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PubMed2003

Whale (Balaenoptera physalus) haemoglobin: primary structure, functional characterisation and computer modelling studies.

The functional properties of haemoglobin from the Mediterranean whale Balaenoptera physalus have been studied as functions of heterotropic effector concentration and temperature. Particular attention has been given to the effect of carbon dioxide and lactate since the animal is specialised for prolonged dives often in cold water. The molecular basis of the functional behaviour and in particular of the weak interaction with 2,3-diphosphoglycerate is discussed in the light of the primary structure and of computer modelling. On these bases, it is suggested that the A2 (Pro-->Ala) substitution observed in the beta chains of whale haemoglobin may be responsible for the displacement of the A helix known to be a key structural feature in haemoglobins that display an altered interaction with 2,3-diphosphoglycerate as compared with human haemoglobin. The functional and structural results, discussed in the light of a previous study on the haemoglobin from the Arctic whale Balaenoptera acutorostrata, give further insights into the regulatory mechanisms of the interactive effects of temperature, carbon dioxide and lactate.

Amino Acid Sequence↗
PubMed2001

Loss of heterozygocity at the thyroid peroxidase gene locus in solitary cold thyroid nodules.

Germline mutations in both alleles of the thyroid peroxidase (TPO) gene have been reported as a frequent cause of congenital hypothyroidism resulting from a total iodide organification defect (TIOD). Because TPO mutations have a prevalence of 1 in 66,000 newborns and is inherited in an autosomal recessive mode the frequency of a heterozygous germline mutation in the TPO gene should reach about 1 in 260 in the population. A somatic TPO mutation coinciding with a somatic loss of one of the TPO alleles or a TPO germline mutation could lead to somatic loss of TPO activity with impairment of thyroid hormone synthesis and decrease of growth control. The latter would lead to increased thyroid epithelial cell proliferation and the subsequent development of a scintigraphically cold thyroid nodule (CTN). To test this hypothesis we studied 40 CTN for the presence of mutations or loss of heterozygosity (LOH) in the TPO gene. For comparisons we also studied LOH in 17 autonomously functioning thyroid nodules (AFTN). Genomic DNA was extracted from nodular and surrounding tissue, polymerase chain reaction (PCR) amplified, sequenced, and analyzed for LOH. In 6 CTNs of 37 informative cases we detected LOH using the genomic markers sRA, D2S2268, and D2S319 within or near the TPO gene locus (2p24-25). In contrast, a genomic marker closer to the centromer (D2S144, 2p24-21) shows LOH in only 1 CTN. We did not detect LOH in AFTN. In none of the cases a germline or somatic mutation in the TPO gene was detectable in the TPO gene. LOH in 6 of 37 CTNs suggests that genetic defects at the TPO or the chromosomal locus 2p24-25 might play a role in the etiology of CTNs. However, we did not find the combination of LOH with a somatic mutation in the TPO gene. It is therefore likely that a gene defect near the TPO locus is part of the neoplastic process in a subgroup of CTNs.

Base Sequence↗
PubMed1993

Carbohydrates and the pathogenesis of Mycoplasma pneumoniae infection and AIDS--some observations and speculations.

Carbohydrate-mediated interactions are prominent in the pathobiology of human infection with Mycoplasma pneumoniae. The mycoplasma uses specific long-chain sialo-oligosaccharides at the host-cell surface as receptors. These sialo-oligosaccharides contain I antigen in their backbones, and they are richly expressed at the primary site of infection--the ciliated bronchial epithelium--and on erythrocytes. The autoantibodies of high titers directed to I antigen, cold hemagglutinins, frequently produced in this infection are therefore antibodies to receptors and may be triggered by an autoimmunogenic mycoplasma-receptor complex in which the lipid-rich microorganism serves as an adjuvant. Current interest is focused on the possibility that the course of infection with human immunodeficiency virus type 1 (HIV-1) may be exacerbated by coinfection with mycoplasmas. One mechanism for such potentiation would be specific interactions between the mycoplasmas and HIV-1 leading to a concentration of the virus at the mycoplasmal surface. The envelope glycoprotein gp120 of HIV-1 is richly decorated with an array of oligosaccharides whose biosynthesis is host cell directed. By an analogy with M. pneumoniae, I propose that recognition of carbohydrates by various mycoplasmal copathogens is a potential mechanism for interactions with HIV-1.

