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991.
The observed stability of a protein is altered when ligands bind, which results in a shift in the melting temperature (T(m)). Binding to the native state in the absence of binding to the denatured state will necessarily lead to an increase in the T(m), while binding to the unfolded state in the absence of native state binding will decrease the T(m) relative to that of the protein in the absence of ligand. These effects are required by the thermodynamics of reversible folding. However, the relationship between binding affinity and the magnitude of the observed temperature shift is not a simple correlation (i.e., a larger shift in T(m) does not necessarily mean tighter binding) and is complicated by interaction with the denatured state. Using exact simulations, the range of behavior for the dependence of the observed T(m) shift on the energetics of ligand binding is investigated here. Specifically, differential scanning calorimetry (DSC) curves are simulated for protein unfolding in the presence of ligands binding to both the native and denatured states. The results have implications for drug screening and the determination of heat capacity changes for protein unfolding. 相似文献
992.
We have recently shown that two of the beta-turns (III and IV) in the ten-stranded, beta-clam protein, cellular retinoic acid-binding protein I (CRABP I), are favored in short peptide fragments, arguing that they are encoded by local interactions (K. S. Rotondi and L. M. Gierasch, Biochemistry, 2003, Vol. 42, pp. 7976-7985). In this paper we examine these turns in greater detail to dissect the specific local interactions responsible for their observed native conformational biases. Conformations of peptides corresponding to the turn III and IV fragments were examined under conditions designed to selectively disrupt stabilizing interactions, using pH variation, chaotrope addition, or mutagenesis to probe specific side-chain influences. We find that steric constraints imposed by excluded volume effects between near neighbor residues (i,i+2), favorable polar (i,i+2) interactions, and steric permissiveness of glycines are the principal factors accounting for the observed native bias in these turns. Longer-range stabilizing interactions across the beta-turns do not appear to play a significant role in turn stability in these short peptides, in contrast to their importance in hairpins. Additionally, our data add to a growing number of examples of the 3:5 type I turn with a beta-bulge as a class of turns with high propensity to form locally defined structure. Current work is directed at the interplay between the local sequence information in the turns and more long-range influences in the mechanism of folding of this predominantly beta-sheet protein. 相似文献
993.
Summers TR Matter JM McKay JM Ronan PJ Larson ET Renner KJ Summers CH 《Hormones and behavior》2003,43(1):245-253
Central serotonin (5-HT) is activated during stressful situations and aggressive interactions in a number of species. Glucocorticoids secreted peripherally during stressful events feed back on central systems and may affect 5-HT mediation of stress-induced behavioral events. To test the neuromodulatory effect of stress hormone secretion, serotonin overflow was measured from the hippocampus of the lizard Anolis carolinensis. Microdialysis was used to collect repeated samples from anesthetized lizards, with perfusate measured by HPLC with electrochemical analysis. Following initially high levels of 5-HT, concentrations stabilized to basal levels after approximately 2 h. Intracortical infusion of 200 ng/ml corticosterone evoked transient increases in 5-HT release of approximately 400%. The effect of corticosterone on 5-HT overflow appears to be dose dependent as 20 ng/ml stimulated an increase of 200%, whereas 2 ng/ml stimulated a 50% increase. Administration of 0.1 and 1 ng/ml GABA via the dialysis probe significantly inhibited 5-HT overflow by 20 and 40%, respectively. The duration of GABA inhibition is greater than the stimulatory response for glucocorticoids. Short-lived glucocorticoid stimulation of 5-HT release suggests a possible mechanism for endocrine mediation of continuously changing social behavioral events. 相似文献
994.
Kim MS Shigenaga J Moser A Feingold K Grunfeld C 《The Journal of biological chemistry》2003,278(11):8988-8995
The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 microg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response. 相似文献
995.
Barrette-Ng IH Ng KK Cherney MM Pearce G Ghani U Ryan CA James MN 《The Journal of biological chemistry》2003,278(33):31391-31400
The Potato II (Pot II) family of proteinase inhibitors plays important roles in the constitutive and inducible defense of plants against predation by a wide range of pests. The structural basis of inhibition by a multidomain Pot II family inhibitor was revealed recently by the structure of the ternary complex between the two-headed tomato inhibitor-II (TI-II) and two molecules of subtilisin Carlsberg. Here we report the 2.15-A resolution crystal structure of the unbound form of TI-II that reveals significant conformational flexibility in the absence of bound proteinase molecules. The four independent copies of unbound TI-II in the asymmetric unit of the unit cell display a range of different conformations when compared with the bound form of the inhibitor, most strikingly in the orientations of the inhibitory domains and in the conformations of the reactive site loops. One of the two linker segments (residues 74 to 79) between the two domains as well as the adjacent beta-strand in Domain I (residues 80-85) is well ordered in all four copies of the unbound inhibitor, even though this region appeared to be disordered in the structure of the ternary complex. Conformational flexibility seen in the reactive site loops of unbound TI-II suggests a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function. 相似文献
996.
