The synthesis of new 6-hydrazinopurines as potential anticancer agents

[Excerpt] Purines, such as adenine, are one of the most important naturally occurring nitrogen heterocycles and they are frequently used as bioactive agents.[1,2] The increasing number of synthetic purines reveals the great potential of these compounds as enzyme inhibitors. Protein Kinases have an important regulatory role in cell proliferation, differentiation and signalling processes. Abnormal signal transduction is responsible for devastating diseases such as cancer. All of the protein kinases identified have in common the cofactor ATP indicating that the adenine nucleus is a very important scaffold for discovery of new anti-cancer agents.[3,4] Previous work identified a modest anticancer activity in a family of 6-arylaminopurines. In the view of these results, it seemed reasonable to assume that some interesting anticancer agents might result by replacement of the phenyl group by a secondary amino group linked to the N-6 atom of the adenine moiety. (...)

Systems biology approaches for the design of novel Saccharomyces cerevisiae winemaking strains for enhanced flavour compounds synthesis

Tese de Doutoramento em Biologia Ambiental e Molecular

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-d pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Desarrollo sustentable de geoestructuras compuestas por suelo loéssico compactado mejorado con agregado de materiales estabilizantes. Sustainable development of compacted loess soil geo-structures with addition of stabilizing materials.

Los suelos estabilizados mediante compactación, permiten obtener materiales con ventajas ténicas y economicas en diferentes tipos de obras de ingeniería. Ejemplos de su uso se tiene en bases viales de autopistas, rutas o calles urbanas, pistas de aterrizaje, barreras de contención para enterramientos sanitarios o lagunas de estabilización, apoyos de plateas para fundación de edificios, losas industriales, entre otras aplicaciones. Las fallas en este tipo de construcciones pueden resultar en catástrofes ambientales, sociales y elevadas pérdidas económicas, por lo que resulta de gran importancia optimizar el diseño e incrementar la seguridad de este tipo de construcciones. Las obras con estas características involucran grandes volúmenes y/o superficies que requieren controles sistemáticos durante su desarrollo, a los fines de garantizar el cumplimiento de las propiedades de los materiales establecidos en la etapa de diseño. De esta forma, es necesario contar con ensayos de campo sencillos, confiables y eficientes que permitan identificar propiedades físicas, mecánicas e hidráulicas. Las geoestructuras generadas mediante la compactación del suelo próximo al sector de construcción pueden funcionar adecuadamente, con reducidos costos de material y transporte. Su estabilización puede ejecutarse en forma natural, o con la incorporación de agregados minerales como bentonita, cal o cemento. Estas incorporaciones mejoran las propiedades hidráulicas y mecánicas del material, optimizando el comportamiento requerido para la obra. Para establecer la forma en la que estos minerales modifican el comportamiento del suelo local compactado deben realizarse investigaciones especiales con los materiales involucrados. En el ámbito internacional existen numerosas investigaciones sobre comportamiento de suelos compactados, no obstante, si bien aportan antecedentes para la planificación de estudios locales, sus resultados no pueden trasladarse de manera directa. Las características propias del suelo local constituye la principal variable debido a la diversidad en las propiedades geotécnicas de cada Región. Esta investigación, se focaliza en el empleo de suelos limosos de la formación loéssica de la zona central de Argentina. Los suelos de la llanura cordobesa poseen comportamientos particulares, los cuales son contemplados en los diseños presentados como resutado de las investigaciones internacionales. Esta particularidad se relaciona con su inestabilidad, lo que los clasifica como suelos colapsables. Los resultados obtenidos en este trabajo podrán ser extendidos a una gran superficie de la Provincia de Córdoba y a la Región Pampeana en general, a los fines de establecer recomendaciones de diseño y construcción para la confección de Pliegos de Especificaciones Técnicas de diferentes tipos de obras públicas y privadas. El estudio contempla la ejecución de un plan experimental a escala de laboratorio y campo. Los materiales corresponden a suelo limosos puros, y diferentes agregados tales como bentonita, cal y cemento. Se planifican ensayos para evaluar el desempeño del material, a partir de la confección de muestras preparadas con diferentes condiciones de compactación (energía, humedad y método), y en forma de mezcla con los distintos tipos de agregados. Se realizarán ensayos de permeabilidad en celdas de pared rígida y flexible, junto a ensayos mecánicos de compresión confinada, simple y triaxial. Para el trabajo experimental de campo se prevé la ejecución de terraplenes de prueba instrumentados con tensiómetros e infiltrómetros para evaluar el comportamiento hidraúlico en el tiempo, junto con ensayos de penetración y plato de carga para la caracterización mecánica. En forma conjunta se propone el desarrollo de modelos numéricos de caracterización hidromecánica. Stabilized soils by compaction, produce materials technical and economic advantages in different types of engineering works. For example, road bases in highways, roads or city streets, containment barriers for sanitary landfill or stabilization ponds, foundation support of building, industrial flat, and other applications. Failures can result in environmental catastrophes, social, and economic loss, so it is important to optimize the design and increase the safety of such buildings. These works involve large surfaces that require systematic tests during construction, so it is necessary to have simple field tests, reliable and efficient to identify physical, mechanical and hydraulic properties. The geo-structures generated by local soil compaction have reduced material and transportation costs. Stabilization can be naturally, or with the addition of mineral aggregates as bentonite, lime and cement. These additions improve the hydraulic and mechanical properties of the material. So, special investigations should be conducted with the materials involved. There are many international studies on compacted soils behavior but their results can not be transferred directly due to the particularities of regional soils. For this research silty soils of central Argentina are the main focus. The soils of Córdoba plains are instability, so are classified as collapsible soils. The results obtained in this work may be extended to a large area of the Province of Cordoba and the Pampas region in general, in order to establish design and construction recommendations. The study includes laboratory and field tests. The materials are pure silty soil, and different aggregates such as bentonite, lime and cement. Tests are planned to evaluate the performance. Laboratory includes rigid and flexible wall cells, confined, triaxial and simple compression tests. For field experimental instrumented embankments will be constructed. A numerical hydromechanical model will be developed.

