Pannexin channels are not gap junction hemichannels

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers, or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term "hemichannels" to describe pannexin oligomers while others use the term "channels" instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus have an assembly and functional state as gap junctional intercellular channels. Here we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channel in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term "hemichannel" be discontinued within the pannexin literature.     

Identification of mitogen-activated protein/extracellular signal-responsive kinase kinase 2 as a novel partner of the scaffolding protein human homolog of disc-large

Human disc-large homolog (hDlg), also known as synapse-associated protein 97, is a scaffold protein, a member of the membrane-associated guanylate kinase family, implicated in neuronal synapses and epithelial-epithelial cell junctions whose expression and function remains poorly characterized in most tissues, particularly in the vasculature. In human vascular tissues, hDlg is highly expressed in smooth muscle cells (VSMCs). Using the yeast two-hybrid system to screen a human aorta cDNA library, we identified mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade, as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. Coimmunoprecipitation assays in both human VSMCs and human embryonic kidney 293 cells, demonstrated that endogenous hDlg physically interacts with MEK2 but not with MEK1. Confocal microscopy suggested a colocalization of the two proteins at the inner layer of the plasma membrane of confluent human embryonic kidney 293 cells, and in a perinuclear area in human VSMCs. Additionally, hDlg also associates with the endoplasmic reticulum and microtubules in these latter cells. Taken together, these findings allow us to hypothesize that hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway, and may improve our understanding of how scaffold proteins, such as hDlg, differentially tune MEK1/MEK2 signaling and cell responses.     

Taxon-specific PCR for DNA barcoding arthropod prey in bat faeces

The application of DNA barcoding to dietary studies allows prey taxa to be identified in the absence of morphological evidence and permits a greater resolution of prey identity than is possible through direct examination of faecal material. For insectivorous bats, which typically eat a great diversity of prey and which chew and digest their prey thoroughly, DNA-based approaches to diet analysis may provide the only means of assessing the range and diversity of prey within faeces. Here, we investigated the effectiveness of DNA barcoding in determining the diets of bat species that specialize in eating different taxa of arthropod prey. We designed and tested a novel taxon-specific primer set and examined the performance of short barcode sequences in resolving prey species. We recovered prey DNA from all faecal samples and subsequent cloning and sequencing of PCR products, followed by a comparison of sequences to a reference database, provided species-level identifications for 149/207 (72%) clones. We detected a phylogenetically broad range of prey while completely avoiding detection of nontarget groups. In total, 37 unique prey taxa were identified from 15 faecal samples. A comparison of DNA data with parallel morphological analyses revealed a close correlation between the two methods. However, the sensitivity and taxonomic resolution of the DNA method were far superior. The methodology developed here provides new opportunities for the study of bat diets and will be of great benefit to the conservation of these ecologically important predators.     

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the "furnace" that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430-435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels.     

Atomic layer deposition of titanium dioxide on cellulose acetate for enhanced hemostasis

TiO₂ films may be used to alter the wettability and hemocompatibility of cellulose materials. In this study, pure and stoichiometric TiO₂ films were grown using atomic layer deposition on both silicon and cellulose substrates. The films were grown with uniform thicknesses and with a growth rate in agreement with literature results. The TiO₂ films were shown to profoundly alter the water contact angle values of cellulose in a manner dependent upon processing characteristics. Higher amounts of protein adsorption indicated by blurry areas on images generated by scanning electron microscopy were noted on TiO₂-coated cellulose acetate than on uncoated cellulose acetate. These results suggest that atomic layer deposition is an appropriate method for improving the biological properties of hemostatic agents and other blood-contacting biomaterials.     

Integrate-and-fire models of insolation-driven entrainment of broadcast spawning in corals

The circa-annual cycle of gametogenesis produces mature gametes at the spawning “season” for successful mass spawning of broadcast corals. We develop a bioenergetic integrate-and-fire model that reveals how annual insolation rhythms can entrain the gametogenetic cycles in tropical hermatypic corals to the appropriate spawning season, since photosynthate is their primary source of energy. In the presence of short-term fluctuations in the energy input, a feedback regulatory mechanism is likely required to achieve coherence of spawning times to within one lunar cycle, in order for subsequent signals such as lunar and diurnal light cycles to unambiguously determine the “correct” night of spawning. The feedback mechanism can also provide robustness against population heterogeneity that may arise due to genetic and environmental effects. We solve the integrate-and-fire bioenergetic model numerically using the Fokker–Planck equation and use analytical tools such as rotation number to study entrainment.     

SPIRE: Systematic protein investigative research environment

The SPIRE (Systematic Protein Investigative Research Environment) provides web-based experiment-specific mass spectrometry (MS) proteomics analysis (https://www.proteinspire.org). Its emphasis is on usability and integration of the best analytic tools. SPIRE provides an easy to use web-interface and generates results in both interactive and simple data formats. In contrast to run-based approaches, SPIRE conducts the analysis based on the experimental design. It employs novel methods to generate false discovery rates and local false discovery rates (FDR, LFDR) and integrates the best and complementary open-source search and data analysis methods. The SPIRE approach of integrating X!Tandem, OMSSA and SpectraST can produce an increase in protein IDs (52-88%) over current combinations of scoring and single search engines while also providing accurate multi-faceted error estimation. One of SPIRE's primary assets is combining the results with data on protein function, pathways and protein expression from model organisms. We demonstrate some of SPIRE's capabilities by analyzing mitochondrial proteins from the wild type and 3 mutants of C. elegans. SPIRE also connects results to publically available proteomics data through its Model Organism Protein Expression Database (MOPED). SPIRE can also provide analysis and annotation for user supplied protein ID and expression data.     

Architecture and development of the Neurospora crassa hypha -- a model cell for polarized growth

Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation -- the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.     

Radioisotopes demonstrate the contrasting bioaccumulation capacities of heavy metals in embryonic stages of cephalopod species

Cephalopods play a key role in many marine trophic food webs and also constitute alternative fishery resources in the context of the ongoing decline in finfish stocks. Most coastal cephalopod species of commercial importance migrate into shallow waters during the breeding season to lay their eggs, and are consequently subjected to coastal contamination. Eggs of common cuttlefish Sepia officinalis, European squid Loligo vulgaris, common octopus Octopus vulgaris and the sepiolid Rossia macrosoma were exposed during embryonic development to dissolved (110m)Ag, (109)Cd, (60)Co, (54)Mn and (65)Zn in order to determine their metal accumulation efficiencies and distribution among different egg compartments. Cuttlefish eggs, in which hard shells enclose the embryos, showed the lowest concentration factor (CF) values despite a longer duration of exposure. In contrast, octopus eggs, which are only protected by the chorionic membrane, accumulated the most metal. Uptake appears to be linked to the selective retention properties of the egg envelopes with respect to each element. The study also demonstrated that the octopus embryo accumulated (110m)Ag directly from the dissolved phase and also indirectly through assimilation of the contaminated yolk. These results raise questions regarding the potential contrasting vulnerability of early life stages of cephalopods to the metallic contamination of coastal waters.     

Engineered single-domain antibodies with high protease resistance and thermal stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(H)Hs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(H)Hs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(H)H pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(H)Hs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.