I. Studies on bacteriophage DNA's and minicircular DNA's in M. lysodeikticus and E. coli 15. II. Flow dichroism of DNA solutions

Part I

Chapter 1.....A physicochemical study of the DNA molecules from the three bacteriophages, N1, N5, and N6, which infect the bacterium, M. lysodeikticus, has been made. The molecular weights, as measured by both electron microscopy and sedimentation velocity, are 23 x 10⁶ for N5 DNA and 31 x 10⁶ for N1 and N6 DNA's. All three DNA's are capable of thermally reversible cyclization. N1 and N6 DNA's have identical or very similar base sequences as judged by membrane filter hybridization and by electron microscope heteroduplex studies. They have identical or similar cohesive ends. These results are in accord with the close biological relation between N1 and N6 phages. N5 DNA is not closely related to N1 or N6 DNA. The denaturation T_m of all three DNA's is the same and corresponds to a (GC) content of 70%. However, the buoyant densities in CsCl of Nl and N6 DNA's are lower than expected, corresponding to predicted GC contents of 64 and 67%. The buoyant densities in Cs₂SO₄ are also somewhat anomalous. The buoyant density anomalies are probably due to the presence of odd bases. However, direct base composition analysis of N1 DNA by anion exchange chromatography confirms a GC content of 70%, and, in the elution system used, no peaks due to odd bases are present.

Chapter 2.....A covalently closed circular DNA form has been observed as an intracellular form during both productive and abortive infection processes in M. lysodeikticus. This species has been isolated by the method of CsC1-ethidium bromide centrifugation and examined with an electron microscope.

Chapter 3.....A minute circular DNA has been discovered as a homogeneous population in M. lysodeikticus. Its length and molecular weight as determined by electron microscopy are 0.445 μ and 0.88 x 10⁶ daltons respectively. There is about one minicircle per bacterium.

Chapter 4.....Several strains of E. coli 15 harbor a prophage. Viral growth can be induced by exposing the host to mitomycin C or to uv irradiation. The coliphage 15 particles from E. coli 15 and E, coli 15 T^- appear as normal phage with head and tail structure; the particles from E. coli 15 TAU are tailless. The complete particles exert a colicinogenic activity on E.coli 15 and 15 T^-, the tailless particles do not. No host for a productive viral infection has been found and the phage may be defective. The properties of the DNA of the virus have been studied, mainly by electron microscopy. After induction but before lysis, a closed circular DNA with a contour length of about 11.9 μ is found in the bacterium; the mature phage DNA is a linear duplex and 7.5% longer than the intracellular circular form. This suggests the hypothesis that the mature phage DNA is terminally repetitious and circularly permuted. The hypothesis was confirmed by observing that denaturation and renaturation of the mature phage DNA produce circular duplexes with two single-stranded branches corresponding to the terminal repetition. The contour length of the mature phage DNA was measured relative to φX RFII DNA and λ DNA; the calculated molecular weight is 27 x 10⁶. The length of the single-stranded terminal repetition was compared to the length of φX 174 DNA under conditions where single-stranded DNA is seen in an extended form in electron micrographs. The length of the terminal repetition is found to be 7.4% of the length of the nonrepetitious part of the coliphage 15 DNA. The number of base pairs in the terminal repetition is variable in different molecules, with a fractional standard deviation of 0.18 of the average number in the terminal repetition. A new phenomenon termed "branch migration" has been discovered in renatured circular molecules; it results in forked branches, with two emerging single strands, at the position of the terminal repetition. The distribution of branch separations between the two terminal repetitions in the population of renatured circular molecules was studied. The observed distribution suggests that there is an excluded volume effect in the renaturation of a population of circularly permuted molecules such that strands with close beginning points preferentially renature with each other. This selective renaturation and the phenomenon of branch migration both affect the distribution of branch separations; the observed distribution does not contradict the hypothesis of a random distribution of beginning points around the chromosome.

