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PubMed2005

Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria.

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.

Adaptation, Physiological↗
PubMed1990

The secD locus of E.coli codes for two membrane proteins required for protein export.

Cold-sensitive mutations in the secD locus of Escherichia coli result in severe defects in protein export at the non-permissive temperature of 23 degrees C. DNA sequence of a cloned fragment that includes the secD locus reveals open reading frames for seven polypeptide chains. Both deletions and TnphoA insertions in this clone have been used in maxicell and complementation studies to define the secD locus and its products. The secD mutations fall into two complementation groups, defining genes we have named secD and secF. These two genes comprise an operon, the first case of two genes involved in the export process being co-transcribed. The DNA sequence of the two genes along with alkaline phosphatase fusion analysis indicates that they code for integral proteins of the cytoplasmic membrane. We suggest that these two proteins may form a complex in the membrane which acts at late steps in the export process.

Amino Acid Sequence↗
PubMed2000

Biophysical and structural characterization of proton-translocating NADH-dehydrogenase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Mitochondrial proton-translocating NADH-dehydrogenase (complex I) is one of the largest and most complicated membrane bound protein complexes. Despite its central role in eukaryotic oxidative phosphorylation and its involvement in a broad range of human disorders, little is known about its structure and function. Therefore, we have started to use the powerful genetic tools available for the strictly aerobic yeast Yarrowia lipolytica to study this respiratory chain enzyme. To establish Y. lipolytica as a model system for complex I, we purified and characterized the multisubunit enzyme from Y lipolytica and sequenced the nuclear genes coding for the seven central subunits of its peripheral part. Complex I from Y lipolytica is quite stable and could be isolated in a highly pure and monodisperse state. One binuclear and four tetranuclear iron-sulfur clusters, including N5, which was previously known only from mammalian mitochondria, were detected by EPR spectroscopy. Initial structural analysis by single particle electron microscopy in negative stain and ice shows complex I from Y. lipolytica as an L-shaped particle that does not exhibit a thin stalk between the peripheral and the membrane parts that has been observed in other systems.

Amino Acid Sequence↗
PubMed1989

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao.

Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.

Hemoglobin A↗
PubMedDate not supplied

Rat liver endothelial cells have a greater capacity than Kupffer cells to endocytose N-acetylglucosamine- and mannose-terminated glycoproteins.

The capacity of rat liver Kupffer and endothelial cells to endocytose glycoproteins with N-acetylglucosamine- or mannose-terminated oligosaccharide chains was studied. For this purpose, agalactoorosomucoid, ahexosaminoorosomucoid and horseradish peroxidase were used as ligands. A reliable determination of the amount of ligand endocytosed in vivo or in vitro was made possible by using the recently developed cold pronase method for the isolation and purification of Kupffer and endothelial cells. Both cell types participated in the uptake of the ligands in vivo as well as in vitro, but their endocytic capacity was several times greater in vivo than in vitro. Under both conditions, endothelial cells possessed a greater capacity to endocytose the ligands than did Kupffer cells. Since the total number of endothelial cells in the liver is at least twice the number of Kupffer cells, the contribution of endothelial cells to the liver uptake of N-acetylglucosamine-terminated glycoproteins in vivo was estimated to be 3 to 7 times higher than that of the Kupffer cells. In vitro experiments showed that the uptake of the glycoproteins followed saturation kinetics and was strongly inhibited at 4 degrees C and in the presence of mannan. Ultrastructural investigations revealed that horseradish peroxidase was taken up by all Kupffer and endothelial cells. These results emphasize the important role liver endothelial cells play in the clearance of specific glycoproteins from the circulation.

Animals↗
PubMed1984

Characterization of the phospholipid requirement of a rat liver beta-glucosidase.

