Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

Computational aspects of feedback in neural circuits

It has previously been shown that generic cortical microcircuit models can perform complex real-time computations on continuous input streams, provided that these computations can be carried out with a rapidly fading memory. We investigate the computational capability of such circuits in the more realistic case where not only readout neurons, but in addition a few neurons within the circuit, have been trained for specific tasks. This is essentially equivalent to the case where the output of trained readout neurons is fed back into the circuit. We show that this new model overcomes the limitation of a rapidly fading memory. In fact, we prove that in the idealized case without noise it can carry out any conceivable digital or analog computation on time-varying inputs. But even with noise, the resulting computational model can perform a large class of biologically relevant real-time computations that require a nonfading memory. We demonstrate these computational implications of feedback both theoretically, and through computer simulations of detailed cortical microcircuit models that are subject to noise and have complex inherent dynamics. We show that the application of simple learning procedures (such as linear regression or perceptron learning) to a few neurons enables such circuits to represent time over behaviorally relevant long time spans, to integrate evidence from incoming spike trains over longer periods of time, and to process new information contained in such spike trains in diverse ways according to the current internal state of the circuit. In particular we show that such generic cortical microcircuits with feedback provide a new model for working memory that is consistent with a large set of biological constraints. Although this article examines primarily the computational role of feedback in circuits of neurons, the mathematical principles on which its analysis is based apply to a variety of dynamical systems. Hence they may also throw new light on the computational role of feedback in other complex biological dynamical systems, such as, for example, genetic regulatory networks.     

Targeting AMPK signaling pathway by natural products for treatment of diabetes mellitus and its complications

Diabetes affects a large population of the world. Lifestyle, obesity, dietary habits, and genetic factors contribute to this metabolic disease. A target pathway to control diabetes is the 5′-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. AMPK is a heterotrimeric protein with α, β, and γ subunits. In several studies, AMPK activation enhanced glucose uptake into cells and inhibited intracellular glucose production. Impairment of AMPK activity is present in diabetes, according to some studies. Drugs used in the treatment of diabetes, such as metformin, are also known to act through regulation of AMPK. Thus, drugs that activate and regulate AMPK are potential candidates for the treatment of diabetes. In addition, many patients encounter important adverse effects, like hypoglycemia, while using allopathic drugs. As a result, the investigation of plant-derived natural drugs that lack adverse side effects and treat diabetes is necessary. Natural products like berberine, quercetin, resveratrol, and so forth have shown significant potential in regulating and activating the AMPK pathway which can lead to manage diabetes mellitus and its complications.     

Using continuous directed evolution to improve enzymes for plant applications

Continuous directed evolution of enzymes and other proteins in microbial hosts is capable of outperforming classical directed evolution by executing hypermutation and selection concurrently in vivo, at scale, with minimal manual input. Provided that a target enzyme’s activity can be coupled to growth of the host cells, the activity can be improved simply by selecting for growth. Like all directed evolution, the continuous version requires no prior mechanistic knowledge of the target. Continuous directed evolution is thus a powerful way to modify plant or non-plant enzymes for use in plant metabolic research and engineering. Here, we first describe the basic features of the yeast (Saccharomyces cerevisiae) OrthoRep system for continuous directed evolution and compare it briefly with other systems. We then give a step-by-step account of three ways in which OrthoRep can be deployed to evolve primary metabolic enzymes, using a THI4 thiazole synthase as an example and illustrating the mutational outcomes obtained. We close by outlining applications of OrthoRep that serve growing demands (i) to change the characteristics of plant enzymes destined for return to plants, and (ii) to adapt (“plantize”) enzymes from prokaryotes—especially exotic prokaryotes—to function well in mild, plant-like conditions.

Continuous directed evolution using the yeast OrthoRep system is a powerful way to improve enzymes for use in plant engineering as illustrated by “plantizing” a bacterial thiamin synthesis enzyme.     

A comparison of polyclonal antisera to pregnanediol 3 alpha glucuronide obtained from three different rabbits and their use in ELISA

The variation in the estimates of urinary pregnanediol glucuronide obtained by ELISA with 12 polyclonal antisera has been assessed. Of the 12 antisera, eight gave values comparable with each other and could be used interchangeably in the ELISA. Thus a large stock of polyclonal antibody which gave consistent values was generated and can now be used in diagnostic ELISA over a long period of time. This procedure is more economical than the development of monoclonal antibodies for the same purpose.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

Effect of an electric field on the motility of Entamoeba histolytica examined by multiple-beam interference microscopy

A recently developed multiple-beam interference microscopic technique has been used to visualize submicroscopic structures of Entamoeba histolytica and their movements in applied external electric fields. The movements were videorecorded and it was found that at low current (120 microA) pseudopods are filled with hyaline ectoplasm. At slightly higher current (about 150 microA), the amoeba stops extending the pseudopods and loosens its attachment to the surface. At higher currents (200 microA), it forms a cyst and remains immobile for a time. Before this stage is reached a narrow ring is formed around the nucleus due to alterations in the proteins to protect it.     

Purification and characterization of a cell-surface lectin (Lectin II) from Agrobacterium radiobacter NCIM 2443

A lectin was isolated from Agrobacterium radiobacter cell surface and purified. It is a monomer of 40 kDa as shown by SDS-PAGE. The lectin has a pI of 9.15 and amino acid composition of the lectin shows that 44% of the amino acids are hydrophobic. The lectin agglutinates rabbit erythrocytes but does not agglutinate human erythrocytes. It does not show specificity for monosaccharides except for D-glucosamine. Fetuin and its N-linked glycopeptide also inhibit the activity of the lectin but greater inhibition is shown by locust bean gum and Nicotiana tobaccum (tobacco) tissue extracts.     

Rice bran lipase: extraction, activity, and stability

A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mM and pH 7, containing 0.5 mM of CaCl(2)). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability.