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1.
Kathleen A. Trychta Emily J. Heathward Agnieszka Sulima Susanne Bäck Mehdi Farokhnia Christopher T. Richie 《Biomarkers》2013,18(8):756-765
AbstractContext: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases.Objective: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein.Materials and methods: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity.Results: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity.Discussion: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases.Conclusion: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction. 相似文献
2.
【背景】细胞焦亡是一种细胞程序性死亡。在古菌和细菌中,gasdermin同源蛋白(GSDM)能够被特定的活化caspase (protease)酶切,从而激活类似于细胞焦亡的效应,产生细胞破碎效果。【目的】合成生物学、代谢工程和生物制造等应用过程中,细胞破碎是不可或缺的一步。利用细胞焦亡法破碎细胞取代传统的破碎方法,可以简化操作、提高生产效益。【方法】在大肠杆菌(Escherichia coli) BW25113中共表达protease和不同来源的GSDM,选择有明显细胞焦亡效应即来源Runella sp.的GSDM进行蛋白截短改造,使其在诱导表达蛋白截短体GSDMJD后能直接激活细胞焦亡效应。对GSDMJD进行过表达优化,获得可控大肠杆菌细胞焦亡菌株。进一步以重组表达蔗糖磷酸化酶为研究模型,验证本系统应用于细胞破碎释放蛋白的效果。【结果】实现了大肠杆菌中细胞焦亡的人为可控。焦亡菌株在诱导表达焦亡相关蛋白2 h后大肠杆菌细胞破碎死亡,内容物释放。将上述系统和超声法应用于制备蔗糖磷酸化酶粗酶液,细胞焦亡法制备的粗酶液的相对酶活显著高于超声法制备的粗酶液。在制备粗酶液的菌液OD600值为2.0时,细胞焦亡法制备的粗酶液相对酶活最高并且相较于超声法制备粗酶液,提高了60%的相对酶活。【结论】细胞焦亡提供了一种更加简单快捷、绿色环保的微生物细胞破碎方式,为合成生物学与代谢工程的发展奠定了基础。 相似文献
3.
W C Claycomb 《Developmental biology》1977,61(2):245-251
[3H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [3H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [3H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication. 相似文献
4.
《Biocatalysis and Biotransformation》2013,31(6):411-426
Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52. 相似文献
5.
Jean Guillon Anita Cohen Nassima Meriem Gueddouda Rabindra Nath Das Stéphane Moreau Luisa Ronga 《Journal of enzyme inhibition and medicinal chemistry》2017,32(1):547-563
Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results showed good antimalarial activity with IC50 in the μM range. In attempting to investigate the large broad-spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotoxicity of these molecules was assessed on the human HepG2 cell line. Structure–activity relationships of these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric G-quadruplexes through a FRET melting assay by our new compounds. 相似文献
6.
Haruhiko Taguchi Akihiro Ohkubo Mitsuo Sekine Kohji Seio Hideaki Kakeya Hiroyuki Osada 《Nucleosides, nucleotides & nucleic acids》2013,32(4-6):647-654
A new phosmidosine analog 10, in which the proline and 8-oxoadenosine moieties were linked by an N-acyl sulfamate linkage, was successfully synthesized by the sulfamoylation of an 8-oxoadenosine derivative 5 followed by coupling with an L-proline derivative 8. An L-alanine-substituted derivative 13 and its derivative 14 without the alanyl residue were also synthesized. The morphological reversion activity of these synthetic compounds in v-src ts NRK cells and their antitumor activity in L1210 and KB cells were studied. As the result, neither L-proline- nor L-alanine-substituted phosmidosine analogs 10 and 13 showed any antitumor activity. Contrary to these results, the derivative 14 lacking the amino acid residue showed potent antitumor activities against cancer cells. 相似文献
7.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):601-610
A series of novel 1,4-substituted semicarbazides 5a–g with a primaquine moiety bridged by a carbonyl group at position 1 and a cycloalkyl, aryl, benzyloxy or hydroxy substituent at position 4 were prepared and biologically evaluated. The synthetic pathways applied for preparation of the title compounds involved benzotriazole as synthetic auxiliary. Primaquine semicarbazides 5a–g and their synthetic precursors benzotriazolecarbonyl semicarbazides 4 were evaluated for cytostatic, antiviral and antioxidative activities. All compounds of the series 5 showed high selectivity towards MCF-7 cells (breast carcinoma) with IC50 values in the low micromolar range and the most active was benzyl derivative 5c (IC50 1?±?0.2 µM). The benzhydryl derivative 5e showed significant cytostatic activities towards all the tested cell lines (IC50 4–18 µM). The same compound was the strongest lipoxygenase inhibitor as well (51%). The highest antioxidant activity was demonstrated for the hydroxy derivative 5g and benzotriazolecarbonyl semicarbazides 4b,c (61.2–68.5%). No antiviral activity was observed against a wide variety of DNA and RNA viruses. 相似文献
8.
