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At least 775 records · Page 43Linked to original sources
PubMed2000

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. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53.6-60.6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a 'hinge', and resulted in a CH2-CH3 hinge peptide longer than any other IgM hinge. Differences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.

Amino Acid Sequence↗
PubMed2001

The intrinsic hypermutability of antibody heavy and light chain genes decays exponentially.

Somatic hypermutation, essential for the affinity maturation of antibodies, is restricted to a small segment of DNA. The upstream boundary is sharp and is probably related to transcription initiation. However, for reasons unknown, the hypermutation domain does not encompass the whole transcription unit, notably the C-region exon. Since analysis of the downstream decay of hypermutation is obscured by sequence-dependent hot and cold spots, we describe a strategy to minimize these fluctuations by computing mutations of different sequences located at similar distances from the promoter. We pool large databases of mutated heavy and light chains and analyse the decay of mutation frequencies. We define an intrinsic decay of probability of mutation that is remarkably similar for heavy and light chains, faster than anticipated and consistent with an exponential fit. Indeed, quite apart from hot spots, the intrinsic probability of mutation at CDR1 can be almost twice that of CDR3. The analysis has mechanistic implications for current and future models of hypermutation.

Animals↗
PubMed2002

Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum.

In the conventional view, the winter adaptation of membrane lipids is induced by temperature decrease. We propose that winter remodelling of membranes in Pyrrhocoris apterus is triggered by short-day photoperiod before the temperature decrease and changes caused by cold temperature represent the later phase of adaptation. The induction of diapause by short-day photoperiod results in an accumulation of phosphatidylethanolamine (PE) molecular species with C(16:0)/C(18:2) acyl chains esterified to sn-1/sn-2 positions of glycerol at the expense of C(18:0)/C(18:2). Proportions of C(16:0)/C(18:2)-PE are enhanced in short-day compared to long-day insects in both thoracic muscles (TM, 15.0 vs. 8.2%) and fat bodies (FB, 24.9 vs. 13.6 %). Proportions of C(16:0)/C(18:2)-PE are further enhanced during cold acclimation (to 26.5% in TM, 33.6 % in FB) at the expense of a more saturated species, C(18:0)/C(18:1)-PE. These changes are less prominent in phosphatidylcholines (PC). The effect of photoperiod seems to be mediated via the corpus allatum. Long-day non-diapause females deprived of their corpus allatum have the phospholipid molecular species profile similar to that found in short-day diapausing females. While the acyl chain remodelling is regulated by both photoperiod and temperature, the head group composition is regulated by temperature only. Similar to most other organisms, the level of PE is higher (50.3 vs. 43.5% in TM, 44.3 vs. 37.8% in FB) and that of PC is lower (35.9 vs. 40.2% in TM, 41.6 vs. 46.1 % in FB) at cold temperatures (</=1 degrees C) compared to warm temperatures (>/=16 degrees C). In contrast to a general rule, the PE is less unsaturated than PC. In both TM and FB, proportions of unsaturated/unsaturated molecular species are consistently high in PC (56.3-67.5% in TM, 59.2-66.6% in FB), while they are consistently low in PE (19.1-26.7% in TM, 12.1-15.1% in FB). An adaptive significance of changes in the phospholipid composition for the low temperature and/or dehydration stress is discussed in relation to known physical properties of phospholipids.

Journal Article↗
PubMedDate not supplied

Molecular identification of uncoupling proteins 2 and 3 in a carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii).

This study investigated the expression of uncoupling proteins 2 and 3 (UCP2 and UCP3) in the carnivorous marsupial Sarcophilus harrisii. The current study used molecular techniques to ascertain whether this species expresses UCP2 and/or UCP3. This species increases nonshivering thermogenesis in response to cold exposure and norepinephrine, although our previous study was unable to demonstrate the presence of brown adipose tissue or uncoupling protein 1. Samples of skeletal muscle and white adipose tissues were taken from five S. harrisii pre- and post-cold acclimation (2 degrees -3 degrees C for 2 wk). The tissues were examined for UCP2 and UCP3 expression through Western blots and reverse transcriptase polymerase chain reaction, with subsequent sequencing to ensure identification of the desired gene. These data suggest that S. harrisii expresses UCP2 but not UCP3. The sequencing of the amplified S. harrisii UCP2 cDNA has revealed a 76% homology with human UCP2 cDNA and a 72% homology with rat UCP2 cDNA. The expression of UCP2 but not UCP3 suggests that UCP2 is conserved from a common ancestor to both the Marsupialia and the Eutheria taxa.

Acclimatization↗
PubMed1999

Cold-induced augmentation of I blood group antigen interactions with galactophilic lectins.

