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PubMed1998

Low-grade pulmonary mucosa-associated lymphoid tissue lymphoma with or without intraclonal variation.

We examined the immunoglobulin heavy chain variable (Ig VH) region genes of 11 low-grade pulmonary mucosa-associated lymphoid tissue (MALT) lymphomas by a two-step polymerase chain reaction (PCR) and sequencing analysis. We observed frequent somatic mutations with the positive selective pressure of the rearranged Ig VH genes in all cases, indicative of postgerminal center cell origin. Eight cases demonstrated intraclonal variations (hypermutation with intraclonal variation type), but the other cases showed only one major clone without intraclonal heterogeneity (hypermutation without intraclonal variation type). The former might reflect the reentry of marginal zone B cells into a germinal center environment leading to further mutations. The latter might be no longer susceptible to hypermutation mechanisms and seemed to be stable. Four cases used Ig VH genes (hv3019b9, VH26, and VH4.21), which are frequently found in a variety of autoantibodies, such as cold agglutinins, rheumatoid factors, and anti-DNA antibodies.

Adult↗
PubMed1995

Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel.

Tryptophans in the gramicidin channel are important for defining the conformation and the orientation with respect to the bilayer normal and for facilitating cation conductance. Here, high-resolution structure and dynamics of these rings are characterized by solid state NMR. Both oriented and unoriented lipid bilayer preparations are used. Fast frozen lipid bilayer preparations of unoriented samples have been used to obtain static characterizations of nuclear spin interaction tensors. The temperature dependence of these unoriented samples and the spectral features of fast frozen oriented samples were used to experimentally define the local motions of the individual indole rings. Local motions were shown to have amplitudes as high as +/- 29 degrees, and the motions were dominated by libration about the chi 2 axis. The high-resolution structure has been achieved by interpreting seven precise (+/- 0.3 degree) orientational constraints from 2H and 15N NMR for each indole ring in light of the motionally averaged interaction tensors. Each of the four indoles is restricted to a unique orientation of the ring with respect to the bilayer normal and one of two possible rotameric states. The side chain torsion angles for each residue are very similar, generating similar electric dipole moment orientations with respect to the channel.

Amino Acid Sequence↗
PubMed2004

Biomolecular cryocrystallography: structural changes during flash-cooling.

To minimize radiation damage, crystal structures of biological macromolecules are usually determined after rapid cooling to cryogenic temperatures, some 150-200 K below the normal physiological range. The biological relevance of such structures relies on the assumption that flash-cooling is sufficiently fast to kinetically trap the macromolecule and associated solvent in a room-temperature equilibrium state. To test this assumption, we use a two-state model to calculate the structural changes expected during rapid cooling of a typical protein crystal. The analysis indicates that many degrees of freedom in a flash-cooled protein crystal are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states. Such cryoartifacts should be most important for strongly solvent-coupled processes, such as hydration of nonpolar cavities and surface regions, conformational switching of solvent-exposed side chains, and weak ligand binding. The dynamic quenching that emerges from the model considered here can also rationalize the glass transition associated with the atomic fluctuations in the protein.

Cold Temperature↗
PubMed1988

Acylation of lysophosphatidylcholine in liver microsomes of thermally acclimated trout.

The specificity of acyl-coenzyme A (CoA): lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) was determined for a range for acyl-CoA substrates differing with respect to chain length and degree of unsaturation in liver microsomes of thermally acclimated (5 and 20 degrees C) rainbow trout, Salmo gairdneri. Absolute levels of oleate incorporation into phosphatidylcholine (PC) were determined at substrate concentrations in the physiological range (12 microM) and higher (64 microM). The specificity of LPCAT was determined by the extent to which competing substrates decreased the incorporation of oleate. LPCAT specificity was significantly influenced by both assay and acclimation temperature at total substrate concentrations of both 72 and 256 microM. A clear preference for 14- and 16-carbon monoenes was exhibited by LPCAT from 20 but not 5 degrees C-acclimated trout. Furthermore, LPCAT from 5 degrees C-acclimated trout preferentially incorporated long chain and polyunsaturated fatty acids (eicosadienoyl, arachidonoyl, erucoyl) and excluded 18-carbon unsaturates at an assay temperature of 5 degrees C compared with 20 degrees C; at 20 degrees C, 18-carbon unsaturates were incorporated more readily than 20-carbon species. Linolenic acid (18:3N3) was generally excluded from incorporation, reflecting a possible mechanism by which this precursor of docasahexaenoic acid (22:6N3, n - 3) remains available for modification. These results indicate that trout liver LPCAT preferentially incorporates fatty acids into PC on the basis of both chain length and degree of unsaturation in a manner consistent with the temperature-induced restructuring of membrane phospholipids.

