Purification and characterization of a thermostable mycelial lectin from basidiomycete Lentinus squarrosulus

Lectins are non-immune carbohydrate-binding proteins or glycoproteins with specific binding sites for certain glycoconjugates. Fungal lectins have been documented for their antitumour, antiproliferative, immunomodulatory, hypotensive and insecticidal effects. In the present study, a mycelial lectin having molecular mass 55 kDa has been purified and characterized from Lentinus squarrosulus. Biological action spectrum of the lectin revealed agglutination of all human blood types (A, B, O, AB), goat, sheep, rabbit and pig erythrocytes. Neuraminidase treatment of blood type O erythrocytes considerably augmented hemagglutination titre. Carbohydrate inhibition studies showed its high affinity to mucin and asialofetuin. Lectin was purified by a combination of ammonium sulphate precipitation, dialysis, ion exchange chromatography and gel filtration chromatography. Optimum pH for lectin activity was observed to be 6.5–8.0 and optimum temperature was 25–30°C. Lectin showed poor pH stability and was stable within pH 7.0–7.5. It was highly thermostable and could withstand temperature upto 70°C. Lectin activity was sensitive to ethylenediaminetetraacetic acid and denaturants.     

Biosynthesis of silver nanoparticles by natural precursor from clove and their antimicrobial activity

Silver nanoparticles (AgNPs) have attracted the attention of researchers because of their unique properties and applications in various fields, such as medicine, catalysis, textile engineering, and pollution treatment. The green synthesis of AgNPs has many advantages, such as less time requirement, highly stable AgNPs, better control over crystal growth, morphology, ease for scale up, and economic viability. Syzygium aromaticum (clove) was used for the extracellular biosynthesis of AgNPs. Eugenols are the active biomolecules present in clove, responsible for the bioreduction of AgNO₃ (Ag⁺) leading to the formation and capping of AgNPs (Ag⁰). One molecule of eugenol releases two electrons and these two electrons will be taken by 2 Ag⁺ ions and these will get reduced to 2 Ag⁰. The synthesis of AgNPs was confirmed by the appearance of brown colour. The synthesized AgNPs were characterised by various techniques, such as UV-VIS spectroscopy, transmission electron microscopy, X-ray diffraction and Fourier transformed infrared spectroscopy. The synthesised AgNPs have λ _max of 440 nm. It was evaluated that the AgNPs were biphasic in nature (cubic + hexagonal) with an average size of 50.0 nm. The synthesized AgNPs showed significant antimicrobial activity against Bacillus cereus NCDC 240 as they are nano-sized and have high surface area to volume ratio. AgNPs inhibit the growth of bacteria by various ways, such as by disrupting the cell membrane of bacteria, uncoupling the oxidative phosphorylation, inhibiting the DNA replication, forming free radicals and affecting the cellular signalling of bacteria leading to cell death.     

Symposium Report: International Symposium on Ciliate Biology,India Habitat Centre,New Delhi,India, 04–06 April 2018

Ciliated protists have attracted wide interest among researchers from the Indian subcontinent in the last few years. An International Symposium on Ciliate Biology (ISCB) 2018 was held on 04–06 April 2018 at the India Habitat Centre, New Delhi, India. The symposium represented a synergy with International Research Coordination Network for Biodiversity of Ciliates (IRCN‐BC), an affiliate society of International Society of Protistologists (ISOP). The symposium provided a platform for Indian and International delegates to exchange knowledge, present their latest research findings, and establish collaborations as well as creating a networking opportunity for undergraduate and postgraduate students. Nine foreign delegates from 5 countries and 300 Indian delegates actively participated in the event which included 22 oral and 57 poster presentations.     

The primary diterpene synthase products of Picea abies levopimaradiene/abietadiene synthase (PaLAS) are epimers of a thermally unstable diterpenol

The levopimaradiene/abietadiene synthase from Norway spruce (Picea abies; PaLAS) has previously been reported to produce a mixture of four diterpene hydrocarbons when incubated with geranylgeranyl diphosphate as the substrate: levopimaradiene, abietadiene, neoabietadiene, and palustradiene. However, variability in the assay products observed by GC-MS of this and orthologous conifer diterpene synthases over the past 15 years suggested that these diterpenes may not be the initial enzyme assay products but are rather the products of dehydration of an unstable alcohol. We have identified epimers of the thermally unstable allylic tertiary alcohol 13-hydroxy-8(14)-abietene as the products of PaLAS. The identification of these compounds, not previously described in conifers, as the initial products of PaLAS has considerable implications for our understanding of the complexity of the biosynthetic pathway of the structurally diverse diterpene resin acids of conifer defense.     

Combined solid state and solution NMR studies of α,ɛ-<Superscript>15</Superscript>N labeled bovine rhodopsin

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of α,ɛ-¹⁵N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state ¹³C,¹⁵N-REDOR and HETCOR experiments of all possible ¹³C′ _i-1 carbonyl/¹⁵N _i -tryptophan isotope labeled amide pairs, and H/D exchange ¹H,¹⁵N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone ¹⁵N nuclei and partially to their bound protons. ¹H,¹⁵N chemical shift assignment was achieved for indole side chains of Trp35^1.30 and Trp175^4.65. ¹⁵N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175^4.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.     

PEPstr: a de novo method for tertiary structure prediction of small bioactive peptides

Among secondary structure elements, beta-turns are ubiquitous and major feature of bioactive peptides. We analyzed 77 biologically active peptides with length varying from 9 to 20 residues. Out of 77 peptides, 58 peptides were found to contain at least one beta-turn. Further, at the residue level, 34.9% of total peptide residues were found to be in beta-turns, higher than the number of helical (32.3%) and beta-sheet residues (6.9%). So, we utilized the predicted beta-turns information to develop an improved method for predicting the three-dimensional (3D) structure of small peptides. In principle, we built four different structural models for each peptide. The first 'model I' was built by assigning all the peptide residues an extended conformation (phi = Psi = 180 degrees ). Second 'model II' was built using the information of regular secondary structures (helices, beta-strands and coil) predicted from PSIPRED. In third 'model III', secondary structure information including beta-turn types predicted from BetaTurns method was used. The fourth 'model IV' had main-chain phi, Psi angles of model III and side chain angles assigned using standard Dunbrack backbone dependent rotamer library. These models were further refined using AMBER package and the resultant C(alpha) rmsd values were calculated. It was found that adding the beta-turns to the regular secondary structures greatly reduces the rmsd values both before and after the energy minimization. Hence, the results indicate that regular and irregular secondary structures, particularly beta-turns information can provide valuable and vital information in the tertiary structure prediction of small bioactive peptides. Based on the above study, a web server PEPstr (http://www.imtech.res.in/raghava/pepstr/) was developed for predicting the tertiary structure of small bioactive peptides.     

CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels

Despite being synthesized in the cytosol without a leader sequence, the soluble 253-residue mammalian protein CLIC4 (Chloride Intracellular Channel 4, or p64H1), a structural homologue of Omega-type glutathione-S-transferase, autoinserts into membranes to form an integral membrane protein with ion channel activity. A predicted transmembrane domain (TMD) near the N-terminus of CLIC4 could mediate membrane insertion, and contribute to oligomeric pores, with minimal reorganization of the soluble protein structure. We tested this idea by reconstituting recombinant CLIC4 in planar bilayers containing phosphatidyethanolamine, phosphatidylserine and cholesterol, recording ion channels with a maximum conductance of approximately 15 pS in KCl under both oxidizing and reducing conditions. The channels discriminated poorly between anions and cations, incompatible with the current "CLIC" nomenclature, and their conductance was modified by the trans (external or luminal) redox potential, as previously observed for CLIC1. We then reconstituted a truncated version of the protein, limited to the first 61 residues containing the predicted TMD. This included a single trans cysteine residue in the putative pore-forming subunits, at the external entrance to the pore. The truncated protein formed non-selective channels with a reduced conductance, but they retained their trans-redox sensitivity, and could still be blocked or inactivated by trans (not cis) thiol-reative dithiobisnitrobenzoic acid. We suggest that oligomers containing the putative TMD are essential components of the CLIC4 pore. However, the pore is inherently non-selective, and any ionic selectivity in CLIC4 (and other membrane CLICs) may be attributable to other regions of the protein, including the channel vestibules.     

A de novo reciprocal t(2;18) translocation with regular trisomy 21

A 4-year-old girl with Down syndrome exhibited an autosomal translocation t(2;18) in addition to trisomy 21. An evaluation of GTG-banded metaphases revealed the karyotype 47,XX,t(2;18),21 that was confirmed by using fluorescent in situ hybridization (FISH) probes. This case represents a very rare coincidence of an autosomal aneuploidy and a structural rearrangement. Her parents showed a normal chromosome complement. The translocation must have been an apparently "balanced" one as the proband presented with typical features of Down syndrome alone. The mechanism of origin of this rearrangement along with a nondisjunctional error and its significance are discussed.