Obesity Preserves Myocardial Function During Blockade of the Glycolytic Pathway

Background: Obesity is defined by excessive accumulation of body fat relative to lean tissue. Studies during the last few years indicate that cardiac function in obese animals may be preserved, increased or diminished. Objective: Study the energy balance of the myocardium with the hypothesis that the increase in fatty acid oxidation and reduced glucose leads to cardiac dysfunction in obesity. Methods: 30-day-old male Wistar rats were fed standard and hypercaloric diet for 30 weeks. Cardiac function and morphology were assessed. In this paper was viewed the general characteristics and comorbities associated to obesity. The structure cardiac was determined by weights of the heart and left ventricle (LV). Myocardial function was evaluated by studying isolated papillary muscles from the LV, under the baseline condition and after inotropic and lusitropic maneuvers: myocardial stiffness; postrest contraction; increase in extracellular Ca2+ concentration; change in heart rate and inhibitor of glycolytic pathway. Results: Compared with control group, the obese rats had increased body fat and co-morbities associated with obesity. Functional assessment after blocking iodoacetate shows no difference in the linear regression of DT, however, the RT showed a statistically significant difference in behavior between the control and the obese group, most notable being the slope in group C. Conclusion: The energy imbalance on obesity did not cause cardiac dysfunction. On the contrary, the prioritization of fatty acids utilization provides protection to cardiac muscle during the inhibition of glycolysis, suggesting that this pathway is fewer used by obese cardiac muscle.

Development of Anatomophysiologic Knowledge Regarding the Cardiovascular System: From Egyptians to Harvey

Our knowledge regarding the anatomophysiology of the cardiovascular system (CVS) has progressed since the fourth millennium BC. In Egypt (3500 BC), it was believed that a set of channels are interconnected to the heart, transporting air, urine, air, blood, and the soul. One thousand years later, the heart was established as the center of the CVS by the Hippocratic Corpus in the medical school of Kos, and some of the CVS anatomical characteristics were defined. The CVS was known to transport blood via the right ventricle through veins and the pneuma via the left ventricle through arteries. Two hundred years later, in Alexandria, following the development of human anatomical dissection, Herophilus discovered that arteries were 6 times thicker than veins, and Erasistratus described the semilunar valves, emphasizing that arteries were filled with blood when ventricles were empty. Further, 200 years later, Galen demonstrated that arteries contained blood and not air. With the decline of the Roman Empire, Greco-Roman medical knowledge about the CVS was preserved in Persia, and later in Islam where, Ibn Nafis inaccurately described pulmonary circulation. The resurgence of dissection of the human body in Europe in the 14th century was associated with the revival of the knowledge pertaining to the CVS. The main findings were the description of pulmonary circulation by Servetus, the anatomical discoveries of Vesalius, the demonstration of pulmonary circulation by Colombo, and the discovery of valves in veins by Fabricius. Following these developments, Harvey described blood circulation.

