Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

<p>Abstract</p> <p>Background</p> <p><it>Anticarsia gemmatalis </it>is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of <it>A. gemmatalis </it>(AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum.</p> <p>Results</p> <p>Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G₀/G₁stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells.</p> <p>Conclusions</p> <p>UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both <it>in vitro </it>bioinsecticide production and applications such as recombinant protein expression or gene delivery.</p

First record of a mosquito iridescent virus in Culex pipiens L. (Diptera: Culicidae)

The mosquito iridescent viruses (MIVs) are large icosahedral DNA viruses that replicate and assemble in the cytoplasm of the host. Paracrystalline arrangements of virions that accumulate in the cytoplasm produce an iridescent color that is symptomatic of acute infections. In August 2010 we found larvae of Culex pipiens with these symptoms in suburban ditches around La Plata city, Argentina. Electron microscope studies, PCR amplification of the Protein (MCP) gene arrangement, DNA sequencing and phylogenetic analysis were carried out

Potential conservation of circadian clock proteins in the phylum Nematoda as revealed by bioinformatic searches

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.Fil: Romanowski, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; ArgentinaFil: Garavaglia, Matías Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Goya, María Eugenia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Golombek, Diego Andres. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Identification of a Wee1-like kinase gene essential for procyclic Trypanosoma brucei survival.

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Fundacion Alberto J. RoemmersFundacion Alberto J. RoemmersConsejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICET)Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET)ANPCyT Agencia Nacional de Promocion Cientifica y Tecnologica, ArgentinaAgencia Nacional de Promoción Científica y Tecnológica (ANPCyT

Sequence comparison between TbWee1 and homolog proteins in other species.

<p>(A) Multiple sequence alignment of the catalytic domains of the putative <i>Trypanosoma brucei</i> Wee1 protein with other Wee1-like kinases. Amino acid sequences were aligned using the ClustalW2 program (<a href="http://www.ebi.ac.uk/clustalw" target="_blank">http://www.ebi.ac.uk/clustalw</a>). Identities are indicated by asterisks below the sequence. Conserved substitutions are marked with two vertical dots and semi-conserved substitutions are marked with a single dot. Dashes represent gaps introduced for optimal alignment. The 11 conserved subdomains are designated by Roman numerals [45,46]. The catalytic and activation segments are indicated with a blue and pink box respectively. A black box indicates the conserved EGD motif. Triangles indicate amino acids that are conserved in all known members of the Wee1 kinase family, but not in other eukaryotic protein kinases. Sequences shown are for Wee1A kinase of <i>Trypanosoma </i><i>brucei</i> (TbWee1, JN083854), humans (HsWee1, NP003381.1), mice (MmWee1, NP033542.2), <i>Schizosaccharomyces </i><i>pombe</i> (SpWee1, NP587933.1), <i>Saccharomyces </i><i>cerevisiae</i> (ScSwe1, NP012348.1), <i>Arabidopsis </i><i>thaliana</i> (AtWee1, NP171796.1), <i>Xenopus laevis</i> (XlWee1, NP001081784.1), and <i>Trypanosoma cruzi</i> (TcWee90, JN573306; TcWee570, JN257712). (B) Comparison of TbWee1 with other protein kinases. The position of the putative protein kinase domain is shown in black and numbers represent the percent amino acid identity with this region of the predicted TbWee1.</p

Morphological phenotypes of Wee1-deficient <i>T. brucei</i> procyclic-form cells.

<p>Samples of the TbWee1-depleted cells taken at different times were stained with DAPI and examined by fluorescence microscopy. (A) Analysis of the numbers of nuclei and kinetoplasts as determined by DAPI staining. Data are presented as the mean percentages ±S.E. of the total population counted (> 200 cells in each of three independent experiments). (B) Wee1-deficient cells viewed by phase-contrast and fluorescence microscopy. N: nucleus, K: kinetoplast.</p

Synchronization of procyclic <i>T. brucei</i> and analysis of TbWee1 protein expression during different stages of the cell cycle.

<p>(A) Cells were synchronized with 0.2 mM hydroxyurea for 12 h, then HU was washed out and the cells were stained with propidium iodide and analyzed for 12 hs by flow cytometry performed every 2 h. (B) The percentages of cells in G1, S and G2/M phases were determined by the ModFitLT software. (C) Protein extracts from S, G2/M and G1 were separated by 12 % SDS-PAGE and electroblotted on nitrocellulose membranes. His-tagged recombinant TbWee1 (10 µg) was used as a positive control. Blots were incubated with anti-TbWee1 polyclonal antibody (1:1000) (lanes 1, 3, 5: 20 µg; lanes 2, 4, 6: 40 µg). An antibody recognizing β-tubulin (1:5000) was used as the load control.</p