Geary Greg G.; McNeill Anne Marie; Ospina Jose A.; Krause Diana N.; Korach Kenneth S.; Duckles Sue P. 《Journal of applied physiology》2001,91(5):2391-2399
Estrogen alters reactivity of cerebral arteries by modifyingproduction of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype isunknown. ER- knockout (ERKO) mice were used to test whether estrogen acts via ER-. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 molater. Estrogen increased levels of endothelial nitric oxide synthaseand cyclooxygenase-1 in vessels from wild-type mice but was ineffectivein ERKO mice. Endothelium-denuded middle cerebral artery segmentsfrom all animals constricted when pressurized. In denuded arteries fromERKO but not wild-type mice, estrogen treatment enhancedconstriction. In endothelium-intact, pressurized arteries fromwild-type estrogen-treated mice, diameters were larger compared witharteries from untreated wild-type mice. In addition, contractileresponses to indomethacin were greater in arteries from wild-typeestrogen-treated mice compared with arteries from untreated wild-typemice. In contrast, estrogen treatment of ERKO mice had no effect ondiameter or indomethacin responses of endothelium-intact arteries. ThusER- regulation of endothelial nitric oxide synthase andcyclooxygenase-1 pathways appears to contribute to effects of estrogenon cerebral artery reactivity. 相似文献
997.
Physiological telemetry is a powerful tool in studying the thermal biology and energetics of elasmobranchs in the laboratory and field. Controlled laboratory studies have increased our understanding of the physiology and behavior of many elasmobranchs, but have focused primarily on small, slow moving species. Extrapolating results from these laboratory studies to free-swimming animals in the field or to other unstudied species may be problematic, due to laboratory constraints or species specific differences. Some elasmobranchs are too large or logistically difficult to maintain in captivity, making them extremely difficult to study in the laboratory, and thus can only be studied in the field. Physiological telemetry offers a bridge between the laboratory and the field providing an opportunity to elucidate similarities and differences. Previous studies have coupled a variety of sensors with ultrasonic transmitters to relay information on epaxial muscle and stomach temperatures of free-swimming lamnid sharks. Even though these studies indicate lamnids exhibit elevated body temperatures, the degree to which these sharks may control body temperature is still not fully understood. Telemetry of heart rate, swimming speed, muscle contraction rate, and tail beat frequency has been used to estimate energy consumption of free-swimming elasmobranchs with varying success. Based on recent advances in technology, several hypotheses regarding thermoregulation, cardiac output, and obligate ram ventilation are discussed. Although many telemetry studies have been restricted by logistical difficulties in conducting long-term tracks, recent developments such as acoustic modems, underwater listening stations and satellite telemetry may significantly increase the amount and types of physiological data that can be collected. These improvements in technology and captive animal husbandry techniques will help to bridge the gap between the laboratory and the field. 相似文献
998.
Barrette-Ng IH Ng KK Mark BL Van Aken D Cherney MM Garen C Kolodenko Y Gorbalenya AE Snijder EJ James MN 《The Journal of biological chemistry》2002,277(42):39960-39966
Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity. 相似文献
999.
Yan Y Haas JP Kim M Sgagias MK Cowan KH 《The Journal of biological chemistry》2002,277(36):33422-33430
Mutation in the BRCA1 gene is associated with an increased risk of breast and ovarian cancer. Recent studies have shown that the BRCA1 gene product may be important in mediating responses to DNA damage and genomic instability. Previous studies have indicated that overexpression of BRCA1 can induce apoptosis or cell cycle arrest at the G(2)/M border in various cell types. Although the activation of JNK kinase has been implicated in BRCA1-induced apoptosis, the role of other members of the mitogen-activated protein kinase family in mediating the cellular response to BRCA1 has not yet been examined. In this study, we monitored the activities of three members of the MAPK family (ERK1/2, JNK, p38) in MCF-7 breast cancer cells and U2OS osteosarcoma cells after their exposure to a recombinant adenovirus expressing wild type BRCA1 (Ad.BRCA1). Overexpression of BRCA1 in MCF-7 cells resulted in arrest at the G(2)/M border; however, BRCA1 expression in U2OS cells induced apoptosis. Although BRCA1 induced JNK activation in both cell lines, there were marked differences in ERK1/2 activation in response to BRCA1 expression in these two cell lines. BRCA1-induced apoptosis in U2OS cells was associated with no activation of ERK1/2. In contrast, BRCA1 expression in MCF-7 cells resulted in the activation of both ERK1/2 and JNK. To directly assess the role of ERK1/2 in determining the cellular response to BRCA1, we used dominant negative mutants of MEK1 as well as MEK1/2 inhibitor PD98059. Our results indicate that inhibition of ERK1/2 activation resulted in increased apoptosis after BRCA1 expression in MCF-7 cells. Furthermore, BRCA1-induced apoptosis involved activation of JNK, induction of Fas-L/Fas interaction, and activation of caspases 8 and 9. The studies presented in this report indicate that the response to BRCA1 expression is determined by the regulation of both the JNK and ERK1/2 signaling pathways in cells. 相似文献
1000.
Rudolph MJ Illig CR Subasinghe NL Wilson KJ Hoffman JB Randle T Green D Molloy CJ Soll RM Lewandowski F Zhang M Bone R Spurlino JC Deckman IC Manthey C Sharp C Maguire D Grasberger BL DesJarlais RL Zhou Z 《Bioorganic & medicinal chemistry letters》2002,12(3):491-495
A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility. 相似文献