Population structure of elephant foot yams (Amorphophallus paeoniifolius (Dennst.) Nicolson) in Asia.

The corms and leaves of elephant foot yams (Amorphophallus paeoniifolius (Dennst.) Nicolson) are important foods in the local diet in many Asian regions. The crop has high productivity and wide agroecological adaptation and exhibits suitability for the agroforestry system. Although the plant is assumed to reproduce via panmixia, a comprehensive study on the genetic background across regions to enhance wider consumer palatability is still lacking. Here, ten informative microsatellites were analyzed in 29 populations across regions in India, Indonesia and Thailand to understand the genetic diversity, population structure and distribution to improve breeding and conservation programs. The genetic diversity was high among and within regions. Some populations exhibited excess heterozygosity and bottlenecking. Pairwise FST indicated very high genetic differentiation across regions (FST = 0.274), and the Asian population was unlikely to be panmictic. Phylogenetic tree construction grouped the populations according to country of origin with the exception of the Medan population from Indonesia. The current gene flow was apparent within the regions but was restricted among the regions. The present study revealed that Indonesia and Thailand populations could be alternative centers of the gene pool, together with India. Consequently, regional action should be incorporated in genetic conservation and breeding efforts to develop new varieties with global acceptance.

Rock inhabiting potassium solubilizing bacteria from Kerala, India: characterization and possibility in chemical K fertilizer substitution.

The role of rock inhabiting bacteria in potassium (K) solubilization from feldspar and their application in crop nutrition through substitution of fertilizer K was explored through the isolation of 36 different bacteria from rocks of a major hill station at Ponmudi in Thiruvananthapuram, Kerala, India. A comprehensive characterization of K solubilization from feldspar was achieved with these isolates which indicated that the K solubilizing efficiency increases with decrease in pH and increase in viscosity and viable cell count. Based on the level of K solubilization, two potent isolates were selected and identified as Bacillus subtilis ANctcri3 and Bacillus megaterium ANctcri7. Exopolysaccharide production, scanning electron microscopic and fourier transform infrared spectroscopic studies with these efficient strains conclusively depicted the role of low pH, increase in viscosity, and bacterial attachment in K solubilization. They were also found to be efficient in phosphorus (P) solubilization, indole acetic acid production as well as tolerant to wide range of physiological conditions. Moreover, the applicability of K containing rock powder as a carrier for K solubilizing bacteria was demonstrated. A field level evaluation on the yield of a high K demanding tuberous vegetable crop, elephant foot yam (Amorphophallus paeoniifolius (dennst.) nicolson) established the possibility of substituting chemical K fertilizer with these biofertilizer candidates successfully.

Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real-time PCR assay.

Conventional and real-time PCR assays were developed for sensitive and specific detection of Phytophthora colocasiae, an oomycete pathogen that causes leaf blight and corm rot of taro. A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. In conventional PCR, the lower limit of detection was 50 pg DNA, whereas in real-time PCR, the detection limit was 12.5 fg for the primer based on Ypt1 gene. The cycle threshold values were linearly correlated with the concentration of the target DNA (range of R(2) = 0.911-0.999). All the primer sets were successful in detecting P. colocasie from naturally infected leaves and tubers of taro. Phytophthora colocasiae was detected from artificially infested samples after 18 and 15 h of postinoculation in conventional and real-time PCR assay, respectively. The developed PCR assay proved to be a robust and reliable technique to detect P. colocasiae in taro planting material and for assessing the distribution of pathogen within fields, thus aid in mitigating taro leaf blight.

Genetic diversity of Phytophthora colocasiae isolates in India based on AFLP analysis.

Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro which is widely distributed in India. Inter and intra-specific genetic diversity among P. colocasiae isolates collected from same field was assessed using amplified fragment length polymorphism (AFLP) marker. Seven primer pairs produced 431 markers, of which 428 (99.2 %) were polymorphic. Considerable genetic variability was displayed by the isolates. The average value of the number of observed alleles, the number of effective alleles, mean Nei's genetic diversity, and Shannon's information index were 1.993, 1.385, 0.261, and 0.420, respectively. Analysis of molecular variance (AMOVA) showed that the majority (85 %) of the diversity were present within populations of P. colocasiae. Dendrogram based on AFLP molecular data using the unweighted pair group method with arithmetic mean (UPGMA) classified the P. colocasiae isolates into two major clusters irrespective of their geographical origin. Clustering was further supported by principle coordinate analysis. Cophenetic correlation coefficient between dendrogram and original similarity matrix was significant (r = 0.816). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.

