Adhesive properties of the purified plasminogen activator Pla of Yersinia pestis

The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays.     

Species-specific features affect the ability of census-derived models to map winter avian distribution

We identify autoecological traits of bird species that influence the accuracy of predictive models of species distribution based on census data obtained from stratified sampling. These models would serve as a complementary approach to the development of regional bird atlases. We model the winter bird abundance of 64 terrestrial bird species in 77 census plots in Central Spain (Madrid province), using regression tree analyses. The predicted distribution of species density derived from statistical models (birds/10 ha) was compared with the published relative abundances depicted by a very accurate regional atlas of wintering birds (birds observed per 10 h). Statistical models explained an average of 41.7% of the original deviance observed in the local bird distribution (range 19.6–79.3%). Significant associations between observed relative abundances (atlas data) and predicted average densities in 1×1 km squares within 10×10 km UTMs were attained for 44 out of 64 species. Interspecific discrepancies between predicted and observed distribution maps decreased with between-year constancy in regional bird distribution and the degree of ecological specialization of species. Therefore, statistical modeling using census localities allowed us to depict geographical variations in bird abundance that were similar to those in the quantitative atlas maps. Nevertheless, bird distributions derived from statistical models are less reproducible in some species than in others, depending on their autoecological traits.     

Intrastriatal infusion of liver growth factor stimulates dopamine terminal sprouting and partially restores motor function in 6-hydroxydopamine-lesioned rats.

Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and may be useful for neuroregenerative therapies. The aim of this work was to investigate the effects of the intrastriatal (IS) infusion of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Tyrosine hydroxylase-positive innervation was significantly increased in the dopamine-denervated striatum of rats receiving intrastriatal LGF infusions (160 ng/day/rat x 15 days) as compared with a vehicle-infused group. There was no evidence of dopaminergic neurogenesis in the striatum or substantia nigra in any experimental group at the times studied. However, in those animals undergoing IS-LGF infusion for 48 hr, we found a significant increase in both microglial proliferation and in the number of microglial cells that acquired the ameboid morphology. This is characteristic of activated microglia/macrophages that has been reported to play an important role in dopamine terminal sprouting. In summary, our study shows that IS infusion of LGF stimulates the outgrowth of tyrosine hydroxylase-positive terminals in the striatum of 6-hydroxydopamine-treated rats. As apomorphine-induced rotational behavior was also reduced in these animals, we propose LGF as a novel factor that, when delivered to the striatum, may be useful in the treatment of Parkinson's disease.     

Divergence with gene flow in Anopheles funestus from the Sudan Savanna of Burkina Faso, West Africa

Anopheles funestus is a major vector of malaria across Africa. Understanding its complex and nonequilibrium population genetic structure is an important challenge that must be overcome before vector populations can be successfully perturbed for malaria control. Here we examine the role of chromosomal inversions in structuring genetic variation and facilitating divergence in Burkina Faso, West Africa, where two incipient species (chromosomal forms) of A. funestus, defined principally by rearrangements of chromosome 3R, have been hypothesized. Sampling across an approximately 300-km east-west transect largely contained within the Sudan-Savanna ecoclimatic zone, we analyzed chromosomal inversions, 16 microsatellite loci distributed genomewide, and 834 bp of the mtDNA ND5 gene. Both molecular markers revealed high genetic diversity, nearly all of which was accounted for by within-population differences among individuals, owing to recent population expansion. Across the study area there was no correlation between genetic and geographic distance. Significant genetic differentiation found between chromosomal forms on the basis of microsatellites was not genomewide but could be explained by chromosome 3R alone on the basis of loci inside and near inversions. These data are not compatible with complete reproductive isolation but are consistent with differential introgression and sympatric divergence between the chromosomal forms, facilitated by chromosome 3R inversions.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Biochemical,physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H₂O₂ and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).

Methods

Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.

Results

Vu-2-Cys-Prx reduces H₂O₂ using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH₂-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys⁵² residue and the amino acids Pro⁴⁵, Thr⁴⁹ and Arg¹²⁸ are conserved as in other 2-Cys-Prx.

General significance

The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.     

