Department of Biochemistry

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    A biosensor generated via high-throughput screening quantifies cell edge Src dynamics
    ( 2011-01-01) Gulyani, Akash ; Vitriol, Eric ; Allen, Richard ; Wu, Jianrong ; Gremyachinskiy, Dmitriy ; Lewis, Steven ; Dewar, Brian ; Graves, Lee M. ; Kay, Brian K. ; Kuhlman, Brian ; Elston, Tim ; Hahn, Klaus M.
    Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high-throughput screening. We made this Src-family kinase (SFK) biosensor by derivatizing a monobody specific for activated SFKs with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and changes to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFKs are activated specifically during protrusion. Activity correlates with velocity, and peaks 1-2 μm from the leading edge. © 2011 Nature America, Inc. All rights reserved.
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    A combined gene signature of hypoxia and Notch pathway in human glioblastoma and its prognostic relevance
    ( 2015-03-03) Irshad, Khushboo ; Mohapatra, Saroj Kant ; Srivastava, Chitrangda ; Garg, Harshit ; Mishra, Seema ; Dikshit, Bhawana ; Sarkar, Chitra ; Gupta, Deepak ; Chandra, Poodipedi Sarat ; Chattopadhyay, Parthaprasad ; Sinha, Subrata ; Chosdol, Kunzang
    Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor - clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O < inf > 2 < /inf > ). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.
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    A comparative study of membrane related phenomena in normal and crown gall tissues of red beet (Beta vulgaris L.)
    ( 1982-11-01) Atchuta Ramaiah, K. V. ; Mookerjee, A.
    Divalent metal ions have been found to protect membranes of red beet crown gall tissue more than their adjacent normal regions from thermal or gamma radiation stress, suggesting the possibility of an alteration on the surface charge accompanying tumor formation in plants. Further, tumor tissue has been observed to possess enhanced membrane ATPase activity, a higher tissue sulfhydryl content and increased protein levels, thus suggesting a model of 'source' (normal tissue) and 'sink' (tumor tissue) relationship. © 1982 Birkhäuser Verlag.
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    A comparative study on in vitro behavior of calcium silicate ceramics synthesized from biowaste resources
    ( 2020-02-01) Palakurthy, Srinath ; Azeem, P. Abdul ; Venugopal Reddy, K. ; Penugurti, Vasudevarao ; Manavathi, Bramanandam
    Calcium silicate ceramics have received significant attention for biomedical applications for their excellent bioactivity and osteoconduction properties. Sol-gel process is extensively used for the fabrication of calcium silicates. In sol-gel process, calcium nitrate tetra hydrate (Ca(NO3)2·4H2O) and tetraethylorthosilicate (TEOS) are used as precursors. However, these precursors are expensive. The objective of this work was to compare in vitro behavior of calcium silicate (CaSiO3) produced using biowaste such as rice husk ash (RHA) and eggshells (coded; NCS) with CaSiO3 prepared using TEOS and Ca(NO3)2·4H2O (coded; CCS). Thermal investigation results revealed that the crystallization temperature for NCS is relatively lower (772°C) than for CCS (870°C). Bioactivity was studied in vitro using simulated body fluid (SBF) with respect to mineralization rate of hydroxyapatite. Mineralization of a greater hydroxyapatite was observed on NCS ceramics than CCS ceramics after incubation for 3, 7, 14 days in SBF solution, which was confirmed using X-ray diffractometer, Fourier transform infrared spectroscopy, scanning electron microscopy-energy dispersive spectroscopy. Degradation studies were conducted in Tris-HCl solution and the test results revealed that NCS ceramics has lower dissolution rate than CCS ceramics. The antimicrobial assay has shown that NCS samples exhibit significant zone of inhibition against Escherichia coli and Staphylococcus aureus which confirmed that the CaSiO3 prepared from RHA and eggshell can prevent bacteria from adhering to the surface. In addition cell culture studies revealed that NCS ceramics possess good cytocompatibility with MG-63 cells and significantly promoted cell proliferation.