Chapter 5....Some physicochemical studies on the minicircular DNA species in E. coli 15 (0.670 μ, 1.47 x 10⁶ daltons) have been made. Electron microscopic observations showed multimeric forms of the minicircle which amount to 5% of total DNA species and also showed presumably replicating forms of the minicircle. A renaturation kinetic study showed that the minicircle is a unique DNA species in its size and base sequence. A study on the minicircle replication has been made under condition in which host DNA synthesis is synchronized. Despite experimental uncertainties involved, it seems that the minicircle replication is random and the number of the minicircles increases continuously throughout a generation of the host, regardless of host DNA synchronization.

Part II

The flow dichroism of dilute DNA solutions (A₂₆₀≈0.1) has been studied in a Couette-type apparatus with the outer cylinder rotating and with the light path parallel to the cylinder axis. Shear gradients in the range of 5-160 sec.^-1 were studied. The DNA samples were whole, "half," and "quarter" molecules of T4 bacteriophage DNA, and linear and circular λb₂b₅c DNA. For the linear molecules, the fractional flow dichroism is a linear function of molecular weight. The dichroism for linear A DNA is about 1.8 that of the circular molecule. For a given DNA, the dichroism is an approximately linear function of shear gradient, but with a slight upward curvature at low values of G, and some trend toward saturation at larger values of G. The fractional dichroism increases as the supporting electrolyte concentration decreases.

Entre a negação e a possibilidade: um estudo discursivo da fala do professor sobre o seu trabalho

Esta pesquisa se propõe a analisar a fala de professores de Língua Portuguesa da rede municipal de ensino do Rio de Janeiro sobre o seu trabalho, inserido num período de mudanças para essa rede de ensino. Para tal, foi realizado um grupo de discussão (GD), um momento coletivo de fala dos professores sobre o seu trabalho, através do qual se verificou a expressiva incidência de enunciados negativos de caráter polêmico, segundo definição de Ducrot (1987). Assim, tais enunciados e seus respectivos pontos de vista subjacentes formaram o corpus da pesquisa. Buscou-se, a partir desse recorte, refletir sobre os discursos que circulam sobre o trabalho docente, relacionando-os aos conceitos de competências (SCHWARTZ, 1998), renormalizações (SCHWARTZ, 2002 e 2007) e trabalho impedido (CLOT, 2006) advindos das Ciências do Trabalho. No que se refere aos estudos das práticas discursivas, enfocou-se as contribuições de Bakhtin sobre dialogismo da linguagem (BAKHTIN, 1981 e 2003), a negação polêmica de Ducrot (1987) e o arcabouço teórico de Maingueneau (2001, 2002 e 2008) para os estudos enunciativos da Análise do Discurso. Os resultados nos possibilitaram apontar discursos circulantes subjacentes acerca do trabalho docente e refletir sobre tais construções de posições enunciativas que, em sua maioria, atribuem a responsabilidade pelos problemas no ensino ao professor. Além disso, identificamos contrastes entre os professores quanto à visão do ensino de Língua Portuguesa para o Ensino Fundamental

The impact of molecular biology on assessment of water quality: advantages and limitations of current techniques

The advent of molecular biology has had a dramatic impact on all aspects of biology, not least applied microbial ecology. Microbiological testing of water has traditionally depended largely on culture techniques. Growing understanding that only a small proportion of microbial species are culturable, and that many microorganisms may attain a viable but non-culturable state, has promoted the development of novel approaches to monitoring pathogens in the environment. This has been paralleled by an increased awareness of the surprising genetic diversity of natural microbial populations. By targeting gene sequences that are specific for particular microorganisms, for example genes that encode diagnostic enzymes, or species-specific domains of conserved genes such as 16S ribosomal RNA coding sequences (rrn genes), the problems of culture can be avoided. Technical developments, notably in the area of in vitro amplification of DNA using the polymerase chain reaction (PCR), now permit routine detection and identification of specific microorganisms, even when present in very low numbers. Although the techniques of molecular biology have provided some very powerful tools for environmental microbiology, it should not be forgotten that these have their own drawbacks and biases in sampling. For example, molecular techniques are dependent on efficient lysis and recovery of nucleic acids from both vegetative forms and spores of microbial species that may differ radically when growing in the laboratory compared with the natural environment. Furthermore, PCR amplification can introduce its own bias depending on the nature of the oligonucleotide primers utilised. However, despite these potential caveats, it seems likely that a molecular biological approach, particularly with its potential for automation, will provide the mainstay of diagnostic technology for the foreseeable future.

Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

Picophytoplankton in freshwater ecosystems: the importance of small-sized phototrophs

About 40 years have passed since the discovery of picophytoplankton; the present knowledge of the taxonomy, physiology and ecology of these tiny photoautotrophic cells offers new perspectives on the importance of the microbial contribution to global biogeochemical cycles and food webs. This review focuses on the relationships among the components of picophytoplankton (picocyanobacteria and the picoplanktic eukaryotes) and biotic and abiotic environmental factors. The dynamics of picophytoplankton in aquatic ecosystems are strictly dependent upon basin size and trophy, temperature, and nutrient and light limitation, but they are also regulated by grazing and viral-induced lysis. The review considers: the pros and cons of the molecular approach to the study of the taxonomy of freshwater Synechococcus spp.; the importance of ecological aspects in understanding the puzzle of picophytoplankton phylogeny (genotype vs ecotype); and the role of biotic vs abiotic interactions in controlling picophytoplankton dynamics. Biotic, top-down control mechanisms are reviewed as well as knowledge of other biological interactions.

Picophytoplankton in freshwater ecosystems: the importance of small-sized phototrophs

About 40 years have passed since the discovery of picophytoplankton; the present knowledge of the taxonomy, physiology and ecology of these tiny photoautotrophic cells offers new perspectives on the importance of the microbial contribution to global biogeochemical cycles and food webs. This review focuses on the relationships among the components of picophytoplankton (picocyanobacteria and the picoplanktic eukaryotes) and biotic and abiotic environmental factors. The dynamics of picophytoplankton in aquatic ecosystems are strictly dependent upon basin size and trophy, temperature, and nutrient and light limitation, but they are also regulated by grazing and viral-induced lysis. The review considers: the pros and cons of the molecular approach to the study of the taxonomy of freshwater Synechococcus spp.; the importance of ecological aspects in understanding the puzzle of picophytoplankton phylogeny (genotype vs ecotype); and the role of biotic vs abiotic interactions in controlling picophytoplankton dynamics. Biotic, top-down control mechanisms are reviewed as well as knowledge of other biological interactions.

Fibroblastos gengivais humanos em co-cultura com Aggregatibacter actinomycetemcomitans lisogênico induzem a liberação de fago

A relação entre bacteriófagos e virulência bacteriana é um modelo muito intrigante e pouco estudado na patogênese periodontal, uma vez que um patógeno periodontal pode ser lisogênico. O objetivo do nosso estudo é determinar a capacidade de fibroblastos gengivais humanos de induzir as cepas lisogênicas de Aggregatibacter actinomycetemcomitans. Dois experimentos foram realizados seguidos da titulação de fago. Os experimentos consistiram da co-cultura com fibroblastos gengivais humanos e três cepas de Aa [Aa29524, Aa2112, Aa29524(Ø2112)], não lisogênica, lisogênica e lisogênica induzida em laboratório, respectivamente. Em três momentos distintos (no experimento 1: 0, 2 e 4 horas; e no experimento 2: 2, 4 e 6 horas), o sobrenadante da co-cultura foi filtrado e cultivado overnight com a bactéria indicadora (Aa29524) e analisado para a capacidade de lisar a célula indicadora. Em ambos os experimentos, o sobrenadante da co-cultura de fibroblastos gengivais humanos com Aa lisogênico e Aa lisogênico induzido em laboratório, ao ser cultivado com a bactéria indicadora, promoveu lise da mesma, resultando no aumento da produção de fago. Pode-se concluir que, nesse estudo os fibroblastos gengivais humanos foram capazes de induzir cepas lisogênicas de Aggregatibacter actinomycetemcomitans.

