A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.     

Pyramidodinium spinulosum sp. nov. (Dinophyceae), a sand‐dwelling non‐motile dinoflagellate from the seafloor (36 m deep) off Mageshima Island,Kagoshima, Japan

A new species of benthic marine dinoflagellate, Pyramidodinium spinulosum Horiguchi, Moriya, Pinto & Terada is described from the deep (36 m) seafloor off Mageshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The life cycle of the dinoflagellate consists of a dominant, attached, dome‐shaped, vegetative form and short‐lasting, motile cell. Asexual reproduction takes place by the formation of two motile cells within each non‐motile cell. The released motile cells swim only for a short period and transform directly into the dome‐shaped vegetative form. The duration of the cell cycle varies and can be extremely long, ranging 5–38 days under culture conditions. The non‐motile cell is enclosed by a cell wall and its surface is covered with many (80 – 130) spines of various length. The dinoflagellate is photosynthetic and contains many (more than 50) discoidal chloroplasts. Phylogenetic analysis reveals that the dinoflagellate is closely related to the type species of the genus Pyramidodinium, P. atrofuscum which also possesses a dominant, attached, non‐motile form. However, P. spinulosum can be clearly distinguished from P. atrofuscum by the cell shape (dome‐shaped vs. pyramid‐shaped) and surface ornamentation (spines vs. wart‐like processes) of the non‐motile form. Based on these morphological differences together with molecular evidence, it was concluded that this organism from a deep water sand sample should be described as a second species of the genus Pyramidodinium, P. spinulosum.     

RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato

Potato apical leaf curl disease is an emerging geminiviral disease in tropics and subtropics. It was reported for the first time in the year 1999 in northern plains of India but quickly spread to almost all potato growing regions of the country largely due to prevalence of warmer weather during early crop growth, thereby favoring whitefly vector. The problem of apical leaf curl disease in India became more severe due to lack of seed indexing for this virus in conventional seed production scheme. Although it accounts for major yield loss, there is no conventional source of resistance available in potato against Tomato Leaf Curl New Delhi Virus-Potato (ToLCNDV-Potato) that causes this disease in potato. In the present study, we have investigated the potential use of RNAi for obtaining resistance against this DNA virus in potato. The replication-associated protein gene (AC1) of the virus was used to obtain pathogen-derived resistance. The AC1 gene was PCR amplified from field-infected potato leaves, cloned and sequenced (JN393309). It showed 93% sequence similarity with the AC1 gene of Tomato Leaf Curl Virus-New Delhi (TOLCV-NDe; DQ169056) virus. Transgenic plants encoding the AC1 gene in three different orientations, viz. sense, antisense and hairpin loop, were raised. Transgenic lines when challenge inoculated with ToLCNDV-Potato showed different levels of resistance for all three constructs. Transgene integration and copy number in selected transgenic lines were determined by qPCR and further confirmed by Southern blot analysis. Though a reduction in viral titer was observed in transgenic lines encoding either antisense or hairpin loop constructs of AC1 gene, the latter transgenics showed most significant results as shown by reduction in the level of symptom expression in glasshouse screening as well as real-time data of in vivo virus concentration. In fact, we obtained a few totally asymptomatic transgenic lines with hairpin loop strategy.     

Habitat suitability modeling for the endangered Bengal slow loris (Nycticebus bengalensis) in the Indo-Chinese subregion of India: a case study from southern Assam (India)

Primates - The Bengal slow loris (Nycticebus bengalensis) is an endangered nonhuman primate distributed in Southeast Asia, including India. The species is facing sharp population decline throughout...     

New spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations

Two simple, sensitive and economical spectrophotometric methods were developed for the determination of nifedipine in pharmaceutical formulations. Method A is based on the reaction of the nitro group of the drug with potassium hydroxide in dimethyl sulphoxide (DMSO) medium to form a coloured product, which absorbs maximally at 430 nm. Method B uses oxidation of the drug with ammonium molybdate and subsequently reduced molybdenum blue is measured at 830 nm. Beer's law is obeyed in the concentration range of 5.0-50.0 and 2.5-45.0 microg ml(-1) with methods A and B, respectively. Both methods have been successfully applied for the assay of the drug in pharmaceutical formulations. No interference was observed from common pharmaceutical adjuvants. The reliability and the performance of the proposed methods are established by point and interval hypothesis tests and through recovery studies.     

Jasmonic acid prevents the accumulation of cyclin B1;1 and CDK-B in synchronized tobacco BY-2 cells

Jasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues. In this report, we focus on the molecular events that occur during JA-induced G2 arrest. We demonstrate that JA prevents the accumulation of B-type cyclin-dependent kinases and the expression of cyclin B1;1, which are both essential for the initiation of mitosis. This feature suggests the existence of an early G2 checkpoint that is affected by JA.     

Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.