Green-tissue-specific, C4-PEPC-promoter-driven expression of Cry1Ab makes transgenic potato plants resistant to tuber moth (Phthorimaeaoperculella, Zeller)

An important strategy for obtaining a safer transgenic plant may be the use of a spatial- or tissue-specific promoter, instead of a constitutive one. In this study, we have used a light-inducible maize PEPC promoter to regulate the cry1Ab gene, aiming to produce transgenic potatoes that are resistant to potato tuber moth (PTM) (Phthorimaea operculella, Zeller). Out of 60 regenerated lines having normal phenotypes, 55 lines were PCR-positive for both the cry1Ab and nptII genes. Southern analysis on three selected putative transgenic lines revealed that they have only a single intact copy of the cry1Ab gene. An investigation of the Cry1Ab protein in the leaves and light-exposed (LE) tubers of the transgenic lines demonstrated the presence of the protein in the foliage and green tubers but not in the light-not exposed (LNE) tubers. A bioassay analysis of excised leaves of nine randomly selected lines showed that eight lines had 100% PTM larval mortality. Confirming results were obtained in six selected lines using the whole plant bioassay in the greenhouse. LE transgenic tubers also exhibited 100% larval mortality; however, the levels of damage to the LNE transgenic tubers were high and statistically the same as those incurred by the non-transgenic ones. Based on the results, we believe that this spatial expression of Cry1Ab using the light-inducible PEPC promoter can control PTM infestation in the field and significantly reduce pollution transmission to storage potatoes.     

Prediction of protein surface accessibility with information theory

A new, simple method based on information theory is introduced to predict the solvent accessibility of amino acid residues in various states defined by their different thresholds. Prediction is achieved by the application of information obtained from a single amino acid position or pair-information for a window of seventeen amino acids around the desired residue. Results obtained by pairwise information values are better than results from single amino acids. This reinforces the effect of the local environment on the accessibility of amino acid residues. The prediction accuracy of this method in a jackknife test system for two and three states is better than 70 and 60 %, respectively. A comparison of the results with those reported by others involving the same data set also testifies to a better prediction accuracy in our case.     

Effect of biotic and abiotic factors on the tunneling behavior of Coptotermes gestroi and Heterotermes tenuis (Isoptera: Rhinotermitidae)

Using bidimensional arenas, the construction and spatial dispersion of tunnels constructed by Coptotermes gestroi and Heterotermes tenuis (Rhinotermitidae) was determined under different laboratory conditions. Workers of both species showed an increase of the tunneled area with the rise in temperature. The activity of workers of C. gestroi also increased with the rise in soil moisture. Primary tunnels showed a high degree of dispersion in the arenas for all treatments. The presence of food did not influence the tunneling pattern of C. gestroi; but for H. tenuis there was a significant decrease in the number of tunnels. In addition, the type of substrate affected the tunneling pattern in both species. The understanding of food searching strategies of subterranean termites could contribute in the improvement of pest management programs based on the use of toxic baits.     

Prediction of protein secondary structure based on residue pair types and conformational states using dynamic programming algorithm

We have used a statistical approach for protein secondary structure prediction based on information theory and simultaneously taking into consideration pairwise residue types and conformational states. Since the prediction of residue secondary structure by one residue window sliding make ambiguity in state prediction, we used a dynamic programming algorithm to find the path with maximum score. A score system for residue pairs in particular conformations is derived for adjacent neighbors up to ten residue apart in sequence. The three state overall per-residue accuracy, Q3, of this method in a jackknife test with dataset created from PDBSELECT is more than 70%.     

Influence of insecticide residues on growth and activity ofStreptococcus diacetilactis andLeuconostoc cremoris

Summary Reconstituted skimmed milk powder containing the insecticides fenvalerate (benzene acetic acid, 4-chloro--1-methylcyano-3-phenoxyphenyl methyl ester), malathion (O,O-dimethyl-S-1, 2-bis-ethoxycarbonyl ethyl phosphorothioate) and DDT (2,2-bis-p-chlorophenyl-1,1,1-trichloroethane) in different concentrations (0, 50,100 and 200 ppm) was separately inoculated withStreptococcus lactis subsp.diacetilactis andLeuconostoc cremoris and incubated at 30°C for 120 h. Fenvalerate was more inhibitory towardsL. cremoris than other insecticides. Malathion strongly inhibited growth ofS. diacetilactis. Fenvalerate inhibited acid production byS. diacetilactis, but had no effect on acid production byL. cremoris. S. diacetilactis was in general more sensitive to all insecticides studied thanL. cremoris. S. diacetilactis produced more acidity and less acetaldehyde than those produced byL. cremoris whether in the presence or absence of insecticide.

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Influence des résidus d'insecticide sur la croissance et l'activité de Streptococcus diacetilactis et de Leuconostoc cremoris

Résumé On a inoculé du lait écrémé, reconstitué à partir de poudre, contenant les insecticides fenvalerate (ester méthylique de l'acide 4-chloro--1-methylcyano-3-phenoxyphenyl benzene acétique), malathion (O,O-diméthyl-S-1,2-bis-éthoxycarbonyl éthyl phosphorothioate) et DDT (2,2-bis-p-chlorophenyl-1,1,1-trichloroéthane) à différentes concentrations (0, 50, 100 et 200 ppm), par leStreptococcus lactis, subsp.diacetilactis ou par leLeuconostoc cremoris. Après incubation à 30°C pendant 120 h, le fenvalerate s'est révélé plus inhibiteur duL. cremoris que les autres insecticides. Le malathion inhibe fortement la croissance deS. diacetilactis. Le fenvalerate inhibe l'acidogénèse parS. diacetilactis mais n'a aucun effet sur l'acidogénèse deL. cremoris. S. diacetilactis est en général plus sensible à tous les insecticides étudiés queL. cremoris. S. diacetilactis est plus acidogène et produit moins d'acétaldehyde que ne le faitL. cremoris que ce soit en présence ou en absence d'insecticide.

