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961.
962.
Vieites M Smircich P Pagano M Otero L Fischer FL Terenzi H Prieto MJ Moreno V Garat B Gambino D 《Journal of inorganic biochemistry》2011,105(12):1704-1711
In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [MIICl2(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [MIICl2(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds. 相似文献
963.
964.
Melissa?de?Freitas Cordeiro-Silva Andressa?Barbosa Marília?Santiago Mariana?Provetti Raquel?Spinassé?Dettogni Thais?Trist?o?Tovar Eliete?Rabbi-Bortolini Iúri?Drumond?LouroEmail author 《Molecular biology reports》2011,38(2):1309-1313
In developed countries deafness has a genetic cause in over 60% of the cases. Contrastingly, in Brazil, it is estimated that
only 16% of all deafnesses are caused by genetic factors. Among hereditary hearing deficiencies, approximately half is caused
by mutations in the Gap Junction Protein Beta-2 (GJB2) gene, which encodes the protein Connexin 26 (Cx26). There are four mutations in this gene that present high prevalence in
specific ethnical groups, namely, 35delG, 167delT, 235delC, and W24X. The 35delG mutation is the most frequent one, occurring in homozygosity or in compound heterozygosity with mutations in the GJB2 and GJB6 genes. This study aims to determine the prevalence of GJB2-35delG, GJB2-167delT, GJB2-235delC, GJB2-W24X, del (GJB6-D13S1830), and del (GJB6-D13S1854) mutations in patients with nonsyndromic deafness in the Espirito Santo State, Brazil. A total of 77 individuals were evaluated,
from which 88.3% presented normal genotypes for all analyzed mutations, 1.3% were compound heterozygotes for 35delG-GJB2/D13S1830-GJB6, 1.3% were compound heterozygotes for 35delG/D13S1854-GJB6, 3.9% were homozygotes for the 35delG mutation and 5.2% were heterozygotes for 35delG/GJB2. The frequency of mutant alleles 35delG/GJB2,
del (D13S1830/GJB6), and del (D13S1854/GJB6) was 7.8, 0.65, and 0.65%, respectively. Mutations 167delT, 235delC, and W24X were not detected. Determining the prevalence of specific mutations related to inherited deafness in a population can contribute
to the development of more efficient and affordable molecular diagnostic protocols, and help in the genetic counseling of
patients and their families. 相似文献
965.
966.
Conventional light microscopy techniques are poorly suited for imaging the vertical cell dimension. This can be accomplished using transmission-through-dye (TTD) imaging, in which cell thickness is directly converted into image intensity in the presence of extracellular dye with strong absorption. We have previously described applications of TTD to living cells using the dye Acid Blue 9 (AB9) to generate contrast. In this work, we investigated the possibility of extending TTD to chemically fixed cells. This would depend on preservation of cell impermeability to the dye; by using a method based on fluorescence quenching, we found that formaldehyde-fixed cells remain impermeable to AB9. Fixation enables imaging of cell surfaces in the presence of high concentrations of AB9, bringing the vertical resolution to several nanometers per pixel; that is at least an order of magnitude better than resolution achievable with live cells. TTD images collected with high-NA objectives are often contaminated by Becke lines resulting from intracellular organelles, and we show how to distinguish them from features on the cell surface. Quantification of cell thickness and volume on fixed cells is also possible during the early stages of fixation; this can be useful, for example, for measuring volume kinetics following rapid introduction of a stimulus. 相似文献
967.
There is considerable interest in exploiting the novel physical and biological properties of microbial exopolysaccharides in industry and medicine. For economic and scientific reasons, large scale production under carefully monitored and controlled conditions is required. Producing exopolysaccharides in industrial fermenters poses several complex bioengineering and microbiological challenges relating primarily to the very high viscosities of such culture media, which are often exacerbated by the producing organism's morphology. What these problems are, and the strategies for dealing with them are discussed critically in this review, using pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides. The role of fermenter configuration in their production is also examined. 相似文献
968.
dos Santos JI Cintra-Francischinelli M Borges RJ Fernandes CA Pizzo P Cintra AC Braz AS Soares AM Fontes MR 《Proteins》2011,79(1):61-78
Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca2+‐dependent hydrolysis of phospholipids. Lys49‐PLA2s are well‐characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49‐PLA2 (BthTX‐II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49‐PLA2 conserves all the residues responsible for Ca2+ coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX‐II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX‐II is not catalytic based on an in vitro cell viability assay and time‐lapse experiments on C2C12 myotube cell cultures, X‐ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX‐II is devoid of catalytic activity, as already observed for Lys49‐PLA2s. Crystallographic studies of the complex BthTX‐II/Ca2+ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca2+ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX‐II is more phylogenetically related to Lys49‐PLA2s than to other Asp49‐PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
969.
Matías G. De Vas Patricio Portal Mariana Schlesinger Héctor N. Torres Silvia Fernández Villamil 《International journal for parasitology》2011,41(1):99-108
Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP+/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations. 相似文献
970.
Morato-Marques M Campos MR Kane S Rangel AP Lewis C Ballinger MN Kim SH Peters-Golden M Jancar S Serezani CH 《The Journal of biological chemistry》2011,286(33):28902-28913
Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs. 相似文献