Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.     

Gene profiling of the red light signalling pathways in roots

Red light, acting through the phytochromes, controls numerousaspects of plant development. Many of the signal transductionelements downstream of the phytochromes have been identifiedin the aerial portions of the plant; however, very few elementsin red-light signalling have been identified specifically forroots. Gene profiling studies using microarrays and quantitativeReal-Time PCR were performed to characterize gene expressionchanges in roots of Arabidopsis seedlings exposed to 1 h ofred light. Several factors acting downstream of phytochromesin red-light signalling in roots were identified. Some of thegenes found to be differentially expressed in this study havealready been characterized in the red-light-signalling pathwayfor whole plants. For example, PHYTOCHROME KINASE 1 (PKS1),LONG HYPOCOTYL 5 (HY5), EARLY FLOWERING 4 (ELF4), and GIGANTEA(GI) were all significantly up-regulated in roots of seedlingsexposed to 1 h of red light. The up-regulation of SUPPRESSOROF PHYTOCHROME A RESPONSES 1 (SPA1) and CONSTITUTIVE PHOTOMORPHOGENIC1-like (COP1-like) genes suggests that the PHYA-mediated pathwaywas attenuated by red light. In addition, genes involved inlateral root and root hair formation, root plastid development,phenylpropanoid metabolism, and hormone signalling were alsoregulated by exposure to red light. Interestingly, members ofthe RPT2/NPH3 (ROOT PHOTOTROPIC 2/NON PHOTOTROPIC HYPOCOTYL3) family, which have been shown to mediate blue-light-inducedphototropism, were also differentially regulated in roots inred light. Therefore, these results suggest that red and bluelight pathways interact in roots of seedlings and that manyelements involved in red-light-signalling found in the aerialportions of the plant are differentially expressed in rootswithin 1 h of red light exposure. Key words: Arabidopsis, gene profiling, microarray, photomorphogenesis, red light, roots     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

The molecular dynamics of osteoclast adhesions

Podosomes are specialized adhesive structures that play a central role in bone resorption. In this article we address the molecular diversity and dynamics of podosomes at different states of organization, ranging from scattered distribution over the entire ventral membrane of non-polarized cells, via formation of podosome clusters and developing rings to the assembly of a peripheral belt, resembling the sealing zone of polarized, bone-resorbing osteoclasts. Based on published data and on our own results, we describe here the spatial relationships between key podosome-associated proteins. Using quantitative microscopy, we show here a dramatic increase in the local levels of F-actin, vinculin, paxillin, and alpha-actinin, which occurs upon the transformation of clustered podosomes into rings and sealing zone-like structures. This change is accompanied by a marked decrease in phosphotyrosine levels in the same region. Therefore, our data suggest that a major change in the molecular composition of podosomes is taking place during osteoclast polarization, a change that may be related to adhesion "reinforcement", associated with the assembly of the bone-resorbing apparatus. Studying the nature of the proteins that undergo de-phosphorylation is critical for the understanding of the mechanisms regulating the processes described above.     

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling

Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/gamma-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by gamma-secretase. These data show that the PS1/gamma-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, gamma-secretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and gamma-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.     

The zoophilic fruitfly Phortica variegata: morphology, ecology and biological niche

Abstract.  Flies belonging to the subfamily Steganinae (Drosophilidae) display unusual zoophilic feeding habits at the adult and/or larval stage. Phortica variegata (Fallén) feeds on tears or eye liquid around the eyes of humans and carnivores. When feeding it is a potential vector of Thelazia callipaeda (Railliet and Henry) eyeworms. Adult and larval stages of this fly may be easily confused with other species belonging to the same genus, and little is known on the biology and ecology of P. variegata . In April–November 2005, a total of 969 P. variegata were collected in an area with a high prevalence of canine thelaziosis. The number of flies collected weekly was then related to climatic and environmental parameters (e.g. temperature, relative humidity and total rainfall) recorded daily at the collection site. The highest number of Phortica were collected during July–August. The sex ratio (number of males : females) rose from ∼ 0.5 during May–July, to ∼ 3.0 in August and 181 during September–October. Distributional data, representing 242 sites at which P. variegata has been collected in Europe, were analysed using a desktop implementation of the genetic algorithm for rule-set prediction (GARP) to model ecological requirements across Europe, as well as in Italy. P. variegata is shown to be mainly active at 20–25 °C and 50–75% RH. The ecological niche model suggests with a high degree of confidence that large areas of Europe are likely to represent suitable habitat for this species, mostly concentrated in central Europe. The results reported here contribute basic knowledge on the ecology and geographical distribution of P. variegata flies, which will be fundamental to gaining a better understanding of their role as vectors of human and animal pathogens.     

CbgA, a protein involved in cortex formation and stress resistance in Myxococcus xanthus spores

CbgA plays a role in cortex formation and the acquisition of a subset of stress resistance properties in Myxococcus xanthus spores. The cbgA mutant produces spores with thin or no cortex layers, and these spores are more sensitive to heat and sodium dodecyl sulfate than their wild-type counterparts.     

Core structure of S2 from the human coronavirus NL63 spike glycoprotein

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.     

Knowledge and attitudes of dentists regarding ageing and the elderly

doi: 10.1111/j.1741‐2358.2011.00534.x
Knowledge and attitudes of dentists regarding ageing and the elderly Objective: This study was aimed to identify the knowledge and attitudes of dentists regarding ageing and the elderly. Background: As the population ages, there is a greater demand for differentiated care from health professionals. However, the treatment needs of the elderly population are not sufficiently addressed, particularly in developing countries. Materials and methods: An official list of all dentists registered in the city of Belo Horizonte, Brazil, was obtained from the Regional Dentistry Council. A questionnaire was delivered to 276 randomly selected dentists; the survey contained questions designed to characterise the sample and identify dentists’ conduct towards elderly patients. A Likert scale was used to identify knowledge regarding care for the elderly and attitudes towards ageing. Results: Women demonstrated more positive attitudes towards the elderly. Graduates who worked in the public sector demonstrated a greater knowledge of geriatric dentistry (p < 0.05). Attending specific continuing education courses had no influence over the dentists’ willingness to treat the elderly population. Significant associations were not found between attitudes and knowledge regarding the elderly. Conclusion: Dentists who worked in the public sector demonstrated a greater knowledge regarding the elderly. This knowledge did not influence attitudes towards ageing. Women demonstrated a more positive attitude regarding ageing and dental care.