Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.     

Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology

Numerous factors influence male fertility. Among these factors is oxidative stress (OS), which has elicited an enormous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various metabolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent antioxidant capacity of the organism is distorted. Spermatozoa are particularly sensitive to ROS as their plasma membrane contains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preventive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in the generation of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance the generation of ROS and thus, cause destructive effects on various cellular organelles like mitochondria, sperm DNA etc. This article analyzes the detrimental effects of OS on male fertility, measurement of OS and effective ways to decrease or eliminate them completely. We have also provided information on oxidative stress in other systems of the body, which may be applied to future research in the field of reproductive biology.     

Cdk4 promotes adipogenesis through PPARgamma activation

Cell cycle regulators such as E2F1 and retinoblastoma (RB) play crucial roles in the control of adipogenesis, mostly by controlling the transition between preadipocyte proliferation and adipocyte differentiation. The serine-threonine kinase cyclin-dependent kinase 4 (cdk4) works in a complex with D-type cyclins to phosphorylate RB, mediating the entry of cells into the cell cycle in response to external stimuli. Because cdk4 is an upstream regulator of the E2F-RB pathway, we tested whether cdk4 was a target for new factors that regulate adipogenesis. Here we find that cdk4 inhibition impairs adipocyte differentiation and function. Disruption of cdk4 or activating mutations in cdk4 in primary mouse embryonic fibroblasts results in reduced and increased adipogenic potential, respectively, of these cells. We show that the effects of cdk4 are not limited to the control of differentiation; cdk4 also participates in adipocyte function through activation of PPARgamma.     

TRPC1-mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells

Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca(2+) channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP(+) showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP(+). MPP(+)-induced alpha-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP(+) was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP(+) neurotoxicity, which was partially dependent on external Ca(2+). Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP(+) treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson's disease-inducing agents.