Showing papers by "Fundación Instituto Leloir published in 2010"

A methyl transferase links the circadian clock to the regulation of alternative splicing

Abstract: Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

Insulin-degrading enzyme sorting in exosomes: a secretory pathway for a key brain amyloid-beta degrading protease.

Abstract: The accumulation of Aβ peptides in the senile plaques is one of the hallmarks of Alzheimer disease (AD) progression. The endocytic pathway has been proposed as a major subcellular site for Aβ generation while the compartments in which Aβ-degrading proteases interact with Aβ are still elusive. It was suggested that extracellular Aβ degradation may take place by plasma-membrane associated proteases or by extracellular proteases, among which insulin-degrading enzyme (IDE) is the most relevant. However, the mechanisms of IDE secretion are poorly understood. In the present study we used N2a cells to explore if IDE is indeed released through exosomes and the effect of exosomes release on extracellular levels of Aβ. We demonstrated that proteolitically active plasma membrane associated-IDE is routed in living N2a cells to multivesicular bodies and subsequently, a major fraction is sorted to exosomes. We described that extracellular IDE levels decrease if the MVBs generation is interfered and may be positively modulated by exosomes release under stress-induced conditions. Our results reinforce the relevance of functional IDE in the catabolism of extracellular Aβ.

Prenatal inflammation impairs adult neurogenesis and memory related behavior through persistent hippocampal TGFβ1 downregulation

Abstract: Prenatal exposure to inflammatory stimuli is known to influence adult brain function. In addition, adult hippocampal neurogenesis is impaired by a local pro-inflammatory microenvironment. On this basis, we hypothesized that a pro-inflammatory insult during gestation would have negative effects on adult neurogenesis in the offspring. Pregnant Wistar rats received subcutaneous injections of lipopolysaccharide (LPS; 0.5mg/kg) or saline every other day from gestational day 14 to 20. The adult offspring prenatally treated with LPS showed a decrease in the proliferating cells and the newborn neurons of the dentate gyrus. Furthermore, prenatal LPS treatment impaired performance in the neurogenesis-dependent novel object recognition test. Maternal care was impaired by prenatal LPS administration but did not contribute to the effects of prenatal LPS on adult neurogenesis. Persistent microglial activation and downregulated expression of transforming growth factor beta-1 (TGFβ(1)) occurred specifically in the adult hippocampus of animals treated prenatally with LPS. Importantly, chronic hippocampal TGFβ(1) overexpression restored neurogenesis as well as recognition memory performance to control levels. These findings demonstrate that prenatal inflammation triggered by LPS impairs adult neurogenesis and recognition memory. Furthermore, we provide a model of reduced adult neurogenesis with long-lasting defined alterations in the neurogenic niche. Finally, we show that the expression of a single cytokine (TGFβ(1)) in the hippocampus can restore adult neurogenesis and its related behavior, highlighting the role of TGFβ(1) in these processes.

A distinctive layering pattern of mouse dentate granule cells is generated by developmental and adult neurogenesis.

Abstract: New neurons are continuously added throughout life to the dentate gyrus of the mammalian hippocampus. During embryonic and early postnatal development, the dentate gyrus is formed in an outside-in layering pattern that may extend through adulthood. In this work, we sought to quantify systematically the relative position of dentate granule cells generated at different ages. We used 5'-bromo-2'-deoxyuridine (BrdU) and retroviral methodologies to birth date cells born in the embryonic, early postnatal, and adult hippocampus and assessed their final position in the adult mouse granule cell layer. We also quantified both developmental and adult-born cohorts of neural progenitor cells that contribute to the pool of adult progenitor cells. Our data confirm that the outside-in layering of the dentate gyrus continues through adulthood and that early-born cells constitute most of the adult dentate gyrus. We also found that substantial numbers of the dividing cells in the adult dentate gyrus were derived from early-dividing cells and retained BrdU, suggesting that a subpopulation of hippocampal progenitors divides infrequently from early development onward.

A balance between circular and linear forms of the dengue virus genome is crucial for viral replication

Abstract: The plasticity of viral plus strand RNA genomes is fundamental for the multiple functions of these molecules. Local and long-range RNA–RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA–RNA interactions and genome cyclization for RNA synthesis. Here, we define a sequence present in the viral 3′UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long-range 5′-3′ interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium toward the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

Abstract: We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1beta (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps Consistent with these findings, we found that biofilms formed by the lasI rhlI P aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P aeruginosa biofilms

A Derivate of the Antibiotic Doxorubicin Is a Selective Inhibitor of Dengue and Yellow Fever Virus Replication In Vitro

Abstract: A doxorubicin derivate, SA-17, that carries a squaric acid amide ester moiety at the carbohydrate (-Ldaunosaminyl) group was identified as a selective inhibitor of in vitro dengue virus (DENV) serotype 2 replication (50% effective concentration [EC50] 0.34 0.20 g/ml [0.52 0.31 M]). SA-17 is markedly less cytostatic than the parent compound, resulting in a selectivity index value of 100. SA-17 also inhibits yellow fever virus 17D (YFV-17D) replication (EC50 3.1 1.0 g/ml [4.8 1.5 M]), although less efficiently than DENV replication, but proved inactive against a variety of enveloped and nonenveloped viruses. SA-17 inhibits in vitro flavivirus replication in a dose-dependent manner, as was assessed by virus yield reduction assays and quantification of viral RNA by means of real-time quantitative reverse transcriptase PCR (RT-qPCR) ( 2t o 3 log reduction). The anti-DENV activity was confirmed using a Renilla luciferase-expressing dengue reporter virus. Time-of-drug-addition studies revealed that SA-17 acts at the very early stages of the viral replication cycle (i.e., virus attachment and/or virus entry). This observation was corroborated by the observation that SA-17, unlike the nucleoside analogue ribavirin, does not inhibit the replication of DENV subgenomic replicons. Preincubation of high-titer stocks of DENV or YFV-17D with >5 g/ml SA-17 resulted in 100% inhibition of viral infectivity (>3 log reduction). SA-17, however, did not prove virucidal. Dengue virus (DENV), of which four serotypes (DENV-1, -2, -3, and -4) are known, and yellow fever virus (YFV) belong to the mosquito-borne cluster of the genus Flavivirus (family Flaviviridae) (25). According to the World Health Organization (WHO), 2.5 billion people, of whom 1 billion are children, are at risk of DENV infection (72). An estimated 50 to 100 million cases of dengue fever, half a million cases of severe dengue disease (i.e., dengue hemorrhagic fever [DHF] and dengue shock syndrome [DSS]), and more than 20,000 deaths occur worldwide each year (69, 72). Every year, increasing numbers of dengue outbreaks/cases are reported. Travelers visiting areas where DENV is endemic (a steadily increasing number) are also at risk of exposure to dengue (70). Dengue fever has been diagnosed in increasing numbers of febrile travelers returning from the tropics, ranging from 2% in the early 1990s to 16% or more recently (4, 5, 33, 59). Due to the nonspecific and self-limiting nature of the milder infections, these data very likely represent an underestimation of the true incidence. In addition, dengue fever represents an emerging problem for troops as well as personnel of nongovernmental organizations (NGOs) deployed in tropical countries where

Mesenchymal stem cells as therapeutic tools and gene carriers in liver fibrosis and hepatocellular carcinoma.

Abstract: Mesenchymal stem (stromal) cells (MSCs) are a source of circulating progenitors that are able to generate cells of all mesenchymal lineages and to cover cellular demands of injured tissues. The extent of their transdifferentiation plasticity remains controversial. Cells with MSC properties have been obtained from diverse tissues after purification and expansion in vitro. These cellular populations are heterogeneous and under certain conditions show pluripotent-like properties. MSCs present immunosuppressive and anti-inflammatory features and high migratory capacity toward inflamed or remodeling tissues. In this study we review available data regarding factors and signaling axes involved in the chemoattraction and engraftment of MSCs to an injured tissue or to a tissue undergoing active remodeling. Moreover, experimental evidence in support of uses of MSCs as vehicles of therapeutic genes is discussed. Because of its regenerative capacity and its particular immune properties, the liver is a good model to analyze the potential of MSC-based therapies. Finally, the potential application of MSCs and genetically modified MSCs in liver fibrosis and hepatocellular carcinoma (HCC) is proposed in view of available evidence.

