The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

Though the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion regulator, Filamin A (FlnA). Endothelial cells (ECs) expressing a Nrp1 mutant devoid of the cytoplasmic domain (nrp1^cytoΔ/Δ) migrated significantly slower in response to VEGF relative to the cells expressing wild-type Nrp1 (nrp1^+/+ cells). The rate of FA turnover in VEGF-treated nrp1^cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1^+/+ ECs, thus accounting for the slower migration rate of the nrp1^cytoΔ/Δ ECs.     

Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.     

Microbial herbivory on the brown tide alga, Aureococcus anophagefferens: results from natural ecosystems, mesocosms and laboratory experiments

Experiments were conducted with natural plankton assemblages from two areas in Great South Bay (GSB) and the Peconic Bays Estuary System, NY, to compare the rates of growth and pelagic grazing mortality of Aureococcus anophagefferens with co-occurring phytoplankton. We hypothesized that A. anophagefferens would experience low mortality rates by microbial herbivores (relative to feeding pressure on other algae) thus providing it with a competitive advantage within the phytoplankton community. In fact, substantial rates of mortality were observed in nearly every experiment in our study. However, mortality rates of A. anophagefferens were less than intrinsic growth rates of the alga during late spring and early summer in Great South Bay, resulting in positive net growth rates for the alga during that period. This timing coincided with the development of a brown tide in this estuary. Similarly, growth rates of the alga also exceeded mortality rates during bloom development in natural plankton assemblages from the Peconic Bays Estuary System held in mesocosms. In contrast to the situation for A. anophagefferens, growth rates of the total phytoplankton assemblage, and another common picoplanktonic phytoplankter (Synechococcus spp.), were frequently less than their respective mortality rates. Mortality rates of A. anophagefferens in both systems were similar to growth rates of the alga during later stages of the bloom. Laboratory studies confirmed that species of phagotrophic protists that consume A. anophagefferens (at least in culture) are present during brown tides but preference for or against the alga appears to be species-specific among phagotrophic protists. We conclude that two scenarios may explain our results: (1) protistan species capable of consuming the brown tide alga were present at low abundances during bloom initiation and thus not able to respond rapidly to increases in the intrinsic growth rate of the alga, or (2) the brown tide alga produced substance(s) that inhibited or retarded protistan grazing activities during the period of bloom initiation. The latter scenario seems less likely given that significant mortality of A. anophagefferens was measured during our field study and mesocosm experiment. However, even a minor reduction in mortality rate due to feeding selectivity among herbivores might result in a mismatch between growth and grazing of A. anophagefferens that could give rise to significant net population growth of this HAB species. Either scenario infers an important role for trophic interactions within the plankton as a factor explaining the development of brown tides in natural ecosystems.     

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.     

Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.     

Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.

The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.     

K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Bone biology and physiology: Part I. The fundamentals

The principles of bone biology and physiology permeate all subspecialty practices in plastic and reconstructive surgery from hand surgery to aesthetic surgery. Despite its importance in our practices, the biology of bone healing and bone physiology rarely surfaces within textbooks, literature reviews, or residency curricula. In this article, the authors present the first of a two-part series reviewing the important concepts of bone biology and bone physiology relevant to plastic surgery in an effort to ameliorate this educational gap.     

Inhibition between invasives: a newly introduced predator moderates the impacts of a previously established invasive predator

1. With continued globalization, species are being transported and introduced into novel habitats at an accelerating rate. Interactions between invasive species may provide important mechanisms that moderate their impacts on native species. 2. The European green crab Carcinus maenas is an aggressive predator that was introduced to the east coast of North America in the mid-1800 s and is capable of rapid consumption of bivalve prey. A newer invasive predator, the Asian shore crab Hemigrapsus sanguineus, was first discovered on the Atlantic coast in the 1980s, and now inhabits many of the same regions as C. maenas within the Gulf of Maine. Using a series of field and laboratory investigations, we examined the consequences of interactions between these predators. 3. Density patterns of these two species at different spatial scales are consistent with negative interactions. As a result of these interactions, C. maenas alters its diet to consume fewer mussels, its preferred prey, in the presence of H. sanguineus. Decreased mussel consumption in turn leads to lower growth rates for C. maenas, with potential detrimental effects on C. maenas populations. 4. Rather than an invasional meltdown, this study demonstrates that, within the Gulf of Maine, this new invasive predator can moderate the impacts of the older invasive predator.