Iventividade, atividade de trabalho e transtornos mentais graves: um ensaio no campo da cognição

Esta dissertação consiste em uma investigação acerca do conceito de atividade dirigida, pertencente à Clínica da Atividade e dos Meios de Trabalho, proposta por Y. Clot e D. Faïta, no sentido de verificar a sua pertinência para uma Psicologia da Inventividade. Trata-se da utilização da categoria de ritornelo para analisar os pressupostos teóricos do conceito de atividade dirigida, no que tange à cognição. A categoria ritornelo está relacionada a um conjunto de materiais apontados como relevantes para se pensar a questão da invenção: trata-se da Esquizoanálise, proposta por G. Deleuze & F. Guattari. A investigação foi realizada a partir de um caso clínico observado pelo pesquisador, que acompanhou um usuário de um Serviço de Saúde Mental, do município do Rio de Janeiro, Brasil, em seu trabalho como padeiro em uma cooperativa de culinária. Apesar de lidar bem com situações mais rotineiras, esse usuário demonstrava ter muita dificuldade em lidar com as variabilidades do meio, com situações que fugiam à sua rotina, apresentando um nível de ansiedade que o impedia de realizar quaisquer tarefas. Criou-se um projeto de atuação clínica que tinha como foco solicitar que ele explicitasse verbalmente o que o afligia, descrevendo todas as situações ansiogênicas, buscando, juntamente com esse paciente, encaminhamentos para tais situações. Ao final de seis meses, o paciente conseguia lidar com os imprevisíveis pertinentes à atividade, laborativa ou não. Foram realizadas duas análises deste caso clínico. Na primeira, utilizou-se a categoria de ritornelo e na segunda, o conceito de atividade dirigida. Finalmente, a segunda análise foi articulada à primeira, com o objetivo de investigar as possibilidades de sinergia entre uma e outra. Dentre as conclusões, observou-se que a construção de um dispositivo dialógico sobre as dificuldades de lidar com o não-rotineiro elemento inerente à vida, e à vida no trabalho pode ser terapeuticamente frutífero na medida em que sua atividade linguageira acerca das dificuldades vividas apresentou-se como objeto de co-análise, alimentando variações no processo de trabalho. Além disso, ritornelo e atividade dirigida articularam-se no nível dos processos de territorialização e desterritorialização propostos por Deleuze & Guattari. A atividade dirigida auxiliou no sentido de ser uma ferramenta relevante para encontrar outros modos de proceder, conectando distantes gêneros, de modo que o mais pleno faça crescer aquele que se encontra precário, atendendo ao 2 e 3 princípios do rizoma postulados por Deleuze & Guattari.

A experiência como fonte de normas: o trabalho de professores da EJA com alunos surdos

A leitura dos documentos oficiais que orientam a educação especial em nosso país Lei de Diretrizes e Bases da Educação Nacional, Plano Nacional de Educacão e Diretrizes para a Educação Especial na Educação Básica- aponta que o atendimento educacional daqueles que possuem necessidades educacionais especiais deve ser realizado, preferencialmente, na rede regular de ensino, tendo como norte os pressupostos da Inclusão, política pública dentro da qual é a escola que deve se adaptar ao aluno e não contrário. Levando em conta esse contexto educacional brasileiro, acrescido do déficit de pesquisas relacionadas à educação escolar de discentes Surdos, em uma perspectiva inclusiva e bilíngue, e de prescrições que orientem o trabalho do professor junto a esse tipo de alunato, o presente trabalho objetiva compreender o que o professor constrói, compartilha, como norma quando há um déficit de prescrições relacionadas à sua tarefa de trabalho. De maneira a alcançar tal objetivo, adotou-se, como dispositivo metodológico, a Instrução ao Sósia, procedimento de coleta de dados que objetiva fazer com que o trabalhador fale sobre experiência de trabalho, sobre aquilo que realmente faz, e não sobre o que deveria fazer, reportando-se, por exemplo, às prescrições (ODONE, 1982; VIEIRA, 2004). Para fundamentação teórica e análise do corpus, partimos, em primeiro lugar, de uma visão discursiva de linguagem, como propõem os estudos foucaultianos (2008); de Giacomoni & Vargas (2010); Narzeti (2010); Maingueneau (2005); Daher (2009). Consideramos, também, a forma como o sujeito institui-se no texto, recorrendo às contribuições teóricas de Koch (1993), Foucault (2008), Ribeiro (2006), Maingueneau (2005) e Koch & Vilela (2001), sobre modalidades discursivas. Em segundo lugar, adota-se uma concepção de trabalho em que ele não é considerado simples execução de tarefas, mas, um processo de (re)construção de normas, em que o trabalhador é convocado a agir, fazendo usos de si, como pressupõe o enfoque ergológico do trabalho (CLOT, 2006; SCHWARTZ, 2002, 2004, 2011; SANTANNA & SOUZA-E-SILVA, 2007; TRINQUET, 2010; TELLES & ALVAREZ, 2004; CUNHA, 2010). Dentro dessa concepção de trabalho, discute-se também, baseados em Mandarino (2006), Tardif (2002), Dias (2008) e Daniellou (2002), sobre o papel do professor e sua atuação, enquanto trabalhador, para a atuação de normas ascendentes. Por último, estabelece-se, a partir dos estudos de Nouroudine (2002), uma relação entre o fazer e o falar sobre o trabalho, ou seja, entre linguagem e trabalho, permitindo ao professor a utilização de recursos linguísticos para abordar o que (não) faz no seu trabalho. Os resultados da análise apontam uma relação entre linguagem e experiência de trabalho e mostram a dificuldade de construção de um coletivo de trabalho

Análise da citotoxicidade do extrato do fruto de juçara (Euterpe oleracea Mart.) do Maranhão em células malignas humanas

A juçara, Euterpe oleracea Mart., fruta indígena da Amazônia Legal, é rica em fitoquímicos com atividades anti-oxidante, antiinflamatória e anti-câncer. Este estudo tem por objetivo analisar os efeitos do extrato hidroalcoólico da casca, caroço e fruto total da juçara em diferentes linhagens de células malignas humana. Os frutos foram coletados no Parque da Juçara, localizado no Maracanã, município de São Luís, seguida da confecção da excicata que se mantém registrada no Herbário Rosa Mochel do Núcleo de Estudos Biológicos da Universidade Estadual do Maranhão. Os extratos hidroalcoólicos da casca, caroço e fruto total foram extraidos no Laboratório de Farmacologia e Psicobiologia da UERJ. As linhagens celulares utilizadas nos ensaios foram MCF-7 (adenocarcinoma de mama), CACO-2 e HT-20 (adenocarcinoma colo retal) e adenocarcinoma na mama (MDA-MB-468). As linhagens foram tratadas com 10, 20 e 40g/mL dos extratos por 24 e 48 horas e feitas às análises. Células MCF-7 controle apresentaram núcleo proeminente com nucléolos evidentes. Após tratamento com o extrato hidroalcoólico da casca da juçara, as células mostraram morfologia arredondada com retração do citoplasma. O ensaio de viabilidade com MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) demonstrou uma redução na viabilidade das células. Após 48 horas, o tratamento das células com 20g/mL do extrato da casca reduziu a viabilidade sendo que o efeito citotóxico do tratamento com 40g/mL do extrato da casca foi potencializado. Células tratadas com 10g/mL do extrato do caroço de juçara apresentavam-se arredondadas com consequente redução no volume celular. A concentração 20g/mL de extrato hidroalcoólico do caroço, causou severa redução no volume das células e ocasionou o surgimento de vacúolos intracelulares. O mesmo foi observado após tratamento com 40g/mL. O tratamento com 40g/mL do extrato hidroalcoólico do fruto total, modificou drasticamente a morfologia das células MCF-7 causando vacuolização e aparente lise com perda do conteúdo citoplasmático e o ensaio da viabilidade com MTT demonstrou redução na viabilidade das células MCF-7 tratadas com 20 e 40g/mL após 24 horas de tratamento. Análises por MET (Microscopia Eletrônica de Transmissão) demonstraram o surgimento de vesículas autofágicas, cuja comprovação deu-se com a identificação da expressão da proteína LC3BII na membrana do autofagossoma pela técnica de Western Blotting. Mediante o demonstrado pelos experimentos, com as linhagens MCF-7 e MDA-MB-468, confirma-se que as frações isoladas do extrato do caroço da juçara, promove modificações celulares indicativas de autofagia a partir de 10g/mL, em 24 horas. O núcleo permaneceu íntegro, não apresentando características de núcleo apoptótico. Os dados são conclusivos para ocorrência de morte celular por autofagia em linhagem celulares de carcinoma de mama MCF-7 quando tratadas com extrato hidroalcoólico da casca, caroço e fruto total da juçara do Maranhão, agente quimiopreventivo no câncer de mama estrogênio-dependente.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Effects of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation and characterization of a prothrombin activator