Networks of High Mutual Information Define the Structural Proximity of Catalytic Sites: Implications for Catalytic Residue Identification

Abstract: Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

Brucella abortus induces the secretion of proinflammatory mediators from glial cells leading to astrocyte apoptosis.

Abstract: Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)−6, IL-1β, tumor necrosis factor (TNF)-α, macrophage chemoattractant protein−1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55−/− mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-α signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.

An Atypical Riboflavin Pathway Is Essential for Brucella abortus Virulence

Abstract: Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis.

Targeting mechanism of the retinoblastoma tumor suppressor by a prototypical viral oncoprotein. Structural modularity, intrinsic disorder and phosphorylation of human papillomavirus E7.

Abstract: DNA tumor viruses ensure genome amplification by hijacking the cellular replication machinery and forcing infected cells to enter the S phase. The retinoblastoma (Rb) protein controls the G1/S checkpoint, and is targeted by several viral oncoproteins, among these the E7 protein from human papillomaviruses (HPVs). A quantitative investigation of the interaction mechanism between the HPV16 E7 protein and the RbAB domain in solution revealed that 90% of the binding energy is determined by the LxCxE motif, with an additional binding determinant (1.0 kcal·mol−1) located in the C-terminal domain of E7, establishing a dual-contact mode. The stoichiometry and subnanomolar affinity of E7 indicated that it can bind RbAB as a monomer. The low-risk HPV11 E7 protein bound 2.0 kcal·mol−1 more weakly than the high-risk HPV16 and HPV18 type counterparts, but the modularity and binding mode were conserved. Phosphorylation at a conserved casein kinase II site in the natively unfolded N-terminal domain of E7 affected the local conformation by increasing the polyproline II content and stabilizing an extended conformation, which allowed for a tighter interaction with the Rb protein. Thus, the E7–RbAB interaction involves multiple motifs within the N-terminal domain of E7 and at least two conserved interaction surfaces in RbAB. We discussed a mechanistic model of the interaction of the Rb protein with a viral target in solution, integrated with structural data and the analysis of other cellular and viral proteins, which provided information about the balance of interactions involving the Rb protein and how these determine the progression into either the normal cell cycle or transformation. Structured digital abstract • MINT-7383794, MINT-7383812, MINT-7383830, MINT-7383868, MINT-7383891, MINT-7384056: E7 (uniprotkb:P03129) and Rb (uniprotkb:P06400) bind (MI:0407) by fluorescence technologies (MI:0051) • MINT-7383923: E7 (uniprotkb:P04020) and Rb (uniprotkb:P06400) bind (MI:0407) by competition binding (MI:0405) • MINT-7383777, MINT-7384078, MINT-7383848, MINT-7384113, MINT-7384096: Rb (uniprotkb:P06400) and E7 (uniprotkb:P03129) bind (MI:0407) by competition binding (MI:0405) • MINT-7383963: Rb (uniprotkb:P06400) and E7 (uniprotkb:P06788) bind (MI:0407) by competition binding (MI:0405) • MINT-7384022, MINT-7384040: E7 (uniprotkb:P03129) and Rb (uniprotkb:P06400) bind (MI:0407) by comigration in non denaturing gel electrophoresis (MI:0404) • MINT-7384004, MINT-7383984: Rb (uniprotkb:P06400) binds (MI:0407) to E7 (uniprotkb:P03129) by pull down (MI:0096)

The biochemical aftermath of anti-amyloid immunotherapy.