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    A conserved R type Methionine Sulfoxide Reductase reverses oxidized GrpEL1/Mge1 to regulate Hsp70 chaperone cycle
    ( 2018-12-01) Allu, Praveen Kumar ; Boggula, Yerranna ; Karri, Srinivasu ; Marada, Adinarayana ; Krishnamoorthy, Thanuja ; Sepuri, Naresh Babu V.
    Cells across evolution employ reversible oxidative modification of methionine and cysteine amino acids within proteins to regulate responses to redox stress. Previously we have shown that mitochondrial localized methionine sulfoxide reductase (Mxr2) reversibly regulates oxidized yeast Mge1 (yMge1), a co-chaperone of Hsp70/Ssc1 to maintain protein homeostasis during oxidative stress. However, the specificity and the conservation of the reversible methionine oxidation mechanism in higher eukaryotes is debatable as human GrpEL1 (hGrpEL1) unlike its homolog yMge1 harbors two methionine residues and multiple cysteines besides the mammalian mitochondria hosting R and S types of Mxrs/Msrs. In this study, using yeast as a surrogate system, we show that hGRPEL1 and R type MSRs but not the S type MSRs complement the deletion of yeast MGE1 or MXR2 respectively. Our investigations show that R type Msrs interact selectively with oxidized hGrpEL1/yMge1 in an oxidative stress dependent manner, reduce the conserved hGrpEL1-Met146-SO and rescue the Hsp70 ATPase activity. In addition, a single point mutation in hGrpEL1-M146L rescues the slow growth phenotype of yeast MXR2 deletion under oxidative duress. Our study illustrates the evolutionarily conserved formation of specific Met-R-SO in hGrpEL1/yMge1 and the essential and canonical role of R type Msrs/Mxrs in mitochondrial redox mechanism.
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    A fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells
    ( 2020-02-01) Mukherjee, Ananya ; Singh, Randhir ; Udayan, Sreeram ; Biswas, Sayan ; Reddy, Pothula Purushotham ; Manmadhan, Saumya ; George, Geen ; Kumar, Shilpa ; Das, Ranabir ; Rao, Balaji M. ; Gulyani, Akash
    Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.
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    A GTP-dependent 'push' is generally required for efficient protein translocation across the mitochondrial inner membrane into the matrix
    ( 1998-08-14) Sepuri, Naresh Babu V. ; Gordon, Donna M. ; Pain, Debkumar
    Mitochondrial biogenesis requires translocation of numerous preproteins across both outer and inner membranes into the matrix of the organelle. This trans, location process requires a membrane potential (ΔΨ) and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schulke, N., and Pain, D. (1998) J. Biol. Chem. 273, 14201424). We now demonstrate that GTP is generally required for efficient import of various preproteins, both native and urea-denatured. The GTP participation is localized to a particular stage in the protein import process. In the presence of ΔΨ but no added nucleoside triphosphates, the transmembrane movement of preproteins proceeds only to a point early in their translocation across the inner membrane. The completion of translocation into the matrix is independent of ΔΨ but is dependent on a GTP-mediated 'push.' This push is likely mediated by a membrane-bound GTPase on the cis side of the inner membrane. This conclusion is based on two observations: (i) GTP does not readily cross the inner membrane barrier and hence, primarily acts outside the inner membrane to stimulate import, and (ii) the GTP-dependent stage of import does not require soluble constituents of the intermembrane space and can be observed in isolated mitoplasts. Efficient import into the matrix, however, is achieved only through the coordinated action of a cis GTP- dependent push and a trans ATP-dependent 'pull'.
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    A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates
    ( 1999-08-06) Schülke, Norbert ; Sepuri, Naresh Babu V. ; Gordon, Donna M. ; Saxena, Sandeep ; Dancis, Andrew ; Pain, Debkumar
    Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in the outer mitochondrial membrane and the other (Tim) located in the inner membrane. These translocases reversibly cooperate during protein import. We have previously constructed a chimeric precursor (pPGPrA) consisting of an authentic mitochondrial precursor at the N terminus (Δ1-pyrroline-5- carboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, to protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arrested during translocation across the outer and inner mitochondrial membranes. Consequently, the two membranes of mitochondria become progressively 'zippered' together, forming long stretches in which they are in close contact (Schulke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7314-7319). We now demonstrate that trapped PGPrA intermediates hold the import channels stably together and inhibit mitochondrial protein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisubunit complex that contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and matrix-localized Hsp70. Further characterization of this complex may shed light on structural features of the complete mitochondrial import machinery.