The I antigen appears on human cells in the postnatal period, by addition of N-acetyllactosamine (beta 1-6) branching to the fetal i antigen structure, which is specified by linear oligo N-acetyllactosamine (beta 1-3) chain. Concurrently with the I antigen appearance on adult human erythrocytes most human sera exhibit low levels of anti-I agglutinins. These antibodies induce hemagglutination mainly at low temperatures (4 degrees C) and scantly at body temperature. Therefore they were named "cold agglutinins". We have used these antibodies and several hemagglutinating galactophilic animal, plant, and microbial lectins that also react with the I antigen, to study whether the cold-favored agglutination of the I antigen-bearing cells is a peculiar property of the anti-I antibodies or a special trait of that antigen. It has been found that the interactions of all of the examined lectins, irrespective of their source, with the adult human erythrocytes significantly increased at 4 degrees C, in contrast to those of the same cells with diverse I-insensitive antibodies and lectins, which were significantly higher at room temperature.

Adult↗
PubMed1988

Sequence comparison of wild-type and cold-adapted B/Ann Arbor/1/66 influenza virus genes.

Consensus sequences for both wt and ca B/Ann Arbor/1/66 viral PB2, PB1, PA, NP, M, and NS genes were directly determined from vRNA using a combination of chemical and chain-termination sequencing methods. There were 105 sites of difference between the wt and ca sets of these six RNA genes. The differences resulted in 26 amino acid substitutions distributed over the six proteins. The sequence changes were compared to the sequences of other known influenza type B wt viruses to pinpoint those changes that were unique to the ca B/ann Arbor/1/66 virus. Of the 26 amino acid differences, only 11 were unique to the cold-adapted virus. These unique sites were distributed among five of the six genes. The NS protein had no amino acid substitutions. The sequence changes are discussed in terms of their probable mode of origin and selection, and in terms of their importance to the cold-adapted, temperature-sensitive, and attenuation phenotypes of ca B/AA/1/66 virus. The sequence and organization of the PB2 gene and predicted protein are also given. The PB2 gene was 2396 nucleotides long, and it encoded a predicted protein of 770 amino acids with a molecular weight of 88,035 Da for the wt virus and 88,072 Da for the ca virus. Both proteins were predominantly hydrophilic, and each had an overall charge of +24.5 at pH 7.0.

Amino Acid Sequence↗
PubMed1995

Structural analysis of VH4-21 encoded human IgM allo- and autoantibodies against red blood cells.

We have sequenced the variable heavy chain regions of a number of VH4-21 encoded monoclonal IgM anti-Rh(D) antibodies produced in response to deliberate immunization. These were compared with the sequences of similarly encoded IgM anti-I cold agglutinins (CA) derived from patients with lympho-proliferative diseases. The anti-Rh(D) antibodies show evidence of clonal expansion and somatic diversification. Even though they are produced in response to an antigenic stimulus, they demonstrate limited hypermutation in the variable heavy chain (VH) segments and there is no evidence of selective pressure acting on the complementarity determining regions (CDRs). The CA demonstrate a higher rate of mutation and yet this results in a lower ratio of replacement to silent mutations (R:S) in the CDRs than seen in the anti-Rh(D) antibodies. It is not clear whether the different pattern of mutations seen in the CA is related to their auto-reactivity or their tumour origin. In both groups of antibodies the region encoded by the VH4-21 segment can be found in germline configuration at the amino-acid level indicating that other V-gene structures, i.e. light chains or CDRH3s, are crucial to the generation of either specificity. A role of the CDRH3 is indicated by the identification of a motif shared by four CAs and one Rh(D) antibody which also demonstrates CA activity independent of its anti-Rh(D) specificity. Amongst the anti-Rh(D) antibodies there seems to be an obligatory combination with VL having closest homology to the DPL16 germline segment indicating this as particularly important in generation anti-Rh(D) specificity.

Agglutinins↗
PubMed1985

Permeability and morphology of low temperature phases in bilayers of single and of mixtures of phosphatidylcholines.

The properties of subtransitions were studied in aqueous dispersions of saturated phosphatidylcholines (PC) by means of permeability measurements, freeze-fracture electron microscopy, and differential scanning calorimetry (DSC). For dispersions of C16PC, a C16PC analog (2,3-dipalmitoyl-cyclopentano-1-phosphocholine with four methylene residues between the nitrogen and the phosphorus atoms) and C17PC, there was good agreement between phase properties (including subtransitions) as observed by DSC and temperature-related permeability. C16PC and C17PC dispersions also displayed a 'crinkled' surface morphology in the subgel state. The phase diagram for mixtures of C14PC and C16PC was consistent with ideal mixing of these two components in the subgel state and also illustrated the relative independence of the subtransition on acyl chain length as compared to the pre- and main transitions. Together, these results indicate that (i) permeability, DSC and freeze-fracture electron microscopy measurements do correlate reasonably well with the existence of a subgel state, (ii) mixtures of lipids with similar acyl chain lengths can be used to investigate subtransitions, (iii) the development of a subtransition appears to be mainly a function of the non-acyl chain moiety of the phospholipid.

