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1.
The microplasmodia of Physarum polycephalum express three types of β-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane β-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane β-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane β-glucosidase 1 sequence and were highly homologous with the primary structures of fungal β-glucosidases. Notably, the C-terminal half of membrane β-glucosidase 1 contains two calx-β motifs, which are known to be Ca 2+ binding domains in the Drosophila Na +/Ca 2+ exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane β-glucosidase 1 differs from all previously identified family 3 β-glucosidases. In addition to cDNA for membrane β-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane β-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other β-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane β-glucosidase is a new type family 3 enzyme fused with the Calx-β domain. We propose that Calx-β domain may modulate the β-glucosidase activity in response to changes in the Ca2+ concentration. 相似文献
2.
Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Δ 4,7,10,13,16,19). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Δ 9 polyunsaturated fatty acids, linoleic acid (C18:2n-6, Δ 9,12) and -linolenic acid (C18:3n-3, Δ 9,12,15), to eicosadienoic acid (C20:2n-6, Δ 11,14) and eicosatrienoic acid (C20:3n-3, Δ 11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Δ 5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ 8 desaturation pathway. 相似文献
3.
Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change Δ G(P +H − − 3P*) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state 3P* is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates Δ G(P +H − − 3P*) Δ H(P +H − − 3P*) and is consistent with Δ G( 1P* − P +H −) and Δ H( 1P* − 3P*) from previous delayed fluorescence and phosphorescence data, implying Δ G Δ H for all combinations of these states. 相似文献
4.
The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10 7 M −1 · s −1, at 298 °K and pH 7.0, with activation parameters Δ H‡ = 44 kJ · mole −1 and Δ S‡ = +46 J · mole −1 · °K −1. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10 5, 7.5 · 10 5 and 1.0 · 10 6 M −1 · s −1, respectively. Similar measurements made with azurin instead of plastocyanin gave k = 6 · 106 and approx. 2 · 107 M−1 · s−1 for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively. Rate constants of 115 and 80 M−1 · s−1 were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 104 and 8 · 104 M−1 · s−1. The results are discussed in relation to photosynthetic electron transport. 相似文献
5.
Characterization of the human glucagon-receptor-encoding gene ( GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [ 125I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns. 相似文献
7.
The positive ion electrospray mass spectrometry (ESI-MS) of trans-[Ru(NO)Cl)(dpaH) 2]Cl 2 (dpaH=2,2′-dipyridylamine), obtained from the carrier solvent of H 2O–CH 3OH (50:50), revealed 1+ ions of the formulas [Ru II(NO +)Cl(dpaH)(dpa)] + ( m/ z=508), [Ru IIICl(dpaH)(dpa −)] + ( m/ z=478), [Ru II(NO +)(dpa) 2] + ( m/ z=472), [Ru III(dpa) 2] + ( m/ z=442), originating from proton dissociation from the parent [Ru II(NO +)Cl(dpaH) 2] 2+ ion with subsequent loss of NO (17.4% of dissociative events) or loss of HCl (82.6% of dissociative events). Further loss of NO from the m/ z=472 fragment yields the m/ z=442 fragment. Thus, ionization of the NH moiety of dpaH is a significant factor in controlling the net ionic charge in the gas phase, and allowing preferential dissociation of HCl in the fragmentation processes. With NaCl added, an ion pair, {Na[Ru II(NO)Cl(dpa) 2]} + ( m/ z=530; 532), is detectable. All these positive mass peaks that contain Ru carry a signature ‘handprint’ of adjacent m/ z peaks due to the isotopic distribution of 104Ru, 102Ru, 101Ru, 99Ru, 98Ru and 96Ru mass centered around 101Ru for each fragment, and have been matched to the theoretical isotopic distribution for each set of peaks centered on the main isotope peak. When the starting complex is allowed to undergo aquation for two weeks in H 2O, loss of the axial Cl − is shown by the approximately 77% attenuation of the [Ru II(NO +)Cl(dpaH)(dpa)] + ion, being replaced by the [Ru II(NO +)(H 2O)(dpa) 2] + ( m/ z=490) as the most abundant high-mass species. Loss of H 2O is observed to form [Ru II(NO +)(dpa) 2] + ( m/ z=472). No positive ion mass spectral peaks were observed for RuCl 3(NO)(H 2O) 2, ‘caged NO’. Negative ions were observed by proton dissociation forming [Ru II(NO)Cl 3(H 2O)(OH)] − in the ionization chamber, detecting the parent 1− ion at m/ z=274, followed by the loss of NO as the main dissociative pathway that produces [Ru IIICl 3(H 2O)(OH)] − ( m/ z=244). This species undergoes reductive elimination of a chlorine atom, forming [Ru IICl 2(H 2O)(OH)] − ( m/ z=208). The ease of the NO dissociation is increased for the negative ions, which should be more able to stabilize a Ru III product upon NO loss. 相似文献
8.