Abstract: Active and passive immunotherapy in both amyloid-beta precursor protein (APP) transgenic mice and Alzheimer's Disease (AD) patients have resulted in remarkable reductions in amyloid plaque accumulation, although the degree of amyloid regression has been highly variable. Nine individuals with a clinical diagnosis of AD dementia were actively immunized with the Aβ peptide 1-42 (AN-1792) and subjected to detailed postmortem biochemical analyses. These patients were compared to 6 non-immunized AD cases and 5 non-demented control (NDC) cases. All patients were assessed for the presence of AD pathology including amyloid plaques, neurofibrillary tangles and vascular amyloidosis. This effort revealed that two immunotherapy recipients had dementia as a consequence of diseases other than AD. Direct neuropathological examination consistently demonstrated small to extensive areas in which amyloid plaques apparently were disrupted. Characterization of Aβ species remnants by ELISA suggested that total Aβ levels may have been reduced, although because the amounts of Aβ peptides among treated individuals were extremely variable, those data must be regarded as tentative. Chromatographic analysis and Western blots revealed abundant dimeric Aβ peptides. SELDI-TOF mass spectrometry demonstrated a substantive number of Aβ-related peptides, some of them with elongated C-terminal sequences. Pro-inflammatory TNF-α levels were significantly increased in the gray matter of immunized AD cases compared to the NDC and non-immunized AD groups. Immunotherapy responses were characterized by extreme variability. Considering the broad range of biological variation that characterizes aging and complicates the recognition of reliable AD biomarkers, such disparities will make the interpretation of outcomes derived from epidemiologic and therapeutic investigations challenging. Although in some cases the apparent removal of amyloid plaques by AN-1792 was impressive, proportionate alterations in the clinical progression of AD were not evident. The fact that plaque elimination did not alter the trajectory of decline into dementia suggests the likelihood that these deposits alone are not the underlying cause of dementia.

Target transcription binding sites differentiate two groups of MerR-monovalent metal ion sensors.

Abstract: The evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerR-monovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3' and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerR-monovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.

Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.

Abstract: For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

Abstract: Background The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent The physiologic significance of the diversity of the PHD oxygen sensors is intriguing Methodology and Principal Findings We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3 Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter Conclusions and Significance Diversity of PHD isoforms is a conserved feature in evolution As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain

Experimental snapshots of a protein-DNA binding landscape

Abstract: Protein recognition of DNA sites is a primary event for gene function. Its ultimate mechanistic understanding requires an integrated structural, dynamic, kinetic, and thermodynamic dissection that is currently limited considering the hundreds of structures of protein-DNA complexes available. We describe a protein-DNA-binding pathway in which an initial, diffuse, transition state ensemble with some nonnative contacts is followed by formation of extensive nonnative interactions that drive the system into a kinetic trap. Finally, nonnative contacts are slowly rearranged into native-like interactions with the DNA backbone. Dissimilar protein-DNA interfaces that populate along the DNA-binding route are explained by a temporary degeneracy of protein-DNA interactions, centered on “dual-role” residues. The nonnative species slow down the reaction allowing for extended functionality.

Linking the Structure and Thermal Stability of β-Galactoside-Binding Protein Galectin-1 to Ligand Binding and Dimerization Equilibria

Abstract: The stability of proteins involves a critical balance of interactions of different orders of magnitude. In this work, we present experimental evidence of an increased thermal stability of galectin-1, a multifunctional beta-galactoside-binding protein, upon binding to the disaccharide lactose. Analysis of structural changes occurring upon binding of lectin to its specific glycans and thermal denaturation of the protein and the complex were analyzed by circular dichroism. On the other hand, we studied dimerization as another factor that may induce structural and thermal stability changes. The results were then complemented with molecular dynamics simulations followed by a detailed computation of thermodynamic properties, including the internal energy, solvation free energy, and conformational entropy. In addition, an energetic profile of the binding and dimerization processes is also presented. Whereas binding and cross-linking of lactose do not alter galectin-1 structure, this interaction leads to substantial changes in the flexibility and internal energy of the protein which confers increased thermal stability to this endogenous lectin. Given that an improved understanding of the physicochemical properties of galectin-glycan lattices may contribute to the dissection of their biological functions and prediction of their therapeutic applications, our study suggests that galectin binding to specific disaccharide ligands may increase the thermal stability of this glycan-binding protein, an effect that could influence its critical biological functions.

