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101.
Phosphatidylinositol 3-kinase signaling regulates the expression of several genes involved in lipid and glucose homeostasis; deregulation of these genes may contribute to insulin resistance and progression toward type 2 diabetes. By employing RNA arbitrarily primed-PCR to search for novel phosphatidylinositol 3-kinase-regulated genes in response to insulin in isolated rat adipocytes, we identified fatty aldehyde dehydrogenase (FALDH), a key component of the detoxification pathway of aldehydes arising from lipid peroxidation events. Among these latter events are oxidative stresses associated with insulin resistance and diabetes. Upon insulin injection, FALDH mRNA expression increased in rat liver and white adipose tissue and was impaired in two models of insulin-resistant mice, db/db and high fat diet mice. FALDH mRNA levels were 4-fold decreased in streptozotocin-treated rats, suggesting that FALDH deregulation occurs both in hyperinsulinemic insulin-resistant state and hypoinsulinemic type 1 diabetes models. Moreover, insulin treatment increases FALDH activity in hepatocytes, and expression of FALDH was augmented during adipocyte differentiation. Considering the detoxifying role of FALDH, its deregulation in insulin-resistant and type 1 diabetic models may contribute to the lipid-derived oxidative stress. To assess the role of FALDH in the detoxification of oxidized lipid species, we evaluated the production of reactive oxygen species in normal versus FALDH-overexpressing adipocytes. Ectopic expression of FALDH significantly decreased reactive oxygen species production in cells treated by 4-hydroxynonenal, the major lipid peroxidation product, suggesting that FALDH protects against oxidative stress associated with lipid peroxidation. Taken together, our observations illustrate the importance of FALDH in insulin action and its deregulation in states associated with altered insulin signaling.  相似文献   
102.
Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.  相似文献   
103.
Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.  相似文献   
104.
105.
DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.  相似文献   
106.
Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.  相似文献   
107.
108.
Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.Bacterial cytokinesis is a highly coordinated process that is carried out by a multiprotein complex known as the divisome (9, 11, 37, 39). In Escherichia coli, there are at least 10 essential divisomal proteins that carry out the division process. Divisome formation is initiated at the incipient division site by the recruitment of the FtsZ ring (1) which provides a molecular scaffold onto which the other divisional proteins are subsequently loaded (24, 33) (Fig. (Fig.1).1). In E. coli, the first proteins to load after FtsZ are a group of predominantly cytoplasmic proteins (FtsA, ZapA, and ZipA) that stabilize nascent FtsZ protofilaments and tether them to the membrane. The stabilized Z-ring then acts as a platform for recruitment of the remaining essential divisomal proteins, which are all single- or multipass membrane proteins (i.e., FtsE/FtsX, FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, and FtsN). With the exception of FtsI, a transpeptidase that cross-links septal murein, the biochemical function of these proteins is unknown.Open in a separate windowFIG. 1.Schema showing the hierarchical pathway of divisome assembly in E. coli and B. subtilis (adapted from reference 30). For a protein to be recruited to the divisome, all of the proteins upstream from it in the hierarchical recruitment pathway must already be present at the septum. Groups of proteins that form a subcomplex independent of other divisomal proteins, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL, are highlighted by gray boxes. Red lines denote pairwise protein-protein interactions that have been experimentally demonstrated using genetic and/or biochemical approaches. The question mark indicates that the precise location of FtsW in the divisome assembly pathway in B. subtilis is currently unknown. (C) Possible outcomes of a heterologous septal targeting experiment in E. coli in which ZapA-DivIB is employed as the bait and GFP-PBP 2B is the prey. A direct interaction between DivIB and PBP 2B should result in a fluorescent ring at midcell (or a pair of dots when viewed in cross-section) due the recruitment of GFP-PBP 2B to the divisome (left panel). In contrast, a halo of fluorescence should be visible around the cell periphery due to the membrane-bound GFP-PBP 2B if there is no interaction between these two proteins (right panel).Divisomal protein recruitment in both Bacillus subtilis and E. coli occurs in a stepwise manner. For example, for FtsQ to be recruited to the E. coli divisome, all of the