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81.
Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX). 相似文献
82.
Sjöstrand D Carlsson J Paratcha G Persson B Ibáñez CF 《The Journal of biological chemistry》2007,282(17):12734-12740
Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor alpha1, shedding light on the molecular architecture of a multicomponent GDNF receptor. 相似文献
83.
Thuswaldner S Lagerstedt JO Rojas-Stütz M Bouhidel K Der C Leborgne-Castel N Mishra A Marty F Schoefs B Adamska I Persson BL Spetea C 《The Journal of biological chemistry》2007,282(12):8848-8859
In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants. 相似文献
84.
Lindgren K Ormestad M Persson M Martinsson S Svensson LT Mahlapuu M 《The FEBS journal》2007,274(11):2887-2896
The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases. 相似文献
85.
Dag Olav Hessen Jens Petter Nilssen Tone Oksum Eriksen 《International Review of Hydrobiology》1986,71(1):1-10
The relationship between two commonly co-occurring filter-feeders; Bosmina longirostris and Eudiaptomus gracilis at different nutrient levels, was investigated in bag experiments in Lake Gjersjøen. At the start of the experiment, the numerical relationship between Bosmina and Eudiaptomus was less than 1 : 10, but during the experimental period, a strong increase in Bosmina and a corresponding decrease in Eudiaptomus and medium-sized algae (5–25 μm) was observed. The shift from Eudiaptomus to Bosmina was probably due to strong competition on mediumsized algae while Bosmina in addition may utilize smaller algae and bacteria, not available to Eudiaptomus. Their coexistence in the lake at much lower densities is probably due to a strong selective fish predation, suppressing Bosmina. Daphnia longispina, not naturally occurring in the lake, showed strong competitive abilities in a fish-free bag. 相似文献
86.
87.
Uzay Gormus Alka Chaubey Suresh Shenoy Yong Wee Wong Lee Yin Chan Bao Ping Choo Liza Oraha Anna Gousseva Fredrik Persson Lawrence Prensky Ephrem Chin Madhuri Hegde 《Current issues in molecular biology》2021,43(2):958
Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies. 相似文献
88.
Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae 总被引:1,自引:0,他引:1
Berhe A Zvyagilskaya R Lagerstedt JO Pratt JR Persson BL 《Biochemical and biophysical research communications》2001,283(4):837-842
The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense RNA approach was utilized to determine if inhibition of p107 expression would block or perturb adipocyte differentiation. A series of three phosphorothioate oligonucleotides in antisense orientation was generated, designated AS1, AS2, and AS3 along with a sense control oligonucleotide complementary to AS1 and added to postconfluent cells at a concentration of 20 and 50 microM throughout hormonally stimulated differentiation. Treatment of cells with either concentration of the sense, AS1, AS2, or 20 microM AS3 oligonucleotides had little effect on either Oil Red O lipid accumulation or induction of p107 protein levels. In contrast, treatment with 50 microM AS3 inhibited the increase in p107 protein levels and led to a complete block in differentiation as detected by Oil Red O lipid accumulation and inhibition of adipocyte-specific mRNA expression. In addition, treatment with AS3 led to a significant inhibition of cellular proliferation associated with clonal expansion. Combined, these results provide strong evidence supporting a functional role for p107 in 3T3-L1 adipocyte differentiation. 相似文献
89.
Assarsson M Andersson ME Högbom M Persson BO Sahlin M Barra AL Sjöberg BM Nordlund P Gräslund A 《The Journal of biological chemistry》2001,276(29):26852-26859
The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site. 相似文献
90.
We analyze the consequences of intraguild predation and stage structure for the possible composition of a three-species community consisting of resource, consumer, and predator. Intraguild predation, a special case of omnivory, induces two major differences with traditional linear food chain models: the potential for the occurrence of two alternative stable equilibria at intermediate levels of resource productivity and the extinction of the consumer at high productivities. At low productivities, the consumer dominates, while at intermediate productivities, the predator and the consumer can coexist. The qualitative behavior of the model is robust against addition of an invulnerable size class for the consumer population and against addition of an initial, nonpredatory stage for the predator population, which means that the addition of stage structure does not change the pattern. Unless the top predator is substantially less efficient on the bottom resource, it tends to drive the intermediate species extinct over a surprisingly large range of productivities, thus making coexistence generally impossible. These theoretical results indicate that the conditions for stable food chains involving intraguild predation cannot involve strong competition for the bottommost resource. 相似文献