Molecular cloning of the gene encoding a 2Fe-2S ferredoxin from extremely halophilic archaeon Haloarcula japonica strain TR-1

A ferredoxin was purified from extremely halophilic archaeon Haloarcula japonica strain TR-1, and then characterized. The Ha. japonica ferredoxin proved to contain a 2Fe-2S cluster. A part of the gene encoding the ferredoxin was amplified by PCR. Subsequently, the entire ferredoxin gene was cloned from chromosomal DNA of Ha. japonica using the PCR product as a probe. The structural gene consisted of an open reading frame of 387 nucleotides. The deduced amino acid sequence showed 89-98% identities with those of the ferredoxins from other extremely halophilic archaea.     

The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7

The crystal structure of ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was determined by multiple isomorphous replacement supplemented with anomalous scattering effects of iron atoms in the Fe-S clusters, and refined at 2.0 A resolution to a crystallographic R value of 0.173. The structural model contains a polypeptide chain of 103 amino acid residues, 2 [3Fe-4S] clusters, and 31 water molecules; in this model, the cluster corresponding to cluster II in bacterial dicluster ferredoxins loses the fourth iron atom although it may originally be a [4Fe-4S] cluster. The structure of the archaeal ferredoxin consists of two parts: the core fold part (residues 37-103) and the N-terminal extension part (residues 1-36). The "core fold" part has an overall main-chain folding common to bacterial dicluster ferredoxins, containing two clusters as the active center, two alpha-helices near the clusters, and two sheets of two-stranded antiparallel beta-sheet (the terminal and central beta-sheets). The "N-terminal extension" part is mainly formed by a one-turn alpha-helix and a three-stranded antiparallel beta-sheet. The beta-sheet in the N-terminal extension is hydrogen-bonded with the terminal beta-sheet in the core fold to form a larger beta-sheet. The distinct structural feature of this archaeal ferredoxin lies in the zinc-binding center where the zinc ion is tetrahedrally ligated by four amino acid residues (His 16, His 19, and His 34 from the N-terminal extension, and Asp 76 from the core fold). The zinc ion in the zinc-binding center is located at the interface between the core fold and the N-terminal extension, and connects the beta-sheet in the N-terminal extension and the central beta-sheet in the core fold through the zinc ligation. Thus, the zinc ion plays an important role in stabilizing the structure of the present archaeal ferredoxin by connecting the N-terminal extension and the core fold, which may be common to thermoacidophilic archaeal ferredoxins.     

Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization

The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes (a) precipitation by ammonium sulfate, (b) fractionation on Sepharose 4B using a decreasing concentration gradient of ammonium sulfate, (c) gel permeation chromatography on Sephadex G-100, (d) chromatography on hydroxylapatite, and (e) affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was found to be 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific "volume" phi' of the active enzyme at 4.26 M NaCl was found to be 0.680 +/- 0.015 mL/g. The molecular weight of the native enzyme was found to be 84 000 +/- 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The molecular weight of the subunits is 39 000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of two subunits.     

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.     

NADP-glutamate dehydrogenase from the halophilic archaeon Haloferax mediterranei: enzyme purification, N-terminal sequence and stability

BACKGROUND: Renal tract neoplasms account for only 15% of all cases of hematuria and are rare before the age of 40. Few cases of bladder carcinoma diagnosed during pregnancy have been reported. CASE: Adenocarcinoma of the dome of the bladder caused hematuria beginning at 20 weeks' gestation but was not diagnosed until the postpartum period. CONCLUSION: Hematuria during pregnancy is usually associated with infection. However, neoplasms must be included in the differential diagnosis.     

Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112

The complete amino acid sequence of transglutaminase (EC 2.3.2.13) (TGase), which is produced by a microorganism, Streptoverticillium sp. strain s-8112, and catalyzes the acyl transfer reaction between gamma-carboxyamide groups of glutamine residues in proteins and various primary amines, has been established by a combination of fast atom bombardment mass spectrometry and standard Edman degradation of peptide fragments produced by treatment of the TGase with various proteolytic enzymes and purified by a reversed-phase high performance liquid chromatography. The TGase consists of 331 amino acid residues with a chemical molecular weight of 37,863, in agreement with the observed molecular weight (37,869.2 +/- 8.8) determined from its electrospray ionization mass spectrum. The sequence of the enzyme is very different from those of mammalian TGases represented by guinea pig liver enzyme. The enzyme contains a sole Cys residue, which is essential for its catalytic activity. Hydropathy analysis indicated that the secondary structure of the region around the active site Cys residue is similar to those of mammalian TGases. These results suggest that this microbial protein evolved by a different pathway from that of mammalian TGases and acquired acyl transfer activity during the evolutional process.     