The lipid requirement of membrane-bound rat liver beta-glucosidase was investigated using 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. The enzyme was solubilized and delipidated by sequential extraction of a crude lysosomal fraction from rat liver lysosomes with sodium cholate and ice-cold butan-1-ol. Neither saturated nor unsaturated phosphatidylcholine activated this enzyme. In contrast, acidic phospholipids like phosphatidylglycerol (PtdGro) and phosphatidylserine (PtdSer) were effective activators. For the PtdGro series, fatty acid composition was important, with the shorter chain or unsaturated fatty acid-containing PtdGro species being the best activators. Heat-stable factor (HSF) from Gaucher spleen by itself (1-2 micrograms) had no effect on enzyme activity. However, the same amount of HSF when combined with 10 micrograms of PtdSer markedly stimulated beta-glucosidase activity. In the presence of HSF, di-9-cis-octadecenoyl-PtdGro (1 microgram) or -PtdSer (5 micrograms) provided maximum protection of beta-glucosidase against heat (60 degrees C) inactivation. In the absence of phospholipids, HSF had no effect on the rate of inactivation of the enzyme by the suicide inhibitor conduritol B epoxide (t0.5, 12 +/- 0.5 min); the maximum rate of inactivation was achieved in the presence of a mixture of PtdGro (2.5-5 micrograms) and HSF (t0.5, 2.8 min). The combination of PtdSer (10 micrograms) and HSF (1.3 micrograms) lowered the Km for 4-methylumbelliferyl-beta-D-glucopyranoside from 24 to 2.7 mM. Inhibition of the enzyme by the glucocerebrosidase substrate analogues N-hexyl-O-glucosylsphingosine and glucosylsphingosine was influenced by the activator substances. The inclusion of PtdSer and HSF in the beta-glucosidase assay medium lowered the Ki of N-hexyl-O-glucosylsphingosine 20-fold. The same combination of activators decreased the I0.5 of the enzyme for glucosylsphingosine from 89.4 to 7.6 microM. A study of log (Vmax./Km) versus pH indicated that the PtdSer-HSF pair creates the active site of beta-glucosidase, making apparent three ionizable groups on the enzyme with pK values in the range 4.5-5.1.

Animals↗
PubMed2005

Evaluation of peroxisome proliferator-activated receptor-gamma expression in benign and malignant thyroid pathologies.

Impairment of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) function through a dominant negative PAX-8/PPAR-gamma fusion gene or other events resulting in wild-type PPAR-gamma downregulation has been implicated in malignant thyroid cell transformation. The aim of our study was to perform a systematic evaluation of PPAR-gamma mRNA and protein expression in normal thyroid tissue as opposed to benign thyroid pathologies of different functional status and thyroid malignancy, to gain further insights into a putative physiological role of PPAR-gamma in the thyroid and to define whether PPAR-gamma could serve as a marker of thyroid cell differentiation. Ten cold benign (CTN) and 10 toxic (TTN) thyroid nodules and corresponding normal thyroid tissues, 10 follicular thyroid cancers (FTC), 10 papillary thyroid cancers (PTC) and 8 Graves' disease (GD) thyroids were studied by real-time polymerase chain reaction (PCR), immunohistochemistry and reverse transcriptase (RT)-PCR (PAX-8/PPAR-gamma fusion gene). PPAR-gamma mRNA expression was demonstrated in all samples. When comparing benign nodular and normal thyroid tissue of the same patient no significant difference in PPAR-gamma mRNA expression was observed. PPAR-gamma mRNA levels were similar in CTN and FTC. In contrast, PPAR-gamma mRNA expression was downregulated in 9 of 10 PTC and all GD samples, whereby at least 4 fold downregulation (compared with normal and benign nodular thyroid tissues) was observed in the latter. Immunohistochemistry showed an increased, patchy PPAR-gamma nuclear staining in CTNs and TTNs and only faint staining in the corresponding normal thyroid tissues. A diffuse and weak PPAR-gamma staining pattern was observed in all GD samples. No PAX-8/PPAR-gamma rearrangements were detected in any of the 68 thyroid tissue samples. In conclusion PPAR-gamma mRNA and protein expression levels are not concordant in benign thyroid nodular disease. Furthermore there is no clear-cut association of PPAR-gamma mRNA expression with follicular thyroid tumorigenesis. Absence of a PAX-8/PPAR-gamma fusion gene in the series of 68 thyroid samples is in agreement with the suggestion of PAX-8/PPAR-gamma rearrangement being restricted to a subset of follicular thyroid cancers. The marked downregulation of PPAR-gamma in GD warrants further investigation and could be linked, for example, with changes in apoptosis.

Adenoma↗
PubMed1984

Temperature-dependence of the action of nerve blocking agents and its relationship to membrane-buffer partition coefficients: thermodynamic implications for the site of action of local anaesthetics.

The local anaesthetic action of a series of nerve blocking agents was examined at temperatures between 4 degrees C and 20 degrees C in isolated sciatic nerves from cold-adapted frogs. Cooling alone had little effect on the amplitude of the action potential but the conduction velocity was decreased and the duration increased. Cooling had little effect on the local anaesthetic action of the short chain alkanols but that of the long chain alkanols, benzyl alcohol, benzocaine and pentobarbitone was markedly enhanced. The partition of both short and long chain alkanols and of pentobarbitone into a liposome suspension of similar composition to axonal membrane lipids was reduced by cooling from 40 degrees C to 4 degrees C. The results are not compatible with the lipid hypotheses of anaesthetic action. The implications for the nature of the site of action are discussed.

