Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects.

Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167-272 and amino acids 689-741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.

Screening and identification of vip genes in Bacillus thuringiensis strains.

AIMS: To identify known vip genes and to detect potentially novel vip genes in a collection of 507 strains of Bacillus thuringiensis. METHODS AND RESULTS: Following a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy, four restriction patterns were found within the vip1 family: vip1Aa1, vip1Ba1/vip1Ba2 and vip1Ca. In the screening of vip2 genes, patterns similar to those of vip2Aa1, vip2Ba1/vip2Ba2 and vip2Ac1 genes were observed. Patterns for vip3Aa1, vip3Ae2 and vip3Af1 were found among vip3 genes. Two new patterns revealed novel vip1 and vip3A genes. The observed frequency of genes belonging to vip1 and vip2 families was around 10%, whereas 48.9% of the strains showed amplification of vip3 genes. A tendency of vip and cry genes to occur together has been observed in this collection of B. thuringiensis strains. CONCLUSIONS: Ten different patterns of vip genes belonging to the three vip families and two novel vip genes have been identified in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that vip1 and vip2 genes have been identified by PCR-RFLP. Furthermore, the results show that the strategy used in this study can lead to the classification of known vip genes as well as the identification of novel vip genes.

Genomic structure and promoter analysis of pathogen-induced repat genes from Spodoptera exigua.

The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5'-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5'-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5'-untranslated region in the enhancement of gene expression.

Chemical composition and biotechnological properties of a polysaccharide from the peels and antioxidative content from the pulp of Passiflora liguralis fruits.

A new polysaccharide with a high molecular weight (greater than 1 x 106 Da) was extracted and characterized from the peels of Passiflora liguralis (granadilla) fruits. Chemical composition of the biopolymer, performed by using a high pressure anion exchange-pulsed amperometric detector (HPAE-PAD), showed the presence of six different sugar residues: xylose, glucose, galactose, galactosamine, an unknown component, and fucose in the relative ratio of 1:0.5:0.2:0.06:0.05:trace. The optical rotation of this xyloglucan was [alpha](D)(25) degrees C = -186.42 (concentration of 1.4 mg/mL of H(2)O), and the viscosity was dependent on the concentration and pH, showing a maximum value of 1.4 eta at a concentration of 3% in distilled water and a maximum value of 7.0 eta in citrate buffer solution. Thermogravimetric analysis indicated that this biopolymer was very stable at high temperatures, showing a degradation temperature at 280 degrees C. The characterization of the polysaccharide was also investigated by spectroscopic methods (1H NMR and IR) pointing out the complexity of this biopolymer and the presence of sugar residues in alpha-manno, alpha-gluco-galacto, and beta-gluco-galacto configurations. The formation of a biodegradable film using this novel xyloglucan was reported, and the anticytotoxic activity of the polysaccharide was studied in a brine shrimp bioassay. Considerable antioxidant activity (Trolox equivalent antioxidant capacity (TEAC) value of 0.32 microM/mg fresh product) was noted in the lipophilic extracts of Passiflora liguralis fruits, indicating, in this fruit, an alternative source of bioactive compounds.

Pain measurement in the elderly: a review.

Measurement of pain in the elderly is an issue that has received limited attention. The purpose of this review was to analyze and synthesize research findings from 1975 to 1999 that are related to pain measurement in the elderly. Based on best-evidence synthesis criteria, the review led to the selection of 15 studies. These studies used a descriptive and quantitative analytic approach and were not based on a theoretical framework. Comparison of selected pain measurement tools was incorporated in 40% of the included studies. Substantial gaps in knowledge were identified; namely, these included determining the reliability and validity of selected tools for the institutionalized or community-dwelling elder; modifying instruments to overcome barriers such as communication issues, cultural diversity, or cognitive dysfunction; and expanding the scope of pain measurement to other dimensions of the pain experience.

Studies of resistance to chloroquine, quinine, amodiaquine and mefloquine among Philippine strains of Plasmodium falciparum.

One hundred cases of slide-confirmed Plasmodium falciparum malaria admitted to the San Lazaro Hospital, Manila, Philippines were screened for in vitro resistance to chloroquine, quinine, amodiaquine and mefloquine using the microtechnique. 59 of the 100 primary parasite isolates produced schizonts, whereas the remaining 41 isolates did not. 51 of the 59 isolates tested were resistant in vitro to chloroquine and eight were sensitive. In contrast, three of the primary isolates were resistant to quinine, three showed resistance to amodiaquine and four were mefloquine-resistant. 43 of the strains judged chloroquine-resistant in vitro were fully in vitro sensitive to amodiaquine, quinine and mefloquine. One chloroquine-resistant isolate was also resistant to quinine alone. Three isolates that were resistant to chloroquine were also resistant to amodiaquine. An additional three were cross-resistant to chloroquine and mefloquine. A single isolate was found to be resistant to chloroquine, quinine and mefloquine and another was cross-resistant to chloroquine, quinine and amodiaquine. All strains demonstrating in vitro resistance to amodiaquine, quinine or mefloquine also showed in vitro resistance to chloroquine. The parasites in 22 patients showed in vivo resistance to chloroquine therapy. 86% were of the R1 type, 9% were R2 and 5% R3. All 22 patients demonstrating in vivo resistance to chloroquine showed in vitro resistance.

Chloroquine resistant Plasmodium falciparum: effect of rabbit serum and incubation time on the in vitro (microtechnique) prediction of in vivo resistance.

A study of chloroquine resistance of 54 isolates of Plasmodium falciparum is reported. Sixty-four percent of the isolates tested produced schizonts in vitro (micro-technique), whereas the remaining 36 percent did not. The accuracy of the in vitro test to predict in vivo resistance was increased when the primary parasite isolates were cultured in the presence of rabbit serum and when the cultures were allowed to incubate for more than 48 hours. Thirteen isolates of P. falciparum that showed in vitro resistance were confirmed in vivo resistant. Eleven of these cases were identified as R-I and two as R-II. Only one case of in vivo resistance (R-II) was observed among the 19 isolates that failed to produce schizonts in vitro.

Chloroquine and quinine resistant Plasmodium falciparum on the island of Mindoro, Philippines, 1982.

A field study was conducted on the island of Mindoro, Republic of the Philippines in which over 800 persons were screened for malaria and approximately 8% were found positive. The in vitro microtechnique was used to test for sensitivity to chloroquine, amodiaquine, mefloquine and quinine in 20 slide-confirmed P. falciparum cases. Sixteen of these cases were also followed for in vivo chloroquine sensitivity. Four cases showed in vitro resistance to chloroquine; 2 also showed resistance to quinine. All showed in vitro sensitivity to mefloquine and amodiaquine. The results of in vivo test were consistent with either a sensitive (S) or R-1, resistant response to chloroquine. Taken together, the in vitro and in vivo chloroquine tests indicate 4 cases of chloroquine resistance at the R1 level.