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41.
A J Conley S E Graham-Lorence M Kagimoto M C Lorence B A Murry K Oka D Sanders J I Mason 《Biochimica et biophysica acta》1992,1130(1):75-77
We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length. 相似文献
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Testicular secretion of estradiol is necessary for normal spermatogenesis and male reproductive physiology in humans and rodents. The role of estradiol in nonmammalian vertebrates remains unknown, but elevated circulating estradiol has been reported in male lizards, alligators, and various bird species. We have been unable to detect circulating estradiol in male alligators; therefore, we reexamined the question of testicular production of estradiol in alligators using more rigorous assay procedures. A large pool of plasma from a male alligator was extracted and run through an HPLC column. Immunoreactive estradiol-like material eluted coincident with authentic estradiol. By using an ultrasensitive RIA and processing large volumes of male plasma (1000 microl), we were able to measure estradiol. Estradiol in male alligators ranged from 0.23 to 3.14 pg/ml, whereas estradiol in immature female alligators ranged from 14 to 66 pg/ml. Aromatase activity in microsomes from adult alligator ovarian tissue was 36.2 +/- 1.6 pmol mg-1 h-1, whereas activity in testicular microsomes ranged between 0.92 and 2.38 pmol mg-1 h-1. Ovarian aromatase activity was inhibited in a concentration-dependent fashion by Fadrozole, but the essentially background activity of testicular aromatase was not inhibited at any concentration of Fadrozole. Likewise, a comparison of alligator testicular and ovarian aromatase mRNA expression gave a similar result: the ovarian expression was 600-fold higher and brain tissue was 10-fold higher than that of the testis. Circulating estradiol in male alligators is probably of extragonadal origin, and the testis produces little if any of this steroid. 相似文献
44.
Stevenson TO Mercer KB Cox EA Szewczyk NJ Conley CA Hardin JD Benian GM 《Journal of molecular biology》2007,374(4):936-950
unc-94 is one of about 40 genes in Caenorhabditis elegans that, when mutant, displays an abnormal muscle phenotype. Two mutant alleles of unc-94, su177 and sf20, show reduced motility and brood size and disorganization of muscle structure. In unc-94 mutants, immunofluorescence microscopy shows that a number of known sarcomeric proteins are abnormal, but the most dramatic effect is in the localization of F-actin, with some abnormally accumulated near muscle cell-to-cell boundaries. Electron microscopy shows that unc-94(sf20) mutants have large accumulations of thin filaments near the boundaries of adjacent muscle cells. Multiple lines of evidence prove that unc-94 encodes a tropomodulin, a conserved protein known from other systems to bind to both actin and tropomyosin at the pointed ends of actin thin filaments. su177 is a splice site mutation in intron 1, which is specific to one of the two unc-94 isoforms, isoform a; sf20 has a stop codon in exon 5, which is shared by both isoform a and isoform b. The use of promoter-green fluorescent protein constructs in transgenic animals revealed that unc-94a is expressed in body wall, vulval and uterine muscles, whereas unc-94b is expressed in pharyngeal, anal depressor, vulval and uterine muscles and in spermatheca and intestinal epithelial cells. By Western blot, anti-UNC-94 antibodies detect polypeptides of expected size from wild type, wild-type-sized proteins of reduced abundance from unc-94(su177), and no detectable unc-94 products from unc-94(sf20). Using these same antibodies, UNC-94 localizes as two closely spaced parallel lines flanking the M-lines, consistent with localization to the pointed ends of thin filaments. In addition, UNC-94 is localized near muscle cell-to-cell boundaries. 相似文献
45.
