DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of-CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for-CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than-CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Type 2 heat-labile enterotoxin (LT-II)-producing Escherichia coli isolated from ostriches with diarrhea.

The culture supernatant of Escherichia coli, isolated from ostriches with diarrhea in Brazil, caused elongation in Vero cell, rounding in Chinese hamster ovary (CHO) cells and a cytoplasmic vacuolation in ostrich embryo fibroblasts (OEF), but it was not cytotoxic for chicken embryo fibroblasts (CEF). These effects were not neutralized by antiserum to cholera toxin. Polymerase chain reaction assays showed that the ostrich E. coli contained the gene encoding (eltII-A), but not those for type 1 heat-labile enterotoxin (eltA), heat-stable enterotoxins (estA, estB), verocytotoxins (stx-I, stx-II), or cytotoxic necrotizing factors (cnf 1, cnf 2). All isolates belonged to serotype O15:H8. The enteropathogenic relevance of LT-II in ostrich diarrhea remains undetermined.

Avian anticoccidial activity of a novel membrane-interactive peptide selected from phage display libraries.

In the present work, we describe the discovery of PW2, a novel peptide presenting in vitro activity against Eimeria acervulina and E. tenella sporozoites. PW2 was selected from phage display (Ph.D.) peptide libraries by an alternative method of panning using living purified E. acervulina sporozoites as targets. Our results showed that the peptide disrupts the sporozoite pellicle, resembling the effect caused by most natural antimicrobial peptides. PW2 peptide was also effective against fungi and showed low activity against Toxoplasma gondii tachyzoites, but no activity against Trypanosoma cruzi, Crithidia fasciculata epimastigotes, and bacteria. Additionally, the parasiticidal concentrations of PW2 produced a very low lytic effect on mammalian and avian cells. The effectiveness against Eimeria sporozoites and the absence of adverse effects to host cells indicates that PW2 may be used as a model to generate new drugs for the control of avian coccidiosis.

Ultrastructural study of enteropathogenic Escherichia coli Olllab: H2 infection in an infant with acute diarrhea

Escherichia coli enteropatogênica clássica é o mais importante agente etiológico como causa de diarréia aguda nos países subdesenvolvidos, especialmente nos lactentes menores de uma ano de idade. As cepas de Escherichia coli enteropatogênica sao capazes de provocar profundas alteraçoes no citoesqueleto do enterócito, as quais sao denominadas aderência e apagamento das microvilosidades do enterócito, as quais sao denominadas aderência e apagamento das microvilosidades, associadas à formaçao de pedestais. Descreve-se uma cepa de Escherichia coli enteropatogênica 0111ab:H2 isolada das fezes de um lactente com diarréia aguda, no 11§ dia de doença, a qual provocou lesoes de aderência e apagamento das microvilosidades do enterócito e inclusive penetrou no interior do citoplasma do mesmo, assim como invadiu células HeLa em cultura de tecido in vitro e da mesma forma no teste da alça ileal ligada de coelho in vivo, todas as lesoes detectadas em estudo ultra-estrutural. Esta observaçao demonstra que lesoes graves da mucosa do intestino delgado causadas por uma cepa de Escherichia coli enteropatogênica 0111ab:H2 podem ocorrer ainda em um estágio precoce da infecçao entérica.

Incidência de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens de casos de diarréia em crianças, na regiäo de Campinas, SP, Brasil / Incidence of enterotoxigenic Escherichia coli (ETEC) rotaviruses and clostridium perfringens from cases of infantile diarrhea, in the region of Campinas, SP, Brazil

Foi realizada uma pesquisa na regiäo de Campinas, S-, Brasil, sobre a presença de Escherichia coli enterotoxigência (ETEC), rotavírus e clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 especimes fecais examinados quanto a presença de ETEC 27 (20,45%) foram positivos. Destes foram isoladas 41 amostras de ETEC, das quais 40 produziram apenas a enterotoxina termolábil (LT) detectada pelo teste de imuno-hemólise radial modificado. Entre as 1983 amostras de fezes examinadas para rotavírus, 29 (15,84%) foram positivas pelas técnicas de eletroforese em gel de poliacrilamida (PAGE) e ensaio imunoenzimático (EIE), sendo que destas, 15 (51,7%) foram provenientes de materiais coletados nos meses de inverno. Todas as amostras pertenciam ao grupo A e, através da técnica de PAGE, pode-se observar que o tipo eletroforético mais freqüente (9 amostras) foi designado Ib, IIc, IIIb, IVa, de acordo com a classificaçäo por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecçäo da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinaçäo passiva reversa e inoculaçäo intravenosa em camundongos, sendo encontradas 12(10,62%) amostras enterotoxigências. Diante destes resultados é chamada a atençäo sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criaçäo de métodos simplificados que favoreçam a detecçäo e identificaçäo dos grupos de agentes enteropatogênicos estudados na presente pesquisa