Membrane interaction of a new synthetic antimicrobial lipopetide sp-85 with broad spectrum activity

Antimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able todevelop genetic resistance, since their main activity is in the lipid component of the bacterial cell mem-brane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin,and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at theN-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibac-terial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane ofbacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog withmembrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared withPOPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respec-tively. LangmuirBlodgett films have been extracted on glass plates and observed by confocal microscopy,and images are consistent with a strong destabilizing effect on the membrane organization induced bysp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same com-position, where biophysical experiments based on fluorescence are indicative of membrane fusion andpermeabilization starting at very low concentrations of peptide and only if anionic lipids are present.Overall, results described here provide strong evidence that the mode of action of sp-85 is the alterationof the bacterial membrane permeability barrier.

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis

Most of the Abundant Protein Fractions of Embryonic Cerebrospinal Fluid are Produced Out of the Brain Anlagen

The microenvironment of the central nervous system is important for neuronal function and development. During the early stages of embryo development the cephalic vesicles are filled by embryonic cerebrospinal fluid, a complex fluid containing different protein fractions, which contributes to the regulation of the survival, proliferation and neurogenesis of neuroectodermal stem cells. The protein content of embryonic cerebrospinal fluid from chick and rat embryos at the start of neurogenesis has already been determined. Most of the identified gene products are thought to be involved in the regulation of developmental processes during embryogenesis. However, due to the crucial roles played by embryonic cerebrospinal fluid during brain development, the embryological origin of the gene products it contains remains an intriguing question. According to the literature most of these products are synthesised in embryonic tissues other than the neuroepithelium. In this study we examined the embryological origin of the most abundant embryonic cerebrospinal fluid protein fractions by means of slot-blot analysis and by using several different embryonic and extraembryonic protein extracts, immunodetected with polyclonal antibodies. This first attempt to elucidate their origin is not based on the proteins identified by proteomic methods, but rather on crude protein fractions detected by SDS-PAGE analysis and to which polyclonal antibodies were specifically generated. Despite some of the limitations of this study, i.e. that one protein fraction may contain more than one gene product, and that a specific gene product may be contained in different protein fractions depending on post-translational modifications, our results show that most of the analysed protein fractions are not produced by the cephalic neuroectoderm but are rather stored in the egg reservoir; furthermore, few are produced by embryo tissues, thus indicating that they must be transported from their production or storage sites to the cephalic cavities, most probably via embryonic serum. These results raise the question as to whether the transfer of proteins from these two embryo compartments is regulated at this early developmental stage.

Façamos Químicos: a "certidão de nascimento" dos cursos de química de nível superior no Brasil

This work presents a brief retrospective of José de Freitas Machado's prominent role in the creation, development and consolidation of Chemistry undergraduate courses in Brazil. Freitas Machado defended in many occasions the importance of chemical studies for the economic development of this country. We analyze, here, his important paper "Façamos Químicos" [Let's Make Chemists] (1917), seminal for the implantation of Industrial Chemistry undergraduate courses in Brazil.

Caracterización de cristales líquidos por microscopía óptica en sistemas surfactante polietoxilado-alcano-agua

The phase behavior of an alcohol polyethoxylated surfactant with decane and dodecane oil phase varying the water proportion from 5 to 90% to determine compositions in which the formation of liquid crystals and microemulsions ocurred was investigated. Pseudoternary phase diagrams were built to represent the regions of liquid crystals, biphases and microemulsions. Polarized light optical microscopy was used for the analysis and characterization of the separate phases. The micrographs obtained showed characteristics of hexagonal and lamellar phases of liquid crystal, isotropic phases, microemulsions and vesicles. This study is important to propose hypothesis regarding the factors determining the formation and stability of phases composed by surfactant/oil/water systems.

Cytopathology of callus cells infected with grapevine leafroll-associated virus 3

The cytopathology of grapevine (Vitis spp.) callus tissue infected with Grapevine leafroll-associated virus 3 (GLRaV-3), genus Vitivirus was studied in order to investigate the usefulness of callus cultures to study grapevine leafroll-associated viruses. Ultrathin sections were made from in vitro callus obtained from stems and shoots of GLRaV-3 infected grapevine plants. Callus was composed of two types of tissue. Translucent, soft callus was formed and composed of large loosely arranged cells, containing big vacuoles and a thin layer of cytoplasm. Other parts of the callus were brown-coloured and composed of small compactly arranged cells, which showed flexuous and rod-shaped closterovirus-like particles, with 10-12 nm in diameter, at higher magnifications. Groups of vesicles formed by a single membrane were also observed, with sizes ranging from 50-200 nm, containing fine fibrillar material, also typical of closterovirus infections. Virus concentration was monitored by Immunosorbent electron microscopy (ISEM) tests, which showed that in vitro culture of callus tissue from grapevine infected plants, could be used to study the GLRaV viruses through many successive generations, despite the decline in virus concentration after repeated transfers. No virus particles were observed in callus tissue obtained from healthy grapevines.

