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Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme
No full text: Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple
biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes
induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM,
while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorant
Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling
No full textEndothelial nitric-oxide synthase (eNOS) and its bioactive
product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological
inhibition of eNOS attenuates angiogenesis during tissue
repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN’s function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of
secreted cytokines involved in inflammatory responses, and
ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF--induced NFB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRINTRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression
leading to deactivation of the NFB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression
Palladium-catalyzed decarboxylative,decarbonylative and dehydrogenative C(sp2)–H acylation at room temperature
No full textOver the past few decades, an impressive array of C–H activation methodology has been developed for
organic synthesis. However, due to the inherent inertness of the C–H bonds (e.g. ∼110 kcal mol−1 for the
cleavage of C(aryl)–H bonds) harsh reaction conditions have been realized to overcome high energetic transition states resulting in a limited substrate scope and functional group tolerance. Therefore, the development of mild C–H functionalization protocols is in high demand to exploit the full potential of the C–H activation strategy in the synthesis of a complex molecular framework. Although, electron-rich substrates undergo electrophilic metalation under relatively mild conditions, electron-deficient substrates
proceed through a rate-limiting C–H insertion under forcing conditions at high temperature. In addition,
a stoichiometric amount of toxic silver salt is frequently used in palladium catalysis to facilitate the C–H
activation process which is not acceptable from the environmental and industrial standpoint. We report
herein, a Pd(II)-catalyzed decarboxylative C–H acylation of 2-arylpyridines with α-ketocarboxylic acids
under mild conditions. The present protocol does not require stoichiometric silver(I) salts as additives and
proceeds smoothly at ambient temperature. A novel decarbonylative C–H acylation reaction has also
been accomplished using aryl glyoxals as acyl surrogates. Finally, a practical C–H acylation via a dehydrogenative
pathway has been demonstrated using commercially available benzaldehydes and aqueous hydroperoxides. We also disclose that acetonitrile solvent is optimal for the acylation reaction at room temperature and has a prominent role in the reaction outcome. Control experiments suggest that the
acylation reaction via decarboxylative, decarbonylative and dehydrogenative proceeds through a radical pathway. Thus we disclose a practical protocol for the sp2 C–H acylation reaction
ROLE OF AU-RICH ELEMENT (ARE) BINDING PROTEIN HuR IN POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION IN MAMMALIAN CELLS
No full textmiRNAs, the 22 nucleotide long but stable non-coding RNAs, form miRNP
complexes with Argonaute proteins which thereby bind to the 3’UTR of target
messages. miRNPs not only cause translational repression of target genes but
also destabilize them. During stress, RNA binding protein, HuR, binds to the
AU-rich elements of target mRNAs, stabilises them and facilitates their
translation. Interestingly, a reduction in cellular miR-122 level of Huh7 cells
upon amino acid starvation was observed, with an increase in extracellular
vesicle (EV)-associated miR-122 level of starved cells. In this context a new
role of ELAV protein HuR was identified. HuR accelerates the EVs-mediated
export of miRNAs in human cells. The protein is both necessary and sufficient
for the export of corresponding miRNAs.
The mechanism behind this phenomenon was studied and it was found
that in amino acid starved cells, HuR and miRNPs bind to the common target
mRNAs in a mutually exclusive manner. HuR replaces miRNPs from target
messages. HuR also reversibly binds with miRNAs and replaces them from
Ago2 on endoplasmic reticulum. This HuR-miR-122 binding is specific for
miR-122 and in this context and RRMIII of HuR is necessary for this HuRmiRNA
binding.
In this study it was also found that HuR undergoes ubiquitination on
multivesicular bodies (MVBs). This ubiquitination further helps in HuRmiRNA
unbinding on MVBs and the resulting HuR-unloaded miRNAs get
exported out via EVs. It was known that, the 110 amino acids long segment
required for ubiquitination of HuR, spans to its hinge region. Interestingly, the
deletion mutant of HuR devoid of hinge region, failed to promote EV-mediated
2
miR-122 export. Therefore, both HuR-miR-122 binding and ubiquitinationmediated
release are necessary for HuR-mediated extracellular export of miR122
via EVs.
