Cytotoxicity of Diepoxybutane and Epichlorohydrin in Relation to Stages of the Cell Cycle

Work conducted in the Millard Biochemistry Research Laboratory examines the dual nature of molecules as carcinogens and anti-tumor agents through the molecular mechanisms of duplex DNA damage by bifunctional alkylating agents. Diepoxybutane (DEB) and epichlorohydrin (ECH) are polar molecules that form covalent DNA interstrand lesions by cross-linking the N7 position of deoxyguanosine residues. A recent experiment indicated that ECH preferentially targets nuclear DNA over mitochondrial DNA, whereas DEB shows similar rates of lesion formation for both loci. It was concluded that preferential targeting of nuclear DNA results from relatively poor uptake of ECH across the mitochondrial membrane. The objective of my honors research was to determine if the cytotoxicities of DEB and ECH vary according to the presence of the nuclear envelope in 6C2 chicken erythro-progenitor cells. The cytotoxicity of DEB and ECH was compared between cells randomly distributed throughout the cell cycle (Go/G, and S » G2/M) and cells enriched in G2/M stages. Results indicated that ECH is more cytotoxic than DEB in both unsynchronized control 6C2 cells and synchronized 6C2 cells enriched in G2/M stages of the cell cycle. Treatment with either bifunctional alkylating agent induced greater cytotoxicity in 6C2 cells enriched in G2/M stages than in unsynchronized control 6C2 cells, suggesting that the presence of the nuclear envelope-or any plasma membrane-may inhibit the reactivity of DEB and ECH.

Neurobiology and clinical implications of lucid dreaming

Several lines of evidence converge to the idea that rapid eye movement sleep (REMS) is a good model to foster our understanding of psychosis. Both REMS and psychosis course with internally generated perceptions and lack of rational judgment, which is attributed to a hyperlimbic activity along with hypofrontality. Interestingly, some individuals can become aware of dreaming during REMS, a particular experience known as lucid dreaming (LD), whose neurobiological basis is still controversial. Since the frontal lobe plays a role in self-consciousness, working memory and attention, here we hypothesize that LD is associated with increased frontal activity during REMS. A possible way to test this hypothesis is to check whether transcranial magnetic or electric stimulation of the frontal region during REMS triggers LD. We further suggest that psychosis and LD are opposite phenomena: LD as a physiological awakening while dreaming due to frontal activity, and psychosis as a pathological intrusion of dream features during wake state due to hypofrontality. We further suggest that LD research may have three main clinical implications. First, LD could be important to the study of consciousness, including its pathologies and other altered states. Second, LD could be used as a therapy for recurrent nightmares, a common symptom of depression and post-traumatic stress disorder. Finally, LD may allow for motor imagery during dreaming with possible improvement of physical rehabilitation. In all, we believe that LD research may clarify multiple aspects of brain functioning in its physiological, altered and pathological states.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ingestion rates of Artemia nauplii for different larval stages of Macrobrachium rosenbergii

The functional response between ingestion rate and food concentration was determined for each larval stage of Macrobrachium rosenbergii. Artemia franciscana nauplii were supplied at 2,4, 6, 8, 10 and 12 per milliliter. The nauplii were counted by sight using a Pasteur pipette and transferred to Petri dishes containing 40 ml of brackish water (12 parts per thousand) lying on the top of black plastic. One larva at each stage was individually placed into each Petri dish containing different food density. After 24 h, each larva was removed from the Petri dish and the leftover nauplii were counted. The amount consumed was determined by the difference between the initial and final number of nauplii. Ingestion rate (I) increased as food density (P) increased and was defined by the model I=I-m(1-e(-kP)). The results suggest four levels of ingestion during larval development. The first level includes stages II, III and IV, with average maximum consumption of about 40 nauplii/day; the second level includes stages V and VI, with consumption of approximately 55 nauplii/day; the third level includes stages VII and VIII, with consumption of 80-100 nauplii/day. The fourth level includes stages IX, X and XI, in which the high values for maximum ingestion (Im) exceed the load capacity of the medium. The low values for constant k (that may correspond to the adaptability of the food to prey characteristics, such as, size, mobility, etc.) obtained for stages IX, X and XI indicated that Artemia is not an adequate prey and there is necessity of a supplementary diet. The best relationship between predator and prey seemed to occur during stage IV Results obtained in the present work may subsidize future researches and serve as a guideline for practical considerations of feeding rates. (C) 2003 Elsevier B.V. B.V. All rights reserved.

The preimaginal stages of the ensign wasp Evania appendigaster (Hymenoptera, Evaniidae), a cockroach egg predator

Evania appendigaster is a cosmopolitan wasp that deposits eggs in the oothecae of some species of cockroaches; its larvae then consume the cockroach eggs and embryos. It is a candidate for the biological control of cockroaches, but little is known about its basic biology. Here we describe the external morphology of all immature stages of E. appendigaster and compare them with the larvae of related species. The life cycle of E. appendigaster includes three larval instars, each with 13 body segments. Their mouthparts were generally reduced, except for the mandibles, which were always sclerotized and toothed, and were especially robust in second-instar larvae. Antennal and mouthpart sensilla were basiconic and difficult to observe. Larvae of E. appendigaster are similar in form to other described evaniid larvae, but quite different from the two available descriptions of larvae of gasteruptiid and aulacid wasps. Further descriptions of evaniid larvae will be useful in determining how widespread this morphology is within the family, and in understanding phylogenetic relationships within Hymenoptera.

Ontogenetic Variation in Ammonia Excretion during the Early Life Stages of the Amazon River Prawn, Macrobrachium amazonicum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biological aspects of the immature stages of Ceraeochrysa everes (Banks) (Neuroptera: Chrysopidae)

Para o estabelecimento de um programa de controle biológico, o conhecimento de alguns aspectos biológicos e de comportamento dos inimigos naturais é de extrema importância. Os aspectos biológicos do desenvolvimento larval e pupal do predador Ceraeochrysa everes (Banks) foram estudados. Larvas oriundas de adultos da geração F1 foram mantidas em laboratório a 25 ± 21°C, 70 ± 10 % UR e fotofase 14 horas, sendo alimentadas com ovos de Sitotroga cerealella (Olivier). A duração e viabilidade do período embrionário, estágios imaturos de desenvolvimento e o período de ovo a adulto foram avaliados. O período embrionário foi em média de 5,0 dias, enquanto que as durações médias para o primeiro, segundo e terceiro instares foram de 5,1 ± 0,03; 4,3 ± 0,05 e 4,5 ± 0,05 dias, respectivamente, com viabilidade superior a 90 %. Os estágios larval, pré-pupal e pupal apresentaram duração média de 13,9 ± 0,07; 5,7 ± 0,07 e 9,6 ± 0,12 dias, respectivamente. A duração do ciclo biológico foi de 34 ± 0,11 dias em média. Ovos de S. cerelella não foram adequados para a manutenção de C. everes em laboratório, por interferir no desenvolvimento do predador.

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mechanism of infection and colonization of Rhipicephalus sanguineus eggs by Mertarhizium anisopliae as revealed by scanning eletron microscopy and histopathology

O presente trabalho teve como objetivo verificar a forma de penetração do fungo Metarhizium anisopliae em ovos do carrapato Rhipicephalus sanguineus, assim como as lesões infringidas no interior do ovo. A aderência e penetração do fungo foram estudadas por meio da microscopia eletrônica de varredura e a ação do fungo nos tecidos internos avaliada em secções histológicas convencionais. Para observação destes eventos, realizaram-se infecções experimentais em 11 grupos de ovos do R. sanguineus contendo 25 mg cada. Os ovos foram banhados durante 3 minutos, sob agitação manual, em suspensão com concentração de 10(8) conídios/mL. Nos grupos controle o banho foi realizado apenas no veículo da suspensão. Os ovos foram processados para análise histopatológica e microscopia eletrônica de varredura nos seguintes tempos após a infecção: 1 e 18h, e um, dois, três, quatro, cinco, seis, sete, nove e onze dias. Observou-se grande germinação de conídios em 67% dos ovos 18h após a inoculação e o fungo penetrou em 92,6% dos ovos 5 dias após a infecção. A extrusão do patógeno ocorreu em 87% dos ovos 7 dias após a infecção, chegando a 100% no 9º dia. Nas análises histopatológicas não foram observadas lesões dignas de nota, porem deve-se ressaltar que houve significativa redução (53,9%) na eclosão a partir dos ovos infectados.

Susceptibility of adult and larval stages of the horn fly, Haematobia irritans, to the entomopathogenic fungus Metarhizium anisopliae under field conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Behavior of Leishmania major metacyclic promastigotes during the course of infection and immune response development in resistant versus susceptible hosts

Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR) em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos) de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro negativamente carregadas, enquanto que promastigotas metacíclicos não se aderem à coluna e podem ser recuperados com facilidade. Condições ótimas de cromatografia foram validadas por análise estatística. O rendimento médio de parasitas obtidos após separação em colunas de lã de vidro e a viabilidade dos promastigotas foram estimados por microscopia óptica. Os promastigotas metacíclicos tiveram um rendimento médio de 43,5% a 57,5%. Camundongos BALB/c (susceptíveis) e camundongos C57BL/6 (resistentes) apresentaram padrões distintos de lesões cutâneas, os primeiros com lesões mais agressivas, induzidas por promastigotas metacíclicos. As respostas à reação de HTR foram maiores nos grupos de camundongos C57BL/6, submetidos à infecção com promastigotas metacíclicos. Estes resultados indicam que o novo método poderia ser integrado aos protocolos existentes para estudar a metaciclogênese de parasitas do gênero Leishmania in vivo.