Acclimatization↗
PubMed1977

The relation of clinical and laboratory findings to the survival of patients with macroglobulinaemia.

The relation of clinical and laboratory findings at the time clinical presentation to subsequent survival was investigated in 204 patients with macroglobulinaemia. It was found that the ten following clinical and laboratory parameters were of prognostic significance and a further twelve were not. The best prognoses were found amongst male patients, patients aged 50-69 years and those with serum IgM paraprotein concentrations of 20-39 g/1, type lambda paraproteins, no Bence-Jones proteinuria or cryoglobulinaemia, only one paraprotein, serum cold haemagglutinin activity, serum albumin above 40 g/1 and serum urea below 10 mmol/1.

Adolescent↗
PubMed1998

Structure of a non-psychrophilic trypsin from a cold-adapted fish species.

The crystal structure of cationic trypsin (CST) from the Atlantic salmon (Salmo salar) has been refined at 1.70 A resolution. The crystals are orthorhombic, belong to space group P212121, with lattice parameters a = 65.91, b = 83.11 and c = 154.79 A, and comprise four molecules per asymmetric unit. The structure was solved by molecular replacement with AMoRe and refined with X-PLOR to an R value of 17.4% and Rfree of 21.5% for reflections |F| > 3sigmaF between 8.0 and 1.7 A resolution. The four non-crystallographic symmetry (NCS) related molecules in the asymmetric unit display r.m.s. deviations in the range 0.31-0.74 A for main-chain atoms, with the largest differences confined to two loops. One of these is the calcium-binding loop where the electron-density indicates a calcium ion for only one of the four molecules. In order to find structural rationalizations for the observed difference in thermostability and catalytic efficiency of CST, anionic salmon trypsin (AST) and bovine trypsin (BT), the three structures have been extensively compared. The largest deviations for the superimposed structures occur in the surface loops and particularly in the so-called 'autolysis loop'. Both the salmon enzymes possess a high methionine content, lower overall hydrophobicity and enhanced surface hydrophilicity, compared with BT. These properties have so far been correlated to cold-adaptation features, while in this work it is shown that the non-psychrophilic cationic salmon trypsin shares these features with the psychrophilic anionic salmon trypsin.

Adaptation, Physiological↗
PubMed2002

Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells.

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Antigen-Presenting Cells↗
PubMed2004

The folding transition state of the cold shock protein is strongly polarized.

The cold shock protein CspB from Bacillus subtilis consists of a three-stranded (beta1-beta3) and a two stranded (beta4-beta5) sheet, which form a closed beta barrel structure. CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 degrees C). The transition state of folding is native-like, as judged by the Tanford betaT value of > or =0.9. By using a mutational approach and Phi value analysis, we find that the folding transition state of CspB is energetically polarized. Despite the high betaT value, most Phi values are low. Values close to 1 were found for only a few residues, particularly in strand beta1 (Lys5, Val6, Lys7, Asn10). The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands beta1 and beta2. Lys5 and Val6 in beta1 interact with residues in beta4, and their high Phi values indicate that an energetic linkage between beta1 and beta4 and thus between the two sheets exists already in the transition state. We compared our experimental Phi values with theoretical predictions of the folding pathway of cold shock proteins. Several of them suggest that the entire first sheet is formed in the transition state, and some identify the beta1-beta4 pairing as a crucial step in folding. Alternative paths that involve formation of the second sheet and beta3-beta5 pairing reactions were, however, suggested as well. The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure. They did not identify the key residues with the high Phi values within these structural elements.

Bacillus subtilis↗
PubMed2004

Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract.

