The binding of zinc and copper ions to nerve growth factor is differentially affected by pH: implications for cerebral acidosis

It has recently been shown that transition metal cations Zn2+ and Cu2+ bind to histidine residues of nerve growth factor (NGF) and other neurotrophins (a family of proteins important for neuronal survival) leading to their inactivation. Experimental data and theoretical considerations indicate that transition metal cations may destabilize the ionic form of histidine residues within proteins, thereby decreasing their pK(a) values. Because the release of transition metal cations and acidification of the local environment represent important events associated with brain injury, the ability of Zn2+ and Cu2+ to bind to neurotrophins in acidic conditions may alter neuronal death following stroke or as a result of traumatic injury. To test the hypothesis that metal ion binding to neurotrophins is influenced by pH, the effects of Zn2+ and Cu2+ on NGF conformation, receptor binding and NGF tyrosine kinase (trkA) receptor signal transduction were examined under conditions mimicking cerebral acidosis (pH range 5.5-7.4). The inhibitory effect of Zn2+ on biological activities of NGF is lost under acidic conditions. Conversely, the binding of Cu2+ to NGF is relatively independent of pH changes within the studied range. These data demonstrate that Cu2+ has greater binding affinity to NGF than Zn2+ at reduced pH, consistent with the higher affinity of Cu2+ for histidine residues. These findings suggest that cerebral acidosis associated with stroke or traumatic brain injury could neutralize the Zn2+-mediated inactivation of NGF, whereas corresponding pH changes would have little or no influence on the inhibitory effects of Cu2+. The importance of His84 of NGF for transition metal cation binding is demonstrated, confirming the involvement of this residue in metal ion coordination.     

Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor

The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

Principal components in three-ball cascade juggling

To uncover the underlying control structure of three-ball cascade juggling, we studied its spatiotemporal properties in detail. Juggling patterns, performed at fast and preferred speeds, were recorded in the frontal plane and subsequently analyzed using principal component analysis and serial correlation techniques. As was expected on theoretical grounds, the principal component analysis revealed that maximally four instead of the original six dimensions (3 balls × 2 planar coordinates) are sufficient for describing the juggling dynamics. Juggling speed was shown to affect the number of dimensions (four for the fast condition, two for the preferred condition) as well as the smoothness of the time evolution of the eigenvectors of the principal component analysis, particularly around the catches. Contrary to the throws and the zeniths, and regardless of juggling speed, consecutive catches of the same hand showed a markedly negative lag-one serial correlation, suggesting that the catches are timed so as to preserve the temporal integrity of the juggling act. Received: 1 April 1999 / Accepted in revised form: 9 August 1999     

Molecular dynamics simulations of human rhinovirus and an antiviral compound

The human rhinovirus 14 (HRV14) protomer, with or without the antiviral compound WIN 52084s, was simulated using molecular dynamics and rotational symmetry boundary conditions to model the effect of the entire icosahedral capsid. The protein asymmetrical unit, comprising four capsid proteins (VP1, VP2, VP3, and VP4) and two calcium ions, was solvated both on the exterior and the interior to fill the inside of the capsid. The stability of the simulations of this large system (~800 residues and 6,650 water molecules) is comparable to more conventional globular protein simulations. The influence of the antiviral compound on compressibility and positional fluctuations is reported. The compressibility, estimated from the density fluctuations in the region of the binding pocket, was found to be greater with WIN 52084s bound than without the drug, substantiating previous computations on reduced viral systems. An increase in compressibility correlates with an entropically more favorable system. In contrast to the increase in density fluctuations and compressibility, the positional fluctuations decreased dramatically for the external loops of VP1 and the N-terminus of VP3 when WIN 52084s is bound. Most of these VP1 and VP3 loops are found near the fivefold axis, a region whose mobility was not considered in reduced systems, but can be observed with this simulation of the full viral protomer. Altered loop flexibility is consistent with changes in proteolytic sensitivity observed experimentally. Moreover, decreased flexibility in these intraprotomeric loops is noteworthy since the externalization of VP4, part of VP1, and RNA during the uncoating process is thought to involve areas near the fivefold axis. Both the decrease in positional fluctuations at the fivefold axis and the increase in compressibility near the WIN pocket are discussed in relationship to the antiviral activity of stabilizing the virus against uncoating.     

Apoptotic cell debris and phosphatidylserine-containing lipid vesicles induce apolipoprotein J (clusterin) gene expression in vital fibroblasts

The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.     

Accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data: A simulation study

The exchange-transferred NOE method to determine the three-dimensional structure of peptides bound to proteins, or other macromolecular systems, is becoming increasingly important in drug design efforts and for large or multicomponent assemblies, such as membrane receptors, where structural analysis of the full system is intractable. The exchange-transferred nuclear Overhauser effect spectroscopy (etNOESY) method allows the determination of the bound-state conformation of the peptide from the intra-molecular NOE interactions between ligand protons. Because only ligand–ligand NOEs are generally observable, the etNOESY method is restricted to fewer NOEs per residue than direct protein structure determination. In addition, the averaging of relaxation rates between free and bound states affects the measured cross-peak intensities, and possibly the accuracy of distance estimates. Accordingly, the study reported here was conducted to examine the conditions required to define a reliable structure. The program CORONA was used to simulate etNOE data using a rate-matrix including magnetic relaxation and exchange rates for two peptide–protein complexes derived from the reference complex of cAMP-dependent protein kinase ligated with a 24-residue inhibitor peptide. The results indicate that reasonably accurate peptide structures can be determined with relatively few NOE interactions when the interactions occur between non-neighboring residues. The reliability of the structural result is suggested from the pattern of NOE interactions. A structure with an accuracy of approximately 1.3 Å rms difference for the main-chain atoms can be obtained when etNOE interactions between non-neighboring residues occur over the length of the peptide. The global precision is higher (approximately 0.9 Å rms difference) but is not correlated to global accuracy. A local definition of precision along the backbone appears to be a good indicator of the local accuracy.     

Interactions among shrub cover and the soil microclimate may determine future Arctic carbon budgets

Arctic and Boreal terrestrial ecosystems are important components of the climate system because they contain vast amounts of soil carbon (C). Evidence suggests that deciduous shrubs are increasing in abundance, but the implications for ecosystem C budgets remain uncertain. Using midsummer CO₂ flux data from 21 sites spanning 16° of latitude in the Arctic and Boreal biomes, we show that air temperature explains c. one‐half of the variation in ecosystem respiration (ER) and that ER drives the pattern in net ecosystem CO₂ exchange across ecosystems. Woody sites were slightly stronger C sinks compared with herbaceous communities. However, woody sites with warm soils (> 10 °C) were net sources of CO₂, whereas woody sites with cold soils (< 10 °C) were strong sinks. Our results indicate that transition to a shrub‐dominated Arctic will increase the rate of C cycling, and may lead to net C loss if soil temperatures rise.     

Microbial rhodopsins on leaf surfaces of terrestrial plants

The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10(26) microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.     

Two randomized controlled clinical trials to study the effectiveness of prednisolone treatment in preventing and restoring clinical nerve function loss in leprosy: the TENLEP study protocols

Background

Nerve damage in leprosy often causes disabilities and deformities. Prednisolone is used to treat nerve function impairment (NFI). However, optimal dose and duration of prednisolone treatment has not been established yet. Besides treating existing NFI it would be desirable to prevent NFI. Studies show that before NFI is clinically detectable, nerves often show subclinical damage. Within the ‘Treatment of Early Neuropathy in LEProsy’ (TENLEP) study two double blind randomized controlled trials (RCT) will be carried out: a trial to establish whether prednisolone treatment of 32 weeks duration is more effective than 20 weeks in restoring nerve function in leprosy patients with clinical NFI (Clinical trial) and a trial to determine whether prednisolone treatment of early sub-clinical NFI can prevent clinical NFI (Subclinical trial).

Methods

Two RCTs with a follow up of 18 months will be conducted in six centers in Asia. In the Clinical trial leprosy patients with recent (< 6 months) clinical NFI, as determined by Monofilament Test and Voluntary Muscle Test, are included. The primary outcomes are the proportion of patients with restored or improved nerve function. In the Subclinical trial leprosy patients with subclinical neuropathy, as determined by Nerve Conduction Studies (NCS) and/or Warm Detection Threshold (WDT), and without any clinical signs of NFI are randomly allocated to a placebo group or treatment group receiving 20 weeks prednisolone. The primary outcome is the proportion of patients developing clinical NFI. Reliability and normative studies are carried out before the start of the trial.

Discussion

This study is the first RCT testing a prednisolone regimen with a duration longer than 24 weeks. Also it is the first RCT assessing the effect of prednisolone in the prevention of clinical NFI in patients with established subclinical neuropathy. The TENLEP study will add to the current understanding of neuropathy due to leprosy and provide insight in the effectiveness of prednisolone on the prevention and recovery of NFI in leprosy patients. In this paper we present the research protocols for both Clinical and Subclinical trials and discuss the possible findings and implications.

Trial registration

Netherlands Trial Register: NTR2300Clinical Trial Registry India: CTRI/2011/09/002022