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PubMed2004

An allelic series for the leptin receptor gene generated by CRE and FLP recombinase.

Body weight regulation is mediated through several major signaling pathways, some of which have been delineated by positional cloning of spontaneous genetic mutations in mice. Lepr(db/db) mice are obese due to a defect in the signaling portion of the leptin receptor, which has led to extensive study of this highly conserved system over the past several years. We have created an allelic series at Lepr for the further examination of LEPR signaling phenotypes using both the FLP /frt and CRE /loxP systems. By inserting a frt-PGK-neo-frt sequence in Lepr intron 16, we have generated a conditional gene repair Lepr allele ( Lepr-neo) that elicits morbid obesity, diabetes, and infertility in homozygous mice, recapitulating the obesity syndrome of Lepr(db/db) mice. Thus, in vivo excision of the PGK-neo cassette with a FLP recombinase transgene restores the lean and fertile phenotype to Lepr(flox/flox) mice. In the same construct, we have also inserted loxP sites that flank Lepr coding exon 17, a region that encodes a JAK docking site required for STAT3 signaling. CRE-mediated excision of Lepr coding exon 17 from Lepr with a frameshift in subsequent exons results in a syndrome of obesity, diabetes, and infertility in LeprDelta17/Delta17 mice, which is indistinguishable from Lepr(neo/neo) and Lepr(db/db) mice. We conclude that suppression of Lepr gene expression by PGK-neo is phenotypically equivalent to deletion of the Lepr signaling motifs, and therefore the Lepr(neo/neo) mouse may be used to investigate conditional gene repair of Lepr signaling deficiency.

Alleles↗
PubMed1977

Metabolic and endocrine studies in a case of lipoatrophic diabetes.

A 20-yr-old female with congenital lipoatrophic diabetes was studied, with the following findings: (1) Serum insulin levels increased after both oral glucose and intravenous arginine administration; there was no growth hormone response to the latter. (2) The infusion of insulin (0.1 units and 0.5 units/kg) during the fed state and following at 110-hr fast produced only minimal changes of various fuels measured, with the exception of a decrease in the branched-chain amino acids. (3) There was a minimal production of ketones during the 110-hr fast. (4) Matabolic expenditure was markedly increased during the postabsorptive state (65-75 kcal/hr/sg m); it fell into the normal range during the 110-hr fast (31-35 kcal/hr/sq m). (5) Following meals, the patient experienced complaints ranging from cold and shivering to feeling hot with gross diaphoresis. These findings were associated with intermittent lability of her skin temperature, which varied 1 degree - 2 degrees F during a 3-hr period. (6) Progressive increases in doses of regular insulin before each meal resulted in up to a total of 9000 units/day being required before normal blood glucose levels were achieved. (7) A 2-wk therapeutic trial of pimozide provided no significant changes in a variety of hormones and fuels in the basal state or following insulin perturbations. (8) A variety of pituitary hormones and pituitary target organ hormones were studied in both the hypothyroid (Hashimoto's thyroiditis) and euthyroid state (following thyroid replacement). All the hormone responses were normal except that growth hormone did not rise during the slow wave sleep in either thyroid state.

Adult↗
PubMed2004

Induction of baroresistance by hydrogen peroxide, ethanol and cold-shock in Saccharomyces cerevisiae.

The acquisition of tolerance to high hydrostatic pressure of 220 MPa (HHP) in response to a 0.4 mM hydrogen peroxide, 6% ethanol and cold-shock (10 degrees C) pretreatment for different lengths of times was studied in the yeast Saccharomyces cerevisiae. The protection conferred by these different treatments was similar ( approximately 3 log cycles) and time-dependent. Analysis of the induction of the most pressure up-regulated genes under these conditions was investigated by RT-PCR. Our results revealed that the cell stress response to HHP shares common features with hydrogen peroxide and ethanol stresses, but differs in some way to cold-shock.

Actins↗
PubMed1979

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.

Animals↗
PubMed1982

Fetal myosin heavy chains in regenerating muscle.