Calorimetry, Differential Scanning↗
PubMed1994

13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.

Binding of the medium-chain fatty acids (MCFA), octanoic (OCT) and decanoic (DEC) acid, to human serum albumin (HSA) has been studied by 13C NMR spectroscopy. NMR spectra at 35 degrees C showed an apparently homogeneous binding environment (a single, narrow resonance for the 13C-enriched carboxyl carbon) at different mole ratios and pH values. Changes in the chemical shift of this peak with mole ratio and protein concentration demonstrated rapid equilibration (< or = msec) of bound and unbound MCFA and permitted a direct quantitation of bound/unbound MCFA. Spectra of OCT/HSA mixtures at 6 degrees C revealed at least three distinct binding sites that fill sequentially. The observed heterogeneity of binding at low temperature, compared to 35 degrees C, is attributed to a slower exchange rate of OCT between binding sites. The highest affinity sites for both OCT and DEC have properties similar to those of binding sites for longer-chain fatty acids, such as the close proximity of the fatty acid carboxylate to basic amino acid residue(s). Interestingly, chemical shift data showed that the first mole of OCT and DEC either bind differently to the same site or bind to different sites on HSA. The rapid desorption of MCFA from HSA binding sites has implications for dietary regimens with medium chain triglycerols.

Binding Sites↗
PubMed2000

On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions.

Poly-N-isopropylacrylamide (PNIPAM) is a chemical isomer of poly-leucine, having the polar peptide group in the side-chain rather than in the backbone. It has been demonstrated experimentally that PNIPAM dissolved in aqueous solution undergoes a collapse transition from coil to globule on increasing temperature above the &theta;-point. By a careful reviewing of existing experimental data, we emphasize that such coil to globule collapse has to be considered an intramolecular first-order transition, analogous to the cold renaturation of small globular proteins. The main theoretical approaches to the coil to globule collapse in homopolymers are discussed briefly, and a critical comparison between the existing models is performed. We point out that, as a general result, the coil to globule collapse is expected to be a first-order transition for rigid and semi-rigid macromolecules. Finally, taking advantage of the analogy between the coil to globule collapse of PNIPAM and the cold renaturation of small globular proteins, we try to clarify some important and intriguing aspects of protein thermodynamics. This leads to the conclusion that the amphiphilic nature of polypeptide chain plays the fundamental role for the existence of two temperature-induced conformational transitions.

Acrylic Resins↗
PubMed1994

Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior.

Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes. These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMedDate not supplied

[Characteristics of the effect of 2-pyrrolidone acetamide (piracetam) on energy coupling in rat skeletal muscle mitochondria during cold stress].

Effects of 2-pyrrolidone acetamide (piracetam) on energetic state of mitochondria were studied in skeletal muscles of intact and ovaryectomized rats under conditions of drastic cooling (4 min swimming at 10 degrees). Readministration of piracetam was shown to cause uncoupling of oxidation and phosphorylation in mitochondrial respiratory chain (a decrease in respiratory control by Chance), as well as the drug increased the rate of O2 consumption in presence of oligomycin and decreased the uncoupler stimulating action on respiration in the group of intact animals. The alterations observed were most distinct in rats with cold stress, which was followed by piracetam administration, except of the rate of respiration stimulation, distinctly increased in presence of uncoupler. Piracetam did not exhibit any noticeable effects on the patterns of respiration studied in skeletal muscles of ovaryectomized rats. Protective properties of piracetam in cooling are discussed.

Animals↗
PubMed2001

Survival of poliovirus in New Zealand green-lipped mussels, Perna canaliculus, on refrigerated and frozen storage.

Poliovirus survival in live and frozen mussels during storage was assessed by both viral culture and molecular methods. Live New Zealand green-lipped mussels were incubated overnight at 20 degrees C in an aerated tank of filtered seawater seeded with the poliovirus 2 (PV2) vaccine strain. An extraction and concentration method that preserved viral infectivity was used to recover PV2 taken up by the mussels at day 0, at day 2 after storage at 4 degrees C, and at days 7, 14, and 28 after storage at -20 degrees C. This method allowed both culture and molecular analysis to be carried out. Presence of intact PV2 in each batch of mussels was determined by a pan-enterovirus specific reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by dot-blot hybridization. Survival of infectious PV2 was determined by the monolayer plaque assay. After 48 h at 4 degrees C, infectious PV2 levels were 81% of the original level detected in the mussels. Infective virus levels then declined to 66, 53, and 44% after storage at -20 degrees C for 7, 14, and 28 days, respectively. Generic RT-PCR methods were 10 times more sensitive than cell culture techniques for virus detection but did not give information on virus infectivity. The survival of infectious pathogenic viruses in fresh and frozen mussels on storage constitutes a potential health risk and so is a major concern for public health authorities.

