulting from isosteric sequence changes; substitutions that decrease packing efficiency; and substitutions which give rise to subtle but distinct shifts in both main-chain and side-chain elements of the structure. This analysis provides a rational basis to test ideas on the factors that confer thermal stability in proteins through a combination of mutagenesis, calorimetry, and structural studies.

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Protein thermostability above 100 degreesC: a key role for ionic interactions.

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Insights into the molecular basis of thermal stability from the analysis of ion-pair networks in the glutamate dehydrogenase family.

The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion-pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology-based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion-pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion-pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough.

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.

Structure-function relationships of lipoprotein lipase: mutation analysis and mutagenesis of the loop region.

The molecular models of two microbial lipases and human pancreatic lipase (PL) have suggested the existence of common structural motifs including a buried active site shielded by an amphipathic surface loop. In an effort to explore the role of residues comprising the loop of lipoprotein lipase (LPL), we have used site-directed mutagenesis to generate three new LPL variants. In variant LPLM1 we deleted 18 amino acids leaving a loop of only 4 residues which resulted in an LPL protein inactive against triolein substrates. In contrast, two other LPL variants with only partial deletions, involving the apical section of the loop [LPLM2 (-8 amino acids) and LPLM3 (-2 amino acids)] manifested normal lipolytic activity. These findings indicate a critical requirement for the maintenance of charge and periodicity in the proximal and distal segments of the LPL loop in normal catalytic function. This is further highlighted by the detection of a mutation in the proximal section of the loop in a patient with LPL deficiency at position 225 which results in a substitution of threonine for isoleucine. The intact catalytic activity of the partial deletion variants (LPLM2 and LPLM3) further suggests that the apical residues of the loop contribute minimally to the functional motifs of the active site. We support this postulate by showing that the conserved glycine in the apical turn section (G229) can be substituted by glutamine, lysine, proline, or threonine without significantly affecting catalytic activity.

Design and inhibitory properties of synthetic Bowman-Birk loops.

A cyclic tridecapeptide based on the sequence of an anti-tryptic loop of a Bowman-Birk inhibitor was synthesized, and demonstrated to be active as an inhibitor of trypsin. Molecular modeling of this sequence suggested an improved sequence which demonstrated an order of magnitude improvement in the inhibitory constant.

Subcellular distribution of asialoglycoprotein receptor in liver regeneration.

Asialoglycoprotein (ASGP) receptor specific activity was determined directly using a radiolabelled ligand (asialoorosomucoid) binding assay in various fractions of rat liver cells before and 24 hours after two-thirds partial hepatectomy. The specific activity of ASGP receptor in the plasma membrane fraction was significantly reduced 24 hours after partial hepatectomy. In an attempt to identify a subgroup of receptor involved in intercellular recognition the calcium-dependent ASGP receptor was re-assayed after pre-incubation in ethylenediaminetetra-acetic acid. This revealed significant amounts of additional receptor in microsomal preparations but not in plasma membrane fractions. Cryptic receptor levels were not affected by partial hepatectomy. Plasma membrane-bound receptor was significantly decreased in regenerating liver.

Extended C-terminal tail of wheat histone H2A interacts with DNA of the "linker" region.

The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.

The C-domain in the H1 histone is structurally conserved.

The C-domain of H1 is conserved in composition and not in sequence. The following regularities have been identified: the distribution of lysine, alanine and proline is non-random; alanine occurs in doublets and at intervals of 4-6 significantly more often than expected for random sequences of equal composition; and lysine also deviates from random distribution in that doublets are under-represented and intervals of 2-7 are over-represented. Lysine preferentially occurs in singlets and alanine in doublets rather than triplets or quadruplets. This discourages the formation of helices without neutralization of lysine charges. When lysine residues are paired with DNA phosphate residues, helices are highly probable. Interproline spacing promotes short helical segments. The regularities arising from the conservation of composition and non-random residue distribution suggests that C-domains adopt similar structures and in fact are structurally conserved.

Crystallization and preliminary diffraction studies of Erythrina trypsin inhibitor.

Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.

Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase in dual porphyria.

The urinary and faecal porphyrin excretory profiles of dual porphyria are said to represent the superimposition of those found in porphyria cutanea tarda on those seen in variegate porphyria. To test this hypothesis the enzymes responsible for these conditions, protoporphyrinogen oxidase (variegate porphyria) and uroporphyrinogen decarboxylase (porphyria cutanea tarda) were measured in vitro in haemolysates and lymphoblasts of 10 subjects with dual porphyria in order to clarify the enzymatic defects. Mean protoporphyrinogen oxidase activity in lymphoblasts from subjects with dual porphyria was decreased by 45% from 0.82 +/- 0.10 to 0.45 +/- 0.09 nmol protoporphyrin/mg protein/h (P less than 0.001). Uroporphyrinogen decarboxylase activity was also significantly reduced from 0.12 +/- 0.05 nmol 7-, 6-, 5- and 4-carboxyl porphyrin/mg protein/h in lymphoblasts from normal subjects to 0.08 +/- 0.02 nmol in lymphoblasts of subjects with dual porphyria (P less than 0.01). There was a similar 27% decrease in mean uroporphyrinogen decarboxylase activity of haemolysates from the same dual porphyria group (P less than 0.01). Mean activity of this enzyme in 5 patients with variegate porphyria did not differ significantly from that in normal subjects. These findings may well provide a rational basis for the abnormal porphyrin excretory profiles found in subjects with dual porphyria.