There are several lines of evidence for the existence of a distinct class of myosins in developing muscle. Using various biochemical and immunological approaches, Whalen et al. recently suggested that two myosin heavy chain isozymes appear sequentially in rat muscle development, preceding the definitive adult myosins. It is unknown whether these myosins are present only in developing fast muscles or whether they also occur in developing slow muscles. Pyrophosphate of gel electrophoresis studies have suggested that fast-twitch and slow-twitch muscles synthesize the same fetal myosin isozymes early in development. Immunocytochemical studies with antibodies directed against adult fast and slow myosins show differences in myosin composition between fetal muscle fibres but interpretation of these findings is complicated by cross-reactions of these antibodies with fetal isomyosins. We have used a more direct immunocytochemical approach to identify the myosin types present in developing muscle fibres. An antibody specific for bovine fetal myosin and cross-reactive with rat fetal myosin has been prepared. We report here that the fetal myosin heavy chains recognized by this antibody show a heterogeneous fibre distribution in fetal and neonatal rat muscle, disappear progressively during postnatal development and are transiently expressed in regenerating muscle.

Animals↗
PubMed1999

A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII.

Little is known about the mechanism of the transition between native proteins and partially folded intermediates. Complete assignments of 2D 1H-NOESY spectra of CHABII at 5 degrees C, pH 6.3, 5.5, 4.6 and 4.0, reveal that lowering of pH results in an extensive but gradual disappearance of NOEs, implying a gradual disruption of tight side-chain packing. Moreover, a tertiary packing core is identified at 5 degrees C and pH 4.0, characterized by persistent long-range NOEs. Thus, we suggest that severe disruption of tight side-chain packing of CHABII can occur at a stage where its secondary structure and tertiary topology remain highly native-like.

Acids↗
PubMed2006

Evaluation of endothelial and inducible nitric oxide synthase mRNA expression in the lung of broiler chickens with developmental pulmonary hypertension due to cold stress.

To clarify the effect of cold-induced pulmonary hypertension on endothelial and inducible nitric oxide synthase (eNOS and iNOS) mRNA expression in the lung of broiler chickens, semi quantitative reverse transcription-PCR was performed on total RNAs isolated from lungs of the broiler chickens exposed to 5 weeks of cold stress. The eNOS gene was expressed increasingly with the increasing age during the rearing period. Comparing the treatment group with its related control group eNOS was expressed significantly only at d 21. Expression of iNOS mRNA also increased in both control and treatment groups with increasing age until d 28 and then decreased at d 35 and 42. Comparing the treatment group with its control group, iNOS mRNA level was significantly higher at 21 d of age in the cold-exposed chickens. It was concluded that, although cold exposure could significantly increase eNOS and iNOS gene expression, cold-induced pulmonary hypertension is not associated with significant variations of eNOS and iNOS expression in the lungs of broiler chickens.

Aging↗
PubMed2005

Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Cold Temperature↗
PubMed2000

Developmental expression of Y-box protein 1 mRNA and alternatively spliced Y-box protein 3 mRNAs in spermatogenic cells in mice.

Y-box proteins bind DNA and RNA and are characterized by a cold shock domain and a carboxyl-terminus containing clusters of aromatic and basic residues that alternate with clusters of acidic residues. Y-box proteins 1 and 3 in mouse testis were cloned here by 3' rapid amplification of cDNA ends (RACE) using a degenerate primer. Northern blots and reverse transcription-polymerase chain reaction (RT-PCR) established that the levels of Y-box protein 1 and 3 mRNAs are regulated individually: (i) Y-box protein 1 mRNA is strongly expressed in kidney, whereas Y-box protein 3 mRNA is strongly expressed in heart and muscle; (ii) Y-box protein 1 and 3 mRNAs are weakly expressed in early prepubertal testis and strongly expressed in pachytene spermatocytes, round spermatids, and elongated spermatids; and (iii) prepubertal testes and meiotic and haploid spermatogenic cells express two alternatively spliced Y-box protein 3 mRNAs encoding isoforms with different carboxyl termini, whereas somatic tissues primarily express one form. Sucrose gradients reveal that approximately 27% of both Y-box protein 3 mRNAs are translationally active in adult testis. In conclusion, spermatogenic cells in mice express five isoforms of Y-box proteins including Y-box protein 1, and two isoforms each of Y-box proteins 2 and 3. This multiplicity is intriguing because Y-box proteins are thought to activate transcription and repress translation in spermatogenic cells.

