Deciphering the factors responsible for the stability of a GFP variant resistant to alkaline pH using molecular dynamics simulations

Charged amino acids having ionizable side chains play crucial roles in maintaining the solubility and stability of a protein. These charged amino acids are mostly exposed on protein surface and participate in electrostatic interactions with neighboring charged amino acids as well as with solvent. Therefore, the change in the solvent pH affects the protein stability in most cases. Previously, we reported a GFP variant, GFP14R having 14 surface lysines replaced with arginines, that showed enhanced stability under alkaline pH. Here, we analyzed the factors that contribute to the stability of the GFP14R under alkaline pH quantitatively using molecular dynamics simulations. Protonation state of the charged amino acids of GFP14R and control GFP under neutral pH and alkaline pH were modeled, and molecular dynamics simulations were performed. This comparative analysis revealed that the GFP14R with more arginine frequency on the surface maintained the stability under both pH conditions without much change in their salt-bridge interactions as well as the hydrogen bond interactions with solvent. On the other hand, these interactions were significantly reduced for the control GFP under alkaline pH due to the deprotonated lysine side chains. These results suggest that the advantageous property of arginine over lysine can be considered one of the parameter for the protein stability engineering under alkaline pH conditions.     

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

GSTM1-null allele predicts rapid disease progression in nondialysis patients and mortality among South Indian ESRD patients

Molecular and Cellular Biochemistry - Neuroblastoma (NB) is the common pediatric tumor of the sympathetic nervous system characterized by poor prognosis. Owing to the challenges such as high tumor...     

Host selection and responses to forest fragmentation in acorn weevils: inferences from dynamic occupancy models

Prior studies on species‐specific responses to habitat alteration have demonstrated that niche breadth is positively associated with patch occupancy rates in landscapes fragmented by agriculture. However, these studies generally have focused on vertebrates and relied upon data collected at a single point in time, neglecting dynamic processes that could alter inferences. We studied the effects of host selection and forest fragmentation on population dynamics of acorn weevils Curculio, the primary insect seed predators of oaks in North America. Detection/non‐detection data were collected from 174 red and white oaks in 19 forested fragments from 2005–2008. We used dynamic multi‐season site‐occupancy models within a Bayesian framework to explore variation in patch (tree‐level) occupancy dynamics of three species of weevils that vary in their specialization, i.e. their relative selection of red and white oak as hosts: C. pardalis (white oak specialist), C. sulcatulus (generalist) and C. proboscideus (generalist). Contrary to expectations, the specialist exhibited greater estimated rates of occupancy than generalists. However, red oak trees occupied by the white oak specialist appeared to function as sink populations maintained by frequent colonization following local extinction. Specialists also exhibited greater relative variation in occupancy and relative abundance on their host trees among years. Generalists exhibited lower local extinction and colonization rates than the specialist. Occupancy and vital rates of weevils on a host tree increased with acorn production and were significantly influenced by neighborhood forest density. Our results suggest that across much of their range in the eastern United States acorn weevils exist in fragmented, temporally dynamic landscapes, with generalists occurring on a lower proportion of usable trees but buffered by access to more suitable patches and greater patch‐specific survival. More generally, our results demonstrate that estimates of specialization derived from occupancy data may be misleading in the absence of patch‐specific information on vital rates.     

Genetic association of Glutathione peroxidase-1 (GPx-1) and NAD(P)H:Quinone Oxidoreductase 1(NQO1) variants and their association of CAD in patients with type-2 diabetes

Coronary artery disease (CAD) is a major health concern and the leading cause of death in individuals with type-2 diabetes mellitus (T2DM). Glutathione peroxidase-1 (GPx-1) and NAD(P)H: quinone oxidoreductase (NQO1) are known for its broad range of detoxification. The role of functional variants of these genes in the development of various disorders is proven. Hereby, we investigated the possible role of these variants in the development of CAD in T2DM patients of South Indian population. In this case-control study, a total of 539 patients (T2DM = 241; T2DM-CAD = 298) and 285 controls were included. The C198T GPx-1 and C609T NQO1 single-nucleotide polymorphisms were analyzed by PCR-RFLP. Further, these genotypes were correlated with blood lipid profile. Regression analysis showed that GPx1-C/T genotype is associated with a 1.35-fold increase (95% CI = 1.000-1.824; P = 0.048) and GPx1-T/T genotype is associated with a 1.76-fold increase (95% CI = 1.011 to 3.066; P = 0.046) to the T2DM development. Increased odds ratio showed that NQO1-T/T genotype had a higher occurrence of CAD in diabetic patients with CAD (95% CI = 1.003-2.674, P = 0.049) than T2DM patients without CAD. The level of triglycerides alone showed significant increase for GPx-1-C/T and -T/T genotypes in Tukey's Post hoc analysis (177.1 ± 19.2 vs. 184 ± 23.5; P = 0.039 and 177.1 ± 19.2 vs. 190 ± 22.4; P = 0.006) among the patients with T2DM-CAD. Our work concludes that GPx-1 variants might contribute to the development of diabetes and both GPx-1 and NQO1 variants confirm the association of CAD in people with T2DM of South Indian population.     