Cloning and characterization of cDNA encoding an elicitor of Phytophthora colocasiae.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. A cDNA encoding elicitor, the major secreted extracellular glycoprotein of Phytophthora colocasiae, a pathogen of taro (Colocasia esculenta) plants, was isolated, sequenced and characterized. The expression of the corresponding elicitor gene during the disease cycle of P. colocasiae was analyzed. Elicitor was shown to be expressed in mycelium grown in culture media, whereas it was not expressed in sporangiospores and zoospores. In planta, during infection of taro, particularly during the biotrophic stage, expression of elicitor was down-regulated compared to in vitro. The highest levels of expression of elicitor were observed in in vitro grown mycelium and in late stages of infection when profuse sporulation and leaf necrosis occur. The elicitation of the suspension-cultured taro cells was effective in the induction of the enzyme activity of l-phenylalanine-ammonia lyase, peroxidase and lipoxygenase as well as the expression of defense-related endochitinase gene. All these biological activities were exerted within a low concentration range. The glycoprotein represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance. It may also be useful to engineer broad disease protection in taro plant against Phytophthora leaf blight.

Purification and characterization of elicitor protein from Phytophthora colocasiae and basic resistance in Colocasia esculenta.

An elicitor was identified in the fungus Phytophthora colocasiae. The molecular weight of the purified elicitor was estimated by means of gel filtration chromatography and SDS-PAGE and was estimated as 15kDa. Protease treatment severely reduced its activity, allowing the conclusion that the elicitor is proteinaceous. Infiltration of a few nanograms of this proteinaceous elicitor into taro leaves caused the formation of lesions that closely resemble hypersensitive response lesions. The elicitation of the cells was effective in the induction of the activity of lipoxygenase. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, after the infiltration of the elicitor protein. After few days, systemic acquired resistance was also induced. Thus, taro plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence. The obtained results give important information regarding the plant-pathogen interactions, mainly as subsidy for taro improvement against Phytophthora leaf blight.

Identification and characterization of differentially expressed genes in the resistance reaction in taro infected with Phytophthora colocasiae.

Leaf blight disease caused by Phytophthora colocasiae represents a major constraint to the growth and yield of taro (Colocasia esculenta L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecule such as salicylic acid, jasmonic acid, and ethylene. Activation of plant defenses is associated with changes in the expression of large number of genes. To gain a better understanding of defense responses, virulent race of P. colocasiae was used to inoculate the taro cultivar UL-56 (compatible) and its nearly isogenic line Muktakeshi (incompatible). We have employed suppressive subtractive hybridization (SSH), cDNA libraries, Northern blot analysis, high throughput DNA sequencing, and bioinformatics to identify the defense-related genes in taro induced by P. colocasiae infection. Two putative resistance genes and a transcription factor were identified among the upregulated sequences. The expression of several candidate genes including lipid transfer proteins (LTPs), and other pathogenesis-related genes were evaluated following 8-48 h of appearance of symptom in compatible and incompatible interactions. Results confirmed the higher overall expression of these genes in Muktakeshi (resistant) compared to UL-56 (susceptible). This study constitutes the first attempt to characterize the taro differential transcriptome associated with host-pathogen interactions from different genotypes. All the generated ESTs have been submitted to GenBank for further functional studies.

Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion.

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma.

Identification of a novel series of tetrahydrodibenzazocines as inhibitors of 17beta-hydroxysteroid dehydrogenase type 3.

A novel series of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3) inhibitors has been identified. These inhibitors, based on a dibenzazocine core, exhibited picomolar to low nanomolar inhibition of 17beta-HSD3 in cell-free enzymatic as well as in cell-based transcriptional reporter assays.

Synthesis and biological activity of N-aryl-2-aminothiazoles: potent pan inhibitors of cyclin-dependent kinases.

N-Aryl aminothiazoles 6-9 were prepared from 2-bromothiazole 5 and found to be CDK inhibitors. In cells they act as potent cytotoxic agents. Selectivity for CDK1, CDK2, and CDK4 was dependent of the nature of the N-aryl group and distinct from the CDK2 selective N-acyl analogues. The N-2-pyridyl analogues 7 and 19 showed pan CDK inhibitory activity. Elaborated analogues 19 and 23 exhibited anticancer activity in mice against P388 murine leukemia. The solid-state structure of 7 bound to CDK2 shows a similar binding mode to the N-acyl analogues.

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent.

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.

1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases: highly potent 2,6-Difluorophenacyl analogues.

Structure-activity studies of 1H-pyrazolo[3,4-b]pyridine 1 have resulted in the discovery of potent CDK1/CDK2 selective inhibitor 21h, BMS-265246 (CDK1/cycB IC(50)=6 nM, CDK2/cycE IC(50)=9 nM). The 2,6-difluorophenyl substitution was critical for potent inhibitory activity. A solid state structure of 21j, a close di-fluoro analogue, bound to CDK2 shows the inhibitor resides coincident with the ATP purine binding site and forms important H-bonds with Leu83 on the protein backbone.

1H-Pyrazolo[3,4-b]pyridine inhibitors of cyclin-dependent kinases.

1H-Pyrazolo[3,4-b]pyridine 3 (SQ-67563) has been shown to be a potent, selective inhibitor of CDK1/CDK2 in vitro. In cells 3 acts as a cytotoxic agent with the ability to block cell cycle progression and/or induce apoptosis. The solid state structure of 3 bound to CDK2 shows 3 resides coincident with the ATP purine binding site and forms important H-bonding interactions with Leu83 on the protein backbone.

Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities.

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC(50) values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase alpha (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.