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81.
The Hsp70 family of molecular chaperones acts to prevent protein misfolding, import proteins into organelles, unravel protein aggregates, and enhance cell survival under stress conditions. These activities are all mediated by recognition of diverse hydrophobic sequences via a C-terminal substrate-binding domain. ATP-binding/hydrolysis by the N-terminal ATPase domain regulates the interconversion of the substrate-binding domain between low and high affinity conformations. The empty state of the substrate-binding domain has been difficult to study because of its propensity to bind nearly any available protein chain, even if only modestly hydrophobic. We have generated a new stable construct of the substrate-binding domain from the Escherichia coli Hsp70, DnaK, which has enabled us to compare the empty and peptide-bound conformations using NMR chemical shift analysis and hydrogen-deuterium exchange. We have determined that the empty state is, overall, quite similar to the peptide-bound state, contrary to a previous report. Peptide binding leads to a subtle alteration in the packing of the alpha-helical lid relative to the beta-subdomain. Significantly, we have shown that the chemical shifts of the substrate-binding domain and the ATPase domain do not change when they are placed together in a two-domain construct, whether or not peptide is bound, suggesting that, in the absence of nucleotide, the two domains of E. coli DnaK do not interact. We conclude that the isolated substrate-binding domain exists in a stable high affinity state in the absence of influence from a nucleotide-bound ATPase domain.  相似文献   
82.
The chemokine receptor CCR5 came into worldwide prominence a decade ago when it was identified as one of the major coreceptors for HIV infectivity. However, subsequent studies suggested an important modulatory role for CCR5 in the inflammatory response. Specifically, CCR5 has been reported to directly regulate T cell function in autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. Moreover, T cell-mediated immune responses are proposed to be critical in the pathogenesis of autoimmune and viral liver diseases, and recent clinical and experimental studies have also implicated CCR5 in the pathogenesis of autoimmune and viral liver diseases. Therefore, in this brief review, we highlight the evidence that supports an important role of CCR5 in the pathophysiology of T cell-mediated liver diseases with specific emphasis on autoimmune and viral liver diseases.  相似文献   
83.
Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 μg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.  相似文献   
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85.
Genetic mutations disrupting the function of signaling lymphocytic activation molecule-associated protein (SAP) lead to T cell intrinsic defects in T cell-dependent Ab responses. To better understand how SAP enables Th cells to help B cells, we first assessed whether molecules important for B cell help are dysregulated in SAP-deficient (SAP knockout (KO)) mice. CD40 ligand (CD40L) expression was enhanced on unpolarized SAP KO T cells; however, Th2 polarization returned their CD40L expression to wild-type levels without rescuing their ability to help B cells. CD40L also localized normally to the site of contact between SAP KO T cells and Ag-bearing B cells. Finally, CD40L-deficient Th cells and SAP KO Th cells differed in their abilities to help B cells in vitro. These data argue that Ab defects caused by SAP deficiency do not result from a loss of CD40L regulation or CD40L function on CD4 T cells. SAP KO Th cells additionally displayed normal patterns of migration and expression of ICOS and CXCR5. Global gene expression was remarkably similar in activated SAP KO vs wild-type T cells, prompting us to investigate whether SAP is necessary for "programming" T cells to become B cell helpers. By restricting SAP expression during differentiation, we determined that SAP is not required during the first 5 days of T cell activation/differentiation to generate Th cells capable of helping B cells. Instead, SAP is necessary for very late stages of differentiation or, most likely, for allowing Th cells to communicate during cognate T:B interactions.  相似文献   
86.
The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.  相似文献   
87.
The stochastic nature of biochemical networks   总被引:3,自引:0,他引:3  
Cell behaviour and the cellular environment are stochastic. Phenotypes vary across isogenic populations and in individual cells over time. Here we will argue that to understand the abilities of cells we need to understand their stochastic nature. New experimental techniques allow gene expression to be followed in single cells over time and reveal stochastic bursts of both mRNA and protein synthesis in many different types of organisms. Stochasticity has been shown to be exploited by bacteria and viruses to decide between different behaviours. In fluctuating environments, cells that respond stochastically can out-compete those that sense environmental changes, and stochasticity may even have contributed to chromosomal gene order. We will focus on advances in modelling stochasticity, in understanding its effects on evolution and cellular design, and on means by which it may be exploited in biotechnology and medicine.  相似文献   
88.
In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.  相似文献   
89.
The cell death cascades in different brain regions namely hippocampus and frontal cortex of rats fed with 10% (v/v) ethanol for 12 weeks, was examined. After Western blotting, different cell death associated proteins displayed differential activation in the two regions observed. In hippocampus, activated caspase-3 and caspase-7 resulted in subsequent cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). Cytochrome c release to cytosol and apoptosis inducing factor (AIF) translocation to nucleus was marginal. B-cell leukemia/lymphoma-2 (Bcl-2) translocation to cytosol was significant whereas Bcl-2-associated X protein (Bax) and Bcl-associated death protein (Bad) were largely located in cytosol. Further, upregulation of N-methyl d-aspartate receptor subunit 1 (NMDAR1), N-methyl d-aspartate receptor subunit 2B (NMDAR2B), N-methyl d-aspartate receptor subunit 2C (NMDAR2C) and activation of calpains were observed. In frontal cortex, caspase-3 activation, cleavage of PARP-1 and nuclear translocation of AIF were more pronounced. Moreover, cytochrome c release to cytosol, Bcl-2 translocation to cytosol was evident. However, levels of Bax, Bad, NMDA receptor subunits, and calpains were unaffected. Apoptosis was further substantiated by in situ staining for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Results of the current study revealed that frontal cortex exhibits a higher level of ethanol-induced apoptosis relative to hippocampus. DNA polymerase beta assay and immunoblot showed significant loss in base excision repair in ethanol treated group.  相似文献   
90.
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