Estudos sôbre a alimentação mineral da mandioca (Manihot utilissima Pohl.)

1. The present work was carried out to study the effects of mineral nutrients in the yield as well as in the composition of cassava roots. The variety "Branca de Sta. Catarina" was grown by the sand culture method, the following treatments being used: N0 P0 K0, N0 P1 Kl, N1 P0 K1, N2 P1 K0, N2 P1 K1, N1 P2 K1, and N1 P1 K2, where the figures 0, 1, and 2 denote the relative proportion of a given element. The nutrients were given as follows: N = 35 grams of ammonium nitrate per pot loaded with 120 pounds of washed sand; P1 = 35 grams of monocalcium phosphate; Kl = 28 grams of sulfate of potash. Besides those fertilizers, each pot received 26 grams of magnesium sulfate and weekly doses of micronutrients as indicated by HOAGLAND and ARNON (1939). To apply the macronutrients the total doses were divided in three parts evenly distributed during the life cycle of cassava. 2. As far yield of roots and foliage are concerned, there are a few points to be considered: 2.1. the most striking effect on yield was verified when P was omitted from the fertilization; this treatment gave the poorest yields of the whole experiment; the need of that element for the phosphorylation of the starchy reserves explains such result; 2.2. phosphorus and nitrogen, under the experimental conditions, showed to be the most important nutrients for cassava; the effect of potassium in the weight of the roots produced was much less marked; it is noteworthy to mention, that in absence of potassium, the roots yield decreased whereas the foliage increased; as potassium is essential for the translocation of carbohydrates it is reasonable to admit that sugars produced in the leaves instead of going down and accumulate as starch in the roots were consumed in the production of more green matter. 3. Chemical analyses of roots revealed the following interesting points: 3.1. the lack of phosphorus brought about the most drastic reduction in the starch content of the roots; while the treatment N1 P1 K1 gave 32 per cent of starch, with NI PO Kl the amount found was 25 per cent; this result can be explained by the requirement of P for the enzymatic synthesis of starch; it has to be mentioned that the decrease in the starch content was associated with the remarkable drop in yield observed when P was omitted from the nutrient medium; 3.2. the double dosis of nitrogen in the treatment N2 P1 K1, gave the highest yields; however the increase in yield did not produce any industrial gain: whereas the treatment N1 P1 K1 gave 32 per cent of starch, by raising the N level to N2, the starch content fell to 24 per cent; now, considering the total amount of starch present in the roots, one can see, that the increase in roots yield did not compensate for the marked decrease in the starch content; that is, the amount of starch obtained with N1 P1 K1 does not differ statistically from the quantity obtained with N2 P1 K1; as far we know facts similar to this had been observed in sugar beets and sugar cane, as a result of the interaction between nitrogen and sugar produced; the biochemical aspect of the problem is very interesting: by raising the amount of assimilable nitrogen, instead of the carbohydrates polymerize to starch, they do combine to the amino groups to give proteinaceous materials; actually, it did happen that the protein content increased from 2.91 to 5.14 per cent.

Fertilizer policy in the developing countries: the case of Brazil

This paper deals initially with the role of mineral fertilizers in increasing agricultural production: the relationship between the two variables is illustrated within global, regional national and local contexts. The pattern and trends in fertilizer usage in Brazil are presented next, namely: increase in consumption in the period 1950/72; regional distribution; consumption as related to crops and cultivated land. It is shown that in less than a quarter or century fertilizer use has increased in the country nearly 12 fold, whereas world consumption was raised 7 fold, thus exceeding estimates based in several criteria. Steps taken to secure the raise in fertilizer consumption above the historical trend are discussed: research experience for outlining fertilization recomendations; the transfer of the knowledge to the farmer by the extension work both official and private; the credit policy and special incentives for the purchase of fertilizer; the national policy for minumum proces of agricultural products; the implantation of a national fertilizer industry. It is considered that the Brazilian experience adapted to similar local conditions in other developing countries, presents a possibility for achieving beneficial results without inflationary reflexes in the economy.

