Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of “deviant” Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide array of metabolic functions (reviewed in Ref. 1). Research addressing the biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter vinelandii led to the discovery of an operon (iscA^nifnifUSVcysE1) involved in the biosynthesis of [Fe-S] clusters (reviewed in Ref. 2). Subsequent experiments led to the finding of two more systems involved in the de novo biosynthesis of [Fe-S] clusters, the isc and the suf systems (3, 4). Like Escherichia coli, the genome of Salmonella enterica serovar Typhimurium encodes for the isc and suf [Fe-S] cluster biosynthesis machinery.Recent studies have identified a number of additional or non-isc/-suf-encoded proteins that are involved in bacterial [Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY, an iron-binding protein believed to be involved in iron trafficking and iron delivery (5–7); YggX, an Fe²⁺-binding protein that protects the cell from oxidative stress (8, 9); ErpA, an alternate A-type [Fe-S] cluster scaffolding protein (10); NfuA, a proposed intermediate [Fe-S] delivery protein (11–13); YtfE, a protein proposed to be involved in [Fe-S] cluster repair (14, 15); and CsdA-CsdE, an alternative cysteine desulferase (16).Analysis of the metabolic network anchored to thiamine biosynthesis in S. enterica identified lesions in three non-isc or -suf loci that compromise Fe-S metabolism as follows: apbC, apbE, and rseC (17–21). This metabolic system was subsequently used to dissect a role for cyaY and gshA in [Fe-S] cluster metabolism (6, 22, 23). Of these, the apbC (mrp in E. coli) locus was identified as the predominant site of lesions that altered thiamine synthesis by disrupting [Fe-S] cluster metabolism (17, 18).ApbC is a member of the ParA subfamily of proteins that have a wide array of functions, including electron transfer (24), initiation of cell division (25), and DNA segregation (26, 27). Importantly, ATP hydrolysis is required for function of all well characterized members of this subfamily, and all members contain a “deviant” Walker A motif, which contains two lysine residues instead of one (GKXXXGK(S/T)) (28). ApbC has been shown to hydrolyze ATP (17).Recently, five proteins with a high degree of identity to ApbC have been shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence alignments of the central portion of these proteins and bacterial ApbC are shown in Fig. 1. HCF101 was demonstrated to be involved in chloroplast [Fe-S] cluster metabolism (29, 30). The CFD1, Npb35, and huNbp35 (formally Nubp1) proteins were demonstrated to be involved in cytoplasmic [Fe-S] cluster metabolism (31, 32). Ind1 was demonstrated to be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme NADH:ubiquinone oxidoreductase (33). There is currently no report of any of these proteins hydrolyzing ATP.Open in a separate windowFIGURE 1.Protein sequence alignments of members of the ApbC/Nbp35 subfamily of ParA family of proteins. Protein alignments were assembled using the Clustal_W method in the Lasergene® software and show only the central portion of the proteins, which have the highest sequence conservation. The three boxed areas highlight the Walker A box, conserved Ser residue, and CXXC motif. Proteins listed are as follows: ApbC (S. enterica serovar Typhimurium LT2), CFD1 (S. cerevisiae), Nbp35 (S. cerevisiae), HCF101 (Arabidopsis thaliana), huNpb35 (formally Nubp1) (Homo sapiens), and Ind1 (Candida albicans).Biochemical analysis of ApbC indicated that it could bind and transfer [Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate isomerase (34). Additional genetic studies indicated that ApbC has a degree of functional redundancy with IscU, a known [Fe-S] cluster scaffolding protein (35, 36).In this study we investigate the correlation between the biochemical properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and [Fe-S] cluster transfer rates) and the in vivo function of this protein. This is the first detailed kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of deviant Walker A proteins and the first functional analysis of a member of the ever expanding family of ApbC/Nbp35 proteins. Data presented indicate that noncomplementing variants have distinct biochemical properties that place them in three distinct classes.     

The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Activates RhoA Signaling in Skeletal Muscle

Obscurin is a large (∼800-kDa), modular protein of striated muscle that concentrates around the M-bands and Z-disks of each sarcomere, where it is well positioned to sense contractile activity. Obscurin contains several signaling domains, including a rho-guanine nucleotide exchange factor (rhoGEF) domain and tandem pleckstrin homology domain, consistent with a role in rho signaling in muscle. We investigated the ability of obscurin''s rhoGEF domain to interact with and activate small GTPases. Using a combination of in vitro and in vivo approaches, we found that the rhoGEF domain of obscurin binds selectively to rhoA, and that rhoA colocalizes with obscurin at the M-band in skeletal muscle. Other small GTPases, including rac1 and cdc42, neither associate with the rhoGEF domain of obscurin nor concentrate at the level of the M-bands. Furthermore, overexpression of the rhoGEF domain of obscurin in adult skeletal muscle selectively increases rhoA expression and activity in this tissue. Overexpression of obscurin''s rhoGEF domain and its effects on rhoA alter the expression of rho kinase and citron kinase, both of which can be activated by rhoA in other tissues. Injuries to rodent hindlimb muscles caused by large-strain lengthening contractions increases rhoA activity and displaces it from the M-bands to Z-disks, similar to the effects of overexpression of obscurin''s rhoGEF domain. Our results suggest that obscurin''s rhoGEF domain signals at least in part by inducing rhoA expression and activation, and altering the expression of downstream kinases in vitro and in vivo.     