Alternative Splicing↗
PubMed2002

Nasal cell micronuclei, cytology and clinical symptoms in stainless steel production workers exposed to chromium.

The objective of the present study was to determine whether workers in stainless steel production with low exposure to various forms of chromium show an increase in micronucleated nasal cells or an excess of nasal symptoms or disease. Altogether, 48 workers employed in a stainless steel production chain were studied, 29 of them in the steel melting shop with exposure to hexavalent chromium (Cr(6+)), 14 in the sintering and crushing departments of the ferrochromium plant with exposure to trivalent chromium (Cr(3+)) and five in the mine with exposure to chromite ore (Cr(3+)). Thirty-nine workers from the cold rolling mill, with very low exposure to chromium, served as referents. All the subjects were never smokers with a minimum of 14 years employment in the same department. There were no significant differences between the exposure groups and the referents regarding the mean frequency of centromere-negative or centromere-positive micronuclei (studied by pancentromeric fluorescence in situ hybridization), nasal diseases and symptoms or mucociliary clearance of the nasal cavity. No statistically significant differences in the incidence of cell atypia or inflammatory cells were detected between the exposed workers and the reference group, except for an increase in lymphocytes among the chromite ore workers. Anterior rhinoscopy indicated slight inflammatory changes in nasal mucosa and secretion more often in the Cr(6+) and Cr(3+) groups than in the referents, the Cr(6+)-exposed workers showing more livid or oedemic epithelium. In conclusion, the stainless steel production workers, with low exposure to dusts or fumes containing hexavalent or trivalent chromium, did not show clinical changes in the nasal mucosa or an increase in nasal cell micronuclei or symptoms of nasal diseases, except for slight changes in the nasal epithelium and secretion.

Adult↗
PubMed2000

Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme.

Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.

Amino Acid Sequence↗
PubMed2006

A class 1 hemoglobin gene from Alnus firma functions in symbiotic and nonsymbiotic tissues to detoxify nitric oxide.

Actinorhizal symbiosis is as important in biological nitrogen fixation as legume-rhizobium symbiosis in the global nitrogen cycle. To understand the function of hemoglobin (Hb) in actinorhizal symbiosis, we characterized a Hb of Alnus firma, AfHb1. A cDNA that encodes nonsymbiotic Hb (nonsym-Hb) was isolated from a cDNA library of A. firma nodules probed with LjHb1, a nonsym-Hb of Lotus japonicus. No homolog of symbiotic Hb (sym-Hb) could be identified by screening in the cDNA library or by polymerase chain reaction (PCR) using degenerate primers for other sym-Hb genes. The deduced amino acid sequence of AfHb1 showed 92% sequence similarity with a class 1 nonsym-Hb of Casuarina glauca. Quantitative reverse transcriptase-PCR analysis showed that AfHb1 was expressed strongly in the nodules and enhanced expression was detected under cold stress but not under hypoxia or osmotic stress. Moreover, AfHfb1 was strongly induced by the application of nitric oxide (NO) donors, and the application of a NO scavenger suppressed the effect of NO donors. Acetylene reduction was strongly inhibited by the addition of NO donors. AfHb1 may support the nitrogen fixation ability of members of the genus Frankia as a NO scavenger.

Alnus↗
PubMed2004

Ureteral necrosis after kidney transplantation: risk factors and impact on graft and patient survival.