Antonietta Nicotra 《Invertebrate reproduction & development.》2013,57(1):121-123
Summary Free swimming Paracentrotus lividus spermatozoa show a significant rate of protein synthesis which remains nearly linear over a period of 90 min. This protein synthetic activity is scarcely affected by emetine but completely suppressed by chloramphenicol, suggesting its possible mitochondrial origin. 相似文献
9.
《Free radical research》2013,47(10):1184-1195
AbstractNRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H2O2, and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished. 相似文献
10.
Manuela Cabiati Benedetta Svezia Marco Matteucci Luca Panchetti Silvia Burchielli Maria-Aurora Morales 《Journal of receptor and signal transduction research》2017,37(4):379-385
Purpose: Eight A2AR variants are reported in humans while no A2AR isoforms in pigs. The aim of this study was to evaluate potential isoforms presence in cardiac pig tissue to better define possible involvement of A2AR in the cardiovascular pathophysiology.Materials and methods: In adult male minipigs (n?=?4) left ventricular dysfunction (LVD) was induced by pacing at 200 bpm in the right ventricular (RV) apex. In these animals and in sham operated pigs (C-SHAM, n?=?4) cardiac tissue was collected from LV-septal wall (LV-SW)-close to pacing site-and from lateral (opposite) site (LV-OSW). A2AR specific primers, derived from Sus scrofa AY772412 sequence, were used for Real-Time PCR. The DNA was sequenced using the Sanger method. Histological analysis was also performed.Results: In LV-SW of LVD minipigs the A2AR melting curves were characterized by a sharp peak between 87 and 91?°C (short isoform, 1–94?bp) on the right of the principal peak corresponding to a long A2AR isoform (GenBank: JQ229674.1) 1–213?bp. As for C-SHAM only one peak was observed in LV-OSW region of LVD animals. The short isoform had an alternative promoter region and a specific translated protein. Histology showed in LVD-LV-SW prominent Purkinje cells compared to LV-OSW and C-SHAM. No difference in A2AR expression was observed between LVD animals and C-SHAM although a slight decrease was observed in LVD-LV-OSW.Conclusions: The presence of two different isoforms in the myocardium close to the insertion of pacing is suggestive of a differential state-specific expression of A2AR in cardiac tissue. 相似文献
11.
Sergio Cocucci 《Plant biosystems》2013,147(3-5):459-468
Abstract Changes of glycolytic substrates level during ripening of the castor bean seed. — The changes of the concentration of carbohydrates and of the main glycolytic substrates in the castor bean seed during the ripening phase were investigated. The following results were obtained: The level of unphosphorylated sugars and of acid hydrolysable polysaccharides remains almost unchanged, with a tendency to a rise during the ripening phase. The slight increase of these compounds, together with the transition of the R. Q. from high to low values, might be interpreted as an indication of a shift of the seed from the a metabolism of fat synthesis to one of conversion of lipids into sugars, such as is observed in the germinating castor bean seeds. Hexose monophosphate level sharply decreases during the last period of maturation. However, the level of these substrates does not fall so low as to suggest a severe limitation for the pentose-P pathway activity. Fructose diphosphate, DOAP, GAP, 3 PGA, 2 PGA, PEP and pyruvate levels consistently increase during the ripening process. This indicates that the drop of oxygen uptake observed in this phase cannot be due to a lack of glycolytic substrates. On the other hand, the ratios between some substrates are shifted, during ripening, from values close to the theoretical equilibrium constants to quite different values. This finding, when correlated with the one of the strong decrease of the glycolytic flow, strongly suggests a severe inactivation of the glycolyting enzymes during ripening. The increase of pyruvate in tissues showing a decreasing respiratory activity indicates a fall of the oxidative capacity of mitochondria. This might be due to a lack of ADP, or other high energy bond acceptor, following a block of synthetic processes. However, no decrease of ADP level, and an increase of the ADP/ATP ratio during ripening is observed, Among the alternative hypothesis: a) lack or excess of oxalacetate; b) increase of concentration of some Krebs cycle inhibitor; c) inactivation of mitochondrial enzymes, the latter is thought most probable, in view of the finding of a sharp decrease of some other enzyme activities during ripening, of the above mentioned interpretation of the shift of the ratios between glycolytic substrates, and of the very low level of mitochondrial activity in preparation from the mature castor bean seed. These results when correlated with those from parallel investigations on the biochemistry of castor bean seed maturation and germination suggest, as a working hypothesis, that the respiratory metabolic inactivation accompanyng seed repening is due to a general block of the metabolism of ribonucleic acid and thus protein synthesis. 相似文献
12.
