Search

We report the potential role of

CDCl3 is the most frequently used solvent for the NMR investigation of organic compounds. Busy chemistry labs need to investigate hundreds of compounds daily. While 1H NMR investigation takes a couple of minutes, recording 13C NMR spectra necessitates hours of signal averaging due to the low abundance and low sensitivity of 13C nuclei. The longer acquisition time for 13C NMR results in a loss of precious spectrometer time in a shared multi-user environment. A regular 5 mm o.d. NMR tube is the most commonly used tube for NMR in organic chemistry labs and is also the cheapest option. We show that for analytes soluble in the CDCl3 solvent using a regular 5 mm o.d. NMR tube, the speed of 13C observation can be enhanced by a factor of two by resorting to a sample preparation method that employs a biphasic system made of H2O or D2O at the top of another layer of CDCl3. By using the biphasic system of two immiscible solvents, the analyte can be concentrated in the CDCl3 layer (within the more sensitive volume of the NMR coil), resulting in the improvement of the signal to noise ratio (SNR) by a factor of up to 1.8 for 13C and 2D 1H–13C HSQC spectra, which results in more than two-fold reduction in the experimental time. 1H NMR and other 2D NMR also get a sensitivity boost. The amount of CDCl3 required for sample preparation can also be reduced by 40% using this biphasic system (CDCl3/H2O). Sample preparation in such an immiscible biphasic system is effortless and straightforward. The performance of such biphasic samples is closer to that of Shigemi tubes and better than that of 3 mm o.d. tubes. more...

CDCl

Two dimensional (2D) NMR display better resolution than one-dimensional (1D) 1H NMR. However, 2D NMR does not display a straightforward quantitative aspect due to J-dependent polarization/coherence transfer. 1D 1H NMR is versatile for quantification; however, it displays significant spectral overlap in biological or organic complex mixtures, which forbids quantification of a large number of signals in 1D 1H NMR. The significant variations in 1H13C scalar couplings, T1, T2, and pulse imperfections are the main problems. Although T1, T2 can be suitably chosen to minimize their adverse effect on quantification, the large variations in 1H - 13C couplings lead to variations in cross peak intensity, which is more influenced by the amount of polarization transfer rather than the quantity of metabolites or amount of analytes in a complex mixture. In the present work, we show that spatial encoding of the polarization transfer periods can be executed in 1H13C HSQC using sweep frequency pulses in the presence of a magnetic field gradient. As a result, uniform transfer of polarization from 1H to 13C over a range of 1H - 13C couplings can be performed, subsequently improving the quantitative aspect of HSQC or improve the intensity of cross-peaks, which are mistuned in regular HSQC more...

NMR is a fast method for obtaining a holistic snapshot of the metabolome and also offers quantitative information without separating the compounds present in a complex mixture. Identification of the metabolites present in a plant extract sample is a crucial step for all plant metabolomics studies. In the present work, we used various two dimensional (2D) NMR methods such as J-resolved NMR, total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence sensitivity enhanced NMR spectroscopy for the identification of 36 common metabolites present in Coriandrum sativum L. seed extract. The identified metabolites belong to the following classes: organic acids, amino acids, and carbohydrates. 1 H NMR spectra of such complex mixtures in general display tremendous signal overlap due to the presence of a large number of metabolites with closely resonating multiplet signals. This signal overlapping leads to ambiguity in an assignment, and hence, identification of metabolites becomes tedious or impossible in many cases. Therefore, the utility of pure-shift proton spectrum along the indirect (F1 ) dimension of the F1 -PSYCHE-TOCSY spectrum is demonstrated for overcoming ambiguity in assignment of metabolites in crowded spectral regions from Coriandrum sativum L. seed extract sample. Because pure-shift NMR methods yield ultrahigh resolution spectrum (i.e., a singlet peak per chemical site) along one or more dimensions, such spectra provide better identification of metabolites compared with regular 2D TOCSY where signal overlap and peak distortions lead to ambiguity in the assignment. Nine metabolites were unambiguously assigned by pure-shift F1 -PSYCHE-TOCSY spectrum, which was unresolved in regular TOCSY spectrum. more...

A diagonal suppressed F1 decoupled total correlation spectroscopy(TOCSY) experiment is developed for analyses of complex mixtures. In 2D homonuclear correlation, assignment of the cross peaks is crucial for structure elucidation. However, when cross peaks are close to the diagonal peaks in overcrowded spectral regions, their assignment becomes tedious. In complex mixtures, the presence of multiple spectra along with broad and complex proton multiplets owing to homonuclear scalar couplings degrade the resolution to the extent that assignment of various cross peaks becomes tedious or impossible. Herein, a diagonal suppressed total correlation technique with F1 decoupling is presented to improve the resolution of the cross peaks. The resolution of the cross peaks is improved by both diagonal suppression as well as the collapse of the multiplets to singlets. Application of the method to a few mixtures of organic compounds reveals better identification of the cross peaks relative to other TOCSY variants. more...

