Human complement C3 is a substrate for transglutaminases. A functional link between non-protease-based members of the coagulation and complement cascades

In this study, we report the finding of functional cross-talk between two non-protease components of the complement and coagulation cascades. We show that complement C3, a central component of the complement system, is associated with the fibrin clot and that C3 becomes covalently cross-linked to other proteins during coagulation. Enzymatic incorporation of dansylcadaverine and dansyl-PGGQQIV into C3 by coagulation factor XIIIa and tissue transglutaminase demonstrated that C3 is a transglutaminase substrate. This suggested that coagulation factor XIIIa covalently cross-links C3 to clot components during coagulation. Using mass spectrometry, we verified that C3 indeed is covalently associated with the fibrin clot in a ratio of 0.05:1 relative to the known coagulation factor XIIIa substrate α2-antiplasmin.     

PHYLOGENETIC CONSTRAINTS IN KEY FUNCTIONAL TRAITS BEHIND SPECIES’ CLIMATE NICHES: PATTERNS OF DESICCATION AND COLD RESISTANCE ACROSS 95 DROSOPHILA SPECIES

Species distributions are often constrained by climatic tolerances that are ultimately determined by evolutionary history and/or adaptive capacity, but these factors have rarely been partitioned. Here, we experimentally determined two key climatic niche traits (desiccation and cold resistance) for 92–95 Drosophila species and assessed their importance for geographic distributions, while controlling for acclimation, phylogeny, and spatial autocorrelation. Employing an array of phylogenetic analyses, we documented moderate‐to‐strong phylogenetic signal in both desiccation and cold resistance. Desiccation and cold resistance were clearly linked to species distributions because significant associations between traits and climatic variables persisted even after controlling for phylogeny. We used different methods to untangle whether phylogenetic signal reflected phylogenetically related species adapted to similar environments or alternatively phylogenetic inertia. For desiccation resistance, weak phylogenetic inertia was detected; ancestral trait reconstruction, however, revealed a deep divergence that could be traced back to the genus level. Despite drosophilids’ high evolutionary potential related to short generation times and high population sizes, cold resistance was found to have a moderate‐to‐high level of phylogenetic inertia, suggesting that evolutionary responses are likely to be slow. Together these findings suggest species distributions are governed by evolutionarily conservative climate responses, with limited scope for rapid adaptive responses to future climate change.     

STIM1 is necessary for store-operated calcium entry in turning growth cones

J. Neurochem. (2012) 122, 1155-1166. ABSTRACT: Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.     

Phosphorus availability and microbial respiration across different tundra vegetation types

Phosphorus (P) is an important nutrient in tundra ecosystems that co-limits or in some cases limits primary production. The availability of P is largely driven by soil characteristics, e.g., pH, organic carbon, and abundance of P-sorbing elements such as aluminium (Al) or iron (Fe). We tested how vegetation and soil properties relate to P availability across different tundra vegetation types. The different soil P fractions in the organic horizon were measured and plant foliar nitrogen (N) to P ratio and a plant bioassay was used as indicators of plant nutrient status. Microbial bioassays were used to study microbial respiration kinetics and in response to carbon, N, and P amendments. The distribution of P fractions differed significantly across vegetation types; labile fractions of P were less abundant in meadow sites compared to heath sites. Calcium-phosphates seemed to be an important P-fraction in meadows, but were only found in lower concentrations in the heath. There were only small differences in NaOH–extractable P between the vegetation types and this correlated with the distribution of oxalate-extractable Al. Plant N:P ratios and the plant bioassay indicated decreasing P availability from dry heath to mesic heath to mesic meadow. The microbial bioassay suggests that the heterotrophic microbial community is C-limited with N as a secondary limiting nutrient although there were indications that microbial P availability was lower in the meadow sites. Overall, we suggest that the observed variations in soil P across vegetation types are affecting both plant and microbial function although the differences seem to be relatively small.     

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery.     

Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.     

Postharvest biocontrol of Monilinia laxa,Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains

The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructicola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preliminary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhibited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8, respectively. For both strains, 10⁸ CFU ml^?1 was the most active concentration, although L1 showed good activity at a concentration of 10⁷ CFU ml^?1. Isolate L8 at the concentration of 10⁷ CFU ml^?1 was ineffective against M. fructicola and M. fructigena, showing no difference between treated fruit and the control, excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 10⁷ CFU ml^?1 reduced the brown rot infections with respect to the control. The increase in population density of A. pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to control brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullulans as active ingredients for the development of biofungicides for postharvest application against three Monilinia species that are responsible for high economic losses in stone fruit crops.     

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

Background

The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.

Methodology/Principal Findings

By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves.

Conclusions

Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases.     

Investigation of trypanothione reductase inhibitory activity by 1,3,4-thiadiazolium-2-aminide derivatives and molecular docking studies

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH(3); MI-4-OCH(3) and MI-4-NO(2)) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO(2) showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO(2). A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO(2) enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas' disease.     

Conditional peripheral membrane proteins: facing up to limited specificity

Regulated relocalization of signaling and trafficking proteins is crucial for the control of many cellular processes and is driven by a series of domains that respond to alterations at membrane surfaces. The first examples of these domains--conditional peripheral membrane proteins--included C1, C2, PH, PX, and FYVE domains, which specifically recognize single tightly regulated membrane components such as diacylglycerol or phosphoinositides. The structural basis for this recognition is now well understood. Efforts to identify additional domains with similar functions that bind other targets (or participate in unexplained cellular processes) have not yielded many more examples of specific phospholipid-binding domains. Instead, most of the recently discovered conditional peripheral membrane proteins bind multiple targets (each with limited specificity), relying on coincidence detection and/or recognizing broader physical properties of the membrane such as charge or curvature. This broader range of recognition modes presents significant methodological challenges for a full structural understanding.