BACKGROUND: Ischemia, the main cause of ureteral necrosis in renal transplantation, cannot alone explain the late occurrence of some fistulas beyond the first postoperative month. The aim of this study, performed on a cohort of 1,629 consecutive kidney transplantations, was to analyze the risk factors implicated in the occurrence of ureteral necrosis and its impact on graft and patient survival. METHODS: Between January 1990 and December 2001, 1,629 renal transplantations were performed in the authors' center. All biologic and clinical data were computerized in a cross-audited and validated data bank (Données Informatisées et Validées en Transplantation). The parameters studied were donor age, gender, cause of death and serum creatinine before procurement; and recipient age, gender, initial disease, panel reactive antibody, retransplantation, cold ischemia time, delayed graft function, human leukocyte antigen incompatibilities, induction and maintenance immunosuppression, right or left kidney, number of arteries, site of transplantation and the presence or not of a double-J stent. The follow-up parameters were the number and timing of acute rejection episodes, cytomegalovirus (CMV) infection (viremia, polymerase chain reaction), and acute pyelonephritis. Ureteral histologic analysis was performed in 25 cases (necrosis, leukocyte infiltration, and CMV or BK virus inclusions). Uni- and multivariate statistical tests were used (alpha risk at 5%). All of the patients with ureteral necrosis had undergone neoureterocystostomy or ureteral anastomosis with the native ureter but with a systematic double-J stent. RESULTS: Ureteral necrosis occurred in 52 of the 1,629 patients (3.2%) and was significantly and independently correlated with donor age (P=0.041) and delayed graft function (P=0.016). CMV infections were also higher in the necrosis group (P=0.001), but donor CMV status was not statistically different between the two groups (36.2% vs. 36.7%). Ureteral histologic studies showed CMV and BK virus inclusions in 4 and 2 cases, respectively, and arterial and venous thrombosis in 4 and 16 cases, respectively. No pattern of ureteral rejection was observed. Ureteral necrosis did not affect the 10-year patient and graft survival, which were 87% and 66%, respectively, for the necrosis group and 86% and 58%, respectively, for the control group (P=not significant). CONCLUSIONS: The authors' data provide new information concerning a classic surgical complication after kidney transplantation. The link they have identified between the occurrence of ureteral necrosis, donor age, and delayed graft function reemphasizes the interdependence between surgical and medical complications in kidney transplantation.

Adult↗
PubMed2004

Identification of members of the metabolically active microbial populations associated with Beggiatoa species mat communities from Gulf of Mexico cold-seep sediments.

In this study, the composition of the metabolically active fraction of the microbial community occurring in Gulf of Mexico marine sediments (water depth, 550 to 575 m) with overlying filamentous bacterial mats was determined. The mats were mainly composed of either orange- or white-pigmented Beggiatoa spp. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from three different sediment depths (0 to 2, 6 to 8, and 10 to 12 cm) that had been subjected to reverse transcription-PCR amplification. Domain-specific 16S PCR primers were used to construct 12 different 16S crDNA libraries containing 333 Archaea and 329 Bacteria clones. Analysis of the Archaea clones indicated that all sediment depths associated with overlying orange- and white-pigmented microbial mats were almost exclusively dominated by ANME-2 (95% of total Archaea clones), a lineage related to the methanogenic order Methanosarcinales. In contrast, bacterial diversity was considerably higher, with the dominant phylotype varying by sediment depth. An equivalent number of clones detected at 0 to 2 cm, representing a total of 93%, were related to the gamma and delta classes of Proteobacteria, whereas clones related to delta-Proteobacteria dominated the metabolically active fraction of the bacterial community occurring at 6 to 8 cm (79%) and 10 to 12 cm (85%). This is the first phylogenetics-based evaluation of the presumptive metabolically active fraction of the Bacteria and Archaea community structure investigated along a sediment depth profile in the northern Gulf of Mexico, a hydrocarbon-rich cold-seep region.

Base Sequence↗
PubMed1998

Vestimentiferan on a whale fall.