PURPOSE: Overactive bladder symptoms due to various etiologies have been successfully treated with capsaicin by desensitization of the temperature sensitive vanilloid receptor TRPV1. Recently another temperature sensitive receptor, TRPM8, activated by menthol and cool temperatures (8C to 28C) was described that may be the proposed cool receptor, at least in part mediating the bladder response in the diagnostic ice water test. We defined the sites of mRNA and protein expression of TRPM8 and TRPV1 in the rat and human genitourinary tract. MATERIALS AND METHODS: Prostate, testis, penis, bladder and dorsal root ganglion tissue was obtained from rats. Prostate, testicle, seminiferous tubules, corpus cavernosum, glans, overlying glans skin, scrotal skin and bladder were obtained from human patients. Reverse transcription-polymerase chain reaction was done using species specific primers for TRPM8 and TRPV1. Immunofluorescence staining for TRPM8 was performed in rat tissues as well as in cultured human urothelial cells. RESULTS: TRPM8 and TRPV1 mRNA were detected in all rat tissues. Human samples demonstrated TRPM8 mRNA in prostate, testicle, seminiferous tubules, scrotal skin and bladder. No TRPM8 mRNA was identified in human corpus cavernosum, glans or overlying glans skin. Separation of layers in human bladder demonstrated mRNA for TRPM8 only in the urothelium and not in the detrusor. Immunofluorescence location of TRPM8 was found in rat prostate, DRG and bladder, and in human urothelial cells in culture. TRPV1 mRNA was detected in all human genitourinary tract tissues. CONCLUSIONS: These results demonstrate that mRNA and protein for TRPM8 exist in multiple genitourinary organs in the rat and human, and it may be considered a possible new target, as is TRPV1, for the pharmacological treatment of detrusor overactivity or other urological disorders.

Animals↗
PubMed2006

Cold denaturation of ubiquitin at high pressure.

Cold-induced conformational transition of ubiquitin was studied at pH 4.5 under a constant pressure of 2 kbar using variable pressure one-dimensional 1H and two-dimensional 15N/1H NMR spectroscopy as well as IR spectroscopy. Although a tendency for preferential stabilization of a peculiar locally disordered and partially hydrated conformer I, identical with that previously found with variable-pressure NMR at 0 degrees C, is recognized, the transition of the folded conformer N to the unfolded conformer U occurs largely cooperatively with decreasing temperature, reaching near completion at - 21 degrees C. NMR spectral features as well as the analysis of NMR relaxation parameters indicate that the polypeptide chain is almost fully unfolded, fairly well-hydrated and floppy at - 21 degrees C, whereas the IR spectrum shows a substantial decrease of the beta-sheet. The Gibbs energy change from the folded state (a mixture of N and I) to the unfolded state at 2 kbar obtained from the 1H NMR data is fitted well with a single DeltaCp value of 2.43 +/- 0.13 (kJ/K mol) for the entire temperature range between - 21 and 90 degrees C, covering both the cold denaturation and heat denaturation, showing that the two denatured states actually belong to a single thermodynamic phase of the protein. The DeltaCp value determined at 2 kbar is substantially smaller than the DeltaCp determined at 1 bar (3.8-5.8 (kJ/Kmol), which is consistent with the fact that the denaturation takes place from a mixture of N and I at 2 kbar rather than from pure N at 1 bar.

Cold Temperature↗
PubMed1987

Effects of increased doses of 1,25 dihydroxyvitamin D3 on matrix and DNA synthesis in condylar cartilage of suckling mice.

