A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha -adrenoceptor stimulation

The purpose of this study was to investigate the potential roleof mitogen-activated protein (MAP) kinase in smooth muscle contractionby monitoring MAP kinase activation, caldesmon phosphorylation, andcontractile force during agonist stimulation. Isometric tension inresponse to KCl and phenylephrine (PE) was measured from strips offerret aorta. MAP kinase activation was monitored by Western blot usinga phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmonantibodies. We report here that treatment of smooth muscle strips withPD-098059, a specific inhibitor of MAP kinase kinase, did notdetectably modify the KCl-evoked contraction but significantlyinhibited the contraction to PE in the absence of extracellularCa²⁺. In this experimentalcondition, where the contraction occurs in the absence of increases in20-kDa myosin light chain phosphorylation, PD-098059 also inhibitedsignificantly MAP kinase and caldesmon phosphorylation. Collectively,these results demonstrate a direct cause-and-effect relationshipbetween MAP kinase activation and Ca²⁺-independent smooth musclecontraction and support the concept of caldesmon phosphorylation as themissing link between both events.

     

Heavy drinking relates to positive valence ratings of alcohol cues

A positive family history of alcohol use disorders (FH) is a robust predictor of personal alcohol abuse and dependence. Exposure to problem-drinking models is one mechanism through which family history influences alcohol-related cognitions and drinking patterns. Similarly, exposure to alcohol advertisements is associated with alcohol involvement and the relationship between affective response to alcohol cues and drinking behavior has not been well established. In addition, the collective contribution that FH, exposure to different types of problem-drinking models (e.g. parents, peers) and personal alcohol use have on appraisal of alcohol-related stimuli has not been evaluated with a large sample. We investigated the independent effects of FH, exposure to problem-drinking models and personal alcohol use on valence ratings of alcohol pictures in a college sample. College students (n = 227) completed measures of personal drinking and substance use, exposure to problem-drinking models, FH and ratings on affective valence of 60 alcohol pictures. Greater exposure to non-familial problem-drinkers predicted greater drinking among college students (beta = 0.17, P < 0.01). However, personal drinking was the only predictor of valence ratings of alcohol pictures (beta = -0.53, P < 0.001). Personal drinking level predicted valence ratings of alcohol cues over and above FH, exposure to problem-drinking models and demographic characteristics. This suggests that positive affective responses to alcohol pictures are more a function of personal experience (i.e. repeated heavy alcohol use) than vicarious learning.     

Cross-Talk between Signaling Pathways Can Generate Robust Oscillations in Calcium and cAMP

Background

To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli.

Methodology/Principal Findings

Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP) and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate ‘bursting’ oscillations of calcium and may enable better filtering of noise.

Conclusion

We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.     

Phenotypic characterization of chronic inflammation in a rare case of endobronchial carcinoma

This report presents a phenotypical characterization of the immune cell infiltrate in a rare case of endobronchial carcinoma. A patient initially treated for an adenocarcinoma of the esophagus developed an endobronchial carcinoma surrounded by gastric metaplasia distal to a suspected gastrobronchial fistula, 11 years after esophagectomy. Our hypothesis is that the sustained exposure of the bronchial mucosa to a mixed acid and pancreatobiliary refluxate led to chronic inflammation and promoted malignant transformation. We performed an immunohistochemical study of the tumor microenvironment evaluating the density of CD3⁺, CD8⁺ T lymphocytes, CD20⁺ B lymphocytes, CD68⁺ macrophages and FoxP3⁺ regulatory T cells. Quantification of immune cell density was completed using a novel software-based analysis method. Our results suggest that, within all the tissues analyzed, FoxP3⁺ regulatory T cells were present at their highest density in the malignant and metaplastic tissues. The endobronchial metaplasia biopsied several years prior to the detection of the endobronchial adenocarcinoma was already densely infiltrated by B cells and macrophages, when compared to the immune cell infiltrate of the endobronchial carcinoma. Altogether, these observations support the current understanding of carcinogenesis promoted by chronic inflammation.     

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.     

Spatial heterogeneity in the Mediterranean Biodiversity Hotspot affects barcoding accuracy of its freshwater fishes

Incomplete knowledge of biodiversity remains a stumbling block for conservation planning and even occurs within globally important Biodiversity Hotspots (BH). Although technical advances have boosted the power of molecular biodiversity assessments, the link between DNA sequences and species and the analytics to discriminate entities remain crucial. Here, we present an analysis of the first DNA barcode library for the freshwater fish fauna of the Mediterranean BH (526 spp.), with virtually complete species coverage (498 spp., 98% extant species). In order to build an identification system supporting conservation, we compared species determination by taxonomists to multiple clustering analyses of DNA barcodes for 3165 specimens. The congruence of barcode clusters with morphological determination was strongly dependent on the method of cluster delineation, but was highest with the general mixed Yule‐coalescent (GMYC) model‐based approach (83% of all species recovered as GMYC entity). Overall, genetic morphological discontinuities suggest the existence of up to 64 previously unrecognized candidate species. We found reduced identification accuracy when using the entire DNA‐barcode database, compared with analyses on databases for individual river catchments. This scale effect has important implications for barcoding assessments and suggests that fairly simple identification pipelines provide sufficient resolution in local applications. We calculated Evolutionarily Distinct and Globally Endangered scores in order to identify candidate species for conservation priority and argue that the evolutionary content of barcode data can be used to detect priority species for future IUCN assessments. We show that large‐scale barcoding inventories of complex biotas are feasible and contribute directly to the evaluation of conservation priorities.     

The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits

Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.     

Immunodetection of Rho-like plant proteins with Rac1 and Cdc42Hs antibodies

A few small GTP-binding proteins of the Ras superfamily have been identified in plants, including members of the Rho family: the proteins belonging to this group are known in mammalian and yeast cells to be involved in the control of polarity, cell morphogenesis and movement by regulating cytoskeleton organization. An investigation into where some Rho-like proteins are located in plant cells was made. The antibodies used, anti-Cdc42Hs and anti-Rac 1, were raised against conserved characteristic sequences of Cdc42Hs and Rac1 mammalian proteins respectively. In fixed cells, Cdc42Hs antibody recognized epitopes generally co-localized with microtubules which may be implicated in the establishment of cell polarity, whereas the proteins recognized by Rac1 antibody seemed to be associated with organelle membranes. The same anti-bodies were used in Western blots of proteins from tobacco BY-2 and lucerne A2 suspension cells: Cdc42Hs antibody recognized three bands whereas Rac1 antibody revealed only one band of 18 kDa Mr. A [³⁵S]GTP overlay revealed four bands of the same Mr as those recognized in Western blots by Cdc42Hs and Rac1 antibodies.Key words: Rho G-proteins, Cdc42, Rac1, Immunofluorescence, plant cells.      

Control of autophagy initiation by phosphoinositide 3‐phosphatase jumpy

The majority of studies on autophagy, a cytoplasmic homeostatis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3‐kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3‐phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI‐1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.