Leishmaniasis in Bolivia: II. The involvement of Psychodopygus yucumensis and Psychodopygus llanosmartinsi in the selvatic transmission cycle of Leishmania braziliensis braziliensis in a lowland subandean region

An epidemiological survey of the vectors of cutaneous leishmaniasis ("espúndia" type) was caried out in the Alto Beni region of Bolivia, an area of Andean foothills at the Eastern limit of the Amazonian lowlands. The climate is typical wet tropical (15ºS latitude). Anthropophilic phlebotomine sandfly species were sampled at 20 sites, all forested. The importance of species from the Psychodopygus group, already suspected as a vector in the transmission of Leishmania from the braziliensis complex, was confirmed by: 1) the aggressiveness and diversity of the species encountered (83% of catches, nine species), 2) the discovery of a new anthropophilic species, P. yucumensis and 3) the isolation of a strain of Leishmania braziliensis braziliensis indistinguishable from human strains from the same area, from two species, P. llanosmartinsi and P. yucumensis.

Estudis funcionals i de plegament dels dominis d'activació de les procarboxipeptidases

Els dominis d’activació (ADs) de les procarboxipeptidases de la subfamília A/B sempre han sorprès ja que representen una quarta part del proenzim. S’han realitzat alguns estudis per intentar descobrir-ne alguna possible funció alternativa, però no han estat fructífers. El descobriment de l’elevada velocitat de plegament del domini d’activació de la procarboxipeptidasa A2 humana, (ADA2h), emperò, va portar a proposar la possibilitat de que realitzessin una funció d’assistència al plegament del domini enzimàtic. Posteriorment, l’anàlisi del plegament d’ADA2h a pH baix va revelar la capacitat d’aquest domini per formar fibres amiloides, a més de demostrar que un increment de l’estabilitat proteica podia prevenir la formació d’aquests agregats. La profunda caracterització del plegament d’ADA2h va fer que aquesta proteïna fos un bon model amiloidogènic, de manera que es van proposar un seguit d’experiments que s’han desenvolupat en el present treball per tal de conèixer millor aquest procés. S’han dut a terme estudis cinètics d’agregació per tal de valorar la contribució dels diferents aminoàcids de la seqüència polipeptídica, utilitzant 29 variants puntuals d’ADA2h. Es va eliminar la contribució de l’estabilitat mitjançant la utilització d’urea, i per dicroïsme circular conjuntament amb un aparell de flux detingut, es van obtenir dues velocitats diferents, v1 i v2, que corresponen a la formació d’un intermediari i a la seva reorganització, respectivament. Experiments complementaris utilitzant espectroscòpia d’infraroig (IR) revelaren la reorganització de l’estat natiu (en aquest cas) per a donar la forma agregada. Les cinètiques d’IR van mostrar que ADA2h forma l’estructura _ típica de les fibres amiloides, previ desplegament les seves hèlixs-_. Finalment, s’han realitzat estudis de biocomputació per tal d’esbrinar possibles funcions alternatives dels ADs. Les superposicions estructurals semblen mostrar similaritat dels ADs amb dominis de reconeixement d’RNA (RRM). Aquesta hipòtesi s’ha comprovat experimentalment amb ADA4h, mostrant una dèbil, però existent, unió a RNA.

