The hemoglobin system of Antarctic and non-Antarctic notothenioid fishes.

Studies of the hemoglobin system of fish of the suborder Notothenioidei have been extended to non-Antarctic species of Pseudaphritis urvillii and Notothenia angustata. The two species belong to families that were the first to diverge within the suborder. The degree of amino acid sequence identity with Antarctic notothenioids and other non-Antarctic fish species is analyzed with respect to phyletic and ecological diverence.

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus.

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms.

Tracking the evolutionary loss of hemoglobin expression by the white-blooded Antarctic icefishes.

The blood of Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) is completely devoid of hemoglobin. Icefishes have developed compensatory adaptations that reduce oxygen demand and enhance oxygen transport. Oxygen delivery to tissues occurs by carrying the gas physically dissolved in the plasma.

DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus.

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology.

Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization.

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation.

Decreased mitochondrial carnitine translocase in skeletal muscles impairs utilization of fatty acids in insulin-resistant patients.

Insulin resistance (IR) and its health consequences (diabetes, hypertension, cardiovascular disease, obesity etc.) affect between 25 and 35% of Westernized populations. Decreased fatty acid (FA) oxidation in skeletal muscle is implicated in obesity-related IR.

The Antarctic Psychrobacter sp. TAD1 has two cold-active glutamate dehydrogenases with different cofactor specificities. Characterisation of the NAD+-dependent enzyme.

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties.

Stability and conformational dynamics of metallothioneins from the antarctic fish Notothenia coriiceps and mouse.

The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N.

Molecular dynamics analysis of a second phosphate site in the hemoglobins of the seabird, south polar skua. Is there a site-site migratory mechanism along the central cavity?

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M.

Design of cassette vectors permitting cloning of all types of human TCR variable alpha and beta regions.

T cell clones are an irreplaceable asset for the study of immune responses relevant to human pathologies. Such cells, however, cannot always be maintained in long-term culture. In order to reconstitute functional human T cell receptors (TCRs) into stable and fast growing hybridoma T cells, we developed a general approach based on a versatile cassette system, which allows cloning of all types of human T cell receptor variable alpha and beta region genes fused to murine constant regions.

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.

Enzymatic synthesis of oligosaccharides by two glycosyl hydrolases of Sulfolobus solfataricus.

The importance of carbohydrates in a variety of biological functions is the reason that interest has recently increased in these compounds as possible components of therapeutic agents. Thus, the need for a technique allowing the easy synthesis of carbohydrates and glucoconjugates is an emerging challenge for chemists and biologists involved in this field. At present, enzymatic synthesis has resulted in the most promising approach for the production of complex oligosaccharides.

Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature.

The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations.

Structural characterization and thermal stability of Notothenia coriiceps metallothionein.

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs.

A novel member of the bacterial-archaeal regulator family is a nonspecific dna-binding protein and induces positive supercoiling.

In hyperthermophilic Archaea genomic DNA is from relaxed to positively supercoiled in vivo because of the action of the enzyme reverse gyrase, and this peculiarity is believed to be related to stabilization of DNA against denaturation. We report the identification and characterization of Smj12, a novel protein of Sulfolobus solfataricus, which is homologous to members of the so-called Bacterial-Archaeal family of regulators, found in multiple copies in Eubacteria and Archaea. Whereas other members of the family are sequence-specific DNA- binding proteins and have been implicated in transcriptional regulation, Smj12 is a nonspecific DNA-binding protein that stabilizes the double helix and induces positive supercoiling.

L-Glutamate dehydrogenase from the antarctic fish Chaenocephalus aceratus. Primary structure, function and thermodynamic characterisation: relationship with cold adaptation.

In order to study the molecular mechanisms of enzyme cold adaptation, direct amino acid sequence, catalytic features, thermal stability and thermodynamics of the reaction and of heat inactivation of L-glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (suborder Notothenioidei, family Channichthyidae) were investigated. The enzyme shows dual coenzyme specificity, is inhibited by GTP and the forward reaction is activated by ADP and ATP. The complete primary structure of C.

Structural and functional analysis of the two haemoglobins of the antarctic seabird Catharacta maccormicki characterization of an additional phosphate binding site by molecular modelling.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis.

Identification of a new subset of cells exhibiting dendritic phenotypes in patients affected by breast proliferative disorders.

We report that a subset of circulating cells reacting with a monoclonal antibody raised against a protein marker is significantly increased in the peripheral blood of women carrying benign or malignant breast diseases, particularly in patients under 55 years of age with ductal mammary carcinomas. These cells were statistically (confidence level of 99%) less represented in a control population including healthy women or women carrying carcinomas of origin other than breast. Double staining analysis showed that they harbor markers of dendritic cells and exhibit endo- cytic activity, as determined by their ability to internalize FITC-dextran particles.

Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii.

A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated.

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens.

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP).

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da.

Identification and molecular characterization of the first alpha -xylosidase from an archaeon.

We here report the first molecular characterization of an alpha-xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 degrees C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to alpha-glucosidases, alpha-xylosidases, glucoamylases and sucrase-isomaltases of the glycosyl hydrolase family 31.