Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Preparation and biological evaluation of some 1,2-O-isopropylidene-D-hexofuranose esters

The synthesis and biological evaluation of some new glycose esters bearing the 1,2-O-isopropylidene-d-hexofuranose functionality and belonging to the 3-O-acyl-d-allose and 6-O-acyl-d-glucose series are reported. When the results concerning cell growth inhibition are compared, it appears that the 6-O-acyl-d-glucose derivatives are more active than the 3-O-acyl-d-allose compounds. Within both 6-O-acyl-d-glucose and 3-O-acyl-d-allose derivatives, butyric esters displayed the highest inhibitory effects. Inhibition of cell growth is not associated with high induction levels of erythroid differentiation, despite the fact that pivaloates induce erythroid differentiation to an extent similar to that exhibited by previously reported molecules [Bioorg. Med. Chem. Lett.1999, 9, 3153-3158].     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Banding pattern of A and B chromosomes of Prochilodus lineatus (Characiformes, Prochilodontidae), with comments on B chromosomes evolution

B chromosomes in Prochilodus lineatus, a migratory neotropical fish, were analyzed in a comparative study among populations from the Dourada lagoon (State of Paraná, Brazil) and from Mogi-Guaçu river (State of São Paulo, Brazil). The data on C-banding and fluorescent in situ hybridization with a satellite DNA probe (SATH1), indicate that the small metacentric B chromosome might correspond to an isochromosome. On the other hand, both populations presented a distinct set of B chromosomes, differentiated either by their number and by the presence of variant B types in the population from Mogi-Guaçu river. The present results indicate that the B chromosomes of P. lineatus should have an ancient origin, and have undergone a differential evolutionary pathway among distinct populations.     

Molecular modeling of the human eukaryotic translation initiation factor 5A (eIF5A) based on spectroscopic and computational analyses

The eukaryotic translation initiation factor 5A (eIF5A) is a protein ubiquitously present in archaea and eukarya, which undergoes a unique two-step post-translational modification called hypusination. Several studies have shown that hypusination is essential for a variety of functional roles for eIF5A, including cell proliferation and synthesis of proteins involved in cell cycle control. Up to now neither a totally selective inhibitor of hypusination nor an inhibitor capable of directly binding to eIF5A has been reported in the literature. The discovery of such an inhibitor might be achieved by computer-aided drug design based on the 3D structure of the human eIF5A. In this study, we present a molecular model for the human eIF5A protein based on the crystal structure of the eIF5A from Leishmania brasiliensis, and compare the modeled conformation of the loop bearing the hypusination site with circular dichroism data obtained with a synthetic peptide of this loop. Furthermore, analysis of amino acid variability between different human eIF5A isoforms revealed peculiar structural characteristics that are of functional relevance.     

Characterization of the calpain/calpastatin system in human hemopoietic cell lines

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.     

HLA class I antigen and HLA-A, -B, and -C haplotype frequencies in Uruguayans

HLA class I antigens were determined for 959 unrelated Uruguayans. The predominant HLA alleles were A2, Cw4, and B35, and the most frequently observed two-loci haplotypes were A2-B44 and B35-Cw4. The most frequent three-loci HLA haplotype was A2-Cw5-B44. We compared the Uruguayan sample with similar data from other populations.     

Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly

The Ena/VASP and WASP family of proteins play distinct roles in actin cytoskeleton remodeling. Ena/VASP is linked to actin filament elongation, whereas WASP plays a role in filament nucleation and branching mediated by Arp2/3 complex. The molecular mechanisms controlling both processes are only emerging. Both Ena/VASP and WASP are multidomain proteins. They both present poly-Pro regions, which mediate the binding of profilin-actin, followed by G-actin-binding (GAB) domains of the WASP-homology 2 (WH2) type. However, the WH2 of Ena/VASP is somewhat different from that of WASP, and has been poorly characterized. Here we demonstrate that this WH2 binds profilin-actin with higher affinity than actin alone. The results are consistent with a model whereby allosteric modulation of affinity drives the transition of profilin-actin from the poly-Pro region to the WH2 and then to the barbed end of the filament during elongation. Therefore, the function of the WH2 in Ena/VASP appears to be to "process" profilin-actin for its incorporation at the barbed end of the growing filament. Conformational changes in the newly incorporated actin subunit, resulting either from nucleotide hydrolysis or from the G- to F-actin transition, may serve as a "sensor" for the processive stepping of Ena/VASP. Conserved domain architecture suggests that WASP may work similarly.     

Overexpression of inducible nitric oxide synthase and cyclooxygenase-2 in rat zinc-deficient lung: Involvement of a NF-kappaB dependent pathway.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of pulmonary diseases. The goal of this study was to measure the response of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 enzymes (COX-2) in lung with moderate zinc deficiency. Adult male Wistar rats were divided into two groups receiving (1) a zinc-deficient diet (ZD) or (2) a zinc-adequate control diet. After 2 months of treatment, the zinc-deficient group showed a significant pulmonary edema. This was associated to a reduction of protein thiols and to a significant increase of metallothionein and glutathione disulfide levels. In addition, a higher serum and lung NO production in ZD group was positively related to the higher activity and expression of iNOS and COX-2 found in lungs. Western blot analysis revealed increased IkappaBalpha degradation, an indicator of NF-kappaB activation in ZD lungs. Anatomopathologic analysis of ZD lungs showed an increase of connective tissue fibers with an influx of polymorphonuclear cells. These cells and type II cells from the alveoli showed specific immunohistochemical signals for iNOS. The conclusion is that, during the development of zinc-deficiency, iNOS activity increases in lung and contributes to lung injury. Zinc deficiency implications must be taken into account to design therapies and public health interventions involving targeted zinc supplementation for high-risk subjects or certain diseases, such as asthma.