Molecular variation at a candidate gene implicated in the regulation of fire ant social behavior

The fire ant Solenopsis invicta and its close relatives display an important social polymorphism involving differences in colony queen number. Colonies are headed by either a single reproductive queen (monogyne form) or multiple queens (polygyne form). This variation in social organization is associated with variation at the gene Gp-9, with monogyne colonies harboring only B-like allelic variants and polygyne colonies always containing b-like variants as well. We describe naturally occurring variation at Gp-9 in fire ants based on 185 full-length sequences, 136 of which were obtained from S. invicta collected over much of its native range. While there is little overall differentiation between most of the numerous alleles observed, a surprising amount is found in the coding regions of the gene, with such substitutions usually causing amino acid replacements. This elevated coding-region variation may result from a lack of negative selection acting to constrain amino acid replacements over much of the protein, different mutation rates or biases in coding and non-coding sequences, negative selection acting with greater strength on non-coding than coding regions, and/or positive selection acting on the protein. Formal selection analyses provide evidence that the latter force played an important role in the basal b-like lineages coincident with the emergence of polygyny. While our data set reveals considerable paraphyly and polyphyly of S. invicta sequences with respect to those of other fire ant species, the b-like alleles of the socially polymorphic species are monophyletic. An expanded analysis of colonies containing alleles of this clade confirmed the invariant link between their presence and expression of polygyny. Finally, our discovery of several unique alleles bearing various combinations of b-like and B-like codons allows us to conclude that no single b-like residue is completely predictive of polygyne behavior and, thus, potentially causally involved in its expression. Rather, all three typical b-like residues appear to be necessary.

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Mass spectrometry for the evaluation of cardiovascular diseases based on proteomics and lipidomics.

The identification and quantification of proteins and lipids is of major importance for the diagnosis, prognosis and understanding of the molecular mechanisms involved in disease development. Owing to its selectivity and sensitivity, mass spectrometry has become a key technique in analytical platforms for proteomic and lipidomic investigations. Using this technique, many strategies have been developed based on unbiased or targeted approaches to highlight or monitor molecules of interest from biomatrices. Although these approaches have largely been employed in cancer research, this type of investigation has been met by a growing interest in the field of cardiovascular disorders, potentially leading to the discovery of novel biomarkers and the development of new therapies. In this paper, we will review the different mass spectrometry-based proteomic and lipidomic strategies applied in cardiovascular diseases, especially atherosclerosis. Particular attention will be given to recent developments and the role of bioinformatics in data treatment. This review will be of broad interest to the medical community by providing a tutorial of how mass spectrometric strategies can support clinical trials.

Methodology in structural determination and synthesis of insect pheromone

By means of ethereal washing of insect pheromone glands of female moths, GC-MS detection along with microchemical reactions and electroantennogram (EAG) survey, six economically important insect species were targeted for pheromone identification. The discovery of a natural pheromone inhibitor, chemo-selectivity and species isolation by pheromone will be described. The modified triple bond migration and triethylamine liganded vinyl cuprate were applied for achiral pheromone synthesis in double bond formation. Some optically active pheromones and their stereoisomers were synthesized through chiral pool or asymmetric synthesis. Some examples of chiral recognition of insects towards their chiral pheromones will be discussed. A CaH2 and silica gel catalyzed Sharpless Expoxidation Reaction was found in shortening the reaction time.

Control of the adaptive response of the heart to stress via the Notch1 receptor pathway.

In the damaged heart, cardiac adaptation relies primarily on cardiomyocyte hypertrophy. The recent discovery of cardiac stem cells in the postnatal heart, however, suggests that these cells could participate in the response to stress via their capacity to regenerate cardiac tissues. Using models of cardiac hypertrophy and failure, we demonstrate that components of the Notch pathway are up-regulated in the hypertrophic heart. The Notch pathway is an evolutionarily conserved cell-to-cell communication system, which is crucial in many developmental processes. Notch also plays key roles in the regenerative capacity of self-renewing organs. In the heart, Notch1 signaling takes place in cardiomyocytes and in mesenchymal cardiac precursors and is activated secondary to stimulated Jagged1 expression on the surface of cardiomyocytes. Using mice lacking Notch1 expression specifically in the heart, we show that the Notch1 pathway controls pathophysiological cardiac remodeling. In the absence of Notch1, cardiac hypertrophy is exacerbated, fibrosis develops, function is altered, and the mortality rate increases. Therefore, in cardiomyocytes, Notch controls maturation, limits the extent of the hypertrophic response, and may thereby contribute to cell survival. In cardiac precursors, Notch prevents cardiogenic differentiation, favors proliferation, and may facilitate the expansion of a transient amplifying cell compartment.