AIDS-Related Opportunistic Infections↗
PubMed1979

Studies of the biosynthesis of C50 carotenoids in Halobacterium cutirubrum.

Cells of Halobacterium cutirubrum were grown in the presence of [14C]mevalonate with and without 3 mM nicotine. Growth of cells was inhibited to a maximum extent of 25% but overall incorporation of 14C into total, neutral, or polar lipids was only inhibited about 16% during active growth and to a much lesser extent or not at all in stationary phase. Little effect of nicotine on labelling of squalenes, vitamin MK-8, geranylgeraniol, and phytoene was observed. However, labelling of bacterioruberin and monoanhydrobacterioruberin was extensively inhibited while that of lycopene and bisanhydrobacterioruberin was increased reciprocally by growth in the presence of 3 mM nicotine. Reincubation of the labelled nicotine-grown cells in fresh cold medium without nicotine restored the 14C-labelling of bacterioruberin and monoanhydrobacterioruberin at the expense of lycopene and bisanhydrobacterioruberin. These results confirm our previous findings suggesting that the C50 bacterioruberin is made by addition of a C5-isoprene unit to each end of the C40-lycopene chain, followed by introduction of four hydroxyl groups.

Carotenoids↗
PubMed1993

[Antibody activities of monoclonal immunoglobulins].

Monoclonal immunoglobulins present a normal structure and have an antibody activity. This activity may be directed against exo-antigens especially bacterial, but more frequently against auto-antigens. It may then be symptomatic and therefore explain unusual clinical or biological manifestations in the course of monoclonal dysglobulinemias. The most common auto-antibody activities are directed against red blood cells (responsible for cold agglutinin disease), IgG (leading to the formation of cryoglobulinemia), and myelin (responsible for peripheral neuropathy). Often, the monoclonal auto-antibody activity is asymptomatic and polyspecific, as it is only the monoclonal expression of polyclonal natural auto-antibodies. Genetic study of variable segments of heavy and light chains used by these monoclonal antibodies are useful to precise the role played by antigen in the driving of the lymphoid proliferation. These physiopathologic models concerning monoclonal gammapathies are a useful tool to understand the physiopathology of polyclonal auto-immune disease and the role of auto-antibodies.

Antibodies, Monoclonal↗
PubMed2005

Expression of tropism-related genes in regenerating skeletal muscle of rats treated with cyclosporin-A.

This work was undertaken to provide further insights into the expression of tropism-related genes in regenerating skeletal muscle of adult rats treated with cyclosporin-A (CsA), a calcineurin inhibitor. Rats were treated with CsA for 5 days and, on the 6th day, were submitted to cryolesion of the soleus muscles. CsA treatment continued for 1, 10, and 21 days after cryolesion. Muscles were removed, frozen, and stored in liquid nitrogen. Body and muscle weights, histological sections stained with toluidine blue, and gene expression of the regeneration molecular markers, viz., desmin and neonatal myosin heavy chain, were assessed to confirm that cryolesion and CsA treatment were effective during the allowed regeneration time. Quantitative reverse transcription/polymerase chain reaction demonstrated that myostatin gene expression was not altered by either cryolesion or CsA treatment combined with cryolesion. Calpain-3 gene expression decreased at 1 day after cryolesion and also following CsA treatment combined with cryolesion. However, calpain-3 gene expression was strongly up-regulated (approximately five-fold) 10 days after cryolesion and returned to control levels at day 21. CsA treatment blocked calpain-3 gene expression rise induced by 10 days of cryolesion. Atrogin-1 gene expression was decreased at 1 day after cryolesion and following cryolesion combined with CsA treatment, returning to control levels at day 10. These results suggest that (1) calpain-3 has a differential role in the early and late stages of regeneration in a calcineurin-dependent manner, and (2) atrogin-1 is involved in the early stages of regeneration independently of calcineurin.

Animals↗
PubMed2003

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, Calyptogena.

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33-42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei.