Seeing the Forest through the Trees: towards a Unified View on Physiological Calcium Regulation of Voltage-Gated Sodium Channels

Voltage-gated sodium channels (Na_Vs) underlie the upstroke of the action potential in the excitable tissues of nerve and muscle. After opening, Na_Vs rapidly undergo inactivation, a crucial process through which sodium conductance is negatively regulated. Disruption of inactivation by inherited mutations is an established cause of lethal cardiac arrhythmia, epilepsy, or painful syndromes. Intracellular calcium ions (Ca²⁺) modulate sodium channel inactivation, and multiple players have been suggested in this process, including the cytoplasmic Na_V C-terminal region including two EF-hands and an IQ motif, the Na_V domain III-IV linker, and calmodulin. Calmodulin can bind to the IQ domain in both Ca²⁺-bound and Ca²⁺-free conditions, but only to the DIII-IV linker in a Ca²⁺-loaded state. The mechanism of Ca²⁺ regulation, and its composite effect(s) on channel gating, has been shrouded in much controversy owing to numerous apparent experimental inconsistencies. Herein, we attempt to summarize these disparate data and propose a novel, to our knowledge, physiological mechanism whereby calcium ions promote sodium current facilitation due to Ca²⁺ memory at high-action-potential frequencies where Ca²⁺ levels may accumulate. The available data suggest that this phenomenon may be disrupted in diseases where cytoplasmic calcium ion levels are chronically high and where targeted phosphorylation may decouple the Ca²⁺ regulatory machinery. Many Na_V disease mutations associated with electrical dysfunction are located in the Ca²⁺-sensing machinery and misregulation of Ca²⁺-dependent channel modulation is likely to contribute to disease phenotypes.     

Associations between retinol-binding protein 4 and cardiometabolic risk factors and subclinical atherosclerosis in recently postmenopausal women: Cross-sectional analyses from the KEEPS Study

ABSTRACT: BACKGROUND: The published literature regarding the relationships between retinol-binding protein 4 (RBP4) and cardiometabolic risk factors and subclinical atherosclerosis is conflicting, likely due, in part, to limitations of frequently used RBP4 assays. Prior large studies have not utilized the gold-standard Western Blot analysis of RBP4 levels. METHODS: Full-length serum RBP4 levels were measured by Western Blot in 709 postmenopausal women screened for the Kronos Early Estrogen Prevention Study. Cross-sectional analyses related RBP4 levels to cardiometabolic risk factors, carotid artery intima-media thickness (CIMT), and coronary artery calcification (CAC). RESULTS: The mean age of women was 52.9 (+/- 2.6) years, and the median RBP4 level was 49.0 (interquartile range 36.9-61.5) ug/mL. Higher RBP4 levels were weakly associated with higher triglycerides (age, race, and smoking-adjusted partial Spearman correlation coefficient= 0.10; P=0.01), but were unrelated to blood pressure, cholesterol, C-reactive protein, glucose, insulin, and CIMT levels (all partial Spearman correlation coefficients [less than or equal to]0.06, P>0.05). Results suggested a curvilinear association between RBP4 levels and CAC, with women in the bottom and upper quartiles of RBP4 having higher odds of CAC (odds ratio [95% confidence interval] 2.10 [1.07-4.09], 2.00 [1.02-3.92], 1.64 [0.82-3.27] for the 1st, 3rd, and 4th RBP4 quartiles vs. the 2nd quartile). However, a squared RBP4 term in regression modeling was non-significant (P=0.10). CONCLUSIONS: In these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with elevations in triglycerides and with CAC, but not with other risk factors or CIMT. These data using the gold standard of RBP4 methodology only weakly support the possibility that perturbations in RBP4 homeostasis may be an additional risk factor for subclinical coronary atherosclerosis. Trial Registration: ClinicalTrials.gov number NCT00154180.     

A Review of the International Atomic Energy Agency (IAEA) International Standards for Tissue Banks

The IAEA International Standards for Tissue Banks published in 2003 were based on the Standards then currently in use in the USA and the European Union, among others, and reflect the best practices associated with the operation of a tissue bank. They cover legal, ethical and regulatory controls as well as requirements and procedures from donor selection and tissue retrieval to processing and distribution of finished tissue for clinical use. The application of these standards allows tissue banks to operate with the current good tissue practice, thereby providing grafts of high quality that satisfy the national and international demand for safe and biologically useful grafts. The objective of this article is to review the IAEA Standards and recommend new topics that could improve the current version.