Animals↗
PubMed1982

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed1978

Cold pepsin digestion: a novel method to produce the Fv fragment from human immunoglobulin M.

A strategy for the proteolytic fragmentation of human IgM has been developed. This method is called "cold pepsin digestion" because of its unique feature of achieving restricted peptic cleavages at 4 degrees and pH 4.0. Cold pepsin digestion has been applied successfully to produce an Fv fragment from 14 human IgM proteins. The Fv fragment consists of the heavy chain variable domain (VH) and the light chain variable domain (VL) held together by strong noncovalent interaction. Thus, each Fv fragment contains one intact antigen-binding site and represents the minimal active fragment derivable from an antibody molecule. A series of other structurally and functionally important fragments were also isolated and characterized. Two basic digestion pathways were recognized; these mainly reflect the relative accessibility of five sets of major interdomain cleavage sites.

Binding Sites, Antibody↗
PubMedDate not supplied

Prolonged cold preservation promotes the recipient's cell participating in neointima formation but delays the later graft arteriosclerosis in rat model.

Chronic graft dysfunction is the greatest barrier to long-term graft survival, although the immediate outcome in organ transplantation has been greatly improved. Graft arteriosclerosis is a prominent feature of chronic graft dysfunction. Recipient progenitor cells have been shown to participate in neointimal development in graft arteriosclerosis. The present study investigated the role of recipient endothelial cells in the repair and remodeling after a cold preservation injury in an orthotopic cross-sex abdominal aortic allotransplantation model, namely female Wistar to male Sprague-Dawley rats. Grafts were preserved for 48 hours in 4 degrees C University of Wisconsin (UW) solution for a prolonged cold ischemia (PCI) group or preserved for <1 hour in the control group; or for <1 hour in the presence of feeding with cyclosporine (CyA). A direct in situ polymerase chain reaction (ISPCR) for the SRY gene showed SRY-marked endothelial and smooth muscle-like cells in neointima at 2 weeks in the PCI group, at 4 weeks in the control group, and rarely at 3 months in the CyA group. Staining by H&E showed the aortic graft intima to be thicker in the PCI than in the control group at 4 weeks, but thinning thereafter. The SRY-positive cells correlated with intimal thickness in the PCI and the control group (r = .801 and .825; P < .05 and <.05, respectively), but not in the CyA group (r = .247, P > .5). Our data suggest that prolonged cold preservation promotes recipient cell participation in graft arteriosclerosis after endothelium injury. The early neointimal formation via recipient cells incorporated into arteriosclerotic neointima may delay later intimal thickening. In the aortic allotransplantation model, prolonged cold ischemia may be beneficial for long-term graft survival due to early endothelial replacement. We hypothesize that controlled injury to the graft may serve as a new strategy for treatment of intimal thickening.

Animals↗
PubMed2002

Genetic determinants of delayed graft function after kidney transplantation.

BACKGROUND: Intracellular concentration of reactive oxygen species is held within tight physiological limits by enzymes with scavenging and repair functions. Under extreme conditions such as prolonged cold ischemia, these enzymes may be unable to adequately protect the organ, resulting in reperfusion injury that renders the graft dysfunctional after transplantation. In this study, we investigated normal human variation of some of these inducible enzymes to determine if certain phenotypes could be identified that are associated with a reduced risk of delayed graft function (DGF). METHODS: Polymerase chain reaction was performed to differentiate polymorphisms for manganese superoxide dismutase and three classes of glutathione-S-transferase in donors and recipients of transplants with over 24 hr of cold ischemia. The data attained was analyzed compared with the presence or absence of DGF, defined as the requirement of hemodialysis in the first week after transplantation. RESULTS: Enzyme polymorphisms were defined for 229 recipients and 104 of their respective donors. Patients receiving a kidney from a donor who expressed GSTM1*B either alone or in combination with GSTM1*A experienced significantly lower rates of DGF (P <0.05). No association was found between any enzyme polymorphism in the recipients and the development of DGF. CONCLUSIONS: The identification of a genetic allele, which is protective against reperfusion injury, generates the possibility for defining polymorphisms at the time of tissue typing to give insight to the inherent biological risk of DGF that an organ possesses.

Adult↗