The in vivo effects of high doses of 1,25(OH)2D3 were studied in condylar cartilage of suckling mice. Seven-day-old animals were treated with 20 ng of the hormone for 7 consecutive days. Biochemical assays on collagen content and synthesis were complemented by structural studies using light and electron microscopy. Indirect immunofluorescent methods were used for the localization of type I and II collagens and for fibronectin. This study revealed that the protein content of the condyle decreased substantially following the administration of the hormone. Protein synthesis increased in hormone-treated animals during the first 4 days but was significantly inhibited thereafter. Collagen synthesis, however, was inhibited instantaneously, followed by a decrease in the percentage of cold hydroxyproline of the total protein. Hormone-treated condyles showed a marked decrease in the distribution of type I collagen, no apparent change in the distribution of type II collagen, but an enhanced reactivity for fibronectin especially around hypertrophic chondrocytes. SDS-gel electrophoresis of collagen chains suggested that the hormone did not induce a significant change in the ratios of type I and II collagen chains, yet additional peaks became evident in 1,25(OH)2D3-treated specimens. The decrease in collagen synthesis was accompanied by ultrastructural changes in the appearance of the extracellular collagen bundles. They later appeared as a dense meshwork of collagen fibrils, a feature that was lacking in control tissues. The changes in collagen fibrillogenesis could be explained by our in vitro studies indicating a marked depression of 35S-sulfate incorporation secondary to treatment with 1,25(OH)2D3. The hormone was also found to suppress the incorporation of 3H-thymidine, hence it may be concluded that 1,25(OH)2D3, when used in high concentrations, possesses an inhibitory effect upon both the proliferative activity of the cartilage progenitor cells as well as upon the metabolic activity of the condylar cells as related to collagen and glycosaminoglycans synthesis.

Animals↗
PubMed2006

Psychrophilic superoxide dismutase from Pseudoalteromonas haloplanktis: biochemical characterization and identification of a highly reactive cysteine residue.

A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with beta-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed.

Amino Acid Sequence↗
PubMed2004

Cloning, functional expression, and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan:galactan galactosyltransferase, from common bugle leaves.

Galactan:galactan galactosyltransferase (GGT) is a unique enzyme of the raffinose family oligosaccharide (RFO) biosynthetic pathway. It catalyzes the chain elongation of RFOs without using galactinol (alpha-galactosyl-myoinositol) by simply transferring a terminal alpha-galactosyl residue from one RFO molecule to another one. Here, we report the cloning and functional expression of a cDNA encoding GGT from leaves of the common bugle (Ajuga reptans), a winter-hardy long-chain RFO-storing Lamiaceae. The cDNA comprises an open reading frame of 1215 bp. Expression in tobacco (Nicotiana plumbaginifolia) protoplasts resulted in a functional recombinant protein, which showed GGT activity like the previously described purified, native GGT enzyme. At the amino acid level, GGT shows high homologies (>60%) to acid plant alpha-galactosidases of the family 27 of glycosylhydrolases. It is clearly distinct from the family 36 of glycosylhydrolases, which harbor galactinol-dependent raffinose and stachyose synthases as well as alkaline alpha-galactosidases. Physiological studies on the role of GGT confirmed that GGT plays a key role in RFO chain elongation and carbon storage. When excised leaves were exposed to chilling temperatures, levels of GGT transcripts, enzyme activities, and long-chain RFO concentrations increased concomitantly. On a whole-plant level, chilling temperatures induced GGT expression mainly in the roots and fully developed leaves, both known RFO storage organs of the common bugle, indicating an adaptation of the metabolism from active growth to transient storage in the cold.

Ajuga↗
PubMed2006

Differential gene expression in hemodialysis patients with "cold" zheng.

The aim of the present study was to search for the differential gene expression and measure the serum level of a number of biochemical parameters in the cold zheng (CZ) and non-cold zheng (NCZ) in patients receiving hemodialysis. Hemodialysis (HD) patients were randomly selected from the CZ and NCZ groups. The between-group differences in gene expression were assessed using complementary DNA (cDNA) microarray. Differential gene expression was further validated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Our results demonstrated that the up-regulation of the inflammation-associated genes, ALOX5AP, S100A8 and S100A12, down-regulation of the genes related to immunity (DEFA4), metabolism (GNG11, PYGB, PRKAR2B), and growth/proliferation (HSF2, DDR2, TK1) were found in the CZ group. Furthermore, the CZ HD patients had significantly lower serum albumin levels compared with their NCZ counterparts (3.31 +/- 0.08 g/dL versus 4.18 +/- 0.12 g/dL). It appears reasonable to conclude that up-regulated inflammatory-gene expression (ALOX5AP, S100A8 and S100A12) may play an important role in CZ HD patients.