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation. more...

Phenol and its derivatives are consistently causing harmful effects to an aquatic ecosystem. The present study focused on the isolation and characterization of potential phenol degrading bacterial strains and subsequently optimization of media ingredients for efficient phenol degradation by potential bacterial strains. Bacterial strains were isolated from municipal sewage, Bilaspur (21'47 and 23'8 N 81'14 and 83'15 E). After optimization phenol degradation rate was increased by 1.84 fold for PDB 5 (from 40.37% to 74.67%) and 1.39 fold for PDB 11 (from 58.62% to 81.51%) at 500mg/l initial phenol concentration. PDB 5 and PDB 11 were identified as Streptococcus sp. PDB 5 and Pseudomonas sp. PDB 11 respectively as potential phenol degrading bacterial strains. These strains can further be used in microbially assisted phenol degradation to remove phenol derivatives present in industrial wastewater.Int J Appl Sci Biotechnol, Vol 4(3): 288-293 more...

Correction to: J. Biosci. 40(5), December 2015, 863-871 https://doi.org/10.1007/s12038-015-9570-0 The image of anti-Myc blot of figure 2C (third panel; Malin-Myc [C26S]) was inadvertently used once again for the c-tubulin loading control of figure 2B. The revised figure 2B with the correct image of the c-tubulin loading control is given below. The interpretation and conclusion provided in the article do not change because of the correction. more...

The progressive myoclonic epilepsy of Lafora or Lafora disease (LD) is a neurodegenerative disorder characterized by recurrent seizures and cognitive deficits. With typical onset in the late childhood or early adolescence, the patients show progressive worsening of the disease symptoms, leading to death in about 10 years. It is an autosomal recessive disorder caused by the loss-of-function mutations in the EPM2A gene, coding for a protein phosphatase (laforin) or the NHLRC1 gene coding for an E3 ubiquitin ligase (malin). LD is characterized by the presence of abnormally branched water insoluble glycogen inclusions known as Lafora bodies in the neurons and other tissues, suggesting a role for laforin and malin in glycogen metabolic pathways. Mouse models of LD, developed by targeted disruption of the Epm2a or Nhlrc1 gene, recapitulated most of the symptoms and pathological features as seen in humans, and have offered insight into the pathomechanisms. Besides the formation of Lafora bodies in the neurons in the presymptomatic stage, the animal models have also demonstrated perturbations in the proteolytic pathways, such as ubiquitin-proteasome system and autophagy, and inflammatory response. This review attempts to provide a comprehensive coverage on the genetic defects leading to the LD in humans, on the functional properties of the laforin and malin proteins, and on how defects in any one of these two proteins result in a clinically similar phenotype. We also discuss the disease pathologies as revealed by the studies on the animal models and, finally, on the progress with therapeutic attempts albeit in the animal models. more...

The heat shock response is a highly conserved cellular stress response pathway that protects cells against stress-induced damages. The heat shock response is characterized by the activation of the heat shock factor, a transcription factor, which in turn regulate the expression of heat shock proteins which help the cells to recover from the stress-induced damages. While the mechanism behind the activation of heat shock factor and the expression of heat shock proteins are well studied, our understanding on the other aspects of the heat shock response pathway – mainly the global suppression of transcriptional and the translational processes – has been very limited. Thus, this chapter would focus more on the recent studies elucidating the mechanism behind the heat shock-induced transcriptional and post-transcriptional repression of gene expression. Given the importance of the stress response pathways in cell survival, the chapter would focus on the discoveries made in the mammalian systems and their impact on human health and disease. more...

A diagonal suppressed F

O-GlcNAcylation is an important post-translational modification of proteins and is known to regulate a number of pathways involved in cellular homeostasis. This involves dynamic and reversible modification of serine/threonine residues of different cellular proteins catalyzed by O-linked N-acetylglucosaminyltransferase and O-linked N-acetylglucosaminidase in an antagonistic manner. We report here that decreasing O-GlcNAcylation enhances the viability of neuronal cells expressing polyglutamine-expanded huntingtin exon 1 protein fragment (mHtt). We further show that O-GlcNAcylation regulates the basal autophagic process and that suppression of O-GlcNAcylation significantly increases autophagic flux by enhancing the fusion of autophagosome with lysosome. This regulation considerably reduces toxic mHtt aggregates in eye imaginal discs and partially restores rhabdomere morphology and vision in a fly model for Huntington disease. This study is significant in unraveling O-GlcNAcylation-dependent regulation of an autophagic process in mediating mHtt toxicity. Therefore, targeting the autophagic process through the suppression of O-GlcNAcylation may prove to be an important therapeutic approach in Huntington disease. more...

