Purification of the herpes simplex virus type 1 65-kilodalton DNA-binding protein: properties of the protein and evidence of its association with the virus-encoded DNA polymerase.

Using a combination of conventional column chromatography and velocity sedimentation, we have purified the 65-kilodalton DNA-binding protein (65KDBP) encoded by herpes simplex virus (HSV) greater than 625-fold. The HSV type 1 (HSV-1)-encoded DNA polymerase (pol) cofractionated with 65KDBP through DEAE-Sephacel, Blue Sepharose, and Mono Q columns and was only separated from 65KDBP by sedimentation through a glycerol gradient. Immunoaffinity columns containing monoclonal antibody (MAb) 6898 immunoglobulin effectively bound most of the HSV-1 pol activity which coeluted with 65KDBP. The pattern of reactivities of HSV-1/HSV-2 recombinants with MAbs specific for HSV-1 65KDBP or the HSV-2-infected cell-specific protein ICSP34,35 strongly suggests that these two species are serotype equivalents of the same protein. Taken together, all these data indicate that 65KDBP is a pol-associated protein and the HSV-1 counterpart of HSV-2 ICSP34,35 previously reported to have similar properties (P. J. Vaughan, D. J. M. Purifoy, and K. L. Powell, J. Virol. 53:501-508, 1985). Purified preparations of 65KDBP were capable of binding to double-stranded DNA, as determined by filter retention and mobility shift assays. The protein-DNA complex formed with 65KDBP was distinct from that produced by pol and could be further shifted by the addition of immunoglobulin specific for 65KDBP. These results demonstrate that 65KDBP has been purified substantially free from pol and indicate that DNA binding is an inherent property of the protein.     

Influence of priority effects and pond location on invaded larval amphibian communities

In Florida, the Cuban Treefrog (Osteopilus septentrionalis) is a superb colonist and appears to be a significant driver of amphibian community dynamics. Decline of native anurans has been linked to possible competition with adult O. septentrionalis but interactions during the larval stage are largely unknown. Rearing O. septentrionalis tadpoles along with two native anurans, the Squirrel Treefrog (Hyla squirella) and the Southern Toad (Bufo terrestris) in both experimental artificial ponds and laboratory aquaria, the role of competition as the mechanism driving the dynamics of invaded amphibian communities in Florida was examined. Also examined was the role of priority effects and variation between pond locations in altering interactions between O. septentrionalis and native anuran larvae. Interspecific competition was strong during the larval stage; the presence of O. septentrionalis reduced larval performance and survival of native anurans. Pond location alone had little effect on interspecific interactions, but priority effects were strong. Pond location and priority effects acted together to influence species interactions. The selective influence of different interaction modifiers acted to increase or decrease the impacts of exotic species on native taxa.     

Cbln1 is essential for interaction-dependent secretion of Cbln3

Cbln1 and the orphan glutamate receptor GluRdelta2 are pre- and postsynaptic components, respectively, of a novel transneuronal signaling pathway regulating synapse structure and function. We show here that Cbln1 is secreted from cerebellar granule cells in complex with a related protein, Cbln3. However, cbln1- and cbln3-null mice have different phenotypes and cbln1 cbln3 double-null mice have deficits identical to those of cbln1 knockout mice. The basis for these discordant phenotypes is that Cbln1 and Cbln3 reciprocally regulate each other's degradation and secretion such that cbln1-null mice lack both Cbln1 and Cbln3, whereas cbln3-null mice lack Cbln3 but have an approximately sixfold increase in Cbln1. Unlike Cbln1, Cbln3 cannot form homomeric complexes and is secreted only when bound to Cbln1. Structural modeling and mutation analysis reveal that, by constituting a steric clash that is masked upon binding Cbln1 in a "hide-and-run" mechanism of endoplasmic reticulum retention, a single arginine confers the unique properties of Cbln3.     

Inconsistencies between human genetic cytolocations and those derived using genomic sequence

One result of the publishing of the human genome sequence is the ability to define objects through their position on the consensus sequence. While this has simplified the process of creating order maps for genes on a chromosome, it has created discrepancies between the published cytolocations of human genes, as presented through genetic references, and those locations derived computationally from the genomic sequence. For the 6,830 records with HUGO gene symbols shared between the online version of Mendelian Inheritance in Man and Ensembl, 18% of the records have a discrepancy of at least one cytogenetic band between the datasets. Discordance between data sets at this frequency would have a significant impact on the utility of datasets created by the amalgamation of numerous biological databases.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.     