Amino Acid Sequence↗
PubMed1994

The Cladophora complex (Chlorophyta): new views based on 18S rRNA gene sequences.

Evolutionary relationships among species traditionally ascribed to the Siphonocladales/Cladophorales have remained unclear due to a lack of phylogenetically informative characters and extensive morphological plasticity resulting in morphological convergence. This study explores some of the diversity within the generic complex Cladophora and its siphonocladalaen allies. Twelve species of Cladophora representing 6 of the 11 morphological sections recognized by van den Hoek were analyzed along with 8 siphonocladalaen species using 18S rRNA gene sequences. The final alignment consisted of 1460 positions containing 92 phylogenetically informative substitutions. Weighting schemes (EOR weighting, combinatorial weighting) were applied in maximum parsimony analysis to correct for substitution bias. Stem characters were weighted 0.66 relative to single-stranded characters to correct for secondary structural constraints. Both weighting approaches resulted in greater phylogenetic resolution. Results confirm that there is no basis for the independent recognition of the Cladophorales and Siphonocladales. The Siphonocladales is polyphyletic, and Cladophora is paraphyletic. All analyses support two principal lineages, of which one contains predominantly tropical members including almost all siphonocladalean taxa, while the other lineage consists of mostly warm- to cold-temperate species of Cladophora.

Base Sequence↗
PubMed2004

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots.

The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2, TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.

Aldose-Ketose Isomerases↗
PubMed2003

Characterization of digestive gland esterase-lipase activity of juvenile redclaw crayfish Cherax quadricarinatus.

Investigation of esterase-lipase activity in the digestive gland of redclaw crayfish Cherax quadricarinatus showed that the optimum enzyme activity occurred between 35 and 40 degrees C, with 100 mM NaCl at pH 8.5. Heavy metals completely inhibited and calcium ions partially inhibited enzyme activity. The enzyme activity diminished as the length of the fatty acid chain of substrates increased. Molecular masses for four isozymes were 43, 46, 63 and 118 kDa, respectively, as determined by PAGE.

Animals↗
PubMed2001

RNA anchoring in the vegetal cortex of the Xenopus oocyte.

The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.

Actins↗
PubMed2006

R-Ras3/(M-Ras) is involved in thermal adaptation in the critical period of thermal control establishment.

Neuroanatomically, the body temperature is balanced by the preoptic anterior hypothalamus (PO/AH) and controlled by thermosensitive neurons. Hot or cold exposure during the critical period of temperature control development causes a plastic change in the ratio between hot- and cold-sensitive cells and can modulate temperature tolerance. In this project, mRNA fingerprinting was used to identify the proteins involved in thermal adaptation in 3-day-old chicks. Fifteen genes were induced, among which were NADH dehydrogenase, protocadherin, anolase alpha, 14-3-3epsilon, and R-Ras3. The role of each of these genes is potentially interesting and requires detailed evaluation, but since the present working hypothesis assumed neuronal remodeling, we concentrated on the role of R-Ras3/(M-Ras), which is uniquely expressed in the brain and whose physiological role has not been described. In the present study, R-Ras3 expression during thermal conditioning was investigated by several molecular techniques and its mRNA was found to be induced in the PO/AH with a tenfold peak after 12 h of heat conditioning and a fourfold increase after 6 h of cold conditioning. To improve our understanding of thermal adaptation-related signal transduction, we screened for changes in the expression of transcription factors that were implicated with the Ras gene family, and found that both jun mRNA expression and Jun phosphorylation were induced after 30 min of temperature conditioning. Taken together, the present findings correlate the R-Ras3-jun pathway with thermal-control establishment.

Acclimatization↗
PubMed2005

HIF-1alpha involvement in low temperature and anoxia survival by a freeze tolerant insect.

