Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent

The recruitment of polymorphonuclear leukocytes (PMN) from the vascular space to the alveolar air space is an early event in host defense against pneumococcal pneumonia. Pneumolysin is a virulence factor for Streptococcus pneumoniae, but a specific role for pneumolysin in neutrophil-endothelial cell interactions has not been investigated. Using a Transwell system, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to wild-type S. pneumoniae (D39) and a pneumolysin-deficient mutant (plnA(-)) incubated on the abluminal surface of the monolayer. S. pneumoniae induction of PMN migration was dose dependent and elicited by > or =10(5) D39. Mutants lacking pneumolysin had dramatically reduced potency for eliciting PMN migration compared with the parent strain (5 x 10(6) plnA(-) elicits 18.6% PMN migration vs. 55.5% for 5 x 10(6) D39). The disparity between D39 and plnA(-) persisted in ethanol-fixed bacteria, consistent with the properties of pneumolysin. Neither conditioned medium from D39 nor purified pneumolysin elicited PMN migration to the same extent as the intact D39, suggesting that the role of pneumolysin in eliciting PMN migration requires a more complex interaction between the organism, the endothelium, and the PMN. Both D39 and plnA(-) adhered to, and translocated across, the endothelium in the abluminal to luminal direction and elicited similar levels of IL-8 production. Neither strain elicited upregulation of the endothelial adhesion molecules ICAM-1, VCAM-1, or E-selectin, and they did not cause translocation of NF-kappaB to the nucleus. These findings demonstrate a novel role for pneumolysin in pneumococcus-induced PMN recruitment across the pulmonary endothelium.     

Effect of muscle wrapping on model estimates of neck muscle strength

Of the computational models of the cervical spine reported in the literature, not one takes into account the changes in muscle paths due to the underlying vertebrae. Instead, all model the individual muscle paths as straight-line segments. The major aim of this study was to quantify the changes in muscle moment arm, muscle force and joint moment due to muscle wrapping in the cervical spine. Five muscles in a straight-line model of the cervical spine were wrapped around underlying vertebrae, and the results obtained from this model were compared against the original. The two models were then validated against experimental and computational data. Results show that muscle wrapping has a significant effect on muscle moment arms and therefore joint moments and should not be neglected.     

Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. eIF5A-1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A-2 mRNA (0.7-5.6 kb) and eIF5A-2 protein (20 kDa) varies widely. Whereas eIF5A-2 mRNA was demonstrable in most cells, eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high overexpression of eIF5A-2 mRNA. Multiple forms of eIF5A-2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3'-UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A-1 and eIF5A-2 precursor proteins were modified comparably in UACC-1598 cells and both were similarly stable. When eIF5A-1 and eIF5A-2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A-2 precursor was synthesized at a level comparable to that of eIF5A-1 precursor, indicating that the elements causing inefficient translation of eIF5A-2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A-2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A-1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A-2 protein even in eIF5A-2 mRNA positive cells is, at least in part, due to inefficient translation.     

Diversification of Neoaves: integration of molecular sequence data and fossils

Patterns of diversification and timing of evolution within Neoaves, which includes almost 95% of all bird species, are virtually unknown. On the other hand, molecular data consistently indicate a Cretaceous origin of many neoavian lineages and the fossil record seems to support an Early Tertiary diversification. Here, we present the first well-resolved molecular phylogeny for Neoaves, together with divergence time estimates calibrated with a large number of stratigraphically and phylogenetically well-documented fossils. Our study defines several well-supported clades within Neoaves. The calibration results suggest that Neoaves, after an initial split from Galloanseres in Mid-Cretaceous, diversified around or soon after the K/T boundary. Our results thus do not contradict palaeontological data and show that there is no solid molecular evidence for an extensive pre-Tertiary radiation of Neoaves.     

Systemic lupus erythematosus

Imerslund-Gräsbeck syndrome (IGS) or selective vitamin B₁₂ (cobalamin) malabsorption with proteinuria is a rare autosomal recessive disorder characterized by vitamin B₁₂ deficiency commonly resulting in megaloblastic anemia, which is responsive to parenteral vitamin B₁₂ therapy and appears in childhood. Other manifestations include failure to thrive and grow, infections and neurological damage. Mild proteinuria (with no signs of kidney disease) is present in about half of the patients. Anatomical anomalies in the urinary tract were observed in some Norwegian patients. Vitamin B₁₂ absorption tests show low absorption, not corrected by administration of intrinsic factor. The symptoms appear from 4 months (not immediately after birth as in transcobalamin deficiency) up to several years after birth. The syndrome was first described in Finland and Norway where the prevalence is about 1:200,000. The cause is a defect in the receptor of the vitamin B₁₂-intrinsic factor complex of the ileal enterocyte. In most cases, the molecular basis of the selective malabsorption and proteinuria involves a mutation in one of two genes, cubilin (CUBN) on chromosome 10 or amnionless (AMN) on chromosome 14. Both proteins are components of the intestinal receptor for the vitamin B₁₂-intrinsic factor complex and the receptor mediating the tubular reabsorption of protein from the primary urine. Management includes life-long vitamin B₁₂ injections, and with this regimen, the patients stay healthy for decades. However, the proteinuria persists. In diagnosing this disease, it is important to be aware that cobalamin deficiency affects enterocyte function; therefore, all tests suggesting general and cobalamin malabsorption should be repeated after abolishment of the deficiency.     

Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates

We describe here a protocol for determining the activity of protein kinases on a large set of peptide substrates. Biotin-tagged peptides are arrayed in multiwell plates and incubated in solution with the kinase of interest and radiolabeled ATP. Reactions are then spotted simultaneously onto a streptavidin membrane, which is washed, dried, and analyzed by autoradiography or phosphor imaging. Differences in the extent of radiolabel incorporation into the various peptide substrates provide a measure of the sequence specificity of the kinase. This approach is a faster, more sensitive, and more generally applicable method for determining kinase phosphorylation motifs than older peptide library screening approaches based on Edman sequencing. The procedure is readily adaptable to other applications that require parallel processing of many kinase reactions, such as screening for small molecule inhibitors. In the format described here, preparation of stock plates prior to running the reactions will require about 4 days. Afterwards, the protocol takes approximately 6 hours to perform.     

Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors

Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD.     

Conservation of intrinsic disorder in protein domains and families: I. A database of conserved predicted disordered regions

Many protein regions have been shown to be intrinsically disordered, lacking unique structure under physiological conditions. These intrinsically disordered regions are not only very common in proteomes, but also crucial to the function of many proteins, especially those involved in signaling, recognition, and regulation. The goal of this work was to identify the prevalence, characteristics, and functions of conserved disordered regions within protein domains and families. A database was created to store the amino acid sequences of nearly one million proteins and their domain matches from the InterPro database, a resource integrating eight different protein family and domain databases. Disorder prediction was performed on these protein sequences. Regions of sequence corresponding to domains were aligned using a multiple sequence alignment tool. From this initial information, regions of conserved predicted disorder were found within the domains. The methodology for this search consisted of finding regions of consecutive positions in the multiple sequence alignments in which a 90% or more of the sequences were predicted to be disordered. This procedure was constrained to find such regions of conserved disorder prediction that were at least 20 amino acids in length. The results of this work included 3,653 regions of conserved disorder prediction, found within 2,898 distinct InterPro entries. Most regions of conserved predicted disorder detected were short, with less than 10% of those found exceeding 30 residues in length.     

Advances in the neural bases of orientation and navigation

The ability to locomote in one direction (oriented movement),and the ability to navigate toward a distant goal are relatedbehaviors that are phylogenetically widespread. Orientationbehaviors include finding the source of an odor or acousticsignal, using a sun-compass for guidance, and moving relativeto fluid-dynamic cues. Such abilities might require little morethan directionally selective sensors coupled to a turning mechanism.This type of behavior, therefore, can be implemented by relativelysimple circuits. In contrast, navigation involves both the abilityto detect direction, as well as a map-sense that provides position.Navigation is less common and arguably requires greater braincomputation than does simple orientation, but may be presentin arthropods as well as in vertebrates. Great progress hasbeen made in exploring the biophysical and sensory bases forthese behaviors, and in recent years the locations and the identityof the cellular transducers of the sensory stimuli (for example,geomagnetic fields) have been narrowed in some taxa. Similarly,neurons within the central nervous that most likely functionin higher order computational processes have been identified.For example, direction-selective and position-responsive braincells have been located in the brains of mammals and birds,and these cells might contribute to a cognitive map that canenable navigation. One model organism in which orientation andnavigation has been extensively studied is the sea slug Tritoniadiomedea. This animal orients its crawling to chemical, hydrodynamic,and geomagnetic cues. The brain of Tritonia has 7000 relativelylarge neurons that facilitate circuit analysis. Recent workhas characterized both peripheral and central neural correlatesof orientation signals, as well as the control of thrust andturning, and studies of their field behavior have suggestedhow these disparate orientation systems may be integrated. Thesefindings provide the basis for future studies to determine howthe nervous system combines multiple sensory cues into a complexhierarchy of signals that can direct motor output and thereforeguide navigational tasks.