Segregation of calcium signalling mechanisms in magnocellular neurones and terminals

Every cell or neuronal type utilizes its own specific organization of its Ca(2+) homeostasis depending on its specific function and its physiological needs. The magnocellular neurones, with their somata situated in the supraoptic and paraventricular nuclei of the hypothalamus and their nerve terminals populating the posterior hypophysis (neural lobe) are a typical and classical example of a neuroendocrine system, and an important experimental model for attempting to understand the characteristics of the neuronal organization of Ca(2+) homeostasis. The magnocellular neurones synthesize, in a cell specific manner, two neurohormones: arginine-vasopressin (AVP) and oxytocin (OT), which can be released, in a strict Ca(2+)-dependent manner, both at the axonal terminals, in the neural lobe, and at the somatodendritic level. The two types of neurones show also distinct type of bioelectrical activity, associated with specific secretory patterns. In these neurones, the Ca(2+) homeostatic pathways such as the Na(+)/Ca(2+) exchanger (NCX), the endoplasmic reticulum (ER) Ca(2+) pump, the plasmalemmal Ca(2+) pump (PMCA) and the mitochondria are acting in a complementary fashion in clearing Ca(2+) loads that follow neuronal stimulation. The somatodendritic AVP and OT release closely correlates with intracellular Ca(2+) dynamics. More importantly, the ER Ca(2+) stores play a major role in Ca(2+) homeostatic mechanism in identified OT neurones. The balance between the Ca(2+) homeostatic systems that are in the supraoptic neurones differ from those active in the terminals, in which mainly Ca(2+) extrusion through the Ca(2+) pump in the plasma membrane and uptake by mitochondria are active. In both AVP and OT nerve terminals, no functional ER Ca(2+) stores can be evidenced experimentally. We conclude that the physiological significance of the complexity of Ca(2+) homeostatic mechanisms in the somatodendritic region of supraoptic neurones and their terminals can be multifaceted, attributable, in major part, to their specialized electrical activity and Ca(2+)-dependent neurohormone release.     

Generation of anti-c-met single domain antibody fragment based on human stable frameworks

The size reduction is an important issue in the biomedical application of antibody and single domain antibody fragment is recognized as very attractive tool. However, it is very time-consuming and laborious to generate the fragment antibody with targeted binding function. Here, we investigated the possibility to prepare single domain antibody (sdAb) by a simple grafting method based on stable human consensus framework sequences. The complementarity determining region sequences in V_H domain of anti-c-Met scFv from rabbit were grafted with the human V_H3 consensus framework sequences, which generated the anti-c-Met single domain antibody showing almost same binding activity to its scFv form. The generated single domain antibody could be produced as functional form in oxidizing cytoplasm of E. coli, but produced as inactive form in reducing cytoplasm. The structural analysis of the homology models gave us the insight on the stability of the single domain antibody. In this report, we have demonstrated that the very stable human consensus framework sequence can be used for the generation of active anti-c-Met sdAb via complementarity determining regions grafting. We expect that this kind of grafting method for the generation of sdAb may provide us with the opportunities to prepare sdAbs based on the known antibody sequences.     

Entomopathogenicity of endophytic <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain SRM against larvae of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Noctuidae: Lepidoptera)

An endophytic Serratia marcescens strain SRM (MTCC 8708) isolated from the flowers of summer squash was found to be entomopathogenic against the larvae of Helicoverpa armigera. Natural epizootic of this bacterial strain on the larvae collected from summer squash flowers ranged from 19.9 to 72.3%. Under laboratory conditions, a dose of 6 × 10¹⁰ c.f.u./ml diet induced 66.3% mortality of first instar H. armigera larvae. Similarly all other growth and development parameters of the insect were severely retarded in a dose-dependent manner. The bacterium invaded the entire alimentary canal and haemolymph with successful replacement of all other gut-associated microflora. There was a great reduction in midgut proteinase activity due to inhibition of five major proteinase isozymes by S. marcescens infection. Further, a synergistic interaction between chitinases isolated from this strain and Bacillus thuringiensis Cry1Ac toxin was observed. The present findings suggest that this plant-associated S. marcescens strain SRM could be suitably exploited for the management of H. armigera.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.