The Profile of Cardiac Cytochrome c Oxidase (COX) Expression in an Accelerated Cardiac-Hypertrophy Model 总被引:1,自引:0,他引:1
Kuo WW Chu CY Wu CH Lin JA Liu JY Ying TH Lee SD Hsieh YH Chu CH Lin DY Hsu HH Huang CY 《Journal of biomedical science》2005,12(4):601-610
Summary The contribution of the mitochondrial components, the main source of energy for the cardiac hypertrophic growth induced by
pressure overload, is not well understood. In the present study, complete coarctation of abdominal aorta was used to induce
the rapid development of cardiac hypertrophy in rats. One to two days after surgery, we observed significantly higher blood
pressure and cardiac hypertrophy, which remained constantly high afterwards. We found an early increased level of cytochrome
c oxidase (COX) mRNA determined by in-situ hybridization and dot blotting assays in the hypertrophied hearts, and a drop to the baseline 20 days after surgery. Similarly,
mitochondrial COX protein level and enzyme activity increased and, however, dropped even lower than baseline 20 days following
surgery. In addition, in natural hypertension-induced hypertrophic hearts in genetically hypertensive rats, the COX protein
was significantly lower than in normotensive rats. Taken together, the lower efficiency of mitochondrial activity in the enlarged
hearts of long-term complete coarcted rats or genetically hypertensive rats could be, at least partially, the cause of hypertensive
cardiac disease. Additionally, the rapid complete coarctation-induced cardiac hypertrophy was accompanied by a disproportionate
COX activity increase, which was suggested to maintain the cardiac energy-producing capacity in overloaded hearts. 相似文献
13.
【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。 相似文献
14.
D. Lixandru P. Alexandru A. Mihai A. Roşca C. Ionescu-Tîrgovişte L.I. Braşoveanu 《Free radical research》2017,51(6):604-615
Aim: To investigate peripheral blood monocytes/macrophages (Mo/M?) paraoxonase 2 (PON2) in diabetes and the factors modulating its activity.Methods: One hundred and eighteen patients with newly diagnosed uncomplicated type 2 diabetes mellitus were compared regarding clinical, biochemical and oxidative stress parameters with 80 healthy subjects. The capacity of the peripheral blood mononuclear cells (PBMNC) to release pro-oxidants and to neutralise them was determined by measuring the respiratory burst (RB) and the intracellular antioxidant enzyme PON2. In vitro experiments were conducted on a differentiated monocytes cell line (dU937) that was exposed to serum deprivation followed by addition of isolated lipoproteins (VLDL or LDL).Results: Paraoxonase 2 activity in Mo/M? was significantly lower in type 2 diabetes patients (0.042?±?0.044 vs 0.165?±?0.133U lactonase activity/mg protein in controls, p?.0005) and decreased in the obese in all groups. It was inversely correlated to parameters of adiposity (BMI and Waist Circumference), of glucose control (blood glucose, fructosamine and HbA1c) and insulin resistance (HOMA-IR). In multivariate regression models, 15–34% of the PON2 variance was explained by diabetes. The in vitro addition of VLDL normalised the RB of serum deprived dU937 cells, S? (to 82?±?18% of the cells incubated with serum, S+) and PON2 activity (from 0.524?±?0.061 in S???to 0.298?±?0.048?U/mg protein). In contrast, when LDL was added, the RB remained lower (61?±?12% of S+, p?=?.03) and PON2 higher (0.580?±?0.030?U/mg protein, p?=?.003).Conclusions: The decrease in monocyte/macrophage PON2 enzymatic activity observed in type 2 diabetes cannot be totally explained by abdominal obesity and insulin resistance. The underlying molecular mechanisms need to be identified. 相似文献
15.
