Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...     

Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code

The expanded genetic code in combination with site-directed mutagenesis was used to probe spectroscopic and structural roles of tryptophan (Trp) residues in Aequorea victoria green fluorescent proteins (avGFPs). Nine different halogen-, chalcogen-, and methyl-containing Trp isosteric analogues and surrogates were incorporated into avGFPs containing indole moieties in, and outside of, the chromophore, by the use of the selective pressure incorporation method. Such isosteric replacements introduced minimal local geometry changes in indole moieties, often to the level of single atomic exchange ('atomic mutation') and do not affect three-dimensional structures of avGFPs but induce changes in spectral properties. Our approach offers a new platform to re-evaluate issues like resonance transfer, mechanisms of chromophore formation and maturation, as well as the importance of local geometry and weak sulphur-aromatic interactions for avGFP spectral properties and structural stability. The library of novel tailor-made avGFP mutants and variants generated in this work has demonstrated not only the potentials of the expanded genetic code to study spectroscopic functions, but also a new approach to generate tailor-made proteins with interesting and useful spectral properties.     

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

Characterization of the genome of the mealybug Planococcus lilacinus,a model organism for studying whole-chromosome imprinting and inactivation

The co-occurrence of three chromosome-wide phenomena--imprinting, facultative heterochromatization and diffuse centromere--in the mealybug Planococcus lilacinus makes investigation of the genomics of this species an attractive prospect. In order to estimate the complexity of the genome of this species, 300 random stretches of its DNA, constituting approximately 0.1% of the genome, were sequenced. Coding sequences appear to constitute approximately 53.5%, repeat sequences approximately 44.5% and non-coding single-copy sequences approximately 2% of the genome. The proportion of repetitive sequences in the mealybug is higher than that in the fruit fly Drosophila melanogaster (approximately 30%). The mealybug genome (approximately 220 Mb) is about 1.3 times the size of the fly genome (approximately 165 Mb) and its GC content (approximately 35%) less than that of the fly genome (approximately 40%). The relative abundance of various dinucleotides, as analysed by the method of Gentles and Karlin, shows that the dinucleotide signatures of the two species are moderately similar and that in the mealybug there is neither over-representation nor under-representation of any dinucleotide.     

Evidence for <Emphasis Type="Italic">Wolbachia</Emphasis> symbiosis in microfilariae of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> from West Bengal,India

Wolbachia are symbiotic endobacteria that infect the majority of filarial nematodes, including Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus. Recent studies have suggested that Wolbachia are necessary for the reproduction and survival of filarial nematodes and have highlighted the use of antibiotic therapy such as tetracycline/doxycycline as a novel method of treatment for infections caused by these organisms. Before such therapy is conceived and implemented on a large scale, it is necessary to assess the prevalence of the endosymbiont in W. bancrofti from different geographical locations. We present data from molecular and electron microscopic studies to provide evidence for Wolbachia symbiosis in W. bancrofti microfilariae collected from two districts (Bankura and Birbhum) of West Bengal, India.     

Spatio-temporal analysis of development of basal roots of common bean (Phaseolus vulgaris L.)