Action Potentials↗
PubMed2003

Mutations of the p53 gene in gliomas from Malay patients.

This is the first investigation performed to detect the presence of the p53 mutation in Malay patients with gliomas. The p53 gene was amplified using polymerase chain reaction (PCR) from 33 fresh-frozen tumour tissues from patients histologically confirmed as glioma. Four hot spot areas that lie between exon 5 to 8 were screened for mutation by mean of non-isotopic "cold" single strand conformation polymorphism (SSCP) analysis and direct sequencing. The frequency of p53 gene mutation in gliomas examined was 33% (11 of 33). Five (45.5%) cases had mutation in exon 7, four (36.4%) had mutation in exon 8 and two (18.1%) had mutation in exon 6. Seven (63.6%) of 11 mutations were single nucleotide point mutations of which 5 were missense mutations, 1 was nonsense mutation and 1 was, silent mutation. Three (27.3%) showed insertion mutation and 1 (9.1%) showed deletion mutation. Of the point mutations, 57.1% were transitions and 42.9% were transversions. These results suggested that p53 mutations frequently occur in gliomas and this gene does play an important role in the tumourigenesis process of Malay patients with brain tumours.

Brain Neoplasms↗
PubMed1989

Cryocrystallography of ribosomal particles.

Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains. At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes. However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated. Hence it has become possible to collect a complete data set from one single crystal. Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane. Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity. In several cases soaking in a cryosolvent was required. Crystallographic data were collected with intense synchrotron radiation. Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H. marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B. stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T. thermophilus.

Bacteria↗
PubMed1999

Microbial Diversity in Sediments Collected from the Deepest Cold-Seep Area, the Japan Trench.

: The Japan Trench land slope at a depth of 6,400 m is the deepest cold-seep environment with Calyptogena communities. Sediment samples from inside and beside the Calyptogena communities were collected, and the microbial diversity in the sediment samples was studied by molecular phylogenetic techniques. From DNA extracted directly from the sediment samples, 16S rDNAs were amplified by the polymerase chain reaction method. The sequences of the amplified 16S rDNAs selected by restriction fragment length polymorphism analysis were determined and compared with sequences in DNA databases. The results showed that 33 different bacterial 16S rDNA sequences from the two samples analyzed fell into similar phylogenetic categories, the alpha-, gamma-, delta-, and varepsilon-subdivisions of Proteobacteria, Cytophaga, and gram-positive bacteria; some of the 16S rDNA sequences were common to both samples. delta- and varepsilon-Proteobacteria-related sequences were abundant in both sediments. These sequences are mostly related to sulfate-reducing or sulfur-reducing bacteria and epibionts, respectively. Eight different archaeal 16S rDNA sequences were cloned from the sediments. The majority of the archaeal 16S rDNA sequences clustered in Crenarchaeota and showed high similarities to marine group I archaeal rDNA. A Methanococcoides burtonii-related sequence obtained from the sediment clustered in the Euryarchaeota indicating that M. burtonii-related strains in the area of Calyptogena communities may contribute to production of methane in this environment. From these results, we propose a possible model of sulfur circulation within the microbial community and that of Calyptogena clams in the cold-seep environment.

Journal Article↗
PubMed2003

The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features.

Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9 A resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63 A. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG.

Amino Acid Sequence↗
PubMed1996

Adenovirus-mediated gene transfer using ex vivo perfusion of the heart graft.

A replication-deficient adenovirus was used for ex vivo gene transfer into rat heart grafts under conditions simulating clinical transplantation. The adenoviral vector, AdHCMVsp1LacZ, containing an expression cassette of Escherichiae coli lacZ, was used to perfuse heart grafts during cold ischemia before transplantation. Heart grafts were perfused with University of Wisconsin (UW) solution containing either 0 pfu, 5 x 10(10) pfu, or 1 x 10(11) pfu of viral vector, and were preserved for either 2 or 4 h and then transplanted into syngeneic recipients. The animals were killed at 1, 7, and 14 days after transplantation. The infection rate was assessed by histochemical staining for beta-galactosidase. Using polymerase chain reaction (PCR), viral DNA presence was confirmed in every graft perfused with viral vectors. The protein production from the transfected gene was confirmed by a functional protein assay. An efficient gene transfer was achieved with an infection rate of 1%-1.5% for all cardiac myocytes, as assessed by 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-gal) staining. All studies were negative in the control grafts. Gene expression persisted for at least 10 days after transplantation. We thus conclude that an efficient adenovirus-mediated gene transfection and expression of gene products can be achieved in ex vivo perfusion of the heart graft during cold preservation.

