Characterization and Pathogenic Relationships of Rhizoctonia solani Isolates in a Potato–Rice System and their Sensitivity to Fungicides

Potato is planted after rice in several parts of Punjab in India and both crops are attacked by Rhizoctonia solani Kühn. Potato tubers showing black scurf and rice plants affected by sheath blight were collected from different regions of the state and the isolates of R. solani so obtained were studied to determine their variability and to ascertain their cross-infectivity and response to fungicides. Potato isolates of R. solani did not infect rice plants but some rice isolates were weakly pathogenic on potato, the sclerotia being less firmly attached on tuber surface, indicating a possible unsuccessful attempt of rice isolates to infect potato. Rice isolates (66.6%) grew faster (>20 mm colony growth per 24 h) than those of the potato isolates (15–20 mm growth rate per 24 h). Hyphal width of isolates from both hosts varied from 7.2 to 12.1 μm. Colony growth of most potato isolates (61.2%) was appressed, whereas that of most rice isolates (53.3%) was fluffy. Rice isolates (73.3%) formed larger sclerotia (1.5–2.0 mm in diameter) than those of the potato isolates (0.5–1.0 mm in diameter). Anastomosis studies indicated that potato isolates belonged to AG-3 and AG-5 groups while rice isolates belonged to the AG-1-1-A group. Representative R. solani isolates from the two hosts showed significant variation in response to fungicides (i.e. carbendazim, carboxin, pencycuron, propiconazole and validamycin) based on their ED₅₀ and ED₉₀ values.     

Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives

A new series of betulinic acid derivatives have been synthesized by introducing heterocyclic ring between C-2 and C-3 positions of betulinic acid. Further modifications were also carried out by reduction of C-20(29) unsaturated bond and substitution of C-28 carboxyl group by ester and amide linkage to enhance the selectivity. Compound 11 resulted in IC(50) of 2.44, 2.5, and 2.7 microg/ml on MIAPaCa, PA-1, and SW620 cancer cell lines, respectively. Compound 38 resulted in IC(50) of 0.67 microg/ml on MIAPaCa cell line.     

Place prioritization for biodiversity conservation using probabilistic surrogate distribution data

We analyse optimal and heuristic place prioritization algorithms for biodiversity conservation area network design which can use probabilistic data on the distribution of surrogates for biodiversity. We show how an Expected Surrogate Set Covering Problem (ESSCP) and a Maximal Expected Surrogate Covering Problem (MESCP) can be linearized for computationally efficient solution. For the ESSCP, we study the performance of two optimization software packages (XPRESS and CPLEX) and five heuristic algorithms based on traditional measures of complementarity and rarity as well as the Shannon and Simpson indices of α‐diversity which are being used in this context for the first time. On small artificial data sets the optimal place prioritization algorithms often produced more economical solutions than the heuristic algorithms, though not always ones guaranteed to be optimal. However, with large data sets, the optimal algorithms often required long computation times and produced no better results than heuristic ones. Thus there is generally little reason to prefer optimal to heuristic algorithms with probabilistic data sets.     

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.     

Creation of salt-insensitive 3'(2'),5'-bisphosphate nucleotidase by modeling and mutagenesis approach

3′(2′),5′-Bisphosphate nucleotidase, (EC 3.1.3.7) (BPntase) is a ubiquitous enzyme. Recently, these enzymes have drawn considerable attention as in vivo targets of salt toxicity as well as therapeutic targets of lithium that is used for the treatment of manic-depressive disorders. They belong to the Mg²⁺-dependent Li⁺-sensitive phosphomonoesterase super-family and are highly sensitive to lithium and sodium ions. However, the molecular mechanism of inhibition of this group of enzymes by monovalent cations has not been completely understood. Previously we have identified a BPntase (Dhal2p) from a highly halotolerant yeast Debaryomyces hansenii. Molecular characterization revealed a number of unique features in Dhal2p, indicating this is an extraordinary member of the family. In this study, we have carried out the structure-function analysis of Dhal2p through the combination of molecular modeling and in vitro mutagenesis approach. We have not only provided the explanation for the role played by the functionally important elements that are conserved among the members of this family but also identified important, novel structural elements in this enzyme. Our study for the first time unraveled the role of a flap as well as a loop region in the functioning of this enzyme. Most importantly, mutations in the loop region resulted in the creation of a BPntase that was insensitive to salt.     

Genetic deletion of the tumor necrosis factor receptor p60 or p80 abrogates ligand-mediated activation of nuclear factor-kappa B and of mitogen-activated protein kinases in macrophages.

Tumor necrosis factor (TNF) is a pleiotropic cytokine known to regulate cell growth, viral replication, inflammation, immune system functioning, angiogenesis, and tumorigenesis. These effects are mediated through two different receptors, TNFR1 and TNFR2 (also called p60 and p80, respectively), with p60 receptor being expressed on all cell types and p80 receptor only on cells of the immune system and on endothelial cells. Although the role of p60 receptor in TNF signaling is well established, the role of p80 is less clear. In this report, by using macrophages derived from wild-type mice (having both receptors) and mice in which the gene for either p60 (p60(-/-)), or p80 (p80(-/-)), or both (p60(-/-) p80(-/-)) receptor have been deleted, we have redefined the role of these receptors in TNF-induced activation of nuclear factor (NF)-kappa B and of mitogen-activated protein kinases. TNF activated NF-kappa B in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. These results correlated with the I kappa B alpha degradation needed for NF-kappa B activation. We also found that TNF activated c-Jun N-terminal protein kinase in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF activated p38 MAPK and p44/p42 MAPK in wild-type but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF induced the proliferation of wild-type macrophages, but for p60(-/-) and p80(-/-) macrophages proliferation was lower, and in p60(-/-) p80(-/-) it was absent. Overall, our studies suggest that both types of TNF receptors are needed in macrophages for optimum TNF cell signaling.     

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.