10 research outputs found
Structure Analysis Of Plant Lectin Domains
No full textLectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties leading to wide use in research and biomedical applications. Seeds of leguminous plants are mainly rich sources of lectins, but lectins are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. This thesis primarily concerns with structure solution and analysis of lectins from the legume and β-prism II fold families using X-ray crystallography. Apart from having the property of specifically and reversibly binding to carbohydrates, lectins are also interesting models to study sequence-structure relationships, especially of how minor change in the sequence may bring about major changes in oligomerization and binding.
Chapter 1 gives an overview of different structural types of plant lectins and describes in detail, their carbohydrate binding features. The details of the various experimental procedures employed during the course of this research, are explained in Chapter 2.
Chapter 3 describes the crystal structure of a β-prism II fold lectin (RVL), from Remusatia vivipara, an epiphytic plant of traditional medicinal value, and analysis of its binding properties. This lectin was established to have distinct binding properties and has nematicidal activity against a root-knot nematode with the localization site identified as the high-mannose displaying gut-lining in the nematode. The crystal structure of RVL revealed a new quaternary association of this homodimeric lectin, different from those of reported β-prism II lectins. Functional studies on RVL showed that it fails to bind to simple mannose moieties yet showed agglutination with rabbit blood cells (which have mannose moieties on the surface) and some high mannose containing glycoproteins like mucin and asialofetuin. Further, ELISA and glycan array experiments indicated that RVL has high affinity to N-glycans like trimannose pentasaccharide such as in gp120, a capsid glycoprotein of HIV virus, necessary in virus-association with the host cell. The structural basis for this N-glycan binding was revealed through structure analysis and molecular modelling, and it was demonstrated that there are two distinct binding sites per monomer, making RVL a truly multivalent lectin. Evolutionary phylogeny revealed the divergence in the β-prism II fold proteins with regards to the number of sugar-binding regions per domain, oligomerization and specificity.
Chapter 4 deals with the structural studies on a galactose-specific legume lectin (DLL-II) from Dolichos lablab, a leguminous plant. The lectin was found to be a planar tetramer in the crystal structures of the native and ligand bound forms, as expected from our solution studies and phylogenetic analysis. The protein is a heterotetramer with subunits differing only in the presence or absence of a C-terminal helical region at the core of the tetramer. Due to the static disorder in all the crystals, the central helix could be oriented in either direction. Structure analysis of DLL-II proved to be an interesting endeavour as static disorder compounded with twinning in the crystal made the data processing and structure solution a challenging process. Subsequent structure and sequence alignments led to the identification of an adenine-binding pocket in the hydrophobic core of the tetramer. Based on this, DLL-II lectin was co-crystallized with adenine and the structure revealed the presence of adenine at the predicted binding site.
Chapter 5 describes the identification and analysis of potential plant lectins/lectin-like domains in the genome of Oryza sativa, using bioinformatics approaches. This project was initiated to study the occurrence of legume-lectin like domains (a predominant dicot feature) in O. sativa, which is a monocot. Later, a large scale genome analysis for all types of lectin domains was carried out through exhaustive PSI-BLAST, profile matching by HMMer, CDD and MulPSSM. The final validation was carried out by assessing the carbohydrate binding potential of the domain by examining the sugar binding sites. The primary interest in undertaking this work was to find the occurrence of association of these domains with other domains as in protein receptor kinases, where lectin is the receptor domain. Though primarily initiated as a bioinformatics project, further structural characterization was attempted by cloning, expression and purification of some of the annotated lectin proteins using prokaryotic expression systems. The protein expression was attained in reasonable amounts for a few of the annotated legume lectin homologs, however purification is yet to be achieved as the expressed proteins are insoluble.
A part of the results described in this thesis and the other related projects that the author was involved are reported in the following publications.
1) Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity Bhat GG, Shetty KN, Nagre NN, Neekhra VV, Lingaraju S, Bhat RS, Inamdar SR, Suguna K, Swamy BM. 2010. Glycoconjugate J. 27(3):309-320
2) Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin Thamotharan S, Karthikeyan T, Kulkarni KA, Shetty KN, Surolia A, Vijayan M & Suguna K. 2011. Acta Crystallographica D 67(3):218-227
3) Crystal structure of a β-prism II lectin from Remusatia vivipara Shetty KN, Bhat GG, Inamdar SR, Swamy BM, Suguna K. 2012. Glycobiology 22(1): 56-69.