The action of Pallas' viper (Agkistrodon halys pallas) venom on blood coagulation was examined in vitro and a strong anticoagulant effect was observed. This action was abolished after treatment with a specific inhibitor of phospholipase A(2) activity (p-bromophenacyl bromide), revealing a procoagulant action in low concentrations of treated venom (around 1 mu g/ml). The effect of the venom an haemostasis was further characterized by measuring its ability to activate purified blood coagulation factors. It is concluded that A. halys pallas venom contains prothrombin activation activity. A prothrombin activator (aharin) was purified from the venom by Sephadex G-75 gel filtration and ion-exchange chromatography on a Mono-Q column. It consisted of a single polypeptide chain, with a mol. wt of 63,000. Purified aharin possessed no amidolytic activity on chromogenic substrates. It did not act on other blood coagulation factors, such as factor X and plasminogen, nor did it cleave or clot purified fibrinogen. The prothrombin activation activity of aharin was readily inhibited by ethylenediamine tetracetic acid (a metal chelator), but specific serine protease inhibitors such as diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride had no effect on it. These observations suggest that, like those prothrombin activators from Echis carinatus and Bothrops atrox venoms, the prothrombin activator from A. halys pallas venom is a metalloproteinase. (C) 1998 Elsevier Science Ltd. All rights reserved.

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

Study on the reproductive biology of Pinctada fucata (Gould) (Mollusca: Pteriidae)

Annual cycle of gonad development and spawning in pearl oyster, Pinctada ficata (Gould) in Nakhiloo, Northeast Persian Gulf, was investigated over two years from August 1994 to June 1996. Gonadal condition was assessed by staging criteria to describe gametogenic development from histological preparations of randomly collected individuals of all sizes. A bimodal gametogenic pattern with summer and autumn spawning periods was evident throughout the study. Gametogensis commenced in November-December which proceeded by major gonadal maturation during February-April. Summer spawning was observed from April to July with major spawning at the latter end. During spawning peak in July, low level of gametogensis was noticed. Gametogenic activity was picked up again in August-September which proceeded by autumn spawning from September to December. Towards the end of spawning season, incidence of gonadal inactivation increased. Minimum level of gonadal activity was observed in November. Temperature regime appears to have influential role in regulation of gametogenic and spawning processes. Gonadal development and spawning trends were similar in both sexes. P. radiaata was found to be protandrous hermaphrodite which matured as a male at shell height greater than 20 mm. Biseivality was uncommon and the sex ratio was about 1:1. Ultrastructure of gametes were investigated in the Pictada fucata (Gould). "Auxiliary cells" closely accociated with developing oocytes were observed. Each oocyte seems to be associated with only one secretory cell. which is characterized by an abundant rough endoplasmic reticulum at the onset of vitellogenesis. Contact between this cell and a developing oocytes is maintained by a desmosome-like junction which can be observed when the vitelline coat is formed. these "auxiliary or nursing cells" seem to play a tropic role in vitellogenesis, and may be involved in the formation of the vitelline coat of the oocytes. Oocytic degeneration is observed in this species, it is a continuous phenomenon of varing intensity throughout the year. The ultrastructural changes resulting in lysis of the oocyte are described. Mature spermatozoa consist of a broad, cap-shaped acrosomal vesicle, subacrosomal material, a round nucleus, two triplet substructure centrioles surrounded by four spherical mitochondria, and a flagellum anchored to the distal centriole and plasma membrane. Spermatozoa of Plucata closley resemble to those of other investigated Pteriidae. Changes in proximate composition of soft tissue and gonadal cycle of Pinctada fucata was studied. Mobilization and utilization of stored reserves are apparent during gametogenesis and gonadal maturation. Protein reserves are utilized during spermatogenesis while reserved carbohydrates form the main energy donor in oogenesis. The role of lipid as am.: energy reserve is second to that of carbohydrate.