Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

Molecular genetic analyses of extracellular signaling pathways during murine retinal development

Extracellular signaling pathways initiated by secreted proteins are important in the co-ordination of tissue interactions in multi-cellular organisms, particularly during embryonic development. These signaling cascades direct diverse cellular events, including proliferation, differentiation and migration, in both autocrine and paracrine modes. In adult animals, abnormal function of these proteins often results in degenerative and tumourigenic syndromes. In this study, I have focused on elucidating the role of Bone Morphogenetic Protein (Bmp) signal transduction during neuronal specification and differentiation in the vertebrate embryo, using the mouse retina as a model. Using tissue-specific conditional knock-out approaches, the consequences of genetic loss-of-function of this signaling pathway on retinal physiology were examined. Mutant mice lacking Bmp type I receptor function displayed a range of retinal phenotypes, each of which appeared to be regulated at a different threshold of Bmp receptor activity. Novel essential functions for Bmp signaling were uncovered for retinal neurogenesis, cell survival, and axonal pathfinding at the optic disc. Further, BmprIa and BmprIa exhibited genetic interactions suggestive of functional redundancy. To further characterize the underlying molecular bases for the pleiotropic effects of Bmp receptors, retina-specific loss-of-function mutants of the obligate Bmp-activated transcriptional mediator Smad4 were generated. A comparison of the retina-specific Smad4 mutant phenotypes with those of the Bmp receptor mutant retina revealed that only a subset of retinal phenotypes, namely optic disc axon pathfinding and axial patterning were common for both classes of mutant animals. Thus, these results suggest that, contrary to the classic scheme of Bmp signal transduction, Smad4-independent pathways may be operative downstream of the type I receptors. Indeed, such alternative intracellular signaling cascades may constitute a molecular basis for the multiple cellular responses elicited by Bmp signaling. Finally, I tested whether the potential Bmp pathway targets, the extracellular ligands Fgf9 and Fgf15, mediate essential cellular processes in the retina. The analyses of Fgf9 −/−; Fgf15−/− mutant mice posit a novel shared role for these genes in intra-retinal axon pathfinding. Collectively, these studies have elucidated part of the molecular machinery directing mammalian neuro-retinal development, and provided useful in vivo models to study visual function. ^

Insights into the biology of lymphatic metastasis

Recent data suggest that the generation of new lymphatic vessels (i.e. lymphangiogenesis) may be a rate-limiting step in the dissemination of tumor cells to regional lymph nodes. However, efforts to study the cellular and molecular interactions that take place between tumor cells and lymphatic endothelial cells have been limited due to a lack of lymphatic endothelial cell lines available for study. ^ I have used a microsurgical approach to establish conditionally immortalized lymphatic endothelial cell lines from the afferent mesenteric lymphatic vessels of mice. Characterization of lymphatic endothelial cells, and tumor-associated lymphatic vessels revealed high expression levels of VCAM-1, which is known to facilitate adhesion of some tumor cells to vascular endothelial cells. Further investigation revealed that murine melanoma cells selected for high expression of α4, a counter-receptor for VCAM-1, demonstrated enhanced adhesion to lymphatic endothelial cells in vitro, and increased tumorigenicity and lymphatic metastasis in vivo, despite similar lymphatic vessel numbers. ^ Next, I examined the effects of growth factors that regulate lymphangiogenesis, and report that several growth factors are capable of activating survival and proliferation pathways of lymphatic endothelial cells. The dual protein tyrosine kinase inhibitor AEE788 (EGFR and VEGFR-2) inhibited the activation of Akt and MAPK in lymphatic endothelial cells responding to multiple growth factors. Moreover, oral treatment of mice with AEE788 decreased lymphatic vessel density and production of lymphatic metastasis by human colon cancer cells growing in the cecum of nude mice. ^ In the last set of experiments, I investigated the surgical management of lymphatic metastasis using a novel model of sentinel lymphadenectomy in live mice bearing subcutaneous B16-BL6 melanoma. The data demonstrate that this procedure when combined with wide excision of the primary melanoma, significantly enhanced survival of syngeneic C57BL/6 mice. ^ Collectively, these results indicate that the production of lymphatic metastasis depends on lymphangiogenesis, tumor cell adhesion to lymphatic endothelial cells, and proliferation of tumor cells in lymph nodes. Thus, lymphatic metastasis is a multi-step, complex, and active process that depends upon multiple interactions between tumor cells and tumor associated lymphatic endothelial cells. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Biology of sensory systems

A comprehensive and highly illustrated text providing a broad and invaluable overview of sensory systems at the molecular, cellular and neurophysiological level of vertebrates, invertebrates and prokaryotes. It retains a strong focus on human systems, and takes an evolutionary and comparative approach to review the mechanosenses, chemosenses, photosenses, and other sensory systems including those for detecting pain, temperature electric and magnetic fields etc. It incorporates exciting and significant new insights provided by molecular biology which demonstrate how similar the molecular architecture and physiology of sensory cells are across species and across sensory modality, often indicationg a common ancestry dating back over half a billion years. Written by a renowned author, with extensive teaching experience in the biology of sensory systems, this book includes: - Over 400 illustrations - Self–assessment questions - Full bibliography preceded by short bibliographical essays - Boxes containing useful supplementary material. It will be invaluable for undergraduates and postgraduates studying biology, zoology, animal physiology, neuroscience, anatomy, molecular biology, physiological psychology and related courses.

Characterizing the Role of the Previously Undescribed Protein Caskin2 in Vascular Biology

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Thaptomys Thomas 1915 (Rodentia, Sigmodontinae, Akodontini) with karyotypes 2n = 50, FN = 48, and 2n = 52, FN = 52: two monophyletic lineages recovered by molecular phylogeny

A novel karyotype with 2n = 50, FN = 48, was described for specimens of Thaptomys collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita, 2n = 52, FN = 52, found in other localities. It was hence proposed that the 2n = 50 karyotype could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosomal rearrangements observed, along with the geographic distance, might represent a reproductive barrier between both forms. Phylogenetic analyses using maximum parsimony and maximum likelihood based on partial cytochrome b sequences with 1077 bp were performed, attempting to establish the relationships among the individuals with distinct karyotypes along the geographic distribution of the genus; the sample comprised 18 karyotyped specimens of Thaptomys, encompassing 15 haplotypes, from eight different localities of the Atlantic Rainforest. The intra-generic relationships corroborated the distinct diploid numbers, once both phylogenetic reconstructions recovered two monophyletic lineages, a northeastern clade grouping the 2n = 50 and a southeastern clade with three subclades, grouping the 2n = 52 karyotype. The sequence divergence observed between their individuals ranged from 1.9% to 3.5%.

Use of chromosome microdissection in fish molecular cytogenetics

Chromosome microdissection is a technique in which whole chromosomes or chromosomal segments are dissected under an inverted microscope yielding chromosome-specific sequences. Several protocol modifications introduced during the past 15 years reduced the number of chromosomes required for most applications. This is of particular interest to fish molecular cytogenetics, since most species present highly uniform karyotypes which make impossible the collection of multiple copies of the same chromosome. Probes developed in this manner can be used to investigate chromosome homologies in closely related species. Here we describe a protocol recently used in the gymnotiform species group Eigenmannia and review the major steps involved in the generation of these markers focusing on protocol modifications aiming to reduce the number of required chromosomes.