Discovery of chemosynthetic communities associated with whale bones led to the hypothesis that whale falls may serve as stepping-stones for faunal dispersal between disjunct hydrothermal vents and cold seeps on the ocean floor (1). The initial observation was followed by a faunal inventory that revealed a diverse assemblage of microbes and invertebrates, supported by chemoautotrophic production, living in close proximity to whale remains (2, 3). To date, the conspicuous absence from whale falls of vestimentiferan tubeworms (a predominant constituent of eastern Pacific vent and seep habitats) has been a major objection to the stepping-stone hypothesis (4-5). We report the first evidence of a vestimentiferan tubeworm associated with a whale fall (Fig. 1). The tubeworm, Escarpia spicata, was identified by morphological criteria and DNA sequence data from a portion of the mitochondrial cytochrome oxidase C subunit I (COI) gene. Additionally, the bacterial endosymbiont in the tubeworm possessed a 16S rRNA gene that was similar to that of endosymbionts from vestimentiferans in sedimented cold-seep environments.

Animals↗
PubMed2000

Expression of thioredoxins f and m, and of their targets fructose-1,6-bisphosphatase and NADP-malate dehydrogenase, in pea plants grown under normal and light/temperature stress conditions.

Thioredoxins (Trxs) f and m, as well as their targets chloroplast fructose-1,6-bisphosphatase (FBPase) and NADP+-malate dehydrogenase (NADP-MDH), displayed transcriptional expression in both photosynthetic and non-photosynthetic organs of pea plants (Pisum sativum L. cv. Lincoln) grown for 50 d under normal irradiance. However, whereas Trx m and both target enzymes were poorly expressed in non-photosynthetic tissues, the content of the precursor form of the Trx f-specific mRNA was high in pea roots. In contrast, the translational expression of Trx f was low in this organ. The high FBPase activity in immature seeds, and the low activity of leaves, must be related to high starch synthesis in the first, and with high sucrose formation in the second. The transcriptional expression of FBPase and NADP+-MDH, and to a lesser extent that of Trxs f and m, was inhibited under low irradiance in plants grown under both normal and high temperatures. Pea plants grown at low temperature displayed a high level of mRNAs for Trxs and their targets, especially when the growth was carried out at low light. To a lesser extent, similar behaviour was observed at the protein level. Chloroplasts of mesophyll leaf cells of pea plants grown under saturating light, or under sub-saturating continuous irradiance, showed broken envelopes, distorted structural elements and disorganized starch grains, as a consequence of a photobleaching process and high starch accumulation.

Blotting, Western↗
PubMed2005

Molecular cloning of a novel LOS2 gene from Capsella bursa-pastoris.

A new LOS2 gene was cloned from C. bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris LOS2 gene (designated as Cblos2) was 1694 bp containing a 1332 bp open reading frame (ORF) encoding a 444 amino acid protein. The predicted CbLOS2 protein contained enolase-N domain, enolase domain, conserved putative DNA-binding and repression domains like LOS2 from A. thaliana. Bioinformatic analysis indicated that CbLOS2 had similarity with other enolase proteins. Cold acclimation assay revealed that Cblos2 expressed constitutively in C. bursa-pastoris and was involved in the cold acclimation process, implying CbLOS2 was a bi-functional enolase.

Acclimatization↗
PubMedDate not supplied

[Splenic lympho-plasmocytic sarcomas discovered by exploratory laparotomy in 2 cases of cold agglutinin disease].

The 2 patients have cold agglutinin disease with lymphoid nodules in the bone marrow. A splenectomy was done because of the severity of the hemolytic anemia and because of the increasing splenomegaly. Tumoral nodules with the aspect of lymphoplasmocytic sarcoma or immunocytoma were discovered in the spleen. Those tumors can be considered either as a morphologic aspect of the cold agglutinin disease which is yet considered as a kind of chronic lymphoproliferative syndrom or as the proliferation of an other clone. In this hypothesis, the lymphoplasmocytic sarcoma habe the same signification that the Richter's syndrome in chronic lymphoid leukemia and that the sarcoma appearing in Waldenström's macroglobulinemia or in alpha heavy chain disease.

Abdomen↗