A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels

BACKGROUND: In vertebrates, two types of cholinesterases exist, acetylcholinesterase and butyrylcholinesterase. The function of acetylcholinesterase is to hydrolyse acetylcholine, thereby terminating the neurotransmission at cholinergic synapse, while the precise physiological function of butyrylcholinesterase has not been identified. The presence of cholinesterases in tissues that are not cholinergically innervated indicate that cholinesterases may have functions unrelated to neurotransmission. Furthermore, cholinesterases display a genuine aryl acylamidase activity apart from their predominant acylcholine hydrolase activity. The physiological significance of this aryl acylamidase activity is also not known. The study on the aryl acylamidase has been, in part hampered by the lack of a specific method to visualise this activity. We have developed a method to visualise the aryl acylamidase activity on cholinesterase in polyacrylamide gels. RESULTS: The o-nitroaniline liberated from o-nitroacetanilide by the action of aryl acylamidase activity on cholinesterases, in the presence of nitrous acid formed a diazonium compound. This compound gave an azo dye complex with N-(1-napthyl)-ethylenediamine, which appeared as purple bands in polyacrylamide gels. Treating the stained gels with trichloroacetic acid followed by Tris-HCl buffer helped in fixation of the stain in the gels. By using specific inhibitors for acetylcholinesterase and butyrylcholinesterase, respectively, differential staining for the aryl acylamidase activities on butyrylcholinesterase and acetylcholinesterase in a sample containing both these enzymes has been demonstrated. A linear relationship between the intensity of colour developed and activity of the enzyme was obtained. CONCLUSIONS: A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed

Not Available

Not AvailableChickpea (Cicer arietinum L.) is the second most important grain legume cultivated by resource poor farmers in the arid and semi-arid regions of the world. Drought is one of the major constraints leading up to 50 % production losses in chickpea. In order to dissect the complex nature of drought tolerance and to use genomics tools for enhancing yield of chickpea under drought conditions, two mapping populations—ICCRIL03 (ICC 4958 × ICC1882) and ICCRIL04 (ICC 283 × ICC 8261) segregating for drought tolerance-related root traits were phenotyped for a total of 20 drought component traits in 1–7 seasons at 1–5 locations in India. Individual genetic maps comprising 241 loci and 168 loci for ICCRIL03 and ICCRIL04, respectively, and a consensus genetic map comprising 352 loci were constructed (http://cmap.icrisat.ac.in/cmap/sm/ cp/varshney/). Analysis of extensive genotypic and precise phenotypic data revealed 45 robust main-effect QTLs (M-QTLs) explaining up to 58.20 % phenotypic variation and 973 epistatic QTLs (E-QTLs) explaining up to 92.19 % phenotypic variation for several target traits. Nine QTL clusters containing QTLs for several drought tolerance traits have been identified that can be targeted for molecular breeding. Among these clusters, one cluster harboring 48 % robust M-QTLs for 12 traits and explaining about 58.20 % phenotypic variation present on CaLG04 has been referred as “QTL-hotspot”. This genomic region contains seven SSR markers (ICCM0249, NCPGR127, TAA170NCPGR21, TR11, GA24 and STMS11). Introgression of this region into elite cultivars is expected to enhance drought tolerance in chickpea.Not Availabl

A Systems Biology Approach Identifies a Regulatory Network in Parotid Acinar Cell Terminal Differentiation

<div><p>Objective</p><p>The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.</p><p>Methodology</p><p>A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.</p><p>Results</p><p>Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for <i>Pparg</i> which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving <i>Klf4</i>, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving <i>Prdm1</i>, <i>Sox11</i>, <i>Pax5</i>, miR-200a, and miR-30a) progressively decreases repression of <i>Xbp1</i> gene transcription, in concert with decreased translational repression by miR-214. <i>The transcription factor Xbp1</i> mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with <i>Atf6</i>. Transfection studies show that <i>Xbp1Mist1</i> promoter. In addition, <i>Xbp1</i> and <i>Mist1</i> each activate the parotid secretory protein (<i>Psp</i>) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.</p><p>Conclusion</p><p>This study identifies novel expression patterns of <i>Pparg</i>, <i>Klf4</i>, and <i>Sox11</i> during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an <i>Xbp1</i> driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.</p></div