Francisella tularensis is a gram-negative coccobacillus that is capable of causing severe, fatal disease in a number of mammalian species, including humans. Little is known about the proteins that are surface exposed on the outer membrane (OM) of F. tularensis, yet identification of such proteins is potentially fundamental to understanding the initial infection process, intracellular survival, virulence, immune evasion and, ultimately, vaccine development. To facilitate the identification of putative F. tularensis outer membrane proteins (OMPs), the genomes of both the type A strain (Schu S4) and type B strain (LVS) were subjected to six bioinformatic analyses for OMP signatures. Compilation of the bioinformatic predictions highlighted 16 putative OMPs, which were cloned and expressed for the generation of polyclonal antisera. Total membranes were extracted from both Schu S4 and LVS by spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation, which separated OMs from cytoplasmic (inner) membrane and other cellular compartments. Validation of OM separation and enrichment was confirmed by probing sucrose gradient fractions with antibodies to putative OMPs and inner membrane proteins. F. tularensis OMs typically migrated in sucrose gradients between densities of 1.17 and 1.20 g/ml, which differed from densities typically observed for other gram-negative bacteria (1.21 to 1.24 g/ml). Finally, the identities of immunogenic proteins were determined by separation on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This is the first report of a direct method for F. tularensis OM isolation that, in combination with computational predictions, offers a more comprehensive approach for the characterization of F. tularensis OMPs. 相似文献
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Evetovich TK Conley DS Todd JB Rogers DC Stone TL 《Journal of strength and conditioning research / National Strength & Conditioning Association》2007,21(1):96-99
The purpose of the study was to examine the potential for using the mechanomyographic (MMG) signal as a biofeedback method to enhance muscular relaxation and to improve performance during forearm flexion repetitions to fatigue. Twelve adult (mean +/- SD; age: 22.0 +/- 1.1 years) moderately trained subjects (weight: 82.3 +/- 29.2 kg; height: 165.7 +/- 49.0 cm) were instructed to relax the biceps brachii muscle using MMG biofeedback (BIO) provided by viewing a computer screen graphically displaying the MMG signal and then without using MMG biofeedback (NOBIO). Electromyographic (EMG) and MMG signals were detected midway over the biceps brachii during the relaxation protocol. In subsequent visits to the laboratory, subjects performed as many repetitions as possible at 85% of 1 repetition maximum with BIO and NOBIO using the seated preacher curl exercise. Two-way (biofeedback x gender) mixed factorial analyses of variance revealed significantly (p < 0.05) lower MMG (mean +/- SEM; BIO = 0.6 +/- 0.1 mV; NOBIO = 1.1 +/- 0.2 mV) and EMG amplitudes (BIO = 6.6 +/- 0.6 microV; NOBIO = 9.4 +/- 1.4 microV) for BIO when subjects were instructed to relax the biceps brachii muscle. There was no significant difference in the number of forearm flexion repetitions performed for BIO (mean +/- SD; 7.9 +/- 0.4 reps) vs. NOBIO (8.1 +/- 0.6 reps). The results of the present study revealed that using MMG as a biofeedback technique can enhance the development of muscle relaxation, but is not useful in delaying fatigue during forearm flexion repetitions. Our results may have been influenced by a relatively short training phase designed to teach subjects to use the MMG signal as a biofeedback method. Future studies are needed to determine whether MMG biofeedback can be used for other purposes. If MMG is found to be useful as a biofeedback method, it has some distinct practical advantages over EMG that the strength and conditioning athlete and professional may find appealing. 相似文献
48.
Protein body formation in leaves of Nicotiana benthamiana: a concentration‐dependent mechanism influenced by the presence of fusion tags
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Reza Saberianfar Jussi J. Joensuu Andrew J. Conley Rima Menassa 《Plant biotechnology journal》2015,13(7):927-937
Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin‐like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP‐ and HFBI‐induced PBs and showed that ELP‐induced PBs are larger than HFBI‐induced PBs. The size of ELP‐ and HFBI‐induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration‐dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER‐resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin‐10 by co‐expression with PB‐inducing proteins. 相似文献
49.
Hoorn EJ Walsh SB McCormick JA Fürstenberg A Yang CL Roeschel T Paliege A Howie AJ Conley J Bachmann S Unwin RJ Ellison DH 《Nature medicine》2011,17(10):1304-1309
Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment. 相似文献
50.
Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A 总被引:7,自引:0,他引:7
Winder W. W.; Wilson H. A.; Hardie D. G.; Rasmussen B. B.; Hutber C. A.; Call G. B.; Clayton R. D.; Conley L. M.; Yoon S.; Zhou B. 《Journal of applied physiology》1997,82(1):219-225
Winder, W. W., H. A. Wilson, D. G. Hardie, B. B. Rasmussen,C. A. Hutber, G. B. Call, R. D. Clayton, L. M. Conley, S. Yoon, and B. Zhou. Phosphorylation of rat muscle acetyl-CoA carboxylase byAMP-activated protein kinase and protein kinase A. J. Appl. Physiol. 82(1): 219-225, 1997This studywas designed to compare functional effects of phosphorylation of muscleacetyl-CoA carboxylase (ACC) by adenosine 3,5-cyclicmonophosphate-dependent protein kinase (PKA) and by AMP-activatedprotein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and thensubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresisfollowed by autoradiography. Functional effects of phosphorylation weredetermined by measuring ACC activity at different concentrations ofeach of the substrates and of citrate, an activator of the enzyme. Themaximal velocity(Vmax) and theMichaelis constants(Km) for ATP,acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA.Phosphorylation by AMPK increased theKm for ATP andacetyl-CoA. Sequential phosphorylation by PKA and AMPK, first withoutlabel and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant (Ka) forcitrate activation was increased to the same extent by AMPKphosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with noapparent functional effects on the enzyme. AMPK appears to be the moreimportant regulator of muscle ACC. 相似文献