The influence of abundance on detectability

Plant and animal survey detection rates are important for ecological surveys, environmental impact assessment, invasive species monitoring, and modeling species distributions. Species can be difficult to detect when rare but, in general, how detection probabilities vary with abundance is unknown. We developed a new detectability model based on the time to detection of the first individual of a species. Based on this model, the predicted detection rate is proportional to a power function of abundance with a scaling exponent between zero and one that depends on clustering of individuals. We estimated the model parameters with data from three independent datasets: searches for chenopod shrub species and coins, experimental searches for planted seedlings, and frog surveys at multiple sites in sub‐tropical forests of eastern Australia. Analyses based on the detection time and detection probability suggest that detection rate increases with abundance as predicted. The model provides a way to scale detection rates to cases of low abundance when direct estimation of detection rates is often impractical.     

Comparative feeding kinematics of temperate pond-dwelling tadpoles (Anura,Amphibia)

Several studies have explored various components of feeding kinematics in anuran larvae; however, a direct comparison of feeding kinematics among morphologically similar and sympatric taxa has not been undertaken. We used high-speed videography (500 frames/s) to capture feeding kinematics of Anaxyrus fowleri (Hinckley, 1882) (Fowler’s Toad), Hyla chrysoscelis (Cope, 1880) (Grey Treefrog), Scaphiopus holbrookii (Harlan, 1835) (Eastern Spadefoot Toad), and Lithobates sphenocephalus (Cope, 1889) (Southern Leopard Frog) tadpoles as they foraged from an algal-covered substrate. In total, we filmed 120 feeding sequences from 25 feeding bouts and quantified eight kinematic variables that were common among all four species. Despite relatively similar keratinized feeding structures among taxa, our videography data revealed fundamental differences in how the tadpoles used these structures. One specific difference was in the speed of the gape cycle. Among taxa, S. holbrookii tadpoles had the longest gape cycle and longest time to reach maximum gape, whereas A. fowleri and L. sphenocephalus tadpoles had shorter durations for both variables and did not differ between species. We also found species differences in the magnitude that tadpoles narrow their lower jaw sheath. Irrespective of gape size, the lower jaw sheath of S. holbrookii tadpoles narrowed by approximately 26% of its maximum width—a twofold difference from A. fowleri tadpoles, which narrowed only 13%. Our study revealed that tadpoles with similar oral structures feeding on the same substrate can exhibit major differences in feeding kinematics.     

Influence of shape-memory stent grafts on local aortic compliance

The effect of repair techniques on the biomechanics of the aorta is poorly understood, resulting in significant levels of postoperative complications for patients worldwide. This study presents a computational analysis of the influence of Nitinol-based devices on the biomechanical performance of a healthy patient-specific human aorta. Simulations reveal that Nitinol stent-grafts stretch the artery wall so that collagen is stretched to a straightened high-stiffness configuration. The high-compliance regime (HCR) associated with low diastolic lumen pressure is eliminated, and the artery operates in a low-compliance regime (LCR) throughout the entire cardiac cycle. The slope of the lumen pressure–area curve for the LCR post-implantation is almost identical to that of the native vessel during systole. This negligible change from the native LCR slope occurs because the stent-graft increases its diameter from the crimped configuration during deployment so that it reaches a low-stiffness unloading plateau. The effective radial stiffness of the implant along this unloading plateau is negligible compared to the stiffness of the artery wall. Provided the Nitinol device unloads sufficiently during deployment to the unloading plateau, the degree of oversizing has a negligible effect on the pressure–area response of the vessel, as each device exerts approximately the same radial force, the slope of which is negligible compared to the LCR slope of the native artery. We show that 10% oversizing based on the observed diastolic diameter in the mid descending thoracic aorta results in a complete loss of contact between the device and the wall during systole, which could lead to an endoleak and stent migration. 20% oversizing reaches the Dacron enforced area limit (DEAL) during the pulse pressure and results in an effective zero-compliance in the later portion of systole.

     

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.