Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.     

Synthesis and biological evaluation of gemcitabine-lipid conjugate (NEO6002)

A novel gemcitabine-lipid conjugate 5 was synthesized and tested for its in vivo efficacy and toxicity. Compound 5 was tested in BxPC-3 human pancreatic tumor model in SCID mice and exhibited promising activity and lower toxicity when compared with Gemzar.     

Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides

A new modified stilbene dimer, diptoindonesin D (1), was isolated from the acetone extract of the tree bark of Hopea dryobalanoides, together with seven known compounds, parviflorol (2), (-)-balanocarpol (3), heimiol A (4), hopeafuran (5), (+)-alpha-viniferin (6), vaticanol B (7) and (-)-hopeaphenol (8). Cytotoxic properties of compounds 1-8 were evaluated against murine leukemia P-388 cells. Compound 8 was found to be the most active with IC50 of 5.7 microM.     

Domain-specific effector interactions within the central cavity of human adult hemoglobin in solution and in porous sol-gel matrices: evidence for long-range communication pathways

The water-filled central cavity of human adult hemoglobin (Hb A) is the binding or interaction site for many different allosteric effectors. Oxygen binding titrations reveal that pyrenetetrasulfonate (PyTS), a fluorescent analogue of 2,3-diphosphoglycerate, behaves like an allosteric effector. The ligation state, pH, and concentrations of other effectors (IHP, L35, and chloride) alter PyTS fluorescence for both solution-phase and sol-gel-encapsulated Hb samples. These conditions also alter the resonance Raman spectra and rates of geminate recombination of CO-ligated Hb. Together, these results demonstrate that there are conformational and functional consequences resulting from interactions between specific domains of the central cavity and individual effectors as well as from long-range synergistic effects that are mediated through the central cavity.     

Analysis of glioma cell platinum response by metacomparison of two-dimensional chromatographic proteome profiles

Successful clinical development of cancer treatments is aided by the development of molecular markers that allow the identification of patients likely to respond. In the case of broadly cytotoxic drugs, such as the multinuclear series of platinum chemotherapeutic agents that we are evaluating for the treatment of glioma, one route to marker identification is proteomic profiling. We are using the two-dimensional chromatography system, the ProteomeLab PF2D, to compare proteomic profiles of glioma cells in culture before and after drug treatment. The existing software tools allowed the rapid identification of peaks increased by treatment of a given drug as compared with control untreated cells. To compare across these pairs, we developed new software, called the MetaComparison Tool (MCT). The MCT uses the chromatographic characteristics of peaks as identifiers, an approach that was validated by mass spectrometry of two independent isolations of a peak, from cells that were treated with two different platinum compounds. The MCT made it possible to rapidly query whether a given peak responded to more than one treatment and so allowed the identification of peaks that were specific to a given drug. As a result, this analysis greatly reduced the list of peaks whose isolation and downstream analysis by mass spectrometry is warranted, accelerating the search for protein markers of response.     

Mucoadhesive, thermosensitive, prolonged-release vaginal gel for clotrimazole:beta-cyclodextrin complex

The purpose of this study was to achieve a better therapeutic efficacy and patient compliance in the treatment for vaginitis. Clotrimazole (1%) has been formulated in a vaginal gel using the thermosensitive polymer Pluronic F127 (20%) together with mucoadhesive polymers such as Carbopol 934 and hydroxypropylmethylcellulose (0.2% for both). To increase its aqueous solubility, clotrimazole was incorporated as its inclusion complex with 1:1 molar ratio with beta-cyclodextrin. The inclusion complex was thoroughly characterized using various techniques, including 1H NMR spectroscopy, FT IR spectrophotometry, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and determination of stability constant (k(1:1)). The gelation temperature and rheological behavior of different formulations at varying temperatures were measured. In vitro release profiles of the gels were determined in pH 5.5 citrate buffer. It was observed that complexation with cyclodextrin slowed down the release of clotrimazole considerably. Carbopol 934, on the other hand, was found to interact with beta-cyclodextrin, inducing precipitation. As far as rheological properties are concerned, thermosensitive in situ gelling was obtained with formulations containing drug:cyclodextrin complex rather than with free drug. Thus, the optimum formulation for a controlled-release thermosensitive and mucoadhesive vaginal gel was determined to be clotrimazole:beta-cyclodextrin 1% with 0.2% hydroxypropylmethylcellulose in Pluronic F127 gel (20%) providing continuous and prolonged release of active material above MIC values.     

Engineering novel traits in plants through RNA interference

RNA interference (RNAi) is a homology-dependent gene silencing technology that involves double-stranded RNA directed against a target gene or its promoter region. Using hairpin constructs, double-stranded RNA can be expressed in plants relatively easily, enabling this technology to be applied to a wide range of species to silence the expression of both specific endogenous genes and genes of invading pathogens. RNAi has also been used to engineer metabolic pathways to overproduce secondary products with health, yield or environmental benefits. The application of tissue-specific or inducible gene silencing, with the use of appropriate promoters, and the ability to silence several genes simultaneously should enhance our ability to create novel traits in plants.     

Microgeographic evolution of snail shell shape and predator behavior

Genetic divergence in geographically isolated populations is a prerequisite for allopatric speciation, one of the most common modes of speciation. In ecologically equivalent populations existing within a small, environmentally homogeneous area, an important role for environmentally neutral divergence is often found or inferred. We studied a species complex of conspicuously shaped Opisthostoma land snails on scattered limestone outcrops within a small area of lowland rainforest in Borneo. We used shell morphometrics, mitochondrial and nuclear DNA sequences, and marks of predation to study the factors involved in allopatric divergence. We found that a striking geographic divergence exists in shell morphology, which is partly associated with neutral genetic divergence. We also found geographic differentiation in the behavior of the snails' invertebrate predator and evidence of an evolutionary interaction between aspects of shell shape and predator behavior. Our study shows that adaptation to biotic aspects of the environment may play a more important role in allopatric speciation than previously suspected, even on a geographically very small scale.     

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.