4) Structure of a galactose binding lectin from Dolichos lablab
Shetty KN, Lavanyalatha V, Rao RN, SivaKumar N & Suguna K (Under review)
5) Occurrence of lectin-like domains: Oryza sativa genome analysis. Shetty KN & Suguna K. (Manuscript in preparation
Gestational diabetes and the incidence of diabetes in the 5 years following the index pregnancy in South Indian women
No full textThis study was carried out to examine the incidence of diabetes and the factors associated with this in a cohort of South Indian women 5 years after they were examined for gestational diabetes (GDM). Women (N=630) whose GDM status was determined (Carpenter-Coustan criteria; GDM: N=41) delivered live babies without major anomalies at the Holdsworth Memorial Hospital, Mysore. Of these, 526 women (GDM: N=35) available for follow-up after 5 years underwent a 2-h oral glucose tolerance test and detailed anthropometry. Diabetes was determined using WHO criteria, and Metabolic Syndrome using IDF criteria recommended for south Asian women. The incidence of diabetes (37% versus 2%) and Metabolic Syndrome (60% versus 26%) was considerably higher in women with previous GDM compared to non-GDM women. GDM women who developed diabetes had lower gestational insulin area-under-the-curve (P=0.05). They had larger waist-to-hip ratio, skinfolds, body mass index, and lower 30-min insulin increment at follow-up than other GDM women. In all, history of diabetes in first-degree relatives was independently associated with higher incidence of diabetes (P<0.001). Our findings suggest high diabetes and cardiovascular risks in women with previous GDM. Follow-up of these women after delivery would provide opportunities to modify adverse lifestyle factor
Structural Studies On Winged Bean Agglutinins
No full textLectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties which have led to their wide use in research and biomedical applications. Seeds of leguminous plants are rich sources of lectins, but they are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. The introductory chapter gives a broad overview of lectins from a structural point of view.
The rest of the thesis is primarily concerned with structural studies on lectins from seeds of the winged bean (Psophocarpus tetragonolobus). Winged bean seeds contain a basic lectin (WBAI) (pi > 9.5) and an acidic lectin (WBAII) (pi -5.5). Both these lectins are N-glycosylated homodimers with about 240 amino acid residues per monomer. They show a high affinity for methyl-a-D-galactose at the monosaccharide level but have entirely different affinities for oligosaccharides. WBAI agglutinates human type A and B erythrocytes but not O type, while WBAII binds specifically to the terminally monofucosylated H-antigenic (responsible for O blood group reactivity) determinants on the cell surface. In this context, the current study seeks to characterise the carbohydrate binding site of a saccharide-free form of WBAI and determine the structural basis of carbohydrate recognition in WBAII. The study also aims to identify the factors responsible for the differences in carbohydrate specificities between WBAI and WBAII.
Diffraction data from a saccharide-free crystal form of WBAI and two crystal forms (Form I and II) of WBAII complexed with methyl-a-D-galactose were collected on a MAR imaging plate system mounted on a Rigaku RU200 rotating anode X-ray generator. The data were processed using the MAR-XDS and DENZO/SCALEPACK suites of programs. The structures were solved by the molecular replacement method using AMoRe. The model used in the case of WBAI and Form I of WBAII was the structure of WBAI in complex with methyl-a-D-galactose (PDB coderlWBL), while the structure of Form II of WBAH was solved using a partially refined model of Form I. The refinements and model building were performed using the programs X-PLOR/CNS and O respectively.
A comparison of the structures of the saccharide-free and bound forms of WBAI revealed three water molecules occupying the carbohydrate binding site, which mimic the hydrogen bonded interactions made by the saccharide in the structure of the complex. Also a shift of -0.6 A in the variable loop, towards the saccharide in the structure of the complex was observed. Significant differences in the conformation of a loop involved in crystal packing interactions were also observed. An analysis of protein hydration demonstrates, among other things, the role of water molecules in stabilising the structure of the loops around the carbohydrate binding site.
The crystal structures of the two forms of WBAH were solved at 3.0 A and 3.3. A resolution. The structure of the complex revealed the role of the length of the variable loop in generating the difference in oligosaccharide specificity between WBAI and WB All. The difference in the pi values between the two lectins is caused by substitutions occurring in loops and edges of sheets. A distinct structural difference between WBAH and all the other legume lectins of known structure is in the new disposition of the 34-45 loop with an r.m.s deviation of -6.0A in Coc positions compared to its position in other lectins. This change in conformation is caused by the formation of salt bridges by amino acid residues unique to WB All in the 34-45 loop and its neighbourhood. Thermodynamic studies on the binding of H-antigenic determinant to WBAII showed a predominance of entropic contribution suggesting a hydrophobically driven binding, not yet observed in lectin-sugar interactions. An analysis involving the docking of H-type II trisaccharide (Fuca(l-2)Galf}(l-4)GlcNAc) into the carbohydrate binding site and a comparison with the binding sites of other legume lectins revealed the role of a Tyr in the variable loop and an Asn in the second loop that are unique to WBAII in generating this unique binding property.