Abdomen↗
PubMed2000

Isolation and cDNA-derived amino acid sequences of hemoglobin and myoglobin from the deep-sea clam Calyptogena kaikoi.

The heterodont clam Calyptogena kaikoi, living in the cold-seep area at a depth of 3761 m of the Nankai Trough, Japan, has abundant hemoglobins and myoglobins in erythrocytes and adductor muscle, respectively. Two types of hemoglobins (Hb I and Hb II) were isolated, and the complete amino acid sequences of Hb I (145 residues) and Hb II (137 residues) were obtained with combination of cDNA and protein sequencing. The amino acid sequences of C. kaikoi Hbs I and II differed from homologous chains of the congeneric clam Calyptogena soyoae in eight and five positions, respectively. The distal (E7) His, one of the functionally important residues in hemoglobin and myoglobin, was replaced by Gln in hemoglobins of C. kaikoi. A phylogenetic analysis of clam hemoglobins indicates that the evolutionary rate of Calyptogena hemoglobins is rather faster than those of other clams, suggesting that the mutation rate might be accelerated in the deep-sea animals around the areas of cold seeps or hydrothermal vents. On the other hand, it was found unexpectedly that two myoglobins Mbs I and II, isolated from the red adductor muscle, are identical in amino acid sequence Hbs I and II, respectively. Thus it was assumed that genes for Hbs I and II are also expressed in the muscle of C. kaikoi in substitution for myoglobin gene. This suggests that the major physiological role of globins in C. kaikoi is storage of oxygen under the low oxygen conditions, rather than circulating of oxygen.

Amino Acid Sequence↗
PubMed2000

The effect of metabolic depression on proton leak rate in mitochondria from hibernating frogs.

Futile cycling of protons across the mitochondrial inner membrane accounts for 20 % or more of the total standard metabolic rate of a rat. Approximately 15 % of this total is due to proton leakage inside the skeletal muscle alone. This study examined whether the rate of proton leak is down-regulated as a part of a coordinated response to energy conservation during metabolic depression in cold-submerged frogs. We compared the proton leak rate of skeletal muscle mitochondria isolated from frogs at different stages of hibernation (control, 1 month and 4 months of submergence in normoxia and hypoxia). The kinetics of mitochondrial proton leak rate was unaltered throughout normoxic and hypoxic submergence. The state 4 respiration rates did not differ between control animals and frogs hibernating in normoxia. In contrast, the state 4 respiration rates obtained from frogs submerged in hypoxic water for 4 months were half those of control animals. This 50 % reduction in respiration rate in hypoxic hibernation was due to a reduction in electron transport chain activity and consequent decrease in mitochondrial membrane potential. We conclude that proton leak rate is reduced during metabolic depression as a secondary result of a decrease in electron transport chain activity, but that the proton conductance is unchanged. In addition, we show that the rate of proton leakage and the activity of the electron transport chain are lower in frogs than in rats, strengthening the observation that mitochondria from ectotherms have a lower proton conductance than mitochondria from endotherms.

Animals↗
PubMed2002

An EREBP/AP2-type protein in Triticum aestivum was a DRE-binding transcription factor induced by cold, dehydration and ABA stress.

We characterize one transcription factor of DRE-binding proteins (TaDREB1) that was isolated from a drought-induced cDNA library of wheat (Triticum aestivum L.). TaDREB1 contains one conserved EREBP/AP2 domain, and shows similarity with Arabidopsis thaliana DREB family members in both overall amino-acid sequences and the secondary structure arrangement within the DNA-binding motifs. In yeast one-hybrid system, TaDREB1, can specially activate the genes fused with the promoter containing three tandemly repeated copies of the wild-type DRE sequence: TACCGACAT. In different wheat cultivars, the Ta DREB1 gene is induced by low temperature, salinity and drought; and the expression of Wcs120 that contains DRE motifs in its promoter is closely related to the expression of TaDREB1. These results suggest that TaDREB1 functions as a DRE-binding transcription factor in wheat. We also observed the dwarf phenotype in transgenic rice (T0) overexpressing TaDREB1.

Abscisic Acid↗