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891.
Härterich S Koschatzky S Einsiedel J Gmeiner P 《Bioorganic & medicinal chemistry》2008,16(20):9359-9368
Investigating prototypical interactions between NT(8-13) and the human neurotensin receptor 1 (hNTR1), we created a receptor-ligand model that was validated by site-directed mutagenesis and structure-activity relationship studies. Stabilization of the extracellular loop 1 (EL1) by pi-stacking clusters proved to be important for agonist binding when substitution of six conserved amino acids by alanine resulted in an agonist specific loss of maximal binding capacity. In agreement with our modeling studies, EL1 seems to adopt a clamp-type border area controlling the shape of the binding site crevice. Employing chemically manipulated peptide analogs as molecular probes, the impact of backbone modifications on receptor-ligand interaction, especially the influence on ligand conformation, was examined in binding studies and explained by in silico analysis. 相似文献
892.
Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients 总被引:2,自引:0,他引:2
Chlamydia-like bacteria, obligate intracellular relatives of Chlamydia trachomatis and Chlamydophila pneumoniae, are widely distributed in nature. Using a two-step nested and semi-nested PCR approach targeting the 16S rRNA gene, we found DNA of Chlamydia-like bacteria in respiratory samples from patients with community-acquired pneumonia. Of 387 cases tested, four (1.03%) tested positive if only sequences showing less than 99.9% 16S rRNA gene sequence similarity to known Chlamydiae were considered. These included for the first time Protochlamydia amoebophila, Waddlia chondrophila, and 'Candidatus Rhabdochlamydia porcellionis'-related sequences. This study extends previous findings suggesting an association of Chlamydia-like bacteria with respiratory disease, but a causal link between these microorganisms and respiratory tract infections has yet to be established. 相似文献
893.
The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs. 相似文献
894.
Wesierska-Gadek J Wandl S Kramer MP Pickem C Krystof V Hajek SB 《Journal of cellular biochemistry》2008,105(5):1161-1171
Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer. 相似文献
895.
Pey AL Rodriguez-Larrea D Bomke S Dammers S Godoy-Ruiz R Garcia-Mira MM Sanchez-Ruiz JM 《Proteins》2008,71(1):165-174
It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins. 相似文献
896.
Behavioral and physiological responses to unpredictable changes in environmental conditions are, in part, mediated by glucocorticoids (corticosterone in birds). In polymorphic species, individuals of the same sex and age display different heritable melanin-based color morphs, associated with physiological and reproductive parameters and possibly alternative strategies to cope with variation in environmental conditions. We examined whether the role of corticosterone in resolving the trade-off between self-maintenance and reproductive activities covaries with the size of melanin-based spots displayed on the ventral body side of male barn owls. Administration of corticosterone to simulate physiological stress in males revealed pronounced changes in their food-provisioning rates to nestlings compared to control males. Corticosterone-treated males with small eumelanic spots reduced nestling provisioning rates as compared to controls, and also to a greater degree than did corticosterone-treated males with large spots. Large-spotted males generally exhibited lower parental provisioning and appear insensitive to exogenous corticosterone suggesting that the size of the black spots on the breast feathers predicts the ability to cope with stressful situations. The reduced provisioning rate of corticosterone-treated males caused a temporary reduction in nestling growth rates but, did not affect fledgling success. This suggests that moderately elevated corticosterone levels are not inhibitory to current reproduction but rather trigger behavioral responses to maximize lifetime reproductive success. 相似文献
897.
Schneider A Schuh A Maetzel FK Rückerl R Breitner S Peters A 《International journal of biometeorology》2008,52(6):549-547
Given the accumulating evidence that people with underlying heart disease are a particularly vulnerable group for triggers like changing meteorological parameters, the objective of this longitudinal study was to analyze the influence of weather parameters on blood pressure, arrhythmia and ischemia in cardiovascular patients. A panel study with repeated measurements was conducted in a rehabilitation clinic in Timmendorfer Strand (Baltic Sea, Germany) with 872 cardiovascular patients. Heart rate, blood pressure and electrocardiography changes were measured during repeated bicycle ergometries. Generalized Estimating Equations were used for regression analyses of immediate, delayed and cumulative influences of the daily measured meteorological data. For men, a decrease in air temperature and in water vapor pressure doubled the risk of ST-segment depression during ergometry [odds ratio (OR) for 1 day delay: 1.88 (1.24; 2.83) for air temperature] with a delay of 1-2 days. For women, an increase of their heart rate before the start of the ergometry [same day: 4.36 beats/min (0.99; 7.74) for air temperature] and a 2- to 3-fold higher risk for ventricular ectopic beats [1 day delay: OR 2.43 (1.17; 5.05) for air temperature] was observed with an increase in temperature and water vapor pressure in almost all analyzed time-windows. The study indicates that meteorological parameters can induce changes in heart function which may lead to adverse cardiovascular events especially in susceptible, diseased individuals. The observed effect on ST-segment depression could be a link between the association of weather changes and cardiovascular morbidity and mortality. 相似文献
898.
Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications 总被引:3,自引:0,他引:3
Holtgrefe S Gohlke J Starmann J Druce S Klocke S Altmann B Wojtera J Lindermayr C Scheibe R 《Physiologia plantarum》2008,133(2):211-228
Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues. 相似文献
899.
C(4) plants have up to 10-fold higher apparent CO(2) assimilation rates than the most productive C(3) plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C(4) plants in comparison with those of C(3) plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C(4) plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate, must be considerably higher in C(4) plants because they exceed the apparent rate of photosynthetic CO(2) assimilation, whereas they represent relatively minor fluxes in C(3) plants. While the enzymatic steps involved in the C(4) biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C(4) chloroplasts. In this study, we used comparative proteomics of chloroplast envelope membranes from the C(3) plant pea (Pisum sativum) and mesophyll cell chloroplast envelopes from the C(4) plant maize (Zea mays) to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C(4) photosynthesis. We show that C(3)- and C(4)-type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triosephosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C(4) plants. Several putative transport proteins have been identified that are highly abundant in C(4) plants but relatively minor in C(3) envelopes. These represent prime candidates for the transport of C(4) photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate. 相似文献
900.
Köhle Annegret Sommer Susanne Li Shu-Ming Schilde-Rentschler Lieselotte Ninnemann Helga Heide Lutz 《Molecular breeding : new strategies in plant improvement》2003,11(1):15-24
The bacterial gene ubiC encodes chorismate pyruvate-lyase (CPL), which converts chorismate to 4-hydroxybenzoate (4HB). The ubiC gene was expressed in tobacco (Nicotiana tabacum L., Solanaceae) and potato (Solanum tuberosum L., Solanaceae) under the control of the very strong constitutive plant promotor (ocs3) mas. High accumulation of 4HB glucosides as new, artificial secondary metabolites was observed in the transgenic plants. 4HB glucoside content reached 5.1% of dry weight in tobacco cell cultures and 4.0% of dry weight in the leaves of potato shoots. This is the highest content of an artificial secondary metabolite produced by genetic engineering of plants reported so far. Surprisingly, no growth retardation and no phenotypical changes were observed in the transgenic cell cultures and plants. Glucosylation of 4HB was achieved by endogeneous, constitutively expressed glucosyltransferases. The total amount of 4HB glucoside acccumulated showed a strict linear dependence on the expression level of ubiC. 相似文献