Winter survival for many insect species relies on the ability to endure the freezing of extracellular body fluids. Because freezing impedes oxygen delivery to tissues, one component of natural freeze tolerance is a well-developed anoxia/ischemia resistance. The present study explores the responses of the hypoxia-inducible factor-1alpha (HIF-1alpha) to cold, freezing and anoxia exposures in the freeze tolerant goldenrod gall fly larva, Eurosta solidaginis. Reverse transcription-PCR was used to quantify hif-1alpha transcript levels; transcripts were significantly elevated by approximately 70% in chilled (3 ( composite function)C), frozen (-16 ( composite function)C) and thawed (returned to 3 ( composite function)C) insects, compared with 15 ( composite function)C controls. Transcripts also rose by approximately 3-fold in insects given anoxia exposure under a nitrogen gas atmosphere. Cold and freezing exposure also elevated HIF-1alpha protein content in the larvae and HIF-1alpha levels increased over the winter months in insects sampled from an outdoor population; levels peaked in February at 2.1-fold higher than in September. A partial sequence of HIF-1alpha that covers the bHLH and PAS domains of the protein was obtained from E. solidaginis and sequence analysis revealed that this segment shared 62% identity overall with Drosophila melanogaster HIF-1alpha and higher percent identities within specific domains: 76% within the bHLH domain and 70% within the PAS domain. The data provide the first documentation of a potential role for HIF-1 in regulating the expression of genes that can aid freezing survival in a cold-hardy animal.

Aerobiosis↗
PubMed1990

Neural control of human skin blood flow.

The paper reviews recent studies on neural control of skin blood flow, with special emphasis on the interaction between vasoconstrictor and vasodilator mechanisms. Intraneural electrical stimulation, mental stress, arousal and deep breaths have been found to cause cutaneous reflex vasodilatation in cold and vasoconstriction in warm subjects. The vasoconstrictions are probably due to increases of sympathetic vasoconstrictor nerve traffic, but whether vasodilatation is due to inhibition of vasoconstrictor nerve traffic or active (= impulse-induced) vasodilatation is unclear. The latter alternative is a possibility since electrical stimulation of the lumbar sympathetic chain may result in skin vasodilatation, suggesting that active vasodilatation is present in the skin of the human foot. Body cooling, which causes a reflex increase of cutaneous sympathetic vasoconstrictor activity, leads to reduction of cutaneous axon reflex vasodilatation. This may be due to competition between vasoconstrictor and vasodilator mechanisms at the blood vessel level.

Axons↗
PubMed2004

Molecular characterization of the leopard gecko POMC gene and expressional change in the testis by acclimation to low temperature and with a short photoperiod.

The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and beta-endorphin, was isolated and analyzed in the squamata species, the leopard gecko, Eublepharis macularius. Leopard gecko POMC (lgPOMC) cDNA is composed of 1299bp, excluding the poly(A) tail, and encodes 270 amino acids. The deduced amino acid sequence showed the same structural organization as that of other species and displayed identity with those of other vertebrates: 68% with mud turtles, 57/57% with African clawed frog A/B, 53% with chickens, and 45% with mice. In a phylogenic tree, the lgPOMC clustered with the sequences of the mud turtle POMC and python POMC. The lgPOMC gene comprises three exons and two introns and this structure is consistent with humans, rats, mice, African clawed frog and zebrafish. RT-PCR analysis revealed that the lgPOMC mRNA was expressed only in the whole brain, pituitary, and gonads. To analyze in more detail, a competitive assay system to quantify the expression levels of POMC mRNA was established. We measured the POMC mRNA expression levels in the leopard gecko testes following transfer from a condition of 29 degrees C, 16L/8D to 18 degrees C, 10L/14D over 6 weeks. This 6-week acclimation increased the POMC mRNA expression levels significantly. This suggests that the leopard gecko POMC-derived peptides play a role in the mediation of the effect of environmental factors on reproduction.

Amino Acid Sequence↗
PubMed2003

Performance of PCR-restriction fragment length polymorphism analysis of the Helicobacter pylori ureB gene in differentiating gene variants.

Recently, PCR-restriction fragment length polymorphism (PCR-RFLP) of the urease genes of Helicobacter pylori was evaluated in a meta-analysis; acceptable discriminatory indices of the ureAB and C genes were found. In the present investigation, we found a discriminatory index of 0.95 for 191 unrelated clinical H. pylori isolates with PCR-RFLP typing of the ureB gene (933 bp), combining the results obtained with restriction enzymes HaeIII and Sau3A, and a mixture of the enzymes. We therefore find that PCR-RFLP typing of the ureB gene of H. pylori with restriction enzymes HaeIII and Sau3A is comparable to typing of other H. pylori urease genes.

