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81.
PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2–3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains (“region II,” PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2–3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 μm and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.  相似文献   
82.
Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.  相似文献   
83.
Plant natural products derived from phenylalanine and the phenylpropanoid pathway are impressive in their chemical diversity and are the result of plant evolution, which has selected for the acquisition of large repertoires of pigments, structural and defensive compounds, all derived from a phenylpropanoid backbone via the plant-specific phenylpropanoid pathway. These compounds are important in plant growth, development and responses to environmental stresses and thus can have large impacts on agricultural productivity. While plant-based medicines containing phenylpropanoid-derived active components have long been used by humans, the benefits of specific flavonoids and other phenylpropanoid-derived compounds to human health and their potential for long-term health benefits have been only recognized more recently. In this part of the review, we discuss the diversity and biosynthetic origins of phenylpropanoids and particularly of the flavonoid and stilbenoid natural products. We then review data pertaining to the modes of action and biological properties of these compounds, referring on their effects on human health and physiology and their roles as plant defense and antimicrobial compounds. This review continues in Part II discussing the use of biotechnological tools targeting the rational reconstruction of multienzyme pathways in order to modify the production of such compounds in plants and model microbial systems for the benefit of agriculture and forestry.  相似文献   
84.
Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 μm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.  相似文献   
85.
Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.  相似文献   
86.
In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB25R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance.  相似文献   
87.
Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph1J/sph1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G3 peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (Kd = 0.30 μm). EF-domains bearing the sph1J mutation are inactive. Binding of protein 4.2 to band 3 (Kd = 0.45 μm) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca2+ (but not Mg2+) by three to five times. Calmodulin also binds to the EF-domain (Kd = 17 μm), and Ca2+-calmodulin blocks Ca2+-dependent binding of protein 4.2 but not Ca2+-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.  相似文献   
88.
Entamoeba histolytica. I. Aerobic metabolism   总被引:5,自引:0,他引:5  
The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Qo2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr.It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas.Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica.Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration.Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose.Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K3 greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.  相似文献   
89.
90.
Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.  相似文献   
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