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1.
Paul McDonough D Merrill Dane Connie C W Hsia Cuneyt Yilmaz Robert L Johnson 《Journal of applied physiology》2006,100(2):474-481
In a previous study, our laboratory showed that young dogs born at sea level (SL) and raised from 2.5 mo of age to beyond somatic maturity at a high altitude (HA) of 3,100 m show enhanced resting lung function (Johnson RL Jr, Cassidy SS, Grover RF, Schutte JE, and Epstein RH. J Appl Physiol 59: 1773-1782, 1985). To examine whether HA-induced adaptation improves pulmonary gas exchange during exercise and whether adaptation is reversible when animals return to SL before somatic maturity, we raised 2.5-mo-old foxhounds at HA (3,800 m) for 5 mo (to age 7.5 mo) before returning them to SL. Lung function was measured under anesthesia 1 mo and 2 yr after return to SL and during exercise approximately 1 yr after return. In animals exposed to HA relative to simultaneous litter-matched SL controls, resting circulating blood and erythrocyte volumes, lung volumes, septal volume estimated by a rebreathing technique, and lung tissue volume estimated by high-resolution computed tomography scan were persistently higher. Lung diffusing capacity, membrane diffusing capacity, and pulmonary capillary blood volume estimated at a given cardiac output were significantly higher in animals exposed to HA, whereas maximal oxygen uptake and hematocrit were similar between groups. We conclude that relatively short exposure to HA during somatic maturation improves long-term lung function into adulthood. 相似文献
2.
To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. 相似文献
3.
Insulin-stimulated hexose transport and glucose oxidation in rat adipocytes is inhibited by sphingosine at a step after insulin binding 总被引:1,自引:0,他引:1
D G Robertson M DiGirolamo A H Merrill J D Lambeth 《The Journal of biological chemistry》1989,264(12):6773-6779
Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin. 相似文献
4.
C T Evans C J Corbin C T Saunders J C Merrill E R Simpson C R Mendelson 《The Journal of biological chemistry》1987,262(14):6914-6920
5.
Gerald A. Merrill David Miller John Chirgwin Paul M. Horowitz 《Journal of Protein Chemistry》1992,11(2):193-199
Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme. 相似文献
6.
William P. Norred Elaine Wang Hwansoo Yoo Ronald T. Riley Alfred H. Merrill Jr. 《Mycopathologia》1992,117(1-2):73-78
The effects of fumonisins B1FB1, B2(FB{2}), and the backbone of fumonisin B1 remaining after hydrolysis of the tricarballylic groups with base (HFB1) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK1). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC50 of FB1=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC50 for FB1=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation. 相似文献
7.
Normal hematological and blood chemistry parameters were measured in 45 American bison (Bison bison) that were divided into three age groups for comparison. There was a statistically significant (P less than 0.05) increase with advancing age in mean corpuscular volume, mean corpuscular hemoglobin, absolute neutrophil and eosinophil counts, total protein, globulin, creatinine, and blood urea nitrogen. There was a statistically significant (P less than 0.05) decrease with advancing age in levels of sorbital dehydrogenase, alkaline phosphatase, glucose, sodium, calcium and phosphorus. 相似文献
8.
A serologic survey was conducted on an American bison (Bison bison) herd in Kansas for antibodies against Brucella spp., Leptospira interrogans serovar canicola, pomona, grippotyphosa, icterohaemorrhagiae, and hardjo, Anaplasma spp., bluetongue virus, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. There was an increase in prevalence of bluetongue antibodies from 38% in 1987 to 100% in 1989 in animals greater than or equal to 24-mo-old. Prevalences of antibodies against the other livestock pathogens were either negative or at levels associated with previous vaccination. 相似文献
9.
Quantitation of free sphingosine in liver by high-performance liquid chromatography 总被引:10,自引:0,他引:10
A H Merrill E Wang R E Mullins W C Jamison S Nimkar D C Liotta 《Analytical biochemistry》1988,171(2):373-381
Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt. 相似文献
10.
Site-directed mutagenesis of the charged residues near the carboxy terminus of the colicin E1 ion channel 总被引:3,自引:0,他引:3
Colicin E1 was altered by oligonucleotide-directed mutagenesis at the site of three charged residues on the COOH side of the 35-residue hydrophobic segment in the channel-forming domain. Asp-509 is one of five conserved acidic residues in the channel domain of colicins A, B, E1, Ia, and Ib and is the first charged residue following the hydrophobic segment, followed by the basic residues Lys-510 and Lys-512. Asp-509 and Lys-512 were changed to amber and ochre stop codons, respectively, while Lys-510 was mutated to a Met codon. Proteins truncated after residue 508 or 511, and missing the last 14 or 11 residues, were obtained from a nonsuppressing cell strain harboring the mutant plasmid while full-length colicin molecules with single residue changes at Asp-509 to Leu, Ser, and Gln, and Lys-512 to Tyr, were obtained by using appropriate suppressor strains. The truncated colicins displayed (i) a low cytotoxicity, approximately 1% of intact wild-type colicin, (ii) 10-fold less in vitro channel activity with liposomes, and (iii) reduced labeling of the colicin in liposomes by a phospholipid photoaffinity probe, showing that one or more of the residues following Asn-511 is necessary for both in vivo and in vitro activity and insertion into the bilayer. (iv) The truncated mutants also displayed an altered conformation at pH 6 that allowed greater binding and activity with liposomes at this pH relative to wild type. The cytotoxicity of single residue substitutions at Asp-509 showed a range of cytotoxicities, wild type greater than Ser-509 greater than Gln-509 greater than Leu-509, although none of these changes greatly affected the in vitro channel activity or pH dependence.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献