proteins upstream from it in the hierarchical recruitment pathway shown in Fig. Fig.1A1A must already be present at the septum. However, this pathway is not completely linear; some proteins appear to form subcomplexes prior to their recruitment to the divisome, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL (2, 12, 14, 15). The situation in B. subtilis is more complex and less well understood. For example, B. subtilis DivIB, DivIC, FtsL, and PBP 2B appear to be recruited to the septum as an interdependent group late in the cell cycle (10) (Fig. (Fig.1B).1B). To further complicate matters, once these individual proteins or subcomplexes have been recruited to the divisome, they engage in a complex network of protein-protein interactions with other divisomal proteins (7, 8, 18, 23).The plethora of protein-protein interactions at the bacterial divisome makes it difficult to decipher which molecular epitopes on individual proteins mediate their interaction with other divisomal proteins. Thus, we recently introduced an artificial septal targeting (AST) technique that allowed us to examine interactions between pairs of interacting B. subtilis divisomal proteins in E. coli (30). This technique involves artificially targeting one of the B. subtilis proteins (the “bait”) to the E. coli divisome by fusing it to E. coli ZapA and then using fluorescence microscopy to determine whether it can recruit to the septum a green fluorescent protein (GFP) fusion to a putative interacting partner (the “prey”) (Fig. (Fig.1C).1C). The primary advantage of the AST technique is that it allows direct assessment of the interaction between two B. subtilis divisomal proteins without interference from other members of the divisome.We previously used AST to demonstrate a direct interaction between B. subtilis FtsL and DivIC and between DivIB and PBP 2B (30). The latter finding is consistent with the observation from bacterial two-hybrid studies that B. subtilis DivIB interacts directly with both PBP 2B and FtsL (5) and that the E. coli orthologs of these proteins (FtsI and FtsQ, respectively) also interact strongly (18). The extracellular domain of DivIB is divided into three subdomains, termed α, β, and γ (31). It was recently shown using a combination of nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) that the concave face of the DivIB β domain makes direct contact with the C-terminal head of the FtsL-DivIC heterodimeric coiled coil (25), forming a stabilizing “cap” for these two intrinsically unstable proteins (32). In contrast, the α and γ regions of DivIB are not critical for formation of the DivIB/FtsL/DivIC ternary complex (25).The FtsQ/DivIB-FtsI/PBP 2B interaction appears to be widely conserved in both Gram-negative and Gram-positive bacteria, and therefore we decided to investigate the molecular details of this evolutionarily conserved interaction. By using a combination of AST and site-directed mutagenesis, we show that DivIB and PBP 2B interact exclusively through their extracytoplasmic regions and that this interaction is mediated by residues near the C terminus of DivIB. In combination with the results of previous studies, these new data have allowed us to construct a working model of the DivIB/PBP 2B/FtsL/DivIC complex.  相似文献   
109.
110.
doi: 10.1111/j.1741‐2358.2011.00476.x
Salivary immunity in elderly individuals presented with Candida‐related denture stomatitis Objectives: Elderly individuals with Candida‐related denture stomatitis (DS) present with a reduced defence against Candida albicans. This study evaluated levels of antimicrobial mediators in the elderly DS saliva and salivary neutrophils’ activation characteristics compared with elderly and young without DS. Methods: Salivary peroxidases (SPO) and elastase activities (ELA), nitric oxide (NO), transforming growth factor beta (TGF‐β), IL‐6 and CCL3 production were determined in saliva from elderly with or without DS, and young control individuals. TLR4, CXCR1, CD11b, CD16 and CD32 expression on salivary neutrophils were evaluated. Correlations between number and apoptosis rate of salivary neutrophils, enzymatic activities and cytokine levels were determined. Results: Elderly DS individuals exhibited the lowest SPO and ELA activities. Also, the activity of both enzymes was low in elderly without DS. Although both elderly groups showed higher salivary NO and TGF‐β levels compared to young control groups, elderly DS presented the highest salivary NO, TGF‐β, IL‐6 and CCL3 levels. Decreased percentages of salivary TLR4+ and CD16+ neutrophils were detected in both elderly groups. Although these damages could influence the establishment and persistence of DS, the highest levels of salivary IL‐6 and CCL3 in elderly DS could be preventing more serious complications.  相似文献   
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