TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.     

A new Late-glacial site with Picea abies in the northern Apennine foothills: an exception to the model of glacial refugia of trees

We describe a new palaeobotanical site at Bubano quarry on the easternmost Po plain, northern Italy. Pollen and macrofossils from river and marsh sediments demonstrate the occurrence of Picea in a Pinus sylvestris forest growing in a radius of some tens of kilometres south of the sedimentation place, at the beginning of the Late-glacial interstadial. The Late-glacial and Holocene history of Picea in the northern Apennines is reconstructed on the basis of the palaeobotanical record. The sharp climatic continentality increase eastwards across the northern Apennines from the Tyrrhenian to the Adriatic coast is considered significant for the survival of Picea during the Late-glacial. The most critical phase of survival is related to the moisture changes and consequent Abies competition associated with the last glacial-interglacial transition and the early Holocene. The residual spruce populations expanded during the middle Holocene. The history of Picea in the northern Apennines is a case of ineffective interglacial spread of tree populations from pre-existing stands of LGM (Last Glacial Maximum) and Late-glacial age.     

Inhibiting protein-amyloid interactions with small molecules: a surface chemistry approach

This paper presents a surface-based approach to inhibit the binding of proteins to Alzheimer's-related beta-amyloid (Abeta) fibrils with small molecules. It reports the idea of using an intracellular, disease-related fibril as a material whose surface can be coated with small molecules. Using an ELISA-based assay, molecular surface coatings with thioflavin T are shown to inhibit 65+/-10% of the binding of two different anti-Abeta IgGs to Abeta fibrils. A molecular surface coating with 3,6-diamino acridine was able to inhibit 76+/-10% of the binding of an anti-Abeta IgG to Abeta fibrils. Maximal inhibition of these protein-amyloid interactions appears in the low to mid-micromolar range of small molecule. This demonstration that molecular surface coatings can be used to attenuate the interaction of proteins with these fibrils suggests a potentially new strategy for therapeutics in neurodegenerative amyloid diseases.     

