Cytotoxicity and potential antiviral evaluation of violacein produced by Chromobacterium violaceum

Natural products are an inexhaustible source of compounds with promising pharmacological activities including antiviral action. Violacein, the major pigment produced by Chromobacterium violaceum, has been shown to have antibiotic, antitumoral and anti-Trypanosoma cruzi activities. The goal of the present work was to evaluate the cytotoxicity of violacein and also its potential antiviral properties.The cytotoxicity of violacein was investigated by three methods: cell morphology evaluation by inverted light microscopy and cell viability tests using the Trypan blue dye exclusion method and the MTT assay. The cytotoxic concentration values which cause destruction in 50 percent of the monolayer cells (CC50) were different depending on the sensitivity of the method. CC50 values were > 2.07 ± 0.08 æM for FRhK-4 cells: > 2.23 ± 0.11 æM for Vero cells; > 2.54 ± 0.18 æM for MA104 cells; and > 2.70 ± 0.20 æM for HEp-2 cells. Violacein showed no cytopathic inhibition of the following viruses: herpes simplex virus type 1 (HSV-1) strain 29-R/acyclovir resistant, hepatitis A virus (strains HM175 and HAF-203) and adenovirus type 5 nor did it show any antiviral activity in the MTT assay. However violacein did show a weak inhibition of viral replication: 1.42 ± 0.68 percent, 14.48 ± 5.06 percent and 21.47 ± 3.74 percent for HSV-1 (strain KOS); 5.96 ± 2.51 percent, 8.75 ± 3.08 percent and 17.75 ± 5.19 percent for HSV-1 (strain ATCC/VR-733); 5.13 ± 2.38 percent, 8.18 ± 1.11 percent and 8.51 ± 1.94 percent for poliovirus type 2; 8.30 ± 4.24 percent; 13.33 ± 4.66 percent and 24.27 ± 2.18 percent for simian rotavirus SA11, at 0.312, 0.625 and 1.250 mM, respectively, when measured by the MTT assay

Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

Outbreaks of gastroenteritis have occurred among consumers of raw or undercooked shellfish harvested from faecally polluted waters. A multiplex reverse transcription-polymerase chain reaction (RT-PCR) was applied for the simultaneous detection of hepatitis A virus (HAV), poliovirus (PV) and simian rotavirus (RV-SA11) and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp), PV (394 bp) and RV (278 bp) were identified when positive controls were used. However, when tested on experimentally contaminated raw oysters, this method was not able to detect the three viruses simultaneously. This is probably due to the low concentration of viral RNAs present in oyster extract which were partially lost during the extracts preparation

Partial sequence analysis of type 1 dengue virus coding for the nonstructural hydrophilic protein NS-3

A complementary DNA copy of DEN-1 RNA was synthesized using reverse transcriptase and a random primer. The double-stranded DNA copy was cloned at the Sma I site of the pUC13 vector and was used to transform Escherichia coli JM83. Among the transformants selected for characterization by nucleotide sequence analysis, we report here one that codes for a region of nonstructural hydrophilic protein, NS-3. Computer analysis of this sequence (967 bp) showed abouth 87% conservation at the amino acid level and 79% at the nucleotide level when compared with dengue serotypess 2,3 and 4 and Japanese encephalitis virus. This suggest an important function which is common to all four serotypes. Comparision of the cloned region with sequences of the above strains also suggest conservation of hydrophobic regions

A direct method for cDNA cloning employing translation released mRNA

1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized derectly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of cDNA clone corresponding to chick alfa (1) procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos