Effects of biocontrol agents on lipid and protein composition of Indian mustard seeds from plants infected with Alternaria species

Field experiments were carried out with Indian mustard (Brassica juncea L. Cv RLM 1359) to investigate the influence of biocontrol agents on seeds from plants infected with Alternaria blight. The biocontrol agents viz, Trichoderma harzianum, Pseudomonas fluorescens and Bacillus subtilis were applied as seed treatment/seed treatment coupled with spray on 30 and 60 days after sowing of seeds in experimental fields. The plants treated with different biocontrol agents were more developed than non-treated plants throughout the experiment. Biochemical analysis revealed that application of biocontrol agents resulted in increase in lipid and protein content in seeds from treated plants. The proportion of various lipidic fractions i.e. phospholipids, glycolipids and sterol content in seeds increased with a corresponding decrease in total glycerides. The proportion of 18:3, 20:1 and 22:1 fatty acids increased while that of 18:1 and 18:2 fatty acids decreased in seeds with application of biocontrol agents. There were both qualitative and quantitative differences in the banding patterns of albumin and globulin proteins after application of biocontrol agents. The data suggested that biochemical alterations in the host induced by treatment with biocontrol agents could be associated with defence mechanisms and enhanced growth of the plant.     

Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids

Planta - Endophytes reside in different parts of the poppy plant and perform the tissue-specific functions. Most leaf endophytes modulate photosynthetic efficiency, plant growth, and productivity...     

Correlating single cell motility with population growth dynamics for flagellated bacteria

Many bacteria used for biotechnological applications are naturally motile. Their "bio-nanopropeller" driven movement allows searching for better environments in a process called chemotaxis. Since bacteria are extremely small in size compared to the bulk fluid volumes in bioreactors, single cell motility is not considered to influence bioreactor operations. However, with increasing interest in localized fluid flow inside reactors, it is important to ask whether individual motility characteristics of bacteria are important in bioreactor operations. The first step in this direction is to try to correlate single cell measurements with population data of motile bacteria in a bioreactor. Thus, we observed the motility behavior of individual bacterial cells, using video microscopy with 33 ms time resolution, as a function of population growth dynamics of batch cultures in shake flasks. While observing the motility behavior of the most intensively studied bacteria, Escherichia coli, we find that overall bacterial motility decreases with progression of the growth curve. Remarkably, this is due to a decrease in a specific motility behavior called "running". Our results not only have direct implications on biofilm formations, but also provide a new direction in bioprocess design research highlighting the role of individual bacterial cell motility as an important parameter.     

In vitro formation of organ specific proximate carcinogen of benzo(a)pyrene by rat homogenates

In order to determine the organ specific carcinogenicity of benzo(a)pyrene (B(a)P), its metabolites, formed in vitro by incubation with the homogenates from liver, lungs, kidneys, intestine and brain of rats, were isolated by TLC and spectroscopy. B(a)P was found to be converted into a number of metabolites by different tissue homogenates. The results showed that the proximate carcinogenic metabolite, 7,8-dihydro-7,8-dihydroxy B(a)P was formed only when rat lung and kidney homogenates were incubated with B(a)P in vitro. The UV spectral analysis also confirmed the formation of this metabolite only on incubation of B(a)P with rat lung and kidney homogenates. As the proximate carcinogenic metabolite was only formed by incubating B(a)P with the homogenates from target organs, its organ specific carcinogenicity may be explained.     

Naturally Occurring Genetic Variants in Human Chromogranin A (CHGA) Associated with Hypertension as well as Hypertensive Renal Disease

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.     

Autoreactive T cells in healthy individuals

The presence of autoreactive CD4(+) T cells in the peripheral blood of healthy human subjects was investigated after removal of CD4(+)CD25(+) regulatory T cells (Treg). CD4(+) T cells that were directed against the type 1 diabetes-associated autoantigen glutamic acid decarboxylase 65, the melanocyte differentiation Ag tyrosinase, and the cancer/testis tumor Ag NY-ESO-1 were readily derived from PBMC of healthy individuals. These autoreactive T cells could be visualized, using Ag-specific class II tetramer reagents, in the peripheral blood of most individuals examined. Addition of CD4(+)CD25(+) Treg back to the CD4(+)CD25(-) population suppressed the expansion of the autoreactive T cells. Autoreactive T cells were cloned based on tetramer binding, and expressed characteristic activation markers upon self-Ag stimulation. These results show that autoreactive T cells are present in most healthy individuals and that Treg likely play an important role of keeping these autoreactive T cells in check.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

Role of proteases in the pathophysiology of cardiac disease

Cardiovascular disease is a major cause of death and thus a great deal of effort has been made in salvaging the diseased myocardium. Although various factors have been identified as possible causes of different cardiac diseases such as heart failure and ischemic heart disease, there is a real need to elucidate their role for the better understanding of the cardiac disease pathology and formulation of strategies for developing newer therapeutic interventions. In view of the intimate involvement of different types of proteases in maintaining cellular structure, the role of proteases in various cardiac diseases has become the focus of recent research. Proteases are present in the cytosol as well as are localized in a number of subcellular organelles in the cell. These are known to use extracellular matrix, cytoskeletal, sarcolemmal, sarcoplasmic reticular, mitochondrial and myofibrillar proteins as substrates. Work from different laboratories using a wide variety of techniques has shown that the activation of proteases causes alterations of a number of specific proteins leading to subcellular remodeling and cardiac dysfunction. Inhibition of protease action by different drugs and agents, therefore, has a clinical relevance and is expected to form a part of new treatment paradigm for improving heart function. This review examines the biochemistry and localization of some of the proteases in the cardiac tissue in addition to identification of the sites of action of some protease inhibitors. (Mol Cell Biochem 263: 241–256, 2004)     

Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein "packing" in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core "packing" structure) within the regulated pathway.     

UNC-16 alters DLK-1 localization and negatively regulates actin and microtubule dynamics in Caenorhabditis elegans regenerating neurons

Neuronal regeneration after injury depends on the intrinsic growth potential of neurons. Our study shows that UNC-16, a Caenorhabditis elegans JIP3 homolog, inhibits axonal regeneration by regulating initiation and rate of regrowth. This occurs through the inhibition of the regeneration-promoting activity of the long isoform of DLK-1 and independently of the inhibitory short isoform of DLK-1. We show that UNC-16 promotes DLK-1 punctate localization in a concentration-dependent manner limiting the availability of the long isoform of DLK-1 at the cut site, minutes after injury. UNC-16 negatively regulates actin dynamics through DLK-1 and microtubule dynamics partially via DLK-1. We show that post-injury cytoskeletal dynamics in unc-16 mutants are also partially dependent on CEBP-1. The faster regeneration seen in unc-16 mutants does not lead to functional recovery. Our data suggest that the inhibitory control by UNC-16 and the short isoform of DLK-1 balances the intrinsic growth-promoting function of the long isoform of DLK-1 in vivo. We propose a model where UNC-16’s inhibitory role in regeneration occurs through both a tight temporal and spatial control of DLK-1 and cytoskeletal dynamics.