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    A new ratiometric fluorescence probe as strong sensor of surface charge of lipid vesicles and micelles
    ( 2003-04-24) Singh, Yashveer ; Gulyani, Akash ; Bhattacharya, Santanu
    We report on the ability of a new fluorescent probe, 4-(2-pyren-1-yl-vinyl) pyridine, 1, to respond to micelles and phospholipid vesicles of different surface charge. The probe gets incorporated into micellar and membranous assemblies and shows a large red-shift in the fluorescence emission maxima especially when the surface charge of the organized media is anionic. The effect on the photo-excitation of the probe is very clear and pronounced as it can be easily visualized. The sample color upon photo-excitation changes from blue to orange/red once the probe experiences negatively charged vesicular or micellar surfaces. These results make the probe molecule useful as a reporter for sensing electrostatic environment in biological membranes and related organized assemblies. © 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
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    A new unusual galactose-specific lectin from the seeds of Indian lablab beans
    ( 1998-12-01) Tulasi, Rajasekhar Baru ; Nadimpalli, Siva Kumar
    Seeds of the Indian lablab beans (Dolichos lablab var. typicus) contain a glucose/mannose-specific lectin that was affinity-purified and well characterized from our laboratory1. The seeds were also found to contain a protein that strongly agglutinates only rabbit erythrocytes. The agglutinating activity is inhibited only by galactose and its derivatives. However, this newly found lectin fails to bind on the sepharose-galactose columns, and hence was isolated by gel filtration on Biogel P-200. The lectin eluted from this column with a molecular size of 1,20,000 and was found to be homogeneous in native PAGE. In SDS-PAGE the lectin dissociated into two subunits with apparent molecular masses of 48 and 20 kDa, respectively. Alanine was the only detectable amino terminal amino acid. The lectin was found to be a glycoprotein with 3% neutral sugar and cross-reacts with an antibody to the well-characterized glucose/mannose specific seed lectin.
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    A reciprocal feedback loop between HIF-1α and HPIP controls phenotypic plasticity in breast cancer cells
    ( 2022-02-01) Khumukcham, Saratchandra Singh ; Penugurti, Vasudevarao ; Soni, Anita ; Uppala, Veena ; Hari, Kishore ; Jolly, Mohit Kumar ; Dwivedi, Anju ; Salam PK, Abdul ; Padala, Chiranjeevi ; Mukta, Srinivasulu ; Bhopal, Triveni ; Manavathi, Bramanandam
    While phenotypic plasticity is a critical factor contributing to tumor heterogeneity, molecular mechanisms underlying this process are largely unknown. Here we report that breast cancer cells display phenotypic diversity in response to hypoxia or normoxia microenvironments by operating a reciprocal positive feedback regulation of HPIP and HIF-1α. We show that under hypoxia, HIF-1α induces HPIP expression that establishes cell survival, and also promotes cell migration/invasion, EMT and metastatic phenotypes in breast cancer cells. Mechanistic studies revealed that HPIP interacts with SRP14, a component of signal recognition particle, and stimulates MMP9 synthesis under hypoxic stress. Whereas, in normoxia, HPIP stabilizes HIF-1α, causing the Warburg effect to support cell growth. Concurrently, mathematical modelling corroborates this reciprocal feedback loop in enabling cell-state transitions in cancer cells. Clinical data indicate that elevated levels of HPIP and HIF-1α correlate with unfavorable prognosis and shorter survival rates in breast cancer subjects. Together, this data shows a reciprocal positive feedback loop between HPIP and HIF-1α that was unknown hitherto. It unveils how the tumor microenvironment influences phenotypic plasticity that has an impact on tumor growth and metastasis and, further signifies considering this pathway as a potential therapeutic target in breast cancer.