Earlier work on peanut lectin and WBAI demonstrated that the modes of dimerisation of legume lectins are governed by features intrinsic to the protein. A phylogenetic analysis of the sequences of all legume lectins whose structures are available has been performed to examine the relationship among the various classes of oligomers and classes of sugar specificity. The information thus obtained showed that groups of legume lectins that share a common mode of dimerisation cluster together. A sequence alignment based on structures revealed amino acid residues unique to each of these clusters that may be important in determining the modes of observed dimerisation.
While pursuing structural studies on WBAI and WBAII, the author has also been involved in an ongoing small molecule project in the laboratory, which involves preparation and X-ray structure determination of the complexes of carboxylic acids with amino acids and peptides. The work carried out in the project is described in the appendix
Validation of Purgation Therapy (Bhedhi) in Pacifying Vatha Kuttram
No full textPatient’s with the symptoms of Vatha were enrolled in the study.
• The author had collected, the review literature for Vatham,Virechanam from classical siddha literatures and Modern aspect of purgation therapy from various texts, Published articles.
• Case sheet and proforma was maintained for all 40 cases.
• Derangement of Uyirthathukkal and Udalthathukkal in the disease had been discussed.
• Ennvagithervugal had studied in detail and their interpretation had done.
• Naadi, Neikuri, Manikkadai Nool before and after treatment were discussed.
• There is no obvious change in Naadi before and after treatment was noticed among patients.
• ManikkadaiNool also shows changes in patients before and after treatment but the results were insignificant.
• Based on patient’s satisfactory rate, about 23% of patients showed better and good relief and about 10% of patients reported to have slight relief.
• Neikuri shows better results after treatment of Vatham was decreased in many patients who showed vatham predominant Neikuri pattern before treatment.
• Better results were shown in patients who had taken oil bath 3 days continuously before taking purgation medicine
• One patient with RA Positive had showed the result of RA Negative after 1 week of taking purgation.
• A patient with Trigeminal Neuralgia showed a better relief after getting purgation for 3 days
Effect of thymoquinone on ethanol and high fat diet induced chronic pancreatitis—a dose response study in rats
No full text292-302A significant increase in serum lipase,
amylase, capase-1 and myeloperoxidase activities, oxidative stress index (OSI),
IL-1β and IL-18 was observed in rats receiving ethanol (EtOH) and high fat diet
(HFD). Thymoquinone (TQ) supplementation along with EtOH and HFD significantly
decreased the levels of serum lipase, amylase, capase-1, myeloperoxidase, OSI
and maintained the antioxidant status when compared to untreated EtOH and HFD
fed rats. Among the 4 doses, 100 mg of TQ/kg body weight was found to provide
optimum protective effect on pancreas against EtOH and HFD induced abnormal changes.
Histological observations added more evidence for the anti-inflammatory effect
of TQ
EXPLORING THE MODERN MYTHOLOGY: ANALYSING KAVITA KANE’S KARNA’S WIFE: THE OUTCAST’S QUEEN AND SITA’S SISTER
No full textIn modern-day literature, the study of mythology has presented authors with a vast and intricate framework to craft elaborate narratives that delve deeply into the intricacies of human behaviour, interpersonal connections, and societal standards. This research paper embarks on a profound and comprehensive analysis of two compelling literary works by the esteemed author Kavita Kane, explicitly focusing on Karna’s Wife: The Outcast’s Queen and Sita’s Sister. Through a meticulous and rigorous examination of the character progression depicted in Karna’s Wife, the primary objective of this study is to uncover and elucidate how Kane adeptly illustrates the transformation of Uruvi’s character, the significant relationships that shape her journey, and the innovative perspective she brings to the enigmatic persona of Karna. Simultaneously, the scrutiny of themes and symbolism in Sita’s Sister delves deeply into the core thematic foundations of the novel, the symbolic elements skilfully utilised by Kane to enhance the storyline, and the crucial influence of familial and societal expectations in moulding the characters within the narrative. By closely analysing these critical components, this research paper strives to illuminate the intricate layers of storytelling and the contemporary reinterpretation of classical mythology in Kane’s literary creations, thereby contributing to the enhanced comprehension of the enduring fascination of mythology within present-day literature
Polyphenol Composition of Nutraceutical Concentrate Obtained from Edible Vegetable Oil Seeds
No full textThis Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page
