Equilibrium unfolding of neuronal calcium sensor-1: N-terminal myristoylation influences unfolding and reduces protein stiffening in the presence of calcium

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.     

Clusters of proteins in archaeal and bacterial proteomes using compositional analysis

In silico proteomics complements computational genomics in characterizing genome evolution. Here we examine cluster patterns in archaeal and bacterial proteomes using compositional properties of protein sequences in contrast to the traditionally used sequence alignment procedures. Application of standard Principal Component Analysis to the multi-dimensional data identified cluster patterns. Two types of cluster patterns exist in bacterial proteomes. Proteomes of type I have one major cluster with few isolated points in space revealing an underlying largely homogeneous compositional structure. In type II proteomes two clusters of protein distribution were discernible. The two clusters differ in size and were separated from each other although the boundary was somewhat fuzzy. Proteins falling in the major cluster were labeled as 'typical' and proteins of the minor cluster were called 'atypical'. The atypical proteins were mapped to Cluster of Orthologous Groups. Species distribution in COGs maps with respect to atypical proteins illuminated the biological relationships of extreme diversity among the archaeal members and of diversity among bacteria in relation to their niche. Amino acids that were over-represented in the atypical proteins had higher biosynthetic cost compared to 'typical' ribosomal proteins. However, archaea and bacteria economize by preferring the less costly amino acid to others closely related in chemical structure. Further, over-representation of serine in atypical proteins of archaeal members suggests re-examining these proteomes for the presence of Serine/Threonine phosphatases and kinases in Archaea. Our computational procedure can serve as a useful addition to the existing tools for carrying out in silico proteomics.     

Regulation of heparin-binding EGF-like growth factor expression in Ha-ras transformed human mammary epithelial cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein expression is induced by EGF in MCF-10A nontransformed and Ha-ras transfected human mammary epithelial cells. The anti-EGF receptor (EGFR) blocking monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 were able to inhibit the induction of HB-EGF mRNA levels in MCF-10A cells. However, the Ha-ras transformed MCF-10A cells were more refractory to inhibition by these agents and only a combination of the 225 MAb and PD153035 was able to significantly abrogate HB-EGF induction by EGF. The anti-erbB2 MAb L26 which interferes with heterodimer formation was able to block HB-EGF induction in response to EGF in MCF-10A cells and in the Ha-ras transformed cells only when used in combination with either the 225 MAb or PD153035. The MEK inhibitor PD90859 completely blocked EGF induction of HB-EGF mRNA levels in the nontransformed and Ha-ras transformed MCF-10A cells, which indicates that MAPK is involved in the signaling pathway of HB-EGF induction by EGF. An increase in the levels of HB-EGF may, therefore, be an important contributor to oncogenic transformation that is caused by Ha-ras overexpression in mammary epithelial cells. J. Cell. Physiol. 186:233-242, 2001. Published 2001 Wiley-Liss, Inc.     

Minimal RNA Aptamer Sequences That Can Inhibit or Alleviate Noncompetitive Inhibition of the Muscle-Type Nicotinic Acetylcholine Receptor

Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10¹¹ neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.     

Oral,Nasal and Pharyngeal Exposure to Lipopolysaccharide Causes a Fetal Inflammatory Response in Sheep

Background

A fetal inflammatory response (FIR) in sheep can be induced by intraamniotic or selective exposure of the fetal lung or gut to lipopolysaccharide (LPS). The oral, nasal, and pharyngeal cavities (ONP) contain lymphoid tissue and epithelium that are in contact with the amniotic fluid. The ability of the ONP epithelium and lymphoid tissue to initiate a FIR is unknown.

Objective

To determine if FIR occurs after selective ONP exposure to LPS in fetal sheep.

Methods

Using fetal recovery surgery, we isolated ONP from the fetal lung, GI tract, and amniotic fluid by tracheal and esophageal ligation and with an occlusive glove fitted over the snout. LPS (5 mg) or saline was infused with 24 h Alzet pumps secured in the oral cavity (n = 7–8/group). Animals were delivered 1 or 6 days after initiation of the LPS or saline infusions.

Results

The ONP exposure to LPS had time-dependent systemic inflammatory effects with changes in WBC in cord blood, an increase in posterior mediastinal lymph node weight at 6 days, and pro-inflammatory mRNA responses in the fetal plasma, lung, and liver. Compared to controls, the expression of surfactant protein A mRNA increased 1 and 6 days after ONP exposure to LPS.

Conclusion

ONP exposure to LPS alone can induce a mild FIR with time-dependent inflammatory responses in remote fetal tissues not directly exposed to LPS.     

The Usefulness and Feasibility of Mobile Interface in Tuberculosis Notification (MITUN) Voice Based System for Notification of Tuberculosis by Private Medical Practitioners – A Pilot Project

Introduction

Tuberculosis (TB) is a notifiable disease and health care providers are required to notify every TB case to local authorities. We conducted a pilot study to determine the usefulness and feasibility of mobile interface in TB notification (MITUN) voice based system for notification of TB cases by private medical practitioners.

Methodology

The study was conducted during September 2013 to October 2014 in three zones of Chennai, an urban setting in South India. Private clinics wherein services are provided by single private medical practitioners were approached. The steps involved in MITUN included: Registration of the practitioners and notification of TB cases by them through voice interactions. Pre and post-intervention questionnaires were administered to collect information on TB notification practices and feasibility of MITUN after an implementation period of 6 months.

Results

A total of 266 private medical practitioners were approached for the study. Of them, 184 (69%) participated in the study; of whom 11 (6%) practitioners used MITUN for TB notification. Reasons for not using MITUN include lack of time, referral of patients to government facility, issues related to patient confidentiality and technical problems. Suggestions for making mobile phone based TB notification process user-friendly included reducing call duration, including only crucial questions and using missed call or SMS options.

Conclusion

The performance (feasibility and usefulness) of MITUN voice based system for TB notification in the present format was sub-optimal. Perceived problems, logistical and practical issues preclude scale–up of notification of TB by private practitioners.