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    A small-molecule inhibitor of the DNA recombinase Rad51 from Plasmodium falciparum synergizes with the antimalarial drugs artemisinin and chloroquine
    ( 2019-05-17) Vydyam, Pratap ; Dutta, X. Dibyendu ; Sutram, Niranjan ; Bhattacharyya, Sunanda ; Bhattacharyya, Mrinal Kanti
    Malaria parasites repair DNA double-strand breaks (DSBs) primarily through homologous recombination (HR). Here, because the unrepaired DSBs lead to the death of the unicellular parasite Plasmodium falciparum, we investigated its recombinase, PfRad51, as a potential drug target. Undertaking an in silico screening approach, we identified a compound, B02, that docks to the predicted tertiary structure of PfRad51 with high affinity. B02 inhibited a drug-sensitive P. falciparum strain (3D7) and multidrug-resistant parasite (Dd2) in culture, with IC50 values of 8 and 3 μM, respectively. We found that B02 is more potent against these P. falciparum strains than against mammalian cell lines. Our findings also revealed that the antimalarial activity of B02 synergizes with those of two first-line malaria drugs, artemisinin (ART) and chloroquine (CQ), lowering the IC50 values of ART and CQ by 15- and 8-fold, respectively. Our results also provide mechanistic insights into the anti-parasitic activity of B02, indicating that it blocks the ATPase and strand-exchange activities of PfRad51 and abrogates the formation of PfRad51 foci on damaged DNA at chromosomal sites, probably by blocking homomeric interactions of PfRad51 proteins. The B02-mediated PfRad51 disruption led to the accumulation of unrepaired parasitic DNA and rendered parasites more sensitive to DNA-damaging agents, including ART. Our findings provide a rationale for targeting the Plasmodium DSB repair pathway in combination with ART. We propose that identification of a specific inhibitor of HR in Plasmodium may enable investigations of HR's role in Plasmodium biology, including generation of antigenic diversity.
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    A study on increasing enzymatic stability and activity of Baliospermum montanum hydroxynitrile lyase in biocatalysis
    ( 2020-01-01) Jangir, Nisha ; Preeti, ; Padhi, Santosh Kumar
    HNL catalysis is usually carried out in a biphasic solvent and at low pH to suppress the non-enzymatic synthesis of racemic cyanohydrins. However, enzyme stability under these conditions remain a challenge. We have investigated the effect of different biocatalytic parameters, i.e., pH, temperature, buffer concentrations, presence of stabilizers, organic solvents, and chemical additives on the stability of Baliospermum montanum hydroxynitrile lyase (BmHNL). Unexpectedly, glycerol (50 mg/mL) added BmHNL biocatalysis had produced > 99% of (S)-mandelonitrile from benzaldehyde, while without glycerol it is 54% ee. Similarly, BmHNL had converted 3-phenoxy benzaldehyde and 3,5-dimethoxy benzaldehyde, to their corresponding cyanohydrins in the presence of glycerol. Among the different stabilizers added to BmHNL at low pH, 400 mg/mL of sucrose had increased enzyme's half-life more than fivefold. BmHNL's stability study showed half-lives of 554, 686, and 690 h at its optimum pH 5.5, temperature 20 °C, buffer concentration, i.e., 100 mM citrate-phosphate pH 5.5. Addition of benzaldehyde as inhibitor, chemical additives, and the presence of organic solvents have decreased both the stability and activity of BmHNL, compared to their absence. Secondary structural study by CD-spectrophotometer showed that BmHNL's structure is least affected in the presence of different organic solvents and temperatures.
