A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.

We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.     

Recovery of saltwater crocodiles following unregulated hunting in tidal rivers of the Northern Territory,Australia

Saltwater crocodiles (Crocodylus porosus) in the Northern Territory of Australia were protected in 1971, after a severe population decline resulting from 26 yr of intense commercial hunting. By that time wild saltwater crocodiles were rarely sighted anywhere and they were commercially extinct in areas where they had once been abundant. Standardized monitoring by spotlight surveys started in 1975 and provided relative density indices over time (1975–2009) as a unique record of the post-protection recovery of a wild crocodilian population. We examined the survey data for populations at 12 major tidal rivers, individually and as a single subpopulation. The pattern of recovery in the subpopulation in both abundance and biomass was approximated by logistic curves, predicting 5.26 non-hatchling crocodiles weighing 387.64 kg sighted per kilometer of river in 2010. We predicted potential carrying capacity as 5.58 non-hatchling crocodiles (5.73% higher than 2010) weighing 519.0 kg (25.31% higher than 2010). Individual rivers showed largely different abundance and biomass among rivers. The statistical model that best described the recovery in individual rivers was not always logistic. However, where it was logistic, expected carrying capacity of different rivers showed considerable variation in abundance and biomass. The variation indicates different habitat quality among the rivers. Recovery occurred despite various consumptive uses, particularly a widespread egg-harvest program, which has been an integral part of the incentive-driven conservation program for saltwater crocodiles in the Northern Territory since 1983. We suggest that the saltwater crocodile population of the Northern Territory is achieving full recovery from uncontrolled hunting in 1945–1971. Although saltwater crocodiles are considered an important natural resource, their increase in number, size, and distribution is posing management issues for public safety. Continuation of human–crocodile conflict management through public education and strategic removal of problem crocodiles will be essential. © 2011 The Wildlife Society.     

Protective immunoglobulin A and G antibodies bind to overlapping intersubunit epitopes in the head domain of type 1 reovirus adhesin sigma1

Nonfusogenic mammalian orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, sigma1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L sigma1 to which the MAb binds. The region bound by a previously characterized type 1 sigma1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of sigma1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified sigma1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the sigma1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D sigma1 trimer, the four positions cluster on the side of the sigma1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per sigma1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on sigma1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

Progressive modulation of endothelial phenotype during in vitro blood vessel formation.

"Sprouting" vascular endothelial cells were used as an in vitro model system to study the progressive morphologic and biosynthetic changes associated with the formation of tubular structures. In vitro, sprouting endothelial cells formed spontaneously without the addition of any exogenous factors from cultures of cloned endothelium exhibiting a polygonal/cobblestone phenotype. These phenotypically variant endothelial cells differentiated to form associated cell networks or nodules which gradually reorganized into tubular structures. Concomitant with these morphologic changes, the biosynthesis of extracellular matrix proteins was modulated, as determined by Northern blot analysis, metabolic labeling, and immunocytochemistry. The initial sprouting phase was characterized by the induction of type I collagen synthesis and the appearance of fibronectin containing the ED-A domain, in comparison to their absence in cloned cultures displaying a stable polygonal/cobblestone phenotype. The organizational stage, where the sprouting endothelial cells assembled into tubular structures, was additionally characterized by the expression of type IV collagen. These studies demonstrate that the progression from polygonal/cobblestone to sprouting cultures, and subsequent tubular organization, involves major alterations in extracellular matrix protein expression. This developmental phenomenon, although not completely analogous to blood vessel formation in vivo, nevertheless may be helpful in understanding the role of matrix macromolecules in the angiogenic process.     

Structural insights into the functional role of the Hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype C/D

Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ∼50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT–HCR. The N-terminal sub-domain of BoNT–HCR, Hcn, comprises ∼50% of BoNT–HCR and adopts a β-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD–HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD–HCR. In the crystal structure of a similar protein, BoNT/D–HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may play a role in ganglioside binding.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Overgrowth in a marine epifaumal community: Competitive hierarchies and competitive networks

Summary The frequencies with which organisms of a species overgrew or were overgrown by organisms of other species in a marine epifaunal community were estimated. The ranking of the ability of the major taxonomic groups to overgrow others was basically hierarchical:ascidianssponges>bryozoans>barnacles, polychaetes, tubicolous amphipods, hydroids. In contrast, the ranking of the competitive ability of species in the community did not form a simple linear hierarchy and there was no single competitively dominant species (measured in terms of overgrowth). There were often no significant differences in the ability of species to overgrow each other within the three major taxonomic groups of sponges, ascidians and bryozoans. Such results were common also between the species of large sponges and ascidiams which dominated substrata immersed for periods longer than two years.A lack of a significant difference in the competitive ability of species was usually the result of (a) frequent formation of delay/ties or standoffs and (b) changes in the outcome of interactions due to change in the relative size of interacting colonies. In many two-species interactions the species which had the larger colony in a given encounter had a greater probability of winning.When the range of colony sizes of two species was similar there was often no significant difference between the competitive ability of each species. Such cases without a clearcut winner often represented a backloop in an otherwise hierarchical sequence of competitive ability, i.e. Species A beats Species B, Species B beats Species C, no significant differences in competitive ability between Species C and A. No examples of competitive networks of the form Species A beats Species B, Species B beats Species C, Species C beats Species A were found. Backloops in otherwise hierarchical sequences (no significant differences in competitive ability) occurred most frequently between species within the same major taxonomic groups and were the result of a very even balance in the generalised competitive mechanism of overgrowth.It seems probable that backloops in hierarchical sequences are more commonly due to the absence of clear competitive dominance in interactions between species (reversals in the outcome of overgrowth interactions and standoffs), rather than to direct backloops formed by a specialised or to a generalised competitive mechanism. Network-like arrangements of competitive ability formed by the type of processes described here are likely to contribute significantly to the high levels of species diversity observed in many marine epifaunal communities subject to low levels of physical disturbance.     

Increased expression of the gene for the pro alpha 1(IV) chain of basement-membrane procollagen in cultured skin fibroblasts from two variants of osteogenesis imperfecta.

Fibroblasts from two lethal variants of osteogenesis imperfecta were shown to synthesize increased amounts of type IV procollagen. Previous studies established that one of these variants had a non-functional allele for the pro alpha 2 chain of type I procollagen, whereas the other pro alpha 2(I) allele contained a mutation leading to synthesis of shortened pro alpha 2(I) chains. In the two variants, the relative level of mRNA for pro alpha 1(IV) was 31 and 42% of the level of mRNA for pro alpha 1(I) chains. A value of less than 2% was found for a third lethal and four non-lethal variants of osteogenesis imperfecta. Immunofluorescent staining of fibroblasts from the two variants synthesizing increased amounts of type IV procollagen indicated that a homogeneous population of cells synthesized both type IV and type I procollagen. The results suggest that mutations in the type I procollagen genes that result in osteogenesis imperfecta can be associated with increased expression of the genes for type IV procollagen.