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV^CAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV^CAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV^CAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV^CARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV^CAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV^CAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV^CAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV^CAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV^CAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV^CAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV^CAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV^CAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.Adenoviruses (Ads) are extensively used today as gene transfer vectors for in vitro, ex vivo, and in vivo gene transfer protocols (reviewed in reference 65). Cell entry of human Ad type 5 (Ad5), the serotype most widely used as a gene vector, occurs most efficiently by the receptor-mediated endocytosis pathway (reviewed in references 64 and 65), via the coxsackievirus B-adenovirus receptor (CAR) (3, 77) and αvβ3/αvβ5 integrins (84, 85), although alternative receptors have been described (11, 12, 14, 27). Cell surface expression of CAR differs with different cell types, and this represents one of the major determinants of the efficiency of Ad5-mediated transduction (43). The ubiquitous nature of CAR is responsible for transduction of nontarget tissues by Ad vectors. Paradoxically, many target cells such as dermal fibroblasts, synoviocytes, mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and dendritic cells (DCs), express no or very low levels of CAR at their surface and are relatively resistant to Ad transduction (14, 15, 19). Much work has been done with different strategies to promote the entry of Ad5 into CAR-defective cells. These strategies include (i) the genetic modification of Ad capsid proteins to carry cell ligands (2, 15, 20, 28, 49, 50), (ii) pseudotyping Ad5 vectors with fibers from other serotypes (13, 57, 74, 86), (iii) using bispecific adapters or peptides (25, 40), (iv) chemical modification of Ad (9, 42), and (v) tethering on nanoparticles (7). The limitations to these strategies are that modifications of the Ad capsid are susceptible to negatively affecting the virus growth or viability, due to an alteration of virion assembly, stability, the viral uncoating process, and/or intracellular trafficking (13, 51).Other viruses which are gaining popularity as gene transfer vectors are the baculoviruses (BVs). Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) is an insect virus with a large double-stranded DNA genome packaged in a membrane-enveloped, rod-shaped protein capsid (70). Since the 1980s, the BV-insect cell expression system has been highly exploited for the production of recombinant proteins. In the mid-1990s, it was shown that recombinant BVs carrying reporter genes under cytomegalovirus (CMV) or retroviral Rous sarcoma virus promoter efficiently expressed reporter genes in mammalian cells (6, 22, 38, 41, 44, 69), as well as in avian cells (72) and fish cells (45). Since then, BVs have been reported to transduce numerous cells originating from species as various as humans, bovines, and fish (8, 32, 41, 73). As gene transfer vectors, BVs have been found to be rapidly inactivated by human serum complement (23), but exposing decay-accelerating factor (DAF) at the surface of BV by fusion with the baculoviral envelope glycoprotein can overcome this inactivation (33). BVs also have a good biosafety profile due to their incapacity to replicate in mammalian cells (31).Taking advantage of the ability of BVs to transduce a large repertoire of cells of invertebrate and vertebrate origins, including human primary cells, we investigated whether a recombinant AcMNPV could act as a carrier or macroadapter for Ad5 vectors to enter Ad5-refractory cells. To this aim, we pseudotyped AcMNPV virions with the high-affinity receptor for Ad5, the human CAR glycoprotein (BV^CAR), to enable the formation of complexes between vector particles of BV^CAR and Ad5-green fluorescent protein (Ad5GFP) mediated by Ad5 fiber and CAR interaction. We found that transduction of cell lines which were poorly permissive to Ad5, including human cancer cells and primary cells, was significantly improved using this strategy of piggybacking Ad5 vector on BV^CAR. More importantly, the increase in BV^CAR-Ad5-mediated transduction was obtained with a low range of Ad5 inputs, i.e., at multiplicities of infection (MOI) of less than 50 Ad5 vector particles per cell. We also found that the cell transduction enhancement observed with BV^CAR-Ad5 required the direct coupling of Ad5 to BV^CAR via fiber-CAR binding and that the cell attachment of the complex was mediated by the baculoviral envelope glycoprotein gp64. Kinetic analysis of virus-cell binding showed that the presence of BV^CAR in the complex was beneficial to Ad5 vector, not only in terms of tropism but also in terms of number of cell-bound virions and rate of cell attachment. In addition, the endocytic pathway of BV^CAR-Ad5 did not require Ad5 penton base RGD-integrin interaction. When administered in vivo to nude BALB/c mice, BV^CAR-Ad5 complex showed the same biodistribution as that of control Ad5 vector injected alone. In vitro, transduction of CAR-negative cells by BV^CAR-Ad5 was insensitive to coagulation factor X (FX), in contrast to Ad5 vector alone.Our novel strategy of gene delivery using the BV^CAR-Ad5 duo could be advantageously applied to various situations in vitro or ex vivo, e.g., for transducing Ad5-refractory cells when Ad5 capsid modifications cannot be envisaged, when oncolytic Ads need to be delivered to tumors via nonpermissive cell carriers belonging to the immune system, or when the simultaneous delivery of two transgenes by two separate vectors might be beneficial in terms of timing and/or level of cellular expression of the transgene products.     

Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Bacterial cell curvature through mechanical control of cell growth

The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament‐like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.     

Salvia geminata sp. nov. with remarkable stamen arrangement from southern Yemen, with notes on S. areysiana (Lamiaceae)

The new species Salvia geminata , a dwarf shrublet from rocky coastal slopes of the Mahrah Region in Yemen, is described and illustrated. A remarkable feature of the species is that the upper thecae of the stamens are inflexed and connate, a condition that seems to be previously unknown in the genus. The apparently closely related S. areysiana , previously known only from the area of Jebel Areys in the Abyan Region, is reported also from the Hadramaut Region.     

Reversed optimality and predictive ecology: burrowing depth forecasts population change in a bivalve

Optimality reasoning from behavioural ecology can be used as a tool to infer how animals perceive their environment. Using optimality principles in a 'reversed manner' may enable ecologists to predict changes in population size before such changes actually happen. Here we show that a behavioural anti-predation trait (burrowing depth) of the marine bivalve Macoma balthica can be used as an indicator of the change in population size over the year to come. The per capita population growth rate between years t and t+1 correlated strongly with the proportion of individuals living in the dangerous top 4 cm layer of the sediment in year t: the more individuals in the top layer, the steeper the population decline. This is consistent with the prediction based on optimal foraging theory that animals with poor prospects should accept greater risks of predation. This study is among the first to document fitness forecasting in animals.