目的:探讨小檗碱对大鼠脑缺血/再灌注损伤的保护作用及免疫机制。方法:50只SD大鼠随机分为假手术组(Sham group)、模型组(Model group)、小檗碱低剂量组(BBR-L,25 mg/kg)、小檗碱中剂量组(BBR-M,50 mg/kg)、小檗碱高剂量组(BBR-H,100 mg/kg),每组各10只。采用Longa线栓法建立脑缺血/再灌注大鼠模型,缺血2h后再灌注24 h处理。于造模成功2 h后灌胃给药,假手术组和模型组组按上述方法同体积给予生理盐水。给药24 h后,测定各组大鼠神经功能缺损程度评分及脑梗死率;采用ELISA法检测抗氧化酶SOD和GSH-Px的活性、细胞因子TNF-α、IFN-β、IL-6和NO的含量;采用流式细胞术检测CD4 +、CD8 +及CD4 +/CD8 +血清含量;进一步采用RT-qPCR与Western blot技术检测大鼠脑组织内NF-κB-NLRP3信号轴关键基因及蛋白的表达情况。结果:与假手术组比较,模型组大鼠神经功能缺损程度、脑梗死率均升高(P<0.... 相似文献
9.
The viscous energy dissipation in a two generation model of the human bronchial tree is determined from inspiratory velocity and static pressure data obtained for large Reynolds numbers (10 4 < Re < 10 5). This dissipation is found to be an increasing function of both Re and distance downstream from the inlet of the model. The ratio of the dissipation in the model to the energy dissipation in an equivalent straight pipe system is determined. This ratio, Z*, for the model is compared to values in the literature for lower (laminar) Re. There is more dissipation in the branched model than in a straight pipe ( Z* > 1) and turbulence keeps Z* at roughly a fixed value for large Reynolds numbers (10 4 < Re < 10 5). Z* values for curved pipes are also compared to the branching system values. It is found that the energy dissipation for the branched model behaves similarly to that in curved pipes. 相似文献
10.
目的:探讨CD11c抗原在慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)中的表达及在临床诊断中的价值,以及CD11c抗原表达与患者的遗传学异常及预后参数的相关性。方法:采用多参数流式细胞术(flow cytometry,FCM)检测200例CLL患者、49例套细胞淋巴瘤(mantle cell lymphoma,MCL)患者CD11c的表达率和平均荧光强度(mean fluorescence intensity,MFI);并比较CLL患者CD11c的表达与预后参数ZAP-70和CD38表达的关系;同时采用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测CLL患者的P53缺失、13q14缺失、ATM缺失、6q23缺失、+12以及IGH重排,比较CD11c~+CLL患者与CD11c~-CLL患者遗传学特点。结果:CLL患者中CD11c阳性率为49.5%(99/200),MFI中位值为2.06(1.00~7.34);而MCL患者中CD11c阳性率为6.12%(3/49),MFI中位值为2.00(1.97~2.54)。CD11c在CLL中的表达率明显高于MCL,(x~2=30.62,P0.05)。CD11c~+CLL患者的ZAP-70和CD38阳性率均明显高于CD11c~-CLL患者(x~2=15.472,P0.05;x~2=11.556,P0.05),差异有统计学意义。而CLL患者的CD11c表达率与P53缺失、13q14缺失、ATM缺失、6q23缺失、+12、IGH重排的结果均无统计学差异。结论:CD11c对于辅助诊断CLL有重要价值,尤其有助于CLL和MCL的诊断和鉴别诊断。 相似文献
11.
Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg 2+ or Co(NH 3) 3+6. Measurements of specific 1H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH 3) 3+6, with 10 mM cacodylate (pH *6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10 4 M −1·s −1. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10 3 M 1·s −1. Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein 相似文献
12.
Aberrant p53 protein accumulation was measured immunohistologically in 342 colorectal paraffin-embedded tissue sections from 115 patients (24 with adenocarcinoma, 59 with adenoma and 32 'hospital controls'). Subjective scoring was compared with quantitative cell imaging, including dichotomous (p53 +/p53 -) status, ng p53 mut mg -1 enterocyte protein, and tumour burden and patient body 'burden' of aberrant p53. A total of 62.5% cancer patients, 23.7% adenoma patients and 3.1% hospital controls were accorded p53 + status on the basis of p53 quantification. Quantitative p53 +/p53 - assignment had a stronger inverse association with survival (χ 2=6.17, p=0.013, Kaplan-Meier test) than subjective 'visual estimation' (χ 2=0.57, p=0.449). There was a strong inverse relationship between the p53 'body burden' and the months of post-diagnosis survival (hazard ratio=1.42, p=0.0004, Cox proportional hazards). Absolute quantification for inactivated p53 permits objective and reproducible scoring, adjusts for intra-laboratory immunostaining 'batch effects', corrects for fixation artefacts, and standardizes for inter-laboratory differences in fixation, antibody selection and staining method. Clinically, in situ quantification of p53 will permit more accurate survival prognoses and will inform therapy selection and dose. Ultimately, accurate quantitative tissue/blood p53 correlations may provide a minimally invasive and systemic surrogate measure for these same clinical purposes. 相似文献
13.
We examined the Na +/H + exchanger message in isolated perfused rabbit hearts using Northern blot analysis with cDNA encoding for the rabbit cardiac Na +/H + exchanger. A cDNA probe from the coding region of the rabbit myocardial Na +/H + exchanger hybridized to mRNA of 5 kb under high stringency, and to a second 3.8 kb mRNA species under low stringency. When Northern blots were re-probed with a section of the 3′-untranslated region of the cDNA, the 5 kb message was apparent while the smaller 3.8 kb message was not. If isolated working rabbit hearts were subjected to ischemia we observed increases in the 3.8 kb message. Overall, the results show that a 3.8 kb mRNA product, which is homologous to the amiloride sensitive Na +/H + exchanger, exists in the myocardium and increases during ischemia in the myocardium. 相似文献
15.
目的:研究成人t(8; 21)急性髓系白血病(AML)初诊Ki-67抗原的表达特征及预后意义。方法:采集2012年7月至2019年2月本院57例成人初诊t(8; 21) AML患者的新鲜骨髓标本,采用流式细胞术(FCM)检测CD34和Ki-67抗原,分析Ki-67表达与患者初诊生物学特征、疗效及复发的关系。结果:全部患者中,CD34~+Ki-67~+细胞比例的中位值为30. 5%(范围:10. 0%~65. 8%);通过受试者工作特征(ROC)曲线确定CD34~+Ki-67~+细胞比例的最适分界阈值,CD34~+Ki-67~+细胞高比例与初诊c-KIT基因突变阳性及WT1转录本低水平均明显相关(P=0. 001; P=0. 042)。随访的36例患者中,CD34~+Ki-67~+高比例比低比例患者具有明显更高的1年累积复发(CIR)率(P=0. 035);此外,初诊WT1转录本低水平和微小残留病(MRD)高水平(2个疗程巩固治疗后RUNX1-RUNX1T1转录本水平下降3-log)均与更高的1年CIR率明显相关(P 0. 0001;P=0. 041),初诊c-KIT基因突变阳性和白细胞计数 10×109/L的患者分别有较高的1年CIR率趋势(P=0. 091; P=0. 054)。联合分组显示,MRD高水平同时CD34~+Ki-67~+细胞高比例的患者比其他患者具有明显更高的1年CIR率(P 0. 0001)。结论:初诊骨髓高比例的CD34~+Ki-67~+可能是成人t(8; 21) AML患者预后不良因素,MRD联合初诊CD34~+Ki-67~+细胞比例可能比单纯MRD更好地预测复发。 相似文献
17.
