Calpain-1 cleaves Rad21 to promote sister chromatid separation

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.     

Molecular characterization of distinct YMV (Yellow mosaic virus) isolates affecting pulses in India with the aid of coat protein gene as a marker for identification

The present study was carried out to find out the variations present in different isolates of yellow mosaic virus (YMV) causing yellow mosaic disease of pulses in southern parts of India. The coat protein gene of YMV was amplified using gene specific and deng universal primers with DNA isolated from YMV infected samples. Further, cloning and DNA sequencing of CP gene was carried out. CP gene decrypt sequences revealed that YMV infected samples of Black gram, Cowpea and Green gram were similar to the MYMV-Tamil Nadu isolates. Whereas the YMV infected sample of Horse gram was found to be similar with HYMV. Hence, in the present study, two distinct YMV infecting pulses in Tamil Nadu (MYMV and HYMV species) were identified and it was observed that there exists considerable genetic variation among these species. In addition, Cowpea crop which was earlier supposed not to be susceptible for YMV infection also showed the presence of this virus similar to the MYMV. Overall, the findings of the present study indicate that the CP region is efficient enough to provide a simple, rapid, and reliable method for early detection of YMV infections in pulses, which would help to develop proper management strategies to control these viruses.     

Design of EGFR kinase inhibitors: a ligand-based approach and its confirmation with structure-based studies

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for 100 anilinoquinazolines, inhibiting epidermal growth factor receptor (EGFR) kinase. The studies included molecular field analysis (MFA) and receptor surface analysis (RSA). The cross-validated r2 (r2cv) values are 0.81 and 0.79 for MFA and RSA, respectively. The predictive ability of these models was validated by 28 test set molecules. The results of the best QSAR model were further compared with structure-based investigations using docking studies with the crystal structure of EGFR kinase domain. The results helped to understand the nature of substituents at the 6- and 7-positions, thereby providing new guidelines for the design of novel inhibitors.     

Brevibacillus as a biological tool: a short review

The significance of Brevibacillus has been documented scientifically in the published literature and commercially in heterologous recombinant protein catalogs. Brevibacillus is one of the most widespread genera of Gram-positive bacteria, recorded from the diverse environmental habitats. The high growth rate, better transformation efficiency by electroporation, availability of shuttle vectors, production of negligible amount of extracellular protease, and the constitutive expression of heterologous proteins make some strains of this genus excellent laboratory models. Regarding biotechnological applications, this genus continues to be a source of various enzymes of great biotechnological interest due to their ability to biodegrade low density polyethylene, ability to act as a candidate bio-control agent, and more recently acknowledged as a tool for the overexpression. This article reviews the properties of Brevibacillus spp. as better biological tools with varied applications.     

Use of monoacetyl-4-hydroxyaminoquinoline 1-oxide to probe contacts between guanines and protein in the minor and major grooves of DNA. Interaction of Escherichia coli integration host factor with its recognition site in the early promoter and transposition enhancer of bacteriophage Mu.

Monoacetyl-4-hydroxyaminoquinoline 1-oxide (Ac-HAQO) reacts with DNA to form adducts at the C8- and N2-positions of guanine and with the N6-position of adenine. Only the N2-guanine adduct blocks the 3'-5' exonuclease action of phage T4 DNA polymerase. Piperidine treatment cleaves the DNA at sites bearing C8-guanine adducts. The N2-position of guanine lies in the minor groove of DNA, whereas the C8-position of guanine occupies the major groove. We have taken advantage of these characteristics to employ Ac-HAQO in conjunction with either T4 DNA polymerase or piperidine in a footprinting technique to probe the interaction of the Escherichia coli integration host factor (IHF) with its binding site. We show that when IHF binds to its recognition site both the N2- and C8-positions of guanines are protected from modification by AcHAQO. In addition, the binding of IHF to DNA was prevented when either an N2- or a C8-AQO adduct was present in the binding site. When dimethylsulfate was used as the footprinting reagent, IHF protected against methylation of the N3 position of adenine in the minor groove but not the N7 position of guanine in the major groove. The difference in results obtained with the two reagents is ascribed to their relative sizes. Both DMS and AcHAQO are excluded by IHF from the minor groove, but only the larger AcHAQO molecule is excluded from the major groove.     

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.     

In vitro biotechnological advancements in Malabar nut (Adhatoda vasica Nees): Achievements,status and prospects

Adhatoda vasica Nees, belonging to family Acanthaceae, is a well-known medicinal plant. It is endorsed for its pyrroloquinazoline alkaloids and its derivatives, such as vasicine and vasicinone. Germinating A. vasica seeds is a tedious task; on that account, vegetative propagation is the preferred method for its multiplication. For rapid and large-scale multiplication, germplasm conservation as well as secondary metabolites production, in vitro culture of A. vasica was preferred over conventional propagation by several researchers; however, some major applications of this tissue culture technique are still awaiting to undergo extensive research. The present review, for the first time, illustrates all the major achievements associated with in vitro regeneration of A. vasica, reported till date and highlights the future prospects.     

Ageing of chloroplasts in vitro — III. Comparison of the effects of ageing in vitro and senescence on the red absorption band and primary photochemical reactions of sunflower chloroplasts

A comparison of changes in absorption properties and electron transport activities of chloroplasts ageing in vivo and in vitro is made. Chloroplasts from sunflower leaves senescing in vivo during 7 days in dark do not show a blue shift of the red absorption band; in contrast, the shift becomes apparent within 24 h of in vitro ageing of isolated organelles. Photosynthetic activity by chloroplasts is lost much faster during in vitro than in vivo ageing. During in vitro ageing, the rate of degradation of thylakoid membranes as characterised by the shift in the red absorption band and loss in Hill reaction is further accelerated in chloroplasts isolated from dark-induced senescing leaves, suggesting the influence of the in vivo status of the chloroplasts on their in vitro stability.Abbreviations DCPIP 2,6-dichlorophenol indophenol - PSI Photosystem I - Chl Chlorophyll