The voltage-gated sodium channels are fundamental units that evoke the action potential in excitable cells such as neurons. These channels are integral membrane proteins typically consisting of one α-subunit, which forms the larger central pore of the channel, and two smaller auxiliary β-subunits, which modulate the channel functions. Genetic alterations in the SCN1A gene coding for the α-subunit of the neuronal voltage-gated sodium ion channel, type 1 (NaV 1.1), is associated with a spectrum of seizure-related disorders in human, ranging from a relatively milder form of febrile seizures to a more severe epileptic condition known as the Dravet syndrome. Among the epilepsy genes, the SCN1A gene perhaps known to have the largest number of disease-associated alleles. Here we present a meta-analysis on the SCN1A gene variants and provide comprehensive information on epilepsy-associated gene variants, their frequency, the predicted effect on the protein, the ethnicity of the affected along with the inheritance pattern and the associated epileptic phenotype. We also summarize our current understanding on the pathophysiology of the SCN1A gene defects, disease mechanism, genetic modifiers and their clinical and diagnostic relevance. more...

The heat shock response in human cells is associated with the transcription of satellite III repeats (SatIII) located in the 9q12 locus. Upon induction, the SatIII transcripts remain associated with the locus and recruit several transcription and splicing factors to form the nuclear stress bodies (nSBs). The nSBs are thought to modulate epigenetic changes during the heat shock response. We demonstrate here that the nSBs are induced by a variety of stressors and show stress-specific patterns of induction. While the transcription factor HSF1 is required for the induction of SatIII locus by the stressors tested, its specific role in the transcriptional process appears to be stress dependent. Our results suggest the existence of multiple transcriptional loci for the SatIII transcripts and that their activation might depend upon the type of stressors. Thus, induction of SatIII transcripts appears to be a generic response to a variety of stress conditions. more...

Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin-proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin-laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins. more...

The heat shock response is a conserved defense mechanism that protects cells from physiological stress, including thermal stress. Besides the activation of heat-shock-protein genes, the heat shock response is also known to bring about global suppression of transcription; however, the mechanism by which this occurs is poorly understood. One of the intriguing aspects of the heat shock response in human cells is the transcription of satellite-III (Sat3) long non-coding RNAs and their association with nuclear stress bodies (nSBs) of unknown function. Besides association with the Sat3 transcript, the nSBs are also known to recruit the transcription factors HSF1 and CREBBP, and several RNA-binding proteins, including the splicing factor SRSF1. We demonstrate here that the recruitment of CREBBP and SRSF1 to nSBs is Sat3-dependent, and that loss of Sat3 transcripts relieves the heat-shock-induced transcriptional repression of a few target genes. Conversely, forced expression of Sat3 transcripts results in the formation of nSBs and transcriptional repression even without a heat shock. Our results thus provide a novel insight into the regulatory role for the Sat3 transcripts in heat-shock-dependent transcriptional repression. more...

Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the EPM2A gene coding for the laforin protein phosphatase or the NHLRC1 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each other's function. Using cell biological assays we demonstrate here that (i) malin promotes its own degradation via autoubiquitination, (ii) laforin prevents the auto-degradation of malin by presenting itself as a substrate and (iii) malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each other's stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneity observed in LD. more...

A new water-soluble, multisite-coordinating ligand LH(7) was prepared by the condensation of tris(hydroxymethyl)aminomethane with 2,6-diformyl-p-cresol. LH(7) is a selective chemosensor for Cu(2+), under physiological conditions, with visual detection limits of 20 ppm (ambient light conditions) and 4 ppm (UV light conditions). LH(7) can also be used in biological cell lines for the detection of Cu(2+). more...

The EPM2A gene, defective in the fatal neurodegenerative disorder Lafora disease (LD), is known to encode two distinct proteins by differential splicing; a phosphatase active cytoplasmic isoform and a phosphatase inactive nuclear isoform. We report here the identification of three novel EPM2A splice variants with potential to code for five distinct proteins in alternate reading frames. These novel isoforms, when ectopically expressed in cell lines, show distinct subcellular localization, interact with and serve as substrates of malin ubiquitin ligase—the second protein defective in LD. Two phosphatase active isoforms interact to form a heterodimeric complex that is inactive as a phosphatase in vitro, suggesting an antagonistic function for laforin isoforms if expressed endogenously in significant amounts in human tissues. Thus alternative splicing could possibly be one of the mechanisms by which EPM2A may regulate the cellular functions of the proteins it codes for. more...