DNA, Bacterial↗
PubMed1998

Evolution, organization, and expression of alpha-tubulin genes in the antarctic fish Notothenia coriiceps. Adaptive expansion of a gene family by recent gene duplication, inversion, and divergence.

To assess the organization and expression of tubulin genes in ectothermic vertebrates, we have chosen the Antarctic yellowbelly rockcod, Notothenia coriiceps, as a model system. The genome of N. coriiceps contains approximately 15 distinct DNA fragments complementary to alpha-tubulin cDNA probes, which suggests that the alpha-tubulins of this cold-adapted fish are encoded by a substantial multigene family. From an N. coriiceps testicular DNA library, we isolated a 13.8-kilobase pair genomic clone that contains a tightly linked cluster of three alpha-tubulin genes, designated NcGTbalphaa, NcGTbalphab, and NcGTbalphac. Two of these genes, NcGTbalphaa and NcGTbalphab, are linked in head-to-head (5' to 5') orientation with approximately 500 bp separating their start codons, whereas NcGTbalphaa and NcGTbalphac are linked tail-to-tail (3' to 3') with approximately 2.5 kilobase pairs between their stop codons. The exons, introns, and untranslated regions of the three alpha-tubulin genes are strikingly similar in sequence, and the intergenic region between the alphaa and alphab genes is significantly palindromic. Thus, this cluster probably evolved by duplication, inversion, and divergence of a common ancestral alpha-tubulin gene. Expression of the NcGTbalphac gene is cosmopolitan, with its mRNA most abundant in hematopoietic, neural, and testicular tissues, whereas NcGTbalphaa and NcGTbalphab transcripts accumulate primarily in brain. The differential expression of the three genes is consistent with distinct suites of putative promoter and enhancer elements. We propose that cold adaptation of the microtubule system of Antarctic fishes is based in part on expansion of the alpha- and beta-tubulin gene families to ensure efficient synthesis of tubulin polypeptides.

Amino Acid Sequence↗
PubMed2006

Transcriptional profiling of sunflower plants growing under low temperatures reveals an extensive down-regulation of gene expression associated with chilling sensitivity.

Being able to sow early to maximize the growing season and to escape drought stress has increased the importance of low-temperature tolerance in sunflower. Yet knowledge about the molecular basis of sunflower response to low temperature is still lacking. To address this issue, nylon microarrays containing >8000 putative unigenes were developed and used. Early- and late-flowering genotypes were sown at 15 degrees C and grown until the two-leaf stage when they were subjected to 7 degrees C until the four-leaf stage. The transcriptional profiles of low temperature-grown plants (15 degrees C and 7 degrees C) were compared with those grown under standard conditions (25 degrees C). Two-step ANOVA normalization and analysis models were used to identify the differentially expressed genes. A total of 108 cDNA clones having a P-value <10(-3) were found to be differentially expressed between the low temperature-grown plants (15 degrees C and 7 degrees C) and their corresponding two-leaf- and four-leaf-stage controls across the two genotypes. About 90% of these genes were down-regulated. This includes genes potentially involved in the metabolism of carbohydrate and energy, protein synthesis, signal transduction, and transport function. Comparing gene expression profiles at 15 degrees C and 7 degrees C revealed that only four genes can be considered as differentially expressed, in both genotypes, suggesting that similar genetic programmes underlie the response of sunflower plants to these temperature regimes. The analysis also revealed that early- and late-flowering genotypes respond similarly to low-temperature tolerance as justified by the low number of genes showing a significant genotype x treatment interaction effect. It seems likely that the down-regulation and/or non-induction of genes having a critical role in low-temperature tolerance may be responsible for the sensitivity of sunflower plants to low-temperature tolerance. The results reported provide an initial characterization of the transcriptome activity of sunflower, as a chilling-sensitive plant under suboptimal temperatures, and could be of importance to reveal the potential differences between chilling-sensitive and chilling-tolerant species.

Acclimatization↗