This EV-mediated extracellular export of miRNAs delimits the cellularmiRNA levels not only in starved hepatic cells but also in breast cancer cells and macrophage cells. Therefore, HuR, by modulating extracellular export of miRNAs, controls stress response in amino acid starved Huh7 cells. It further
controls cell senescence in MDA-MB-231 breast cancer cells and proinflammatory
miRNA levels in RAW 264.7 macrophage like cells
Elusive Thiyl Radical Migration in a Visible Light Induced Chemoselective Rearrangement of g-Keto Acrylate Thioesters: Synthesis of Substituted Butenolides
No full textThe first metal/photocatalyst-free, visible
light induced chemoselective rearrangement of gketo
acrylate thioesters via elusive thiyl radical
migration is described. Thioesters bearing at least
one alkyl substitution undergo 1,2-thiyl radical
migration, whereas 1,4-migration has been observed
when the substrate is substituted by aromatics
only. The reactions show excellent functional
group tolerance and are proposed to proceed
through the trans-cis isomerization/intramolecular
lactonization/thiyl radical capture pathway. This
atom-economical process constitutes an efficient
and practical method for the formation of substituted
butenolides
Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-�-induced Neuronal Death
No full textAmyloid-� (A�)-induced neuron death is considered central
to the pathogenesis of Alzheimer’s disease (AD). Among several death modalities, autophagy and apoptosis play important roles in A�-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon A� treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro. Increased Trib3 levels inhibited the activity of the kinase Akt by interacting
with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to A� insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. Wefound that Trib3 indirectly activates unc-51-like
autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from A� insult. Our results suggest that a self-amplifying
feed-forward loop among Trib3, Akt, and FoxO1 in
A�-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD
Physical chemistry in a single live cell: confocal microscopy
No full textA live cell is a complex, yet extremely important container. Understanding the dynamics in a selected
intracellular component is a challenging task. We have recently made significant progress in this
direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal
microscope is B0.2 mm (200 nm). This is nearly one hundred times smaller than the size of a live cell.
Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper,
we discuss how one can image different intracellular components/organelles, record fluorescence
spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics
of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell.
We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer
cells, 3D tumor spheroids and towards drug deliver
Studies on Plant Bio-Molecules and Their Application Potential
Full text linkWinged bean (Psophocarpus tetragonolobus) seeds are rich source of Kunitz-type serine protease inhibitors of about 20 kDa in size with two disulfide bridges. In this work structural changes of winged bean trypsin inhibitor (WbTI-2) and bi-functional chymotrypsin/ trypsin inhibitor (WBCTI) have been studied as a function of temperature. These two proteins completely retained inhibitory properties against trypsin and/ or chymotrypsin even after heating at 70°C. On the basis of circular dichroism studies it appeared that WBCTI and WbTI-2 maintained their canonical structure up to 70°C. But the activity and stability of the secondary structures were found to decrease drastically in presence of dithiothreitol, indicating the role of two disulfide bonds for additional stability of these proteins.
When insecticidal and growth inhibitory potential were evaluated, both the proteins have shown reasonable inhibition of mid-gut proteases of Helicoverpa armigera. In artificial feeding trial, addition of WBCTI and WbTI-2 in diet resulted in significant growth retardation of H. armigera larva. WBCTI also caused delayed pupae formation and higher mortality in H. armigera larvae. Based on this observation, the possibility of WBCTI as an effective insecticidal lead molecule has been studied by generating transgenic tobacco plants. Transgenically expressed WBCTI has shown reasonable inhibition of mid-gut proteases of H. armigera. During infestation of wild type and transgenic tobacco plants with H. armigera larva, transgenic plants were resistant to insect attack to a great extent compared to the wild type plants. This finding supported the deployment of WBCTI as a suitable candidate gene from a non-host plant for producing stress-tolerant transgenic plant.
In the final part of the work, the concept of protein engineering was explored by appending the amylase inhibitory property in WBCTI to convert it to an amylase-trypsin-chymotrypsin trifunctional inhibitor (TFI). An amylase-inhibitory nona-peptide, representing the N-terminus of ragi bi-functional inhibitor (RBI), was attached with WBCTI through PCR. This newly engineered protein was completely functional as it inhibited alpha amylase, trypsin and chymotrypsin. Interestingly it has also shown reasonable inhibition of the mid-gut proteases of H. armigera. So it can be concluded that TFI may become an efficient insecticidal lead molecule, as a single defensive gene product, to combat with the protein as well as carbohydrate digestive systems of the pests