A human skeletal actin · tropomyosin · troponin complex was phosphorylated in the presence of [γ- 32P]ATP, Mg 2+, adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 μM cyclic AMP. In the presence of 10 −7 M Ca 2+ and protein kinase 0.1 mole of [ 32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [ 32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca 2+, 5 · 10 −5 M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [ 32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [ 32P]phosphate. Increasing the Ca 2+ concentration did not significantly decrease the [ 32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstituted human skeletal actomyosin made with the [ 32P]phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca 2+. 相似文献
18.
唾液酸免疫球蛋白型凝集素-15(sialic acid-binding immunoglobulin-type lectin-15,Siglec-15)属于Siglecs家族的一员,是一种新型免疫抑制分子。Siglec-15在多种人类肿瘤细胞和肿瘤相关巨噬细胞中高表达,但Siglec-15在结直肠癌(colorectal cancer,CRC)中的生物学功能及其对免疫微环境的影响尚不明确。本文旨在分析Siglec-15异常表达对CRC细胞功能及CD4 +T细胞、CD8 +T细胞浸润的影响。首先,分析TCGA数据库中结直肠癌与正常组织中 Siglec-15 mRNA表达水平,并对52例人CRC与配对癌旁组织进行免疫组织化学染色(IHC),发现Siglec-15在CRC中的表达水平高于癌旁组织( P<0.01)。CCK8和划痕愈合结果显示,敲低 Siglec-15能抑制人CRC细胞SW480增殖( P<0.01)和迁移( P<0.05)。磁珠分选小鼠脾的CD8 +T细胞并与小鼠CRC细胞MC38共培养,发现MC38细胞过表达 Siglec-15能抑制CD8 +T细胞对其的杀伤以及IFN-γ和TNF-α的分泌( P<0.01)。小鼠荷瘤结果表明,过表达 Siglec-15可以促进小鼠肿瘤生长( P<0.05)。单样本基因集富集分析、荷瘤小鼠肿瘤组织及人结直肠癌组织IHC分析均表明,Siglec-15高表达时,肿瘤微环境中CD4 +T细胞、CD8 +T细胞浸润减少( P<0.05)。综上所述,Siglec-15可能通过促进CRC细胞增殖迁移以及抑制CD4 +T细胞、CD8 +T细胞浸润促进结直肠癌进展。本文为探究Siglec-15在CRC中的免疫抑制作用提供了一些新的实验依据。 相似文献
19.
Tumour-promoting phorbol esters (phorbol-12-myristate-13-acetate, PMA; phorbol-12,13-dibutyrate, PDBu) but not 4β-phorbol, activate protein kinase C. Using human platelets pre-labelled with quin2 or 32PO 4 we examined the effects of these compounds on human platelet cytosolic free Ca 2+ ([Ca 2+] j) and on [ 32]phosphatidic acid ([ 32P]PtdOH). PMA and PDBu, but not 4β-phorbol inhibited thrombin-, PAF- and vasopressin-induced elevation of [Ca 2+], and [ 2+P]PtdOH formation. It is suggested that protein kinase C may act to terminate the transduction processes that link receptor occupancy to cellular activation. 相似文献
20.
This study demonstrates that Ca 2+ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10 −4 M CaCl 2 (high ionized Ca 2+) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)- N, N′-tetraacetic acid (EGTA) (low ionized Ca 2+). In both high and low Ca 2+, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca 2+ (approx. 2.3 nmoles·mg −1·min −1) than in low Ca 2+ (approx. 1.8 nmoles·mg −1·min −1). This difference, when analyzed by the Student's t-test for paired samples was highly significant ( P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na + + K +)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca 2+, measured with the use of CaEGTA buffers, was studied. The Ca 2+-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10 −7 M Ca 2+. This corresponds to an apparent binding constant of 2.2·10 6 M −1, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca 2+ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet. 相似文献
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