Pythium soft rot of ginger: Detection and identification of the causal pathogens, and their control

Ginger is considered by many people to be the outstanding member among 1400 other species in the family Zingiberaceae. Not only it is a valuable spice used by cooks throughout the world to impart unique flavour to their dishes but it also has a long track record in some Chinese and Indian cultures for treating common human ailments such as colds and headaches. Ginger has recently attracted considerable attention for its anti-inflammatory, antibacterial and antifungal properties. However, ginger as a crop is also susceptible to at least 24 different plant pathogens, including viruses, bacteria, fungi and nematodes. Of these, Pythium spp. (within the kingdom Stramenopila, phyllum Oomycota) are of most concern because various species can cause rotting and yield loss on ginger at any of the growth stages including during postharvest storage. Pythium gracile was the first species in the genus to be reported as a ginger pathogen, causing Pythium soft rot disease in India in 1907. Thereafter, numerous other Pythium spp. have been recorded from ginger growing regions throughout the world. Today, 15 Pythium species have been implicated as pathogens of the soft rot disease. Because accurate identification of a pathogen is the cornerstone of effective disease management programs, this review will focus on how to detect, identify and control Pythium spp. in general, with special emphasis on Pythium spp. associated with soft rot on ginger.

Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

Impact of a new mandatory reporting law on reporting and identification of child sexual abuse: A seven year time trend analysis

Child sexual abuse is widespread and difficult to detect. To enhance case identification, many societies have enacted mandatory reporting laws requiring designated professionals, most often police, teachers, doctors and nurses, to report suspected cases to government child welfare agencies. Little research has explored the effects of introducing a reporting law on the number of reports made, and the outcomes of those reports. This study explored the impact of a new legislative mandatory reporting duty for child sexual abuse in the State of Western Australia over seven years. We analysed data about numbers and outcomes of reports by mandated reporters, for periods before the law (2006-08) and after the law (2009-12). Results indicate that the number of reports by mandated reporters of suspected child sexual abuse increased by a factor of 3.7, from an annual mean of 662 in the three year pre-law period to 2448 in the four year post-law period. The increase in the first two post-law years was contextually and statistically significant. Report numbers stabilised in 2010-12, at one report per 210 children. The number of investigated reports increased threefold, from an annual mean of 451 in the pre-law period to 1363 in the post-law period. Significant decline in the proportion of mandated reports that were investigated in the first two post-law years suggested the new level of reporting and investigative need exceeded what was anticipated. However, a subsequent significant increase restored the pre-law proportion, suggesting systemic adaptive capacity. The number of substantiated investigations doubled, from an annual mean of 160 in the pre-law period to 327 in the post-law period, indicating twice as many sexually abused children were being identified.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

Chromatographic separation, fractionation and oxidation of selected beta-lactoglobulin peptides

The literature review elucidates the mechanism of oxidation in proteins and amino acids and gives an overview of the detection and analysis of protein oxidation products as well as information about ?-lactoglobulin and studies carried out on modifications of this protein under certain conditions. The experimental research included the fractionation of the tryptic peptides of ?-lactoglobulin using preparative-HPLC-MS and monitoring the oxidation process of these peptides via reverse phase-HPLC-UV. Peptides chosen to be oxidized were selected with respect to their amino acid content which were susceptible to oxidation and fractionated according to their m/z values. These peptides were: IPAVFK (m/z 674), ALPMHIR (m/z 838), LIVTQTMK (m/z 934) and VLVLDTDYK (m/z 1066). Even though it was not possible to solely isolate the target peptides due to co-elution of various fractions, the percentages of target peptides in the samples were satisfactory to carry out the oxidation procedure. IPAVFK and VLVLDTDYK fractions were found to yield the oxidation products reviewed in literature, however, unoxidized peptides were still present in high amounts after 21 days of oxidation. The UV data at 260 and 280 nm enabled to monitor both the main peptides and the oxidation products due to the absorbance of aromatic side-chains these peptides possess. ALPMHIR and LIVTQTMK fractions were oxidatively consumed rapidly and oxidation products of these peptides were observed even on day 0. High rates of depletion of these peptides were acredited to the presence of His (H) and sulfur-containing side-chains of Met (M). In conclusion, selected peptides hold the potential to be utilized as marker peptides in ?-lactoglobulin oxidation.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

An intelligent approach using support vector machines for monitoring and identification of faults on transmission systems

Power system disturbances are often caused by faults on transmission lines. When faults occur in a power system, the protective relays detect the fault and initiate tripping of appropriate circuit breakers, which isolate the affected part from the rest of the power system. Generally Extra High Voltage (EHV) transmission substations in power systems are connected with multiple transmission lines to neighboring substations. In some cases mal-operation of relays can happen under varying operating conditions, because of inappropriate coordination of relay settings. Due to these actions the power system margins for contingencies are decreasing. Hence, power system protective relaying reliability becomes increasingly important. In this paper an approach is presented using Support Vector Machine (SVM) as an intelligent tool for identifying the faulted line that is emanating from a substation and finding the distance from the substation. Results on 24-bus equivalent EHV system, part of Indian southern grid, are presented for illustration purpose. This approach is particularly important to avoid mal-operation of relays following a disturbance in the neighboring line connected to the same substation and assuring secure operation of the power systems.

Synthesis and Identification of a new class of antileukemic agents containing 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole

Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahydrobenzod]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7 tetrahydrobenzod]thiazole moiety, a series of 2-arlycarboxamide substituted-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetrhydrobenzod]thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with ortho substitution showed poor activity and with meta and para substitution showed good activity. (C) 2010 Elsevier Masson SAS. All rights reserved.

Drosophila “enhancer-trap” transposants: Gene expression in chemosensory and motor pathways and identification of mutants affected in smell and taste ability

We have isolated about a thousandDrosophila P-element transposants that allow thein situ detection of genomic enhancer elements by a histochemical assay for β-galactosidase activity. We summarize the β-galactosidase staining patterns of over 200 such transposants in the adult. Our aim was to identify genes that are likely to be involved in the chemosensory and motor pathways ofDrosophila. Based on β-galactosidase expression patterns in the tissues of our interest, we have chosen some strains for further analysis. Behavioral tests on a subset of the transposants have, in addition, identified several strains defective in their chemosensory responses.

Assessment of genetic diversity and identification of core collection in sandalwood germplasm using RAPDS

Sandalwood is an economically important aromatic tree belonging to the family Santalaceae. The trees are used mainly for their fragrant heartwood and oil that have immense potential for foreign exchange. Very little information is available on the genetic diversity in this species. Hence studies were initiated and genetic diversity estimated using RAPD markers in 51 genotypes of Santalum album procured from different geographcial regions of India and three exotic lines of S. spicatum from Australia. Eleven selected Operon primers (10mer) generated a total of 156 consistent and unambiguous amplification products ranging from 200bp to 4kb. Rare and genotype specific bands were identified which could be effectively used to distinguish the genotypes. Genetic relationships within the genotypes were evaluated by generating a dissimilarity matrix based on Ward's method (Squared Euclidean distance). The phenetic dendrogram and the Principal Component Analysis generated, separated the 51 Indian genotypes from the three Australian lines. The cluster analysis indicated that sandalwood germplasm within India constitutes a broad genetic base with values of genetic dissimilarity ranging from 15 to 91 %. A core collection of 21 selected individuals revealed the same diversity of the entire population. The results show that RAPD analysis is an efficient marker technology for estimating genetic diversity and relatedness, thereby enabling the formulation of appropriate strategies for conservation, germplasm management, and selection of diverse parents for sandalwood improvement programmes.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015