Temporal development of roots is key to the understanding of root system architecture of plants which influences nutrient uptake, anchorage and plant competition. Using time lapse imaging we analyzed developmental patterns of length, growth angle, depth and curvature of Phaseolus basal roots from emergence till 48 h in two genotypes, {"type":"entrez-nucleotide","attrs":{"text":"B98311","term_id":"3000390","term_text":"B98311"}}B98311 and TLP19 with contrasting growth angles. In both genotypes all basal roots appeared almost simultaneously, but their growth rates varied which accounted for differences in root length. The growth angles of the basal roots fluctuated rapidly during initial development due to oscillatory root growth causing local bends. Beyond 24 h, as the root curvature stabilized, so did the growth angle. Therefore growth angle of basal roots is not a very reliable quantity for characterizing root architecture, especially during early seedling development. Comparatively, tip depth is a more robust measure of vertical distribution of the basal roots even during early seedling development.Key words: basal root, kinematics, root architecture, root growth, spatiotemporal analysis, root imagingVertical and horizontal placements of the roots in the soil influence plant performance through acquisition of below ground resources like water and nutrients, plant anchorage and intra- and inter-plant competition.^1–4 Therefore the architecture of the root system plays important roles in regulating plant growth and yield, especially under abiotic stresses.⁵ As a seedling grows to become a mature plant, the root architecture develops continuously in response to various cues e.g., genotypic, environmental, hormonal, etc. Therefore studies of root architecture of plants of different ages are important for understanding the influence of these cues in regulating plant growth.The root scaffold of a plant is comprised of different types of roots with different functions. A mature common bean (Phaseolus vulgaris L.) plant has root system consisting of primary, adventitious, lateral and basal roots. Among these, the basal roots are typically the earliest emerging secondary roots from the hypocotyl⁶ forming a major part of the mature root system. We have recently demonstrated important differences in architectural traits of the basal roots of common bean in the early seedling stage between two contrasting class of genotypes and how auxin-ethylene interplay regulates these traits.⁷ While this study of basal roots at a fixed time allows assessment and comparison of root development up to that point of time, investigation of the temporal events of emergence and growth of the basal roots is important and complementary to the understanding of their architectural traits. Therefore in the present study, we examined the detailed developmental patterns of basal roots through time lapse imaging in two genotypes.We chose two bean genotypes with contrasting basal root growth angles (BRGA) relative to the gravity—{"type":"entrez-nucleotide","attrs":{"text":"B98311","term_id":"3000390","term_text":"B98311"}}B98311 producing basal roots of smaller BRGA (41.7° ± 14°) and TLP19 having roots of larger BRGA (56.4° ± 18°).⁸ The germinated seedling with 2–3 cm radical was transferred to the blue germination paper (Anchor Paper Co., St. Paul, MN), which was suspended in nutrient solution⁷ inside a growth chamber (ACMAS Technocracy Limited, Delhi, India) maintained at 25 ± 1°C. Time lapse photography was carried out for 48 h at 30 min intervals using Nikon D200 digital camera fitted with a macro lens to obtain high resolution digital images of the roots. Imaging started from the visibility of the protrusions of emerging basal root along the root-shoot interface. A computer program was developed in Matlab^® 7.8 (Mathworks, Natick) to analyze the images semi-automatically. From every image the computer program identified the basal roots using contrast of color between the roots (mostly white) and the germination paper (blue). Root midlines were determined following the methodology of Miller et al.⁹ and smoothed using the method of overlapping polynomials. Length of the midline is root length. The angle between gravity and the line connecting the root tip to the base is BRGA.⁷ The vertical distance of the root tip from the base of the lowest emerging root along the gravity vector is tip depth. From the midline, root curvature was also determined using the equation κ=x′y″−y′x″(x′2+y′2)3/2,(1) where [x(x), y(s)] is coordinate of any point along the root midline, s is normalized distance along the midline, and the primes denote derivatives with respect to s. Here positive curvature signifies bending upward and vice versa.     

Structural basis for regulation of the Crk signaling protein by a proline switch

Proline switches, controlled by cis-trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in the Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory conformation, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch, the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers, thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal new SH3 binding surfaces, highlighting the binding versatility and expanding the noncanonical ligand repertoire of this important signaling domain.     

Adrenocortical involvement during diverse stress in soft-shelled turtle Lissemys p. punctata Bonnoterre

Adrenocortical responses to diverse stressful situations (dehydration, formaldehyde treatment and salt loading) were studied in the adult female soft-shelled turtle, Lissenmys p. punctata. Dehydration, formaldehyde treatment (formalin, 1%: 0.1 ml/100 g body weight daily) or salt loading (NaCl, 1%: 0.1 ml/100 g body weight daily) treatments consecutively for 7 days caused hypertrophy of the adrenocortical cells with their nuclear diameter increased, and depletions of adrenal cholesterol and ascorbic acid concentrations followed by decreased acid phosphatase and alkaline phosphatase activities in turtles. Corticosterone levels were elevated in both the adrenal gland and serum of turtles after dehydration and formalin stress, but the hormone level remained unaltered after salt loading in turtles. The results suggest active involvement of adrenal cortex in stress for homeostasis in Lissemys turtles.