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

Cole latent virus (CoLV), genus Carlavirus, was studied by electron microscopy and biochemical approaches with respect both to the ultrastructure of the Chenopodium quinoa infected cells and to its association with chloroplasts. The CoLV was observed to be present as scattered particles interspersed with membranous vesicles and ribosomes or as dense masses of virus particles. These virus particles reacted by immunolabelling with a polyclonal antibody to CoLV. Morphologically, chloroplasts, mitochondria and nuclei appeared to be unaltered by virus infection and virus particles were not detected in these organelles. However, virus particle aggregates were frequently associated with the outer membrane of chloroplasts and occasionally with peroxisomes. Chloroplasts were purified by Percoll gradient, and the coat protein and virus-associated RNAs were extracted and analyzed by Western and Northern blots respectively. Coat protein and CoLV-associated RNAs were not detected within this organelle. The results presented in this work indicate that the association CoLV/chloroplasts, observed in the ultrastructural studies, might be a casual event in the host cell, and that the virus does not replicate inside the organelle.

The Role of Integrins in Enterovirus Infections and in Metastasis of Cancer

Integrins are a family of transmembrane glycoproteins, composed of two different subunits (alpha and beta). Altered expression of integrins in tumor cells contributes to metastasis tendency by influencing on the cells‟ attachment to adjacent cells and their migration. Viral pathogens, including certain enteroviruses, use integrins as receptors. Enteroviruses have also been suggested to be involved in the etiopathogenesis of type 1 diabetes. The study focuses on the role of integrins in the pathogenesis of metastasis to cortical bone and on type 1 diabetes (T1D) and echovirus 1 infection. In the first part of the thesis, the role of different integrins in the initial attachment of MDA-MD-231 breast cancer cells to bovine cortical bone disks was studied. A close correlation between alpha2beta1 and alpha3beta1 integrin receptor expression and the capability of the tumor to attach to bone were observed. In the second part, a possible correlation between susceptibility to enterovirus infections in diabetic children and differences in enterovirus receptor genes, including certain integrins, was investigated. In parallel, virus-specific neutralizing antibodies and diabetic risk alleles were studied. In the diabetic group, an amino acid change was detected in the polio virus receptor and the neutralizing antibody titers against echovirus 30 were lower. However, to obtain statistically sustainable results, a larger number of individuals should be analyzed. Echovirus 1 (EV1) enters cells by attaching to the alpha2I domain of the alpha2beta1 integrin. In the third part EV1 was shown to attach to a chimeric receptor construct of the transferrin receptor and the alpha2I domain and to enter cells through clathrin-mediated endocytosis that is normally not used by the virus. The chimeric receptor was recycled to the plasma membrane, whereas the virus remained in intracellular vesicles. The virus replication cycle was initiated in these cells, suggesting that evolution pressure could possibly cause the virus to evolve to use a different entry mechanism. Moreover, a cDNA microarray analysis of host gene expression during EV1 replication showed that 0.53% of the total genes, including several immediate early genes, were differently expressed.

Propagação vegetativa de cedro-rosa por miniestaquia

O objetivo deste trabalho foi avaliar a técnica de miniestaquia como método de propagação vegetativa de cedro-rosa (Cedrela fissilis), quanto à produção e sobrevivência das minicepas nas sucessivas coletas e quanto ao porcentual de enraizamento e do crescimento em altura e diâmetro do colo das miniestacas. As minicepas foram obtidas a partir de mudas de sementes de cedro-rosa, das quais promoveram-se as coletas sucessivas de miniestacas, sendo essas submetidas a diferentes dosagens do regulador de crescimento AIB para enraizamento. Os resultados demonstraram a eficiência da técnica na propagação vegetativa desta espécie, atingindo-se até 79% de sobrevivência aos 120 dias de idade das mudas, devendo-se destacar que a não-aplicação do AIB proporcionou melhores resultados. Em termos gerais, a miniestaquia de cedro-rosa, a partir de material de origem seminal, é tecnicamente viável, tornando-se uma alternativa para produção de mudas dessa espécie durante todo o ano, principalmente nas situações em que a semente é insumo limitante.