Aged↗
PubMed1998

Differential regulation of uncoupling protein-2 and uncoupling protein-3 gene expression in brown adipose tissue during development and cold exposure.

The expression of the two novel uncoupling proteins genes, UCP2 and UCP3, is differentially regulated during prenatal maturation of brown adipose tissue in the mouse. UCP2 gene is expressed early in prenatal development, when neither UCP1 nor UCP3 gene expression yet occurs. UCP3 mRNA is absent at any stage of fetal life; it appears suddenly at birth and reaches adult levels in a few hours. UCP2 mRNA increased after birth but more slowly than UCP3 and UCP1 mRNA. Short-time exposure of adult mice to cold caused a rise in UCP2 or UCP1 mRNA levels but not in that of UCP3. The postnatal rise in UCP2 gene expression appears to be a response to the thermic stress associated with birth, similarly to UCP1, whereas different biological signals may be responsible for the surge in UCP3 gene expression at birth.

Adipose Tissue, Brown↗
PubMed1994

Inactivation of hepatitis C virus in low pH intravenous immunoglobulin.

The safety from hepatitis C virus of intravenous immunoglobulin prepared by the cold ethanol method of Cohn-Oncley is demonstrated by clearance through the manufacturing process of 9 x 10(6) plaque-forming units of bovine viral diarrhea virus used as a surrogate for hepatitis C virus. Incubation of the intravenous immunoglobulin in its final formulation at pH 4.25 for 21 days at 21 degrees C caused a 10,000-fold decrease in bovine viral diarrhea virus intentionally added and complete inactivation of 1000 chimpanzee infectious doses per ml of hepatitis C virus.

Animals↗
PubMed1996

Involvement of phospholipid molecular species in controlling structural order of vertebrate brain synaptic membranes during thermal evolution.

Fluorescence anisotropy parameter of [p-(6-phenyl)-1,3,5-hexatrienyl]phenyl-propionic acid (DPH-PA) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) embedded in synaptic plasma membranes prepared from brains of cold (5 degrees C) and warm (22 degrees C) adapted fish (Cyprinus carpio L.), rat (Rattus norvegicus) and bird (Branta canadensis), was studied. Fatty acid composition of total lipids as well as molecular species composition of diacyl phosphatidylcholines and phosphatidylethanolamines was also determined. The amount of long-chain polyunsaturated fatty acids decreased with increasing body temperature. There was a near-complete compensation of membrane structural order for environmental/body temperature over the evolutionary scale as seen by DPH-PA. Using TMA-DPH, the compensation was partial with rat and bird. Since DPH-PA and TMA-DPH differ in their charges, it is proposed, that the former reported membrane regions rich in cationic or zwitterionic (neutral) phospholipids and the latter, membrane regions rich in negatively charged phospholipids in the synaptic plasma membranes. Many different molecular species (20-25) of diacyl phosphatidylcholines and diacyl phosphatidylethanolamines were identified. The level of 16:0/22:6 phosphatidylcholine decreased while disaturated phosphatidylcholines increased with increase of environmental/body temperature from the fish through the bird. Level of 1-monoenoic, 2-polyenoic phosphatidylethanolamines also decreased with an increase in environmental/body temperature. Experiments using vesicles made of mixed synthetic phosphatidylcholine vesicles (16:0/16:0, 16:0/18:1, 16:0/22:6 in various proportions) showed that increase in disaturated phosphatidylcholine species does not explain the observed complete adjustment of membrane structural order in synaptic plasma membranes. Change in level of 1-monoenoic, 2-polyenoic phosphatidylethanolamines might be one of the factors involved in controlling the biophysical properties of the membrane according to the temperature.

Animals↗
PubMed1992

The amino acid sequence of the single hemoglobin of the high-antarctic fish Bathydraco marri Norman.

1. Bathydraco marri Norman is a cold-adapted Antarctic teleost (Family: Bathydraconidae), living preferably at depths between 400 and 1200 m. 2. The blood of this species contains a single hemoglobin, in which oxygen binding is pH-regulated (Bohr and Root effects). 3. The complete amino acid sequence of the alpha and beta chains of the hemoglobin of B. marri has been elucidated.

Acylation↗