PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of ΔpepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of ΔpepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.     

Energy deprivation by silibinin in colorectal cancer cells

Small molecules with the potential to initiate different types of programmed cell death could be useful ‘adjunct therapy’ where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨ_m) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT–MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly ‘double-edged sword’ against CRC cells thereby further advocating its clinical effectiveness against this malignancy.     

A STUDY OF ABANDONED ASH PONDS RECLAIMED THROUGH GREEN COVER DEVELOPMENT

Green capping is one of the popular methods to re-vegetate abandoned ash ponds of coal based thermal power plants thereby lowering the risk of contamination to the surrounding environment. It has innumerable advantages such as prevention of dust emission, checking soil erosion, stabilizing the surface areas of ash, preventing potential ground water contamination, and finally, adding native vegetation cover, which is very vital in the long term. During the early nineties and later, various reclamation projects were carried out on fly ash dumps, but until date, there have not been any initiatives to assess the alterations in physicochemical and biological properties of fly ash resulting from implementation of these reclamation projects. In the present study, three abandoned ash ponds, located in India, that were reclaimed during 1998–2003 are investigated. Marked alterations in nutritional status, microbial population, and microbial activities have been observed in reclaimed ash ponds.     

Cryptic species,taxonomic inflation,or a bit of both? New species phenomenon in Sri Lanka as suggested by a phylogeny of dwarf geckos (Reptilia,Squamata, Gekkonidae,Cnemaspis)

We evaluated the status of 16 of 22 recognized Sri Lankan Cnemaspis Strauch species, and flagged overlooked diversity with two mitochondrial (cyt b & ND2) and two nuclear markers (RAG1 & PDC) totalling 2829 base pairs. A fossil-calibrated timetree and sampling of other South Asian Cnemaspis provide insights into the diversification of the genus in peninsular India and Sri Lanka. Phylogenetic analyses consistently inferred two broad clades within South Asian Cnemaspis, with Sri Lankan species in two clades, which we call the podihuna and kandiana clades. Each Sri Lankan clade as a whole is sister to Indian taxa and nested within Indian lineages. Cnemaspis modigliani Das from Indonesia is a member of the kandiana clade. This suggests a minimum of two dispersal events between India and Sri Lanka and one between Sri Lanka/India and South-east Asia. South Asian Cnemaspis date back to at least the Eocene, in Sri Lanka to the early Miocene, with late Miocene diversification in the kandiana clade. All but one of the named species we sampled is likely to be valid, and 10 divergent unnamed lineages may warrant specific recognition. A resolution of Sri Lankan Cnemaspis taxonomy will require thorough sampling and the use of both morphological and molecular data.     

Genetic Predisposition for Development of Nephropathy in Type 2 Diabetes Mellitus

The aim of the study was to explore the association of the angiotensin-converting enzyme (ACE) gene I/D polymorphism and the methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with development of diabetic nephropathy in type 2 diabetes mellitus. Three groups were recruited during 2007–2011: 232 normal controls, 185 type 2 diabetics without nephropathy, and 407 type 2 diabetics with nephropathy. The ACE I/D and MTHFR C677T polymorphisms were examined using PCR and PCR-RFLP methods. We found no significant association of the ACE I/D polymorphism with diabetic nephropathy in genotype, allele, dominant, and recessive models. We observed a significant association of MTHFR C677T with development of diabetic nephropathy in type 2 diabetics. The MTHFR C677T polymorphism plays a significant role in predisposition of renal insufficiency in diabetic patients.     

Effect of short-term heat stress on growth, physiology and antioxidative defence system in wheat seedlings

An experiment was conducted to find out the effect of short-term heat stress on morpho-physiological characters and antioxidants in 10 diverse wheat genotypes. Seed were aseptically grown in test tubes containing filter paper whose lower half was dipped in one-fourth MS media. Heat stress conditions were created by exposing the seedlings at 45 °C for 2 h after 7 days of their germination. Measurements were taken after 3 days of treatment. Heat stress significantly reduced the shoot dry mass, root dry mass, shoot length and root length in all the genotypes. The chlorophyll content and membrane stability index decreased, whereas proline content increased in heat-treated plants. There was significant increase in the activity of catalase, guaiacol peroxidase and superoxide dismutase under stress conditions. The genotypic variations were also significant. On the basis of a coordinated simulation of all these parameters, wheat genotypes Raj 4037 and PBW 373 were identified as tolerant to high temperature stress. The study provides evidence that the tolerant genotypes were equipped with better management of physiological processes along with an efficient antioxidative defence system, sensitivity of which can be evaluated to a sufficient level of certainty at seedling stage.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide as hybrid antiprotozoal agents

A new series of 4-aminochloroquinoline based sulfonamides were synthesized and evaluated for antiamoebic and antimalarial activities. Out of the eleven compounds evaluated (F1–F11), two of them (F3 and F10) showed good activity against Entamoeba histolytica (IC₅₀ <5 μM). Three of the compounds (F5, F7 and F8) also displayed antimalarial activity against the chloroquine-resistant (FCR-3) strain of Plasmodium falciparum with IC₅₀ values of 2 μM. Compound F7, whose crystal structure was also determined, inhibited β-haematin formation more potently than quinine. To further understand the action of hybrid molecules F7 and F8, molecular docking was carried out against the homology model of P. falciparum enzyme dihydropteroate synthase (PfDHPS). The complexes showed that the inhibitors place themselves nicely into the active site of the enzyme and exhibit interaction energy which is in accordance with our activity profile data. Application of Lipinski ‘rule of five’ on all the compounds (F1–F11) suggested high drug likeness of F7 and F8, similar to quinine.