Enraizamento de miniestaca caulinar e foliar na propagação vegetativa de cedro-rosa (Cedrela fissilis Vell.)

No presente trabalho objetivou-se avaliar o enraizamento de cinco diferentes tipos de miniestaca (caulinar, caulinar apical, caulinar intermediária, caulinar apical desfolhada e foliar), na propagação vegetativa de cedro-rosa (Cedrela fissilis) por miniestaquia, a partir de material seminal. Os resultados obtidos quanto ao enraizamento indicaram o melhor desempenho da miniestaca caulinar, com 84% de sobrevivência das mudas aos 90 dias de idade, demonstrando o potencial da miniestaquia como alternativa na produção de mudas de cedro-rosa.

Propagação vegetativa de cedo-australiano (Toona ciliata M. Roemer) por miniestaquia

O método de propagação usual do cedro-australiano (Toona ciliata) é via seminal, entretanto a oferta sazonal das sementes e sua curta viabilidade ao longo do tempo representam um problema para a produção contínua de mudas destinadas à implantação de povoamentos. Este trabalho foi conduzido com o objetivo de avaliar a viabilidade da propagação vegetativa da espécie por miniestaquia e a necessidade da aplicação de acido indolbutírico (AIB) para o enraizamento das miniestacas. A partir de um banco de estacas de origem seminal, foram obtidas brotações para produção de mudas clonais, em três diferentes épocas de coleta (2,5; 4,5; e 5,5 meses após a recepa das mudas). Antes do estaqueamento, as miniestacas tiveram suas bases imersas em quatro concentrações de AIB (0; 1.500; 3.000; e 4.500 mg L-1). Durante o experimento, obtiveram-se 100% de sobrevivência das minicepas e das miniestacas. Houve 100% de enraizamento das miniestacas nas três coletas, não ocorrendo diferença no comprimento de raízes em função das doses de auxina aplicadas. Quanto maior o intervalo entre as coletas e quanto maiores as brotações que originaram as miniestacas, maior a velocidade de crescimento das mudas. Miniestacas de cedro-australiano possuem capacidade de enraizamento, e mudas recepadas apresentam brotação, possibilitando a clonagem da espécie pelo processo de miniestaquia.

Propagação vegetativa do jequitibá-rosa (Cariniana estrellensis (Raddi) Kuntze) por estaquia

O presente trabalho teve como objetivo desenvolver uma metodologia para a propagação vegetativa do jequitibá-rosa (Cariniana estrellensis (Raddi) Kuntze), por meio da técnica de estaquia, avaliando-se a sobrevivência e capacidade produtiva das cepas em coletas sucessivas de estacas em jardim clonal. A sobrevivência, o enraizamento, a altura, o vigor e a biomassa radicular e foliar das estacas, em razão da aplicação de diferentes dosagens do regulador de crescimento ácido indolbutírico (AIB) e do tipo de estaca utilizado. O jardim clonal foi constituído de plantas oriundas a partir de mudas de material seminal, com uma densidade de nove plantas por m², estabelecidas em solo. Foram feitas avaliações quanto ao enraizamento das estacas em dois períodos de tempo diferentes do ano, em casa de vegetação (aos 120 dias), em casa de sombra (aos 140 dias) e em pleno sol (aos 170 dias) após o estaqueamento. A aplicação do AIB não teve efeito na maioria das características avaliadas. No entanto, quanto ao tipo de estaca, as apicais foram as que apresentaram maiores valores para as características estudadas. A sobrevivência das cepas foi de 100% e a produção de brotações mostrou tendência crescente nas coletas sucessivas. Conclui-se que a propagação vegetativa do jequitibá-rosa pela técnica de estaquia é viável, principalmente quando se utilizam estacas apicais, e a aplicação de AIB não mostrou efeitos destacados que indiquem a sua utilização na propagação do jequitibá-rosa pela estaquia.

