Characterization of hemocytes from the yellow fever mosquito,Aedes aegypti

Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.     

Chromatographic determination of 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones in rat plasma: relationship to their anticonvulsant activity

The present investigation was designed to develop an assay suitable for pharmacokinetic studies of new compounds, i.e. the novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one derivatives (2a and 2b), acting as non-competitive AMPA-receptor antagonists. A reversed-phase high-performance liquid chromatographic method has been developed to determine the time-course of plasma concentrations of derivatives 2a and 2b administered intraperitoneally to Sprague-Dawley rats. The separation of compounds studied and a N-methyl-2,3-benzodiazepin-4-one derivative as internal standard (I.S.) from plasma, were carried out by liquid-liquid extraction using diethyl ether. The samples were injected onto the analytical column (Partisil 10 ODS) eluted with acetonitrile/0.01 M acetate buffer (pH 5.3) at a flow-rate of 2 ml/min and detected at 240 nm. Compounds 2a, 2b and I.S. gave retention times of 8.5, 5.25 and 11.1 min, respectively. The selectivity of the method was satisfactory. The mean recovery from spiked rat plasma ranged from 86.7 to 91.6% for 2a, and from 85.1 to 87.0% for 2b. The procedures were validated with a good reproducibility and linear response from 0.0625 to 2 microg/ml, with a regression coefficient of 0.9932 for 2a and 0.9854 for 2b. The lower limit of quantification (LOQ) was taken as 15 ng/ml for the two compounds. 2a and 2b showed no signs of significant degradation in rat plasma during storage at -20 degrees C and following freeze/thaw cycles. Moreover, plasma levels of the tested compounds have been correlated with their anticonvulsant activity, determined in vivo in genetically epilepsy-prone rats. Due to its sensitivity, the method was suitable for application to pharmacokinetic study.     

Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba

Olive oil mill wastewater (OMW) is produced as waste in olive oil extraction. With the purpose of treating this highly polluting waste, a number of experiments were conducted in a laboratory-scale bioreactor with the white rot fungus Phanerochaete flavido-alba (P. flavido-alba). It is known that this fungus is capable of decolorizing OMW in static or semistatic cultures at Erlenmeyer scale and at 30 degrees C. The objective of this work was to prove that P. flavido-alba could decolorize OMW in submerged cultures and that it is capable of reducing OMW toxicity at room temperature (25 degrees C) and in a laboratory-scale bioreactor. In the experiments conducted, manganese peroxidase (MnP) and laccase enzymes were detected; however, unlike other studies, lignin peroxidase was not found to be present. Decoloration obtained after treatment was 70%. The reduction of aromatic compounds obtained was 51%, and the toxicity of the culture medium was reduced by up to 70%. We can therefore state that P. flavido-alba is capable of reducing important environmental parameters of industrial effluents and that prospects are positive for the use of this process at a larger scale, even when working at room temperature.     

Virus bioinformatics: databases and recent applications

Bioinformatics is now used as an umbrella term for almost all aspects of computational biology. Bioinformatics research will have an impact on all of biology, and virology is not immune from these research methods. Although virology has been slower to embrace bioinformatics this is now changing, particularly in the areas of viral sequences databasing and the systematic identification of viral and host homologous proteins. Here we will review some of these recent advances focusing mainly on the herpesvirus.     

Molecular characterization of p53 mutations in primary and secondary liver tumors: diagnostic and therapeutic perspectives

p53 is one of the most mutated genes in human cancer. We have performed the molecular characterization of p53 and have searched for correlations with etiological factors and clinical parameters in primary and secondary liver tumors. A systematic study was carried out, innovative in many respects, to determine the mutational pattern of all 11 exons of p53 and analysis was extended also to exons 1–4 and 9–11 and the exon/intron junctions. Our analyses were performed on case histories of 114 patients from the European area and highlighted p53 mutation patterns different from those reported in the literature for the same tumors. In our case history, different tumors of the same organ showed a different frequency and distribution of mutations. In analyzed tumor types, gene status was a prognostic indicator of survival because patients undergoing liver resection without mutated p53 had a more favorable prognosis than mutated patients. This suggests p53 molecular diagnosis could become a further criterion in the decision for surgery and possible therapies. We describe the ideal conditions for polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing, which we have set in order to optimize yields, sensitivity, and time of what might become a massive molecular screening.     

Calbindin and calretinin immunoreactivities in the retina of a chondrostean,Acipenser baeri

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.     

Distribution of intrachromosomal telomeric sequences (ITS) on Macaca fascicularis (Primates) chromosomes and their implication for chromosome evolution

The intrachromosomal location of the telomeric sequence in the crab-eating macaque, Macaca fascicularis (F. Cercopithecidae, Catarrhini) has been analysed by fluorescent in situ hybridisation with a long synthetic (TTAGGG)(n) probe. A total of 237 metaphases was analysed. As expected, all telomeres hybridised with the probe and 90 intrachromosomal loci with different hybridisation frequencies were also detected. The chromosomal location of interstitial telomeric sequences in M. fascicularis and in Homo sapiens was then compared, 37 sites (41.11%) being found to be conserved. Some of these sequences can be derived from rearrangements, such as inversions (MFA13q23) or fusions (MFA2p13 and MFA13p12), that have taken place during karyotype evolution.     

Multiscale modelling as a tool to prescribe realistic boundary conditions for the study of surgical procedures

This work was motivated by the problems of analysing detailed 3D models of vascular districts with complex anatomy. It suggests an approach to prescribing realistic boundary conditions to use in order to obtain information on local as well as global haemodynamics. A method was developed which simultaneously solves Navier-Stokes equations for local information and a non-linear system of ordinary differential equations for global information. This is based on the principle that an anatomically detailed 3D model of a cardiovascular district can be achieved by using the finite element method. In turn the finite element method requires a specific boundary condition set. The approach outlined in this work is to include the system of ordinary differential equations in the boundary condition set. Such a multiscale approach was first applied to two controls: (i) a 3D model of a straight tube in a simple hydraulic network and (ii) a 3D model of a straight coronary vessel in a lumped-parameter model of the cardiovascular system. The results obtained are very close to the solutions available for the pipe geometry. This paper also presents preliminary results from the application of the methodology to a particular haemodynamic problem: namely the fluid dynamics of a systemic-to-pulmonary shunt in paediatric cardiac surgery.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Molecular diversity among marine picophytoplankton as revealed by psbA analyses

Photosynthetic microorganisms play a crucial role in the marine environment. In vast areas of the oceans, marine primary productivity is performed by cells smaller than 2-3 micro m (picoplankton). Here, we report on molecular analyses of the conserved photosynthetic psbA gene (coding for protein D1 of photosystem II reaction centre) as a diversity indicator of naturally occurring marine oxygenic picophytoplankton. The psbA genes proved to be good indicators of the presence of a wide variety of photosynthetic marine microbial groups, including new cyanobacterial groups and eukaryotic algae (prasinophytes). Furthermore, using environmental bacterial artificial chromosome (BAC) libraries, we were able to correlate psbA genes with small subunit rRNAs and, therefore, to confirm their phylogenetic affiliation.