Structural study of biologically significant ligands with major birch pollen allergen Betv1 by docking and molecular dynamics simulation

The major birch pollen allergen, Betv1 of Betula verrucosa is the main causative agent of birch pollen allergy in humans. Betv1 is capable of binding several physiological ligands including fatty acids, flavones, cytokinins and sterols. Until now, no structural information from crystallography or NMR is available regarding binding mode of any of these ligands into the binding pocket of Betv1. In the present study thirteen ligands have been successfully docked into the hydrophobic cavity of Betv1 and binding free energies of the complexes have been calculated using AutoDock 3.0.5. A linear relationship with correlation coefficient (R2) of 0.6 is obtained between ΔG(b)s values plotted against their corresponding IC50 values. The complex formed between Betv1 and the best docking pose for each ligand has been optimized by molecular dynamics simulation. Here, we describe the ligand binding of Betv1, which provides insight into the biological function of this protein. This knowledge is required for structural alteration or inhibition of some of these ligands in order to modify the allergenic properties of this protein.     

Role of Moringa oleifera on enterochromaffin cell count and serotonin content of experimental ulcer model

The present study has been undertaken to observe the effect of aqueous extract of M. oleifera (MO) leaf (300mg/kg body weight) on mean ulcer index, enterochromaffin (EC) cells and serotonin (5-hydroxytryptamine; 5-HT) content of ulcerated gastric tissue. Ulceration was induced by using aspirin (500 mg/kg, po), cerebellar nodular lesion and applying cold stress. In all cases increased mean ulcer index in gastric tissue along with decreased EC cell count was observed with concomitant decrease of 5-HT content. Pretreatment with MO for 14 days decreased mean ulcer index, increased both EC cell count and 5-HT content in all ulcerated group, but treatment with ondansetron, a 5-HT3 receptor antagonist, along with MO pretreatment increased mean ulcer index, decreased 5-HT content without any alteration in EC cell count. The results suggest that the protective effect of MO on ulceration is mediated by increased EC cell count and 5-HT levels which may act via 5-HT3 receptors on gastric tissue.     

In vitro anti-oxidant activity, fluorescence quenching study and structural features of carbohydrate polymers from Phyllanthus emblica

The water-extracted carbohydrate polymers (WE) of Phyllanthus emblica are analyzed using chemical, chromatographic, and spectroscopic methods. Anion-exchange-chromatography of WE yielded four fractions (F1-F4) with different chemical compositions and all of them contain phenolics. The major fraction F4 possesses 50% polysaccharide and 26% phenol, and is a glycoconjugate. The antioxidant capacities of WE and F4 are comparable to standard anti-oxidants. Notably, activities of F1-F4 correlate with their phenol content. Evidence for the complexation of F4 with bovine serum albumin is presented by fluorescence quenching measurement. The results also indicate conformational change of protein at high carbohydrate polymer concentration.     

Cell surface phosphorylation by a novel ecto-protein kinase: a key regulator of cellular functions in spermatozoa

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.     

Post-embryonic pericardial cells of <Emphasis Type="Italic">Drosophila</Emphasis> are required for overcoming toxic stress but not for cardiac function or adult development

The Drosophila heart is composed of two cell types: cardioblasts (CB) and pericardial cells (PC). Whereas CBs act to maintain rhythmic contractions, the functions of accessory PCs are not clear. The close association between these two cell types has led to speculation of a cardio-regulatory role for PCs. However, we find that viability and cardiac function are normal in larvae following post-embryonic ablation of PCs by induced cell death. Removal of PCs during the larval instars or before metamorphosis results in viable and fertile adults. Interestingly, such animals have a reduced lifespan and increased sensitivity to toxic chemicals. Thus, although PCs may have an embryonic role in cardiogenesis, they do not appear to play a part later in cardiac function as suggested. However, the role of PCs in the uptake and sequestering of toxins, their sensitivity to toxic stress and the decreased lifespan of animals without PCs indicate the importance of PCs in organismal homeostasis. D.D. is supported by a CSIR-SPM fellowship. This work was funded by the JNCASR, the Council of Scientific and Industrial Research, and the Department of Biotechnology, Government of India.     

Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.     

Attention Deficit/Hyperactivity Disorder Symptoms and Cognitive Abilities in the Late-Life Cohort of the PATH through Life Study

Attention Deficit/Hyperactivity Disorder (ADHD) is a common neuropsychiatric disorder that has not been well studied in older adults. In this study we examined relationships between ADHD symptoms and cognitive ability and compared them between middle-age (MA; 48–52 years) and older-age (OA; 68–74 years) adults sampled from the same population. ADHD, mood disorder symptoms and cognitive abilities were assessed in a large population-based sample (n = 3443; 50% male). We measured current ADHD symptoms using the adult ADHD Self-Report Scale (ASRS), which we found to have the same underlying structure in both cohorts. Older adults reported significantly lower levels of ADHD symptoms and 2.2% of the OA cohort scored equal or above the ASRS cut-off score of 14 (which has been previously associated with ADHD diagnosis) compared with 6.2% of MA adults. Symptom levels were not significantly different between males and females. Using multi-group structural equation modelling we compared ADHD symptom–cognitive performance relationships between the two age groups. Generally higher ADHD symptoms were associated with poorer cognitive performance in the MA cohort. However, higher levels of inattention symptoms were associated with better verbal ability in both cohorts. Surprisingly, greater hyperactivity was associated with better task-switching abilities in older adults. In the OA cohort ADHD symptom–cognition relationships are indirect, mediated largely through the strong association between depression symptoms and cognition. Our results suggest that ADHD symptoms decrease with age and that their relationships with co-occurring mood disorders and cognitive performance also change. Although symptoms of depression are lower in older adults, they are much stronger predictors of cognitive performance and likely mediate the effect of ADHD symptoms on cognition in this age group. These results highlight the need for age-appropriate diagnosis and treatment of comorbid ADHD and mood disorders.     