Thermostabilisation of human serum butyrylcholinesterase for detection of its inhibitors in water and biological fluids

The ability of gelatine-trehalose to convert the normally fragile, dry human serum BChE into a thermostable enzyme and its use in the detection of cholinesterase inhibitors in water and biological fluids is described. Gelatine or trehalose alone is unable to protect the dry enzyme against exposure to high temperature, while a combination of gelatine and trehalose were able to protect the enzyme activity against prolonged exposure to temperature as high as +50°C. A method for rapid, simple and inexpensive means of screening for cholinesterase inhibitors such as carbamates and organophosphates in water, vegetables and human blood has been developed.<br>A capacidade da gelatina-trehalose em converter a frágil BChE do soro humano em uma enzima termoestável e seu uso na descoberta de inibidores de colinesterase em água e fluidos biológicos é apresentado. A Gelatina ou trehalose são incapazes de proteger a enzima seca BchE do soro humano contra exposição a elevadas temperaturas, enquanto que uma combinação de gelatina e trehalose são capazes de proteger a atividade de enzima contra exposição prolongada a temperaturas elevadas e da ordem de 50° C. Um método barato, simples e rápido de screening para inibidores de colinesterase tal como carbamatos e organofosfatos em água, verduras e sangue humano foi desenvolvido

<i>Xbp1</i> Regulates <i>Mist1</i> Expression during Parotid Differentiation.

<p>(A) Log2 plot of microarray data for <i>Xbp1</i> and <i>Mist1</i>. (B) Expression of <i>Xbp1</i> and <i>Mist1</i> is highly correlated across parotid differentiation. Plot of Log2 <i>Xbp1</i> vs. Log2 <i>Mist1</i> shows a linear trend with R² = 0.9538. (C) Luciferase assay shows activation of <i>Mist1</i> promoter by <i>Xbp1</i> in ParC5 cells. Increasing amount of <i>Xbp1</i>-S (spliced <i>Xbp1</i>) cDNA/well (0.25 μg, 0.5 μg, and 1 μg) were co-transfected with a luciferase expression plasmid driven by a <i>Mist1</i> promoter. Significant increase in luciferase expression was observed for all concentrations of <i>Xbp1</i>-S (p = 0.017, p = 0.01, and p = 0.05 respectively) (n = 3).</p

MicroRNA Expression Changes between Time Points.

<p>MicroRNA Expression Changes between Time Points.</p

Transient Activation of <i>Pparg</i> during Parotid Acinar Cell Differentiation.

<p>(A) Network showing transcription factor <i>Pparg</i> and known downstream target genes found in DE Cluster 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.g002" target="_blank">Fig 2</a>). DE Cluster 4 contains 106 genes (including <i>Pparg</i>) with a unique expression pattern; higher expression only in stages 2 and 3. The Metacore knowledge-base identifies 18 of these as <i>Pparg</i> target genes. A green arrow indicates activation of transcription while red arrow indicates inhibition. A grey line means the interaction is uncharacterized. Although a red arrow connects <i>Pparg</i> and <i>ACP5</i>, some publications list the interaction as activating [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref067" target="_blank">67</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125153#pone.0125153.ref068" target="_blank">68</a>] indicating it could be context dependent. (B) Log2 expression of <i>Pparg</i> from microarray data. (C) qPCR data confirming the expression profile of <i>Pparg</i>. RNA samples from independent animals were collected at three time points (E20, P5, and P25). Expression was normalized to <i>Arbp</i>, and data showed significant change in expression by ANOVA. n = 3.</p