A broad range of neurodegenerative disorders result from the cytotoxicity conferred by aberrantly folded mutant proteins. Intriguingly, the cytotoxicity and aggregation property of a few mutant proteins are known to be modulated by the flanking sequences. One of such modulators is the proline repeat tract. Using a mammalian cellular model, we show here that proline repeat tract, both in cis- and in trans-positions, ameliorate the cytotoxicity of wide range of misfolded proteins coded by synthetic constructs. We further show that the proline repeat tract could possibly confer protection against the cytotoxicity of misfolded proteins by altering their conformation at the time of their synthesis. Thus, our study elucidates the mechanism by which the proline repeat tract might ameliorate the toxicity of misfolded proteins, and opens up new therapeutic modalities for disorders caused by cytotoxic misfolded proteins. more...

The essential oil of Elsholtzia blanda Benth. collected from Jorhat (Assam) has been analyzed by GC, GC/MS, GC/FTIR, IR, 1H- and 13C-NMR spectroscopy. Geranyl acetate was found to be the major constituent (71%). Twenty-eight other components were also characterized in the oil with linalool (5.27%), geraniol (3.77%), (E)-β-ocimene (2.96%), β-caryophyllene (1.75%), α-bergamotene (1.70%), (Z)-β-ocimene (1.38%), acetophenone (1.13%) and linalyl acetate (1.11%) being the next most abundant compounds. more...

The essential oil obtained from the aerial parts of Hyptis suaveolens Poit collected in the Kumaun Region of India was found (by GC/MS) to differ chemically from that reported from chemotypes collected in South America and Malaysia. The main components of this Indian oil were sabinene (41.0%), β-pinene (9.8%), β-caryophyllene (7.9%), and limonene (6.8%), accompanied by more than 50 other compounds including (Z)-α-trans-bergamotol (2.0%), caryophyllene oxide (5.8%), ar-abietatriene (4.4%), and ar-abietatrienol (0.4%). more...

Proteins with homopolymeric repeat tracts are very common in the human proteome. Intriguingly, some but not all repeat tracts show length variation in the population and, in a few, the expansion of repeat tract beyond the normal length is associated with neurodegenerative and developmental disorders. In this study we have addressed questions such as why some amino acid residues are favored in longer repeat tracts and why repeat tracts show terminal bias. Using cell biological assays for repeat tracts fused to green fluorescent protein we show here that homopolymeric repeats that are beyond their naturally occurring length in the proteome are cytotoxic in nature. This toxicity is further modulated by the length of the peptide that bears the repeat and the spatial location of the repeat within the peptide. Thus, the cellular toxicity appears to be one of the selective processes that regulate the evolution of homopolymeric repeats in the proteome. more...

Background Inflammation is one of the major components of atherosclerosis which is the underlying disorder that leads to various diseases including coronary artery disease (CAD). Genes that are involved in the inflammatory processes are therefore good candidates for the risk of CAD. Variations in the genes involved in various molecular pathways of inflammation have been implicated to exaggerated atherosclerosis and the risk of cardiovascular diseases. In this study, we performed a genetic association study on the single nucleotide polymorphisms (SNPs) present in the genes CD14 (−159 C/T), TNFα (−308 G/A), IL-1α (−889 C/T), IL-6 (−174 G/C), PSMA6 (−8 C/G), and PDE4D (SNP83 T/C, respectively) in order to discern their possible role in the susceptibility to CAD in a North Indian population. Methods Angiographically proven CAD patients (n = 210) and age, sex and ethnically matched normal healthy controls (n = 232) were recruited for this case-control study. Genotypes were determined by PCR–RFLP method. Chi-square and logistic regression analyses were performed to compare the genotype and allele frequencies between the patient and the control groups. Results None of the SNPs showed significant association with CAD in the study population before and after adjustment for the confounding risk factors like age, sex, hypertension, smoking habit, and diabetes. Conclusion This study was unable to demonstrate any association between the six gene variants tested and CAD in the North Indian population. more...

GC, CG/MS, FTIR, 1H-NMR and 13C-NMR analyses revealed that the essential oil of Elsholtzia eriostachya var. pusilla growing in Kumaun region (India) contained 53% geranial and 39% neral together with 19 other, mainly terpenoid constituents. more...