An Efficient Wavelet Based Approximation Method to Steady State Reaction–Diffusion Model Arising in Mathematical Chemistry

The mathematical model of Rahamathunissa and Rajendran (J Math Chem 44:849–861, 2008) in an amperometric biosensor response is discussed. In this paper, we have applied the shifted second kind Chebyshev wavelets (CW) to obtain the numerical solutions of reaction–diffusion equations containing a nonlinear term related to Michaelis–Menton kinetics of the enzymatic reaction. The application of the shifted second kind CW operational matrices for solving initial and boundary value problems is presented. The obtained numerical results demonstrate efficient and applicability of the proposed method. The power of the manageable method is confirmed. Moreover the use of shifted second kind CW method is found to be simple, efficient, accurate, small computation cost, and computationally attractive.     

A new model for ligand release. Role of side chain in gating the enediyne antibiotic

Antitumor antibiotic chromoproteins such as neocarzinostatin involve a labile toxin that is tightly bound by a protective protein with very high affinity but must also be freed to exert its function. Contrary to the prevalent concept of ligand release, we established that toxin release from neocarzinostatin requires no major backbone conformational changes. We report, herein, that subtle changes in the side chains of specific amino acid residues are adequate to gate the release of chromophore. A recombinant wild type aponeocarzinostatin and its variants mutated around the opening of the chromophore binding cleft are employed to identify specific side chains likely to affect chromophore release. Preliminary, biophysical characterization of mutant apoproteins by circular dichroism and thermal denaturation indicate that the fundamental structural characteristics of wild type protein are conserved in these mutants. The chromophore reconstitution studies further show that all mutants are able to bind chromophore efficiently with similar complex structures. NMR studies on 15N-labeled mutants also suggest the intactness of binding pocket structure. Kinetic studies of chromophore release monitored by time course fluorescence and quantitative high pressure liquid chromatography analyses show that the ligand release rate is significantly enhanced only in Phe78 mutants. The extent of DNA cleavage in vitro corresponds well to the rate of chromophore release. The results provide the first clear-cut indication of how toxin release can be controlled by a specific side chain of a carrier protein.     

A buoyancy-based screen of Drosophila larvae for fat-storage mutants reveals a role for Sir2 in coupling fat storage to nutrient availability

Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet.     

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB

MelB catalyzes the obligatory cotransport of melibiose with Na⁺, Li⁺, or H⁺. Crystal structure determination of the Salmonella typhimurium MelB (MelB_St) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na⁺ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔC_p), and regulatory phosphotransferase EIIA^Glc binding with isothermal titration calorimetry (ITC). We found that MelB_St thermostability is increased by either substrate (Na⁺ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔC_p, suggesting MelB_St hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelB_St dehydration and cavity closure. Lastly, we observed that EIIA^Glc, which has been suggested to trap MelB_St at an outward-open state, readily binds to the MelB_St apo state at an affinity similar to MelB_St/Na⁺. However, it has a suboptimal binding to the ternary state, implying that MelB_St in the ternary complex may be conformationally distant from the EIIA^Glc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na⁺ or melibiose) favors MelB_St at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.     

CCK4 contains the full hormonal information of cholecystokinin in isolated pancreatic acini

In mouse and rat isolated pancreatic acini, the C-terminal tetrapeptide amide of CCK (CCK₄) fully mimicked the actions of the physiological octapeptide hormone (CCK₈) although CCK₄ was 10–100 thousand fold less potent than CCK₈. Parallelism was observed for stimulation of both amylase secretion (including the submaximal secretion observed at supramaximal concentrations of agonist), and stimulation of glucose transport. Furthermore, CCK₄ and CCK₈ were able to comletely inhibit the binding of radioiodinated CCK₃₃ to CCK receptors on acini. Therefore, the CCK₄ sequence appears to be the minimal functional unit which possesses all of the information required to elicit the actions of CCK on the pancreas. The additional 4 amino acids present in CCK₈ increase the affinity of the CCK molecule for pancreatic CCK receptors and thus enhance target organ specificity and sensitivity.     

Hyperglycemic and Hyperlipidemic Conditions Alter Cardiac Cell Biomechanical Properties

Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.     

High Resolution Mapping of Modafinil Induced Changes in Glutamate Level in Rat Brain

Modafinil is marketed in the United States for the treatment of narcolepsy and daytime somnolence due to shift-work or sleep apnea. Investigations of this drug in the treatment of cocaine and nicotine dependence in addition to disorders of executive function are also underway. Modafinil has been known to increase glutamate levels in rat brain models. Proton magnetic resonance spectroscopy (¹HMRS) has been commonly used to detect the glutamate (Glu) changes in vivo. In this study, we used a recently described glutamate chemical exchange saturation transfer (GluCEST) imaging technique to measure Modafinil induced regional Glu changes in rat brain and compared the results with Glu concentration measured by single voxel ¹HMRS. No increases in either GluCEST maps or ¹HMRS were observed after Modafinil injection over a period of 5 hours. However, a significant increase in GluCEST (19±4.4%) was observed 24 hours post Modafinil administration, which is consistent with results from previous biochemical studies. This change was not consistently seen with ¹HMRS. GluCEST mapping allows regional cerebral Glu changes to be measured and may provide a useful clinical biomarker of Modafinil effects for the management of patients with sleep disorders and addiction.