Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis

Human glutamate carboxypeptidase II [GCPII (EC 3.4.17.21)] is recognized as a promising pharmacological target for the treatment and imaging of various pathologies, including neurological disorders and prostate cancer. Recently reported crystal structures of GCPII provide structural insight into the organization of the substrate binding cavity and highlight residues implicated in substrate/inhibitor binding in the S1' site of the enzyme. To complement and extend the structural studies, we constructed a model of GCPII in complex with its substrate, N-acetyl-l-aspartyl-l-glutamate, which enabled us to predict additional amino acid residues interacting with the bound substrate, and used site-directed mutagenesis to assess the contribution of individual residues for substrate/inhibitor binding and enzymatic activity of GCPII. We prepared and characterized 12 GCPII mutants targeting the amino acids in the vicinity of substrate/inhibitor binding pockets. The experimental results, together with the molecular modeling, suggest that the amino acid residues delineating the S1' pocket of the enzyme (namely Arg210) contribute primarily to the high affinity binding of GCPII substrates/inhibitors, whereas the residues forming the S1 pocket might be more important for the 'fine-tuning' of GCPII substrate specificity.     

Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl-phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, alphavbeta6. Reverse immunoprecipitation using anti-beta6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or beta6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of beta6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.     

Parasite plastids: maintenance and functions

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.     

Analysis of telomerase activity and detection of its catalytic subunit,hTERT

The discovery of the enzyme telomerase and its subunits has led to major advances in understanding the mechanisms of cellular proliferation, immortalization, aging, and neoplastic transformation. The expression of telomerase in more than 85% of tumors provides an excellent tool for the diagnosis, prognosis, and treatment of cancer. However, the techniques employed in its detection appear to play a significant role in the interpretation of the results. The telomeric repeat amplification protocol (TRAP assay) has been the standard assay in the detection of telomerase activity and many variations of this technique have been reported. Recent advances in the development of the TRAP assay and the incorporation of techniques that provide a quantitative and qualitative estimate of telomerase activity are assessed in this review. In addition to histological and cytological examination of tissues, distribution patterns of the catalytic subunit of telomerase, hTERT, are frequently used in the prognosis of tumors. The methods involved in the detection of hTERT as a biomarker of cellular transformation are also analyzed.     

Nifs and Sufs in malaria

This review assembles data from three bodies of literature (bacterial genetics, plastid biogenesis and parasitology) that seldom have much direct cross-talk. After overcoming terminological complications to sort out microbial nifS from sufS genes, we connect a bacterial operon, recently found to be involved in iron metabolism, the formation of [Fe-S] clusters and oxidative stress to a potentially important gene (sufB) carried on the degenerate plastid genome of malaria and related parasites.     

Recent advances in the MOBJ algorithm for training artificial neural networks

This paper presents a new scheme for training MLPs which employs a relaxation method for multi-objective optimization. The algorithm works by obtaining a reduced set of solutions, from which the one with the best generalization is selected. This approach allows balancing between the training error and norm of network weight vectors, which are the two objective functions of the multi-objective optimization problem. The method is applied to classification and regression problems and compared with Weight Decay (WD), Support Vector Machines (SVMs) and standard Backpropagation (BP). It is shown that the systematic procedure for training proposed results on good generalization neural models, and outperforms traditional methods.     

Direct innervation of GnRH neurons by encephalic photoreceptors in birds

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.     

Behaviour of the Common Moorhen in Rio Grande do Sul, Brazil

This study presents data on behavioural acts performed by the Common Moorhen Gallinula chloropus in southern Brazil, and compares these with the behaviours previously reported for other populations. Focal observations of individuals were conducted in the municipality of Santa Maria, in the central region of the state of Rio Grande do Sul, Brazil. The sampling was done in 2-hour sessions, between January and March of 2007. A total of 20 behavioural acts, grouped in seven categories, were identified and described: locomotion (N = 5 acts), grooming (N = 4), intra-specific behaviour (N = 2), inter-specific behaviour (N = 3), foraging (N = 2), reproduction (N = 2) and rest (N = 2). Among the observed behaviours were acts that are not described in the literature such as greeting of offspring and some feeding acts. Regarding the use of habitat, we observed that this species has a preference for water or aquatic macrophytes, which is contrary to other reports. In the analysis of behavioural daily variation, overall behavioural categories did not vary significantly throughout the day, whereas we observed a significant difference in the use of categories during the periods 11:00 am–1:00 pm, 1:00–3:00 pm and 5:00–7:00 pm. The contrasting data between studies indicate that the variation between habitats and ecological interactions may generate different selective pressures on the behaviour of G. chloropus.     

The Brazilian legal framework on the scientific use of animals

Brazil has an exceptionally dynamic research sector in Latin America in health, biotechnology, and pharmacology, backed by defined government policies on science and technology and a health research agenda focusing on important neglected diseases: malaria, leishmaniasis, Chagas disease, turberculosis, leprosy, and dengue. The Brazilian health research policy promotes partnerships and networks among scientists in academic institutions in both wealthy industrialized and disease-endemic countries, and in these efforts the government's guidelines for animal use in biomedical research are considered fundamental to guarantee both animal welfare and the quality of research. Given international discussions of animal experimentation regulations and guidelines, in this article we describe current Brazilian legislation governing the use of animals in scientific investigations. We conclude that, despite advances in the implementation of the 3Rs (reduction, refinement, replacement), the new regulatory framework does not sufficiently incorporate ethical considerations, lacking explicit reference to the 3Rs as well as measures for their full application. The more humane use of animals in research will depend on the approach adopted by Brazil's National Council for the Control of Animal Experimentation to promote the 3Rs and to improve internal regulations as well as data collection and analysis in research institutions. In Brazil as elsewhere, one of the greatest challenges to policymakers is to harmonize the myriad and intertwined legal provisions without hindering biomedical research.     

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.