HR-LCMS Profiling of phytochemical constituents and evaluation of antioxidant, antibacterial, anti-cancerous and anti-inflammatory potentials, plasma biocompatibility and cytotoxicity of Grewia orbiculata Rottler
No full textInfectious diseases are one of the main reasons that are causing a greater number of deaths in the world owing to their strong resistance development and evolution. There is an immediate urgency for the discovery of drugs with a new class or new mode of action to combat these resistant bugs. In the past few decades, we have not been able to find new antibiotics, which are effective on resistant bugs. Instead of searching for synthetic molecules, if we divert our search for alternative sources that are abundant in nature, we can easily find new molecules. Plants are the best as they are known to possess complex molecules that are strong in their potency while being relatively safe for the host and tough on pathogens. With this rationale, the study was conducted to assess the phytochemical constituents of different parts of plant Grewia orbiculataRottler using different solvents and to elucidate the biological activities. From qualitative analysis of all extracts, Methanolic Extract of Bark (MEB) and Ethyl acetate Extract of Leaf (EEL) were found to be rich in total phenolics and total flavonoids. Major phytochemicals found in MEB were Catechin, Epicatechin, and Carnitine and in EEL were Quinin acid, Gallic acid, Catechol, Isoquinoline, Coumaric acid, Kaempferol, and Quercetin of G. orbiculata. Upon testing the biopotentials of these extracts, it was found that among the different solvent extracts of leaves, twigs, buds, and bark, MEB showed the highest biological potential and therapeutic value. The antioxidant property of MEB assessed through DPPH and ABTS assays resulted in an IC50 value of 50 µg/mL and 36 µg/mL, respectively. The metal chelating property of MEB gave a FRAP value of 24 ± 0.093 mmol/g equivalent to that of Tannic acid. Further, MEB was found to possess very good antibacterial activity against human pathogens such as Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, Streptococcus epidermidis, and Mycobacterium smegmatis. In addition, MEB also showed good anti-cancerous property against A549 cells, having IC50 value of 98.73 µg/mL. The anti-inflammatory assay with MEB showed protection of BSA denaturation up to a concentration of 1000 µg/mL. Finally, the biocompatibility assay with blood showed no significant agglutination of RBCs up to a concentration of 200 µg/mL and cytotoxicity of MEB resulted in less than 50 inhibition of HTE cell proliferation at the highest concentration of 320 µg/mL, proving its non-toxic nature towards normal cells. Our study is the first to report and evaluate the therapeutic value of the plant G. orbiculata. MEB was found to possess very good therapeutic potential and can be used as potent antimicrobial agent to treat deadly human infections. © 2022, The Author(s) under exclusive licence to Society for Plant Research
Mutational Analysis in Dystrophin Gene with Dystrophinopathy: A Novel Familial Case Report in Tamil Nadu
No full textMek1Y130C mice recapitulate aspects of human cardio-facio-cutaneous syndrome
No full textThe RAS/MAPK signaling pathway is one of the most investigated pathways, owing to its established role in numerous cellular processes and implication in cancer. Germline mutations in genes encoding members of the RAS/MAPK pathway also cause severe developmental syndromes collectively known as RASopathies. These syndromes share overlapping characteristics, including craniofacial dysmorphology, cardiac malformations, cutaneous abnormalities and developmental delay. Cardio-facio-cutaneous syndrome (CFC) is a rare RASopathy associated with mutations in BRAF, KRAS, MEK1 (MAP2K1) and MEK2 (MAP2K2). MEK1 and MEK2 mutations are found in ∼25% of the CFC patients and the MEK1Y130C substitution is the most common one. However, little is known about the origins and mechanisms responsible for the development of CFC. To our knowledge, no mouse model carrying RASopathy-linked Mek1 or Mek2 gene mutations has been reported. To investigate the molecular and developmental consequences of the Mek1Y130C mutation, we generated a mouse line carrying this mutation. Analysis of mice from a Mek1 allelic series revealed that the Mek1Y130C allele expresses both wild-type and Y130C mutant forms of MEK1. However, despite reduced levels of MEK1 protein and the lower abundance of MEK1 Y130C protein than wild type, Mek1Y130C mutants showed increased ERK (MAPK) protein activation in response to growth factors, supporting a role for MEK1 Y130C in hyperactivation of the RAS/MAPK pathway, leading to CFC. Mek1Y130C mutant mice exhibited pulmonary artery stenosis, cranial dysmorphia and neurological anomalies, including increased numbers of GFAP+ astrocytes and Olig2+ oligodendrocytes in regions of the cerebral cortex. These data indicate that the Mek1Y130C mutation recapitulates major aspects of CFC, providing a new animal model to investigate the physiopathology of this RASopathy.
This article has an associated First Person interview with the first author of the paper