Li Lang Ningning Dong Deyan Wu Xue Yao Chen Zhang 《Journal of enzyme inhibition and medicinal chemistry》2016,31(6):98-105
AbstractHuman lipoxygenases (LOXs) have been emerging as effective therapeutic targets for inflammatory diseases. In this study, we found that four natural 2-arylbenzo[b]furan derivatives isolated from Artocarpus heterophyllus exhibited potent inhibitory activities against human LOXs, including moracin C (1), artoindonesianin B-1 (2), moracin D (3), moracin M (4). In our in vitro experiments, compound 1 was identified as the most potent LOX inhibitor and the moderate subtype selective inhibitor of 12-LOX. Compounds 1 and 2 act as competitive inhibitors of LOXs. Moreover, 1 significantly inhibits LTB4 production and chemotactic capacity of neutrophils, and is capable of protecting vascular barrier from plasma leakage in vivo. In addition, the preliminary structure–activity relationship analysis was performed based on the above four naturally occurring (1–4) and six additional synthetic 2-arylbenzo[b]furan derivatives. Taken together, these 2-arylbenzo[b]furan derivatives, as LOXs inhibitors, could represent valuable leads for the future development of therapeutic agents for inflammatory diseases. 相似文献
16.
Polyribosomes have been isolated from pear seeds which show a capacity for in vitro protein synthesis. Translational capacity increased during stratification with a peak after 13 days of stratification. This increase was greatly diminished in seeds which were incubated at 25°C (“warm stratification”). The peak in protein synthetic activity coincides with the period of stratification required for normal development of excised embryos. 相似文献
17.
【背景】北虫草作为冬虫夏草的代用品,具有与冬虫夏草类似的药理活性,其富含的蛋白质和氨基酸通常作为衡量真菌营养价值的重要指标,从中分离纯化具有潜在临床应用价值的蛋白质或多肽,已成为一个研究热点。【目的】检测沈阳北虫草野生与市售菌株人工培育子实体的蛋白质组成,分析相同培育条件下获得的蛋白种类、数量及其功能的差异,为深入研究鉴定沈阳地区北虫草药用蛋白和针对性驯化提供了蛋白质组学数据基础。【方法】采集沈阳棋盘山野生北虫草菌株,与市售人工栽培北虫草菌株同期分别经组织分离、液体发酵后培育获得子实体,通过蛋白提取、胰酶酶解后,采用非标定量技术液相色谱-质谱联用方法,对野生和市售来源培育的子实体样本进行定量蛋白组的研究。【结果】共鉴定到9 233条特异性肽段和1 923个蛋白,其中含有1 163个可定量蛋白,野生来源培育子实体有214个蛋白表达发生上调,181个蛋白表达发生下调,对这些差异蛋白进行功能富集分析发现,其主要参与能量生产/转换、氨基酸转运/代谢、抗氧化功能。在相同的营养摄取条件下,野生来源培育菌种在各个能量代谢、氨基酸代谢功能中的相关蛋白表达量高于市售来源培育的菌种。野生来源培育菌种的一种抗氧化重要蛋白(Gene Name:ISF_02112)表达量远远高于(Fold Change9)市售来源培育菌种。同时与抗氧化和代谢功能相关的差异蛋白有22个。【结论】沈阳地区北虫草野生菌株经适当人工培育会保留部分优良的生物学特性,2种来源菌株培育的子实体具有丰富及优异抗氧化功能的蛋白,子实体蛋白的抗氧化能力与其整体代谢能力相关。本研究结果为深入研究鉴定北虫草药用蛋白和针对性驯化提供蛋白质组学数据基础。 相似文献
18.
Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G0/G1 phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer. 相似文献
19.
Mark A. Paulik Lawrence L. Hamacher David P. Yarnall Caroline J. Simmons Lidia Maianu Richard E. Pratley W. Timothy Garvey Daniel K. Burns James M. Lenhard 《Journal of cellular biochemistry》1997,65(4):527-541
In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109–121, 178–195, 254–271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTPγS, and GDPβS but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109–121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109–121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis. J. Cell. Biochem. 65:527–541. © 1997 Wiley-Liss Inc. 相似文献
20.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2020,1864(4):129523
BackgroundWolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency.MethodsAlterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy.ResultsOxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles.ConclusionsWfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway.General significanceThis work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance. 相似文献