Direct Interaction between a Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport of Chloroplasts

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.The thylakoid membrane of plant chloroplasts possesses two systems working in parallel for the transport of soluble proteins across the bilayer and into the lumen, namely the chloroplast secretory system and the chloroplast twin arginine translocation (cpTat) system (Müller and Klösgen, 2005; Cline and Theg, 2007; Cline and Dabney-Smith, 2008; Albiniak et al., 2012). For both systems, proteins destined for the thylakoid lumen are encoded by nuclear genes, cytoplasmically translated as higher molecular mass precursor proteins containing targeting sequences, and imported into the chloroplast. However, lumen-targeting sequences on precursors directed to the cpTat system contain obligate twin Arg residues on the amino-proximal side of the hydrophobic core, and the precursors are transported in folded conformations. Both systems require energy to drive the translocation process, but the cpTat system relies solely on the transmembrane potential generated by the protonmotive force (PMF) of photosynthesis, whereas the secretory system also relies on ATP hydrolysis (Cline and Theg, 2007). It is estimated that roughly one-half of the lumen proteins contain twin-Arg signal peptides (Peltier et al., 2004; Sun et al., 2004), several of which are involved in photosynthetic processes, such as the 23-kD subunit of the oxygen evolving complex of PSII (OE23; Ifuku et al., 2011), OE17 (Yi et al., 2006), and subunit T of PSII (Kapazoglou et al., 1995) and subunit N of PSI (Haldrup et al., 1999), making the cpTat system a vital pathway for higher plants.Twin arginine translocation (Tat) systems are also found in bacterial plasma membrane, and both thylakoid and bacteria serve as model systems for studies on the Tat pathway mechanism, each providing insight into different aspects of the mechanism and demonstrating important differences between the two. The cpTat machinery contains three membrane-bound components, Tha4, Hcf106, and cpTatC, with homologous proteins TatA, TatB, and TatC in bacteria, respectively. Tha4 and Hcf106 share sequence and structural homology. They both contain an N-terminal single-transmembrane region followed by a hinge region that connects to an amphipathic α-helix and a divergent C-terminal tail. However, they have distinct functions in the cpTat pathway (Sargent et al., 1999; Dabney-Smith et al., 2003). Hcf106 is largely found in complex with cpTatC, together composing the Tat receptor complex in the thylakoid that migrates as an approximately 700-kD complex by blue native (BN)-PAGE, whereas Tha4 is found as a separate homooligomeric complex of approximately 400 kD or less by BN-PAGE. cpTatC contains six transmembrane regions with the N and C termini on the stromal face of the membrane and serves as the initial receptor of the signal peptide in the receptor complex.Translocation occurs in a cyclical fashion. Precursor binds to the cpTatC-Hcf106 receptor complex in an energy-independent manner. Then, in the presence of the PMF, which mainly consists of ∆pH in isolated thylakoids, Tha4 assembles with the precursor-bound receptor complex to form the active translocase. At this point, transport occurs. After transport, Tha4 dissociates from the receptor complex, thus resetting the system for subsequent rounds of translocation (Mori et al., 1999; Cline and Mori, 2001; Mori and Cline, 2002). This regulated assembly of Tha4 and its tight correlation to transport of the precursor suggests that Tha4 has a critical role in the translocation step.Several models of the Tat translocase propose that Tha4 (TatA) serves as the protein-conducting channel. Several characteristics support this hypothesis, including a regulated assembly mechanism, the requirement for Tha4 only at the translocation step (Cline and Mori, 2001), the molar excess of Tha4 over cpTatC and Hcf106 (Mori et al., 2001; Celedon and Cline, 2012), oligomerization of Tha4 at the translocase (Dabney-Smith and Cline, 2009), and observations of channel-like structures of the Escherichia coli TatA in detergent extracts or even in vivo in E. coli cells (Gohlke et al., 2005; Sargent et al., 2006; Berthelmann et al., 2008). However, none of these studies demonstrate a direct interaction between precursor and Tha4 (TatA).Studies on the E. coli Tat system demonstrate weak cross links between TatA and precursor but did not follow the interaction during active transport, as the UV-inducible cross linking occurred after transport (Maurer et al., 2010). The question still remains how Tha4 (TatA) is directly involved in the translocation event itself. If Tha4 serves the role of protein-conducting channel, one would expect that as the precursor passes through the channel it would interact with Tha4. To test this hypothesis, we have employed an alternative cross-linking strategy involving disulfide exchange cross linking by generating Cys-containing variants of both precursor and Tha4 in pea (Pisum sativum). This method allows probing of the interactions between precursor and Tha4 in the steps immediately prior to and during the transport of precursor, unlike other cross-linking methods employed previously. Through one-to-one disulfide bond formation between single Cys residues placed throughout the mature domain of pOE17 and Tha4, we determined that Tha4 is in direct contact with full-length precursor after its binding the receptor and immediately prior to or during transport across the membrane. BN-PAGE demonstrated that Cys-substituted Tha4 was able to relocate into the approximately 700-kD complex in the presence of Cys-substituted precursor, demonstrating that direct interaction between the two occurs as part of the active translocase. Moreover, site-specific Cys mutations allow us to determine, to our knowledge for the first time, the region of Tha4 in contact with precursor during transport. How these data affect current models for protein transport by the cpTat pathway are discussed.     

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.