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    A transcriptional repressive role for epithelial-specific ETS factor ELF3 on oestrogen receptor alpha in breast cancer cells
    ( 2016-04-15) Gajulapalli, Vijaya Narasihma Reddy ; Samanthapudi, Venkata Subramanyam Kumar ; Pulaganti, Madhusudana ; Khumukcham, Saratchandra Singh ; Malisetty, Vijaya Lakhsmi ; Guruprasad, Lalitha ; Chitta, Suresh Kumar ; Manavathi, Bramanandam
    Oestrogen receptor-α (ERα) is a ligand-dependent transcription factor that primarily mediates oestrogen (E2)-dependent gene transcription required for mammary gland development. Coregulators critically regulate ERα transcription functions by directly interacting with it. In the present study, we report that ELF3, an epithelial-specific ETS transcription factor, acts as a transcriptional repressor of ERα. Co-immunoprecipitation (Co-IP) analysis demonstrated that ELF3 strongly binds to ERα in the absence of E2, but ELF3 dissociation occurs upon E2 treatment in a dose- and time-dependent manner suggesting that E2 negatively influences such interaction. Domain mapping studies further revealed that the ETS (E-twenty six) domain of ELF3 interacts with the DNA binding domain of ERα. Accordingly, ELF3 inhibited ERα's DNA binding activity by preventing receptor dimerization, partly explaining the mechanism by which ELF3 represses ERα transcriptional activity. Ectopic expression of ELF3 decreases ERα transcriptional activity as demonstrated by oestrogen response elements (ERE)-luciferase reporter assay or by endogenous ERα target genes. Conversely ELF3 knockdown increases ERα transcriptional activity. Consistent with these results, ELF3 ectopic expression decreases E2-dependent MCF7 cell proliferation whereas ELF3 knockdown increases it. We also found that E2 induces ELF3 expression in MCF7 cells suggesting a negative feedback regulation of ERα signalling in breast cancer cells. A small peptide sequence of ELF3 derived through functional interaction between ERα andELF3couldinhibit DNA binding activity of ERα and breast cancer cell growth. These findings demonstrate that ELF3 is a novel transcriptional repressor of ERα in breast cancer cells. Peptide interaction studies further represent a novel therapeutic option in breast cancer therapy.
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    A Tunable Palette of Molecular Rotors Allows Multicolor, Ratiometric Fluorescence Imaging and Direct Mapping of Mitochondrial Heterogeneity
    ( 2021-05-17) Raja, Sufi O. ; Sivaraman, Gandhi ; Biswas, Sayan ; Singh, Gaurav ; Kalim, Fouzia ; Kandaswamy, Ponnuvel ; Gulyani, Akash
    Environment-sensitive molecular probes offer the potential for a comprehensive mapping of the complex cellular milieu. We present here a radically new strategy of multiplexing highly sensitive, spectrally tuned fluorescent dyes for sensing cellular microenvironment. To achieve this multicolor, ratiometric cellular imaging, we first developed a series of highly sensitive, tunable molecular rotors for mitochondrial imaging, with emission wavelengths spanning the visible spectrum. These fluorogenic merocyanine dyes are all sensitive to solvent viscosity despite distinctive photophysical features. Our results show that merocyanine dyes can show a rotor-like behavior despite significant changes to the conventional donor-acceptor or push-pull scaffolds, thereby revealing conserved features of rotor dye chemistry. Developing closely related but spectrally separated dyes that have distinct response functions allows us to do ″two-color, two-dye″ imaging of the mitochondrial microenvironment. Our results with multidye, combinatorial imaging provide a direct visualization of the intrinsic heterogeneity of the mitochondrial microenvironment. The overall mitochondrial microenvironment (including contributions from local membrane order) as reported through two-color fluorescence ″ratio″ changes of multiplexed rotor dyes shows dynamic heterogeneity with distinct spatiotemporal signatures that evolve over time and respond to chemical perturbations. Our results offer a powerful illustration of how multiplexed dye imaging allows the quantitative imaging of mitochondrial membrane order and cellular microenvironment.
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    A unique self-assembly-driven probe for sensing a lipid bilayer: Ratiometric probing of vesicle to micelle transition
    ( 2018-01-01) Gulyani, Akash ; Dey, Nilanjan ; Bhattacharya, Santanu
    An amphiphilic pyrene-terpyridine (Pytpy) probe forms novel, fluorescent nanoaggregates in phospholipid membranes. This unique membrane-driven self-assembly of Pytpy shows large Stokes shifts and long-lived fluorescent states and efficiently reports on vesicle-micelle transition through ratiometric changes. Strikingly, Pytpy can even distinguish between bilayer-like domains and more-dynamic micelle-like 'rim' phases that co-exist in mixed assemblies (bicelles).
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    AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila
    ( 2013-01-01) Pathak, Rashmi U. ; Mamillapalli, Anitha ; Rangaraj, Nandini ; Kumar, Ram P. ; Vasanthi, Dasari ; Mishra, Krishnaveni ; Mishra, Rakesh K.
    Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes. © 2013 Landes Bioscience.
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    Active and prospective latent tuberculosis are associated with different metabolomic profiles: clinical potential for the identification of rapid and non-invasive biomarkers
    ( 2020-01-01) Albors-Vaquer, A. ; Rizvi, A. ; Matzapetakis, M. ; Lamosa, P. ; Coelho, A. V. ; Patel, A. B. ; Mande, S. C. ; Gaddam, S. ; Pineda-Lucena, A. ; Banerjee, S. ; Puchades-Carrasco, L.
    Although 23% of world population is infected with Mycobacterium tuberculosis (M. tb), only 5–10% manifest the disease. Individuals surely exposed to M. tb that remain asymptomatic are considered potential latent TB (LTB) cases. Such asymptomatic M. tb.-exposed individuals represent a reservoir for active TB cases. Although accurate discrimination and early treatment of patients with active TB and asymptomatic M. tb.-exposed individuals are necessary to control TB, identifying those individuals at risk of developing active TB still remains a tremendous clinical challenge. This study aimed to characterize the differences in the serum metabolic profile specifically associated to active TB infected individuals or to asymptomatic M. tb.-exposed population. Interestingly, significant changes in a specific set of metabolites were shared when comparing either asymptomatic house-hold contacts of active TB patients (HHC-TB) or active TB patients (A-TB) to clinically healthy controls (HC). Furthermore, this analysis revealed statistically significant lower serum levels of aminoacids such as alanine, lysine, glutamate and glutamine, and citrate and choline in patients with A-TB, when compared to HHC-TB. The predictive ability of these metabolic changes was also evaluated. Although further validation in independent cohorts and comparison with other pulmonary infectious diseases will be necessary to assess the clinical potential, this analysis enabled the discrimination between HHC-TB and A-TB patients with an AUC value of 0.904 (confidence interval 0.81–1.00, p-value < 0.0001). Overall, the strategy described in this work could provide a sensitive, specific, and minimally invasive method that could eventually be translated into a clinical tool for TB control.
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    Adaptation of Mge1 to oxidative stress by local unfolding and altered Interaction with mitochondrial Hsp70 and Mxr2
    ( 2019-05-01) Karri, Srinivasu ; Singh, Swati ; Paripati, Arun Kumar ; Marada, Adinarayana ; Krishnamoorthy, Thanuja ; Guruprasad, Lalitha ; Balasubramanian, Dorairajan ; Sepuri, Naresh Babu V.
    Perturbations in mitochondrial redox levels oxidize nucleotide exchanger Mge1, compromising its ability to bind to the Hsp70, while the Mxr2 enzyme reduces the oxidized Mge1. However, the effects of persistent oxidative stress on Mge1 structure and function are not known. In this study, we show that oxidation-induced selective and local structural adaptations cause the detachment of Mge1 from Hsp70. Notably, persistent oxidative stress causes monomeric Mge1 to aggregate and to generate amyloid-type particles. Mxr2 appears to protect Mge1 from oxidative stress induced aggregation. We conclude that the Mxr2-Mge1-Hsp70 protein triad is finely regulated through structural alterations of Mge1 mediated by redox levels.
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    Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography
    ( 2013-07-01) Shetty, Kartika N. ; Latha, Vakada Lavanya ; Rao, Rameshwaram Nagender ; Nadimpalli, Siva Kumar ; Suguna, Kaza
    Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.