A room temperature ionic liquid as convenient solvent for the oxidative folding of conopeptides

We report the first example of conopeptide oxidation performed in a biocompatible ionic liquid, 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]), which enables the efficient formation of both hydrophilic and poorly water‐soluble conotoxins compared with conventional methods. Moreover, the method features a high‐concentration approach ultimately leading to higher yields at reduced separation effort. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Experimental translocation of juvenile water voles in a Scottish lowland metapopulation

Population density affects dispersal success because residents can hinder or facilitate immigration into a new site, via a “social fence effect” or “social attraction” (or “conspecific attraction”), respectively. These mechanisms can affect the dynamics of fragmented populations and the success of translocations. However, information on the settlement behaviour of dispersers is rare. We conducted a manipulative field experiment using wild water voles, which exist in metapopulations along waterways in Scotland. We translocated 17 young of dispersal age into either an occupied site or a vacant site containing good habitat, which had recently become extinct due to a feral predator (American mink) moving through. We monitored the movements of translocated voles using radio telemetry. Translocated voles were less likely to settle in occupied sites with higher densities of residents, suggesting a possible social fence effect at high density. There was evidence of a social attraction mechanism, because voles never remained at new sites unless another individual arrived soon after translocation, and they were more likely to settle in occupied or colonised sites than vacant ones. Voles remained in the transient phase of dispersal for many days, and often followed a “stepping stone” trajectory, stopping for several days at successive sites. We suggest that trajectories followed by dispersing water voles, the time scale and long dispersal distances found in this species are conducive to locating conspecifics at low density and colonising vacant habitat. These results are encouraging for prospects of metapopulation persistence and future translocation success.     

Technical note: Applicability of tooth cementum annulation to an archaeological population

The use of tooth cementum annulations for age determination has been deemed promising, exhibiting high correlations with chronological age. Despite its apparent potential, to date, the tooth cementum annulations method has been used rarely for estimating ages in archaeological populations. Here we examine the readability of cementum annulations and the consistency of age estimates using a sample of 116 adults from the Iron Gates Gorge Mesolithic/Neolithic series. Our examination of the method pointed to several sources of error that call into question the use of this method for estimating the chronological ages of archaeologically derived dental samples. The poor performance of the method in our analysis might be explained by taphonomic influences, including the effect of chemical and biological agents on dental microstructures. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Inertial sensor-based knee flexion/extension angle estimation

A new method for estimating knee joint flexion/extension angles from segment acceleration and angular velocity data is described. The approach uses a combination of Kalman filters and biomechanical constraints based on anatomical knowledge. In contrast to many recently published methods, the proposed approach does not make use of the earth's magnetic field and hence is insensitive to the complex field distortions commonly found in modern buildings. The method was validated experimentally by calculating knee angle from measurements taken from two IMUs placed on adjacent body segments. In contrast to many previous studies which have validated their approach during relatively slow activities or over short durations, the performance of the algorithm was evaluated during both walking and running over 5 minute periods. Seven healthy subjects were tested at various speeds from 1 to 5 mile/h. Errors were estimated by comparing the results against data obtained simultaneously from a 10 camera motion tracking system (Qualysis). The average measurement error ranged from 0.7 degrees for slow walking (1 mph) to 3.4 degrees for running (5 mph). The joint constraint used in the IMU analysis was derived from the Qualysis data. Limitations of the method, its clinical application and its possible extension are discussed.     

Mobilization of Neural Stem Cells and Generation of New Neurons in 6-OHDA�Clesioned Rats by Intracerebroventricular Infusion of Liver Growth Factor

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson''s disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine–lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases. (J Histochem Cytochem 57:491–502, 2009)     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Metabolic modulation and cellular therapy of cardiac dysfunction and failure

At present the prevalence of heart failure rises along with aging of the population. Current heart failure therapeutic options are directed towards disease prevention via neurohormonal antagonism (β-blockers, angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers and aldosterone antagonists), symptomatic treatment with diuretics and digitalis and use of biventricular pacing and defibrillators in a special subset of patients. Despite these therapies and device interventions heart failure remains a progressive disease with high mortality and morbidity rates. The number of patients who survive to develop advanced heart failure is increasing. These patients require new therapeutic strategies. In this review two of emerging therapies in the treatment of heart failure are discussed: metabolic modulation and cellular therapy. Metabolic modulation aims to optimize the myocardial energy utilization via shifting the substrate utilization from free fatty acids to glucose. Cellular therapy on the other hand has the goal to achieve true cardiac regeneration. We review the experimental data that support these strategies as well as the available pharmacological agents for metabolic modulation and clinical application of cellular therapy.