Polyamine metabolism in Trypanosoma cruzi: studies on the expression and regulation of heterologous genes involved in polyamine biosynthesis

Abstract: Biochemical studies have shown that Trypanosoma cruzi and Toxoplasma gondii are the only eukaryotic organisms so far described which are auxotrophic for polyamines. Both parasites are unable to carry out the de novo biosynthesis of putrescine, and therefore they need the addition of exogenous polyamines to the culture medium for their normal proliferation. Further investigations at the molecular level have demonstrated that the wild-type T. cruzi genome does not contain ornithine or arginine decarboxylase-like nucleic acid sequences, and that the corresponding genes have been presumably lost during evolution. Since T. cruzi behaves as a deletion mutant for ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes, this parasite has been selected to study the regulation of the expression of heterologous genes involved in polyamine biosynthesis in other organisms. The resulting transgenic parasites have been useful tools to analyze the different stages of gene expression after transformation, as well as the mechanisms of drug resistance induction and the post-translational processing of enzyme precursors.

Neuroanatomical distribution of angiotensin-II-like neuropeptide within the central nervous system of the crab Chasmagnathus; physiological changes triggered by water deprivation

Abstract: The angiotensins constitute a neuropeptidergic system that emerged early in evolution. Their classical osmoregulatory and dipsogenic functions and their mnemonic actions have been demonstrated both in vertebrates and in some invertebrates. Previously, we have shown that, in the euryhaline and semiterrestrial crab Chasmagnathus granulatus, water deprivation correlates with an increased level of brain angiotensin-II-like neuropeptide/s (ANGII-like) and improves memory processes through ANGII receptors. We have proposed that the release of brain angiotensins in response to water shortages is an ancient mechanism for coordinating various functions that, together, enable organisms to tolerate this environmental change. Here, we have evaluated the physiological changes in ANGII-like levels in diverse structures of the central nervous system of these animals during water deprivation. The neuroanatomical distribution of ANGII-like is described in the optic lobes and brain of Chasmagnathus granulatus and the physiological changes in ANGII-like distribution in various brain neuropils is evaluated after water deprivation. Our results indicate that ANGII-like is widely distributed, especially in the medial protocerebrum. After 2 h of water deprivation, ANGII-like immunoreactivity increases in the central body and decreases in the olfactory neuropil and, after 6 h of water deprivation, is markedly reduced in several brain areas. Although further experiments are needed to establish that the angiotensinergic system is involved in the balance of body fluids in this crab, our results suggest that ANGII regulates several functions during water shortages.

Estimación del area de las hojas en plantas de trigo bajo diferentes tipos de estrés abiótico

Abstract: En trigo, es posible estimar el area de las hojas (AF) utilizando el producto del largo, el ancho de la lamina (LxA) y un coeficiente de proporcionalidad (bm). Sin embargo, no hay informacion sobre la posibilidad de usar el mismo valor del coeficiente para estimar el area en plantas que sufren estres hidrico, luminico o nutricional. Para estudiar este punto se realizaron dos experimentos en los cuales se aplico sequia, sombreo y deficiencias de N y P a plantas de trigo.El coeficiente bm se calculo a partir de la regresion lineal entre AF y LxA y fue similar entre las plantas control y aquellas que sufrieron sequia o deficiencias de N o P, pero fue distinto en plantas sombreadas. El mayor valor de bm en las plantas sombreadas se debio a una mayor proporcion del sector medio de la lamina, definido por su forma rectangular. La validacion de la posibilidad de usar el bm del control para estimar AF en plantas estresadas se realizo por regresion lineal entre el AF medida y calculada. Se concluye que puede usarse el mismo coeficiente bm para estimar el AF en plantas no estresadas y en plantas que sufren sequia o deficiencias de N o P. El uso del mismo valor del coeficiente bm en plantas sombreadas llevo a una subestimacion del AF, la que fue mas pronunciada a medida que aumento el sombreo.

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Abstract: Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vβ inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vβ region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.