     

Characterization of the defect in the Na(+)-phosphate transporter in vitamin D-resistant hypophosphatemic mice.

Hypophosphatemic vitamin D-resistant rickets is the most common form of vitamin D-resistant rickets in man. The hypophosphatemic mouse model (Hyp) is phenotypically and biochemically similar to the human disease. Biochemically, hypophosphatemia is the hallmark of this disorder. The cause of the hypophosphatemia is thought to be secondary to a defect in the renal and/or intestinal Na(+)-phosphate transporter. The current studies were designed to investigate and characterize the localization of the defect in the Na(+)-phosphate transporter in this disorder. Phosphate uptake by renal brush border membrane vesicles (BBMV) showed a significant decrease in the slope of the initial rate of phosphate uptake in (Hyp) compared with control mice (0.009 versus 0.013, respectively). The slopes representing initial rates of phosphate uptake by jejunal BBMV were similar in (Hyp) and control mice (0.004 and 0.004, respectively). Kinetics of jejunal Na(+)-dependent phosphate uptake showed a Vmax of 0.63 +/- 0.12 and 0.64 +/- 0.12 nmol/mg protein/15 s in (Hyp) and control mice, respectively, whereas Km values were 0.12 +/- 0.08 and 0.2 +/- 0.11 mM, respectively. Similar kinetic analysis in the kidney showed a Vmax of 0.32 +/- 0.06 and 1.6 +/- 0.1 (p less than 0.01) and Km of 0.07 +/- 0.06 and 0.39 +/- 0.05 (p less than 0.02) in (Hyp) and control mice, respectively. Na(+)-dependent D-glucose uptake by BBMVs of intestine and kidney showed typical overshoot phenomena in (Hyp) and control mice. In order to explore these findings further, Na(+)-phosphate transporter expression from intestine and kidney was accomplished by microinjection of 50 ng of poly(A)+ RNA into Xenopus laevis oocytes. Na(+)-dependent phosphate uptake was expressed 6 days after the microinjection of intestinal and kidney poly(A)+ RNA from control mice. However, expression of the transporter from (Hyp) mice occurred only from the intestine, and not from the kidney. The decrease in the expression of the Na(+)-dependent phosphate transporter was not secondary to accelerated efflux of phosphate or decreased metabolism in oocytes injected with poly(A)+ RNA from (Hyp) mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative proteomics profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses

Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.     

Identification of Phytochemicals Targeting c-Met Kinase Domain using Consensus Docking and Molecular Dynamics Simulation Studies

c-Met receptor tyrosine kinase is a proto-oncogene whose aberrant activation is attributed to a lower rate of survival in most cancers. Natural product-derived inhibitors known as “fourth generation inhibitors” constitute more than 60% of anticancer drugs. Furthermore, consensus docking approach has recently been introduced to augment docking accuracy and reduce false positives during a virtual screening. In order to obtain novel small-molecule Met inhibitors, consensus docking approach was performed using Autodock Vina and Autodock 4.2 to virtual screen Naturally Occurring Plant-based Anti-cancer Compound–Activity–Target database against active and inactive conformation of c-Met kinase domain structure. Two hit molecules that were in line with drug-likeness criteria, desired docking score, and binding pose were subjected to molecular dynamics simulations to elucidate intermolecular contacts in protein–ligand complexes. Analysis of molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area studies showed that ZINC08234189 is a plausible inhibitor for the active state of c-Met, whereas ZINC03871891 may be more effective toward active c-Met kinase domain compared to the inactive form due to higher binding energy. Our analysis showed that both the hit molecules formed hydrogen bonds with key residues of the hinge region (P1158, M1160) in the active form, which is a hallmark of kinase domain inhibitors. Considering the pivotal role of HGF/c-Met signaling in carcinogenesis, our results propose ZINC08234189 and ZINC03871891 as the therapeutic options to surmount Met-dependent cancers.     

Preparation of nanoliposomes linked to HER2/neu-derived (P5) peptide containing MPL adjuvant as vaccine against breast cancer

The study was aimed at evaluating antitumor and immunomodulatory effects of liposomal vaccine composed of P5 human epidermal growth factor receptor 2 (HER2)/neu-derived peptide coupled to the surface of high-temperature nanoliposomes containing distearoylphosphocholine:distearoylphosphoglycerol:Chol:dioleoylphosphatidylethanolamine (DOPE) comprising monophosphoryl lipid A (MPL) adjuvant in HER2/neu overexpressing the breast cancer model. BALB/c mice bearing TUBO carcinoma were subcutaneously immunized with formulations containing 10 µg P5 peptide and 25 µg MPL three times with 2-week intervals. To determine immuno responses in immunized mice, the amount of released interferon-γ and IL-4 were measured by the enzyme-linked immunospot method and the flow cytometric analysis on the isolated splenocytes. The results demonstrated that tumor-bearing mice immunized with Lip/DOPE/MPL/P5 formulation had the most released interferon-γ and the highest cytotoxic T lymphocyte responses that led to the lowest tumor size and the longest survival time than those of other formulations. The results achieved by Lip/DOPE/MPL/P5 formulation could make it a suitable candidate to induce effective antigen-specific tumor immunity against breast cancer.     

Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.