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181.
Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations. 相似文献
182.
Dhami GK Dale LB Anborgh PH O'Connor-Halligan KE Sterne-Marr R Ferguson SS 《The Journal of biological chemistry》2004,279(16):16614-16620
Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase. 相似文献
183.
Liquid transport of minute amounts of biomaterials is of paramount importance in many biotechnological applications. One of the challenges is the transport of viscous liquids without heating. Electro hydro dynamic atomization or electrospray is a viable method for the controlled transport of nanoliter volume of viscous liquids as shown for PEG400. Experimental results and the design of a novel spraying configuration, which can be incorporated in an optical microscope, are reported. 相似文献
184.
Belin RJ Sumandea MP Kobayashi T Walker LA Rundell VL Urboniene D Yuzhakova M Ruch SH Geenen DL Solaro RJ de Tombe PP 《American journal of physiology. Heart and circulatory physiology》2006,291(5):H2344-H2353
It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI. 相似文献
185.
Bareetseng AS Kock JL Pohl CH Pretorius EE Strauss CJ Botes PJ Van Wyk PW Nigam S 《Systematic and applied microbiology》2006,29(6):446-449
The distribution of 3-hydroxy oxylipins in Saturnispora saitoi was mapped using immunofluorescence microscopy. Fluorescence was observed on aggregating ascospores, indicating the presence of 3-hydroxy oxylipins on the surface or between ascospores. The oxylipin was identified as 3-hydroxy 9:1 using gas chromatography mass spectrometry. Furthermore, ultrastructural studies using scanning and transmission electron microscopy on ascospores revealed a clear equatorial ledge surrounding oval-shaped ascospores. 相似文献
186.
Trosset JY Dalvit C Knapp S Fasolini M Veronesi M Mantegani S Gianellini LM Catana C Sundström M Stouten PF Moll JK 《Proteins》2006,64(1):60-67
The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions. 相似文献
187.
Heirman I Ginneberge D Brigelius-Flohé R Hendrickx N Agostinis P Brouckaert P Rottiers P Grooten J 《Free radical biology & medicine》2006,40(2):285-294
Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors. 相似文献
188.
189.
190.
Pieter?PypersEmail author Josefien?Delrue Jan?Diels Erik?Smolders Roel?Merckx 《Plant and Soil》2006,284(1-2):217-227
Phosphorus (P) uptake by plant roots depends on P intensity (I) and P quantity (Q) in the soil. The relative importance of Q and I on P uptake is unknown for soils with large P sorption capacities because of difficulties in determining trace levels of
P in the soil solution. We applied a new isotope based method to detect low P concentrations (<20 μg P l−1). The Q factor was determined by assessment of the isotopically exchangeable P in the soil (E-value) and the I factor was determined by measurement of the P concentration in the pore water. A pot trial was set up using four soils with
similar labile P quantities but contrasting P buffering capacities. Soils were amended with KH2PO4 at various rates and pigeon pea (Cajanus cajan L.) was grown for 25 days. The P intensity ranged between 0.0008 and 50 mg P l−1 and the P quantity ranged between 10 and 500 mg P kg−1. Shoot dry matter (DM) yield and P uptake significantly increased with increasing P application rates in all soils. Shoot
DM yield and P uptake, relative to the maximal yield or P uptake, were better correlated with the P concentration in the pore
water (R
2 = 0.83–0.90) than with the E-value (R
2=0.40–0.53). The observed P uptakes were strongly correlated to values simulated using a mechanistic rhizosphere model (NST
3.0). A sensitivity analysis reveals that the effect of P intensity on the short-term P uptake by pigeon pea exceeded the
effect of P quantity both at low and high P levels. However, DM yield and P uptake at a given P intensity consistently increased
with increasing P buffering capacity (PBC). The experimental data showed that the intensity yielding 80% of the maximal P
uptake was 4 times larger in the soil with the smallest PBC compared to the soil with the largest PBC. This study confirms
that short-term P uptake by legumes is principally controlled by the P intensity in the soil, but is to a large extent also
affected by the PBC of the soil.
Section Editor: N. J. Barrow 相似文献