Haloanaerobium lacusroseus sp. nov., an extremely halophilic fermentative bacterium from the sediments of a hypersaline lake

A new extremely halophilic chemoorganotrophic bacterium (strain H200T [T = type strain]) was isolated from the hypersaline sediments of Retba Lake in Senegal. This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grew optimally at 40 degrees C in the presence of 180 to 200 g of NaCl per liter. The DNA base composition was 32 mol% guanine plus cytosine. The fermentation products from glucose were ethanol, acetate, H2, and CO2. Yeast extract was required for growth. The fermentable substrates included D-fructose, galactose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, D-mannitol, glycerol, and Casamino Acids. On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species. The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species. We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus.     

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae

Tricyclic antidepressant overdose produces rapid multisystem complications for the patient. Treatment of the patient requires careful assessment and aggressive nursing interventions in order to produce a positive outcome.     

Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: analysis of structure and thermostability

The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 A resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %). The crystals belong to the space group C2 (a=76.3 A, b=124.3 A, c=60.3 A, beta=128.8 degrees) with two identical monomers in the asymmetric unit. The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map. The overall fold of the monomer of S. solfataricus SOD is similar to that of the other known Fe or Mn-SODs. S. solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus. Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus. However, in contrast to the A. pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter- subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures. The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site. Its involvement in the specific activity of the enzyme is discussed.     

Sorption extraction of cobalt ions from nitrate media by the iminodiacetate chelating resins Lewatit TR-207 and TR-260

Cobalt-ion sorption from nitrate media by the macroporous chelating resins Lewatit TR-207 and TR-260, which contain active functional groups based on iminodiacetic acid, is studied. Both resins are shown to sorb cobalt from nitrate solutions rather efficiently at pH 1.5–2.0, and the degree of cobalt extraction reaches ～100% for a solid to liquid ratio of ≥0.3. Under optimal conditions, the cobalt capacity of the resins is ～160 mg/g. The system under study is equilibrated within 240 min, and >70% Co is extracted during the first hour of sorption. On heating to 40–40°C, cobalt sorption enhances; however, upon further heating to 50–52°C, the extraction of cobalt decreases, which is probably caused by thermal destruction of the resin. The estimated change in the enthalpy of cobalt sorption in the temperature range 21–41°C is about +84 kJ/mol. The results obtained are employed to test the cobalt sorption under dynamic conditions in a laboratory column packed with Lewatit TR-207 resin in the H⁺ form (the working volume of the column is 100 ml) using the real mother liquor (24.5 mg Co/l) of cobalt oxalate precipitation. Quantitative desorption of cobalt is conducted with a solution of HNO₃ (400 g/l). During dynamic tests, about nine specific volumes of the initial mother liquor are passed through the column. After this, the cobalt content in the purified solution is lower than 0.4–0.5 mg/l; i.e., the total degree of cobalt extraction is >98%. In the desorption solution, the cobalt concentration increases to ～0.3 g/l, which corresponds to a more than tenfold degree of cobalt enrichment. Thus, the resins under study can be recommended for the extraction of cobalt microamounts (20–50 mg/l) from foul and reused waters.     

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.     

Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide

The chemical structure of a novel lipid A, which was obtained as a major component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta(1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-position, respectively. The absence of a phosphoryl group at the 4'-position and fatty acyl groups at the 3- and 3'-position, and the stoichiometric presence of 2-aminoethyl phosphate at the 1-position are unique features, distinguishing it from the lipid A of enterobacteria.     

Primary structure of gene H of cattle plague virus strain K]

The paper analyzes the standard legal and methodological assurance of the quality and safety of animal food raw materials and foodstuffs (meat, meat products, fish, shellfish, crayfish and their processing products) by the parasitic purity rates according the requirements under the Russian Federation's laws "On Sanitary and Epidemiological Well-Being of the Population", "On Protection of Consumer's Rights", "On Certification of Products and Services", those of SanPiN, such as 2.3.2.560-96 "Sanitary Requirements for the Quality and Safety of Food Raw Materials and Foodstuffs" and 3.2.569-96 "Prevention of Parasitic Diseases in the Russian Federation".     

Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain

We established four new mouse strains with defective T and B cells as well as defects in innate immunological reactions using an NK cell depletion antibody and showed that all mutant mouse strains efficiently received human peripheral blood leukocyte (PBL) engraftment (hu-PBL-scid mice). Higher levels of human immunodeficiency virus type 1 (HIV-1) replication were observed in these new hu-PBL-scid mice than in conventional hu-PBL-C.B-17-scid mice. In one particular strain, hu-PBL-NOD-scid mice, high levels of HIV-1 viremia (more than 10(6) 50% infectious doses per ml) were detected after infection with HIV-1. The plasma viral load was about 100 to 1,000 times higher than that observed in other hu-PBL-scid mice infected with HIV-1. Although high-level viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected only in hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lungs, and brain. PCR in situ hybridization confirmed that HIV-1-infected cells invaded the brain tissue of the hu-PBL-NOD-scid mice. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent central nervous system invasion with HIV-1. The hu-PBL-NOD-scid mouse represents a useful model for the study of the pathogenesis of HIV-1 in vivo, especially brain involvement, and therapy of primary HIV-1 viremia.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.     

Primary structure of Beijing duck apolipoprotein A-1

The primary structure of Beijing duck apolipoprotein A-1 was determined by sequencing peptide fragments derived from tryptic and endoproteinase Asp-N digestion of the protein, and alignment with homologous chicken apo A-1. All of the peptide fragments were isolated by high-pressure liquid chromatography (HPLC) with a Vydac C18 column using a trifluoroacetic acid (TFA) buffer system. The N-terminus of the protein was determined to be aspartic acid by directly sequencing 52 residues of the intact protein. The C-terminus was alanine. The protein contains 240 amino acid residues. By analysis of the whole protein and its tryptic peptides, a six amino acid (Arg-Tyr-Phe-Trp-Gln-His) prosegment was determined. No cross-reactivity between duck and human apo A-1 with a goat antiserum against human apo A-1 was found. Sequence analysis of apo A-1 of other species indicates that amino acid substitutions in rat are more extensive than in other mammals. Isoleucine residues in apo A-1 are inversely correlated to the homology of human to other species, except dog.     

Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 A resolution

The extremely thermostable superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius was crystallized and the three-dimensional structure was determined by X-ray diffraction methods. The enzyme crystallized in the monoclinic spacegroup C2 with the cell dimensions a=168.1 A, b=91.3 A, c=85.7 A, beta=91.4 degrees. The diffraction limit of these crystals was 2.2 A. The crystals were very stable in the X-ray beam and measured diffraction data of a single crystal had a completeness of 99.5 % up to a resolution of 2.2 A. The crystal structure of S. acidocaldarius superoxide dismutase was solved by Patterson search methods using a dimer of Thermus thermophilus superoxide dismutase as a search model. The asymmetric unit accommodates three dimers. Two dimers form a tetramer by using only local symmetries; the third dimer forms a tetramer as well, however, by using the crystallographic 2-fold symmetry. The three-dimensional structure of the S. acidocaldarius dismutase has typical features of tetrameric dismutases. Secondary structure elements as well as residues important for the catalytic activity of the enzyme were found to be highly conserved. The model was refined at a resolution of 2.2 A and yielded a crystallographic R-value of 17.4 % (Rfree=22.3 %). A structural comparison of the two extremely stable tetrameric dismutases from S. acidocaldarius and Aquifex pyrophilus with the less stable enzyme from T. thermophilus and Mycoplasma tuberculosis revealed the structural determinants which are probably responsible for the high intrinsic stability of S. acidocaldarius dismutase. The most obvious factor which may give rise to the extraordinary thermal stability of S. acidocaldarius dismutase (melting temperature of about 125 degreesC) is the increase in intersubunit ion pairs and hydrogen bonds and, more importantly, the significant reduction of solvent-accessible hydrophobic surfaces, as well as an increase in the percentage of buried hydrophobic residues.     

Primary structure of trypsin inhibitors from Sicyos australis

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.