Caracterização morfológica do ramo, sementes e plântulas de matayba guianensis Aubl. e produção de mudas em diferentes recipientes e substratos

Matayba guianensis Aubl. é uma Sapindaceae de porte arbustivo ou arbóreo de grande ocorrência no Cerrado brasileiro, com papel fundamental no fornecimento de recursos para formigas e abelhas. Apresenta rápido crescimento e é importante para recuperação de áreas degradadas, mas pouco se conhece sobre a sua produção de mudas. Diante disso, o objetivo deste estudo foi descrever a morfologia do ramo e da germinação de sementes e plântulas de Matayba guianensis Aubl. em seu desenvolvimento pós-seminal, bem como definir o tipo de substrato e recipiente adequados para emergência de plântulas e produção de mudas desta espécie. Foram avaliados três tipos de recipientes: saco de polietileno preto; tubete e bandeja de isopor, com cinco tipos de substratos: areia; terra; terra-areia-esterco (1:1:1); substrato comercial e fibra de coco, com quatro repetições de 25 sementes em cada tratamento. O ramo, fruto, semente, plântulas e seus eventos morfológicos foram descritos. O ramo é cilíndrico, com folhas paripinadas e alternas; fruto seco, deiscente e sementes com grande quantidade de arilo; a germinação é criptocotiledonar e hipógea. Sua maior porcentagem de emergência ocorreu no recipiente isopor com 91% das sementes germinadas em substrato comercial, seguida de fibra de coco (88%). O desenvolvimento da raiz e do caule foi maior em tubete e em saco plástico, utilizando-se substrato comercial ou fibra de coco.

SAZONALIDADE E SOLUÇÕES NUTRITIVAS NA MINIESTAQUIA DE Araucaria angustifolia (Bertol.) Kuntze

Objetivou-se avaliar o efeito da sazonalidade e de soluções nutritivas na produção, sobrevivência, enraizamento e vigor radicial de miniestacas juvenis de Araucaria angustifolia, bem como o hábito de crescimento das mudas formadas. As minicepas foram manejadas em minijardim sob sistema semi-hidropônico, em que foram aplicadas duas soluções nutritivas, com diferentes concentrações de nutrientes, fornecidas por gotejamento, durante as quatro estações do ano. Após 11 coletas, as minicepas apresentaram 100% de sobrevivência. A maior produção ocorreu no verão, com 1.356 miniestacas.m-2.ano-1, e a menor no inverno, com 429 miniestacas.m-2.ano-1. As coletas de inverno apresentaram os melhores resultados de enraizamento, com média de 83% em casa de sombra, contra 31% das demais estações. O maior vigor radicial ocorreu nas coletas de primavera e verão e o menor, no inverno. A solução nutritiva mais concentrada propiciou maior produção de miniestacas e melhor vigor radicial, e todas as mudas resultantes da miniestaquia apresentaram hábito ortotrópico de crescimento. A técnica de miniestaquia com propágulos vegetativos de origem seminal mostrou-se potencial para a produção de mudas de araucária, sendo significativamente influenciada pela época do ano e pelas soluções nutritivas empregadas.

O estado da arte da cirurgia do baço, no início do século XXI

In the last fifty years evolution of scientific knowledge on the spleen provoked an entirely new approach to splenic surgery. It was shown that virulence may emerge as a significant consequence of environmental and evolutionary changes of some microbial communities, and devastating pathogenetic results of these changes become visible in human hosts without the splenic function. In other words: the spleen plays a pivotal role in the dynamic balance between biodiversity, microorganisms and immunogenecity in human hosts. Therefore, to preserve the "splenic immunologic repertoire" became an increasing commitment among surgeons. Understanding the integration of these multiple information on spleen, seems central to understand the new splenic surgery. Partial splenectomies (Réglées) - based on anatomical, experimental and clinical studies, developed at the University of Minas Gerais since the fifties - were successfully applied initially to treat the traumatic injuries of the spleen; in a following step, partial splenectomy were used to control hematological diseases. "Réglées" techniques on the spleen have conquered "ethical support, consilience status and clinical governance" to give birth to surgical therapeutic decisions on the spleen, in order to spare the structural integrity of the immune system. Splenic réglées procedures became a seminal achievement of splenic surgical practice. Initial results of "Partial splenectomies" - with conventional surgical armamentaria and techniques - were confirmed and improved by the introduction of techniques based on laparoscopic and endovascular approaches. And current usage of surgical splenic saving procedures propitiated the emergence of an appropriate lexicon for medical communication and became an "end point" of a "long-standing surgical inhibition" over the spleen.