Mo(II) complexes: A new family of cytotoxic agents?

Several molybdenum complexes, [Mo(η³-C₃H₅)X(CO)₂(N-N)] (N-N = 1,10-phenanthroline, phen: X = CF₃SO₃T1, X = Br B1, X = Cl C1; N-N = 2,2′-bipyridyl, X = CF₃SO₃T2, X = Br B2) and [W(η³-C₃H₅)Br(CO)₂(phen)] (W1) have been synthesized and characterized. Their antitumor properties have been tested in vitro against human cancer cell lines cervical carcinoma (HeLa) and breast carcinoma (MCF-7) using a metabolic activity test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT), leading to IC₅₀ values ranging from 3 to 45 μM, approximately. Most complexes exhibited significant antitumoral activity. Complexes B1 and T2 were chosen for subsequent studies aiming to understand their mechanism of action. Cellular uptake of molybdenum and octanol/water partition assays revealed that both B1 and T2 exhibit a selective uptake by cells and intermediate partition coefficients. The binding constants of B1 and T2 with ct DNA, as determined by absorption titration, are 2.08 (± 0.98) × 10⁵ and 3.68 (± 2.01) × 10⁵ M^− 1, respectively. These results suggest that they interact with DNA changing its conformation and possibly inducing cell death, and may therefore provide a valuable tool in cancer chemotherapy.     

Enhancing somatic nuclear reprogramming by Oct4 gain-of-function in cloned mouse embryos

Cloned mouse embryo development to blastocyst stage correlates positively with the expression level of Oct4 (Pou5f1) at the morula stage, as reported previously by our laboratory. However, whether this correlation is based on a cause-effect relationship has remained unclear. To address this question, we artificially increased the level of Oct4 prior and subsequent to somatic cell nuclear transfer, by microinjection of Oct4 mRNA into ooplasts and by transgenic Oct4 induction at the morula stage, respectively. We observed higher developmental rates of cloned embryos to blastocyst when higher levels of Oct4 were superimposed with the initial reprogramming events; whereas increasing Oct4 at later stages of preimplantation development did not have a significant effect on developmental rates. Our results show that supplemental Oct4 facilitates oocyte-mediated reprogramming only during the first cleavages, implying that the higher Oct4 level observed in developmentally competent cloned morulae is a readout of reprogramming events that successfully took place earlier.     

Salivary gland-specific gene expression in the malaria vector Anopheles gambiae

Molecular studies on the tissue-specific gene expression in the salivary glands of Anopheles gambiae may provide useful tools for the development of new strategies for the control of the most efficient malaria vector in the sub-Saharan Africa. We summarize here the results of a recent investigation focused on the isolation of secreted factors and putative receptors from the salivary glands of An. gambiae. Using the Signal Sequence Trap technique we have identified the first cDNAs specifically expressed in the An. gambiae salivary glands. Among these, four are exclusively expressed in female glands and encode factors presumably involved in blood-feeding, whereas two other cDNAs seem to be expressed both in male and in female glands and are likely implicated in sugar-feeding. Homologues of genes previously identified in the yellow fever mosquito Aedes aegypti, like the apyrase and D7, as well as novel salivary gland-specific cDNAs, were identified. The isolation and characterization of promoter sequences from the corresponding genes may prove useful for the expression of anti parasitic agents in the salivary glands of transgenic mosquitoes.     

Green fluorescent protein-based system for analysis of E-selectin-mediated adhesion

Numerous cell-based or cell-free systems for study of selectin adhesion use radiolabeled tracers. However, in addition to handling problems associated with the use of radioisotopes, these assays have difficulty relating a number of counts to a number of adherent cells. Here, we describe an assay that uses the natural fluorescence of the green fluorescent protein (GFP) to measure binding of cells to E-selectin. We elaborated an adhesion system composed of a cell monolayer expressing E-selectin ligand to which monodispersed fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are added. Due to GFP autofluorescence, adhered cells can be easily distinguished from cell monolayers by fluorescence microscopy, and adhesion can be measured by cytofluorometry. We applied this GFP-based adhesion assay to measure the adherence of a pancreatic tumor cell line and found that the binding parameters of these cells satisfy a number of E-selectin-specific criteria.     

Flow cytometric analysis of genome size variation in some Passiflora species

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.     

Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.     

Influence of carbon, nitrogen and phosphorous sources on glucoamylase production by Aspergillus awamori in solid state fermentation

It was the objective of the present study to increase the production of glucoamylase by Aspergillus awamori through solid state fermentation, using wheat bran as the main carbon source and (NH4)2SO4, urea, KH2PO4, glucose, maltose and starch as additional nitrogen, phosphorus, and carbon sources. The production of glucoamylase is strongly influenced by N and C sources. A 100% increase was observed when the (NH4)2SO4 was replaced by urea, with C/N = 4.8, using maltose as the additional carbon source. C/P ratios in a range of 5.1 to 28.7 did not induce glucoamylase production under the studied conditions.     

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.     

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging. In isolated mitochondria, superoxide production on the matrix side of the membrane is particularly high during reversed electron transport to complex I driven by oxidation of succinate or glycerol 3-phosphate. Reversed electron transport and superoxide production from complex I are very sensitive to proton motive force, and can be strongly decreased by mild uncoupling of oxidative phosphorylation. Both matrix superoxide and the lipid peroxidation product 4-hydroxy-trans-2-nonenal can activate uncoupling through endogenous UCPs (uncoupling proteins). We suggest that superoxide releases iron from aconitase, leading to a cascade of lipid peroxidation and the release of molecules such as hydroxy-nonenal that covalently modify and activate the proton conductance of UCPs and other proteins. A function of the UCPs may be to cause mild uncoupling in response to matrix superoxide and other oxidants, leading to lowered proton motive force and decreased superoxide production. This simple feedback loop would constitute a self-limiting cycle to protect against excessive superoxide production, leading to protection against aging, but at the cost of a small elevation of respiration and basal metabolic rate.