YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis

YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an α-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.     

Plasma Kallikrein Promotes Epidermal Growth Factor Receptor Transactivation and Signaling in Vascular Smooth Muscle through Direct Activation of Protease-activated Receptors

The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.     

Multiple Asian pig origins revealed through genomic analyses

Previous mitochondrial DNA (mtDNA) studies have suggested that European and Asian pig populations were derived through multiple domestication events. We investigated whether domestic pig populations were derived from distinct ancestors within their respective regions, using eight domestic breeds (five European and three Asian), and also European and Asian wild boar populations. Genomic analyses utilized 21 microsatellite markers (MS) selected for their distribution across the pig genome in addition to the mtDNA D-loop region. The number of alleles per MS loci ranged from 8 (Sw2008) to 16 (S0097 and S0218). Few significant departures from Hardy–Weinberg equilibrium were detected, suggesting the absence of heterozygote deficiencies. Analyses within populations revealed observed mean heterozygosity from 0.48 (Erhualian) to 0.68 (Dutch WB) and an expected mean heterozygosity from 0.53 (Hampshire) to 0.80 (Japanese WB) with effective alleles ranging from 2.28 (Hampshire) to 3.74 (French WB). Wild boar populations demonstrated a higher level of heterozygosity than domestic breeds. Genetic differentiation estimated by fixation indices (F_ST) ranged from 0.021 (Yorkshire and Duroc) to 0.410 (Meishan and Hampshire) and was consistent with previous mtDNA analysis. Both phylogenetic and principal component analyses revealed a distinct separation of European and Asian derived populations with tight clustering of the European domestic breeds. Conversely, the use of both MS and mtDNA clarified that the Asian populations were comprised of three groups, one represented by Erhualian and Meishan breed, the second represented by Lanyu pigs and the third represented by the Asian wild boars. The current findings support the hypothesis that Asian domestic populations were derived from multiple Asian ancestral origins whereas the European domestic populations represent a single ancestral European lineage.     

Disparate Geography of Consumption,Production, and Environmental Impacts

International trade transfers social and environmental impacts across national borders. The consumption of forest products often takes place far away from industrial production sites, and mills procure raw material from remote forests. Finland produces about 10% of forest products that are traded internationally, with the majority of its exports destined for other European countries. Here we report and analyze data that demonstrate that international leakage, in relative terms, increased faster than the production of commodities. The international consumption of products made in Finland increased, and an increase in wood imports from Russia provided the raw material for most of the incremental production. The international consumption‐production system translated the increasing global demand for Finnish products into increased harvests in Russia, until the Russian customs duties started to increase in 2007. We argue that national and regional policies for the promotion of sustainable consumption and production must be analyzed and assessed from an international, holistic perspective.     

Feasibility of guided cognitive behaviour therapy (CBT) self-help for childhood anxiety disorders in primary care

Anxiety disorders in childhood are common, disabling and run a chronic course. Cognitive behaviour therapy (CBT) is effective but expensive and trained therapists are scarce. Guided self-help treatments may be a means of widening access to treatment. This study aimed to examine the feasibility of guided CBT self-help in primary care for childhood anxiety disorders, specifically in terms of therapist adherence, patient and therapist satisfaction and clinical gain.Participants were children aged between five and 12 years referred to two primary child and adolescent mental health services (PCAMHSs) in Oxfordshire, UK, who met diagnostic criteria for a primary anxiety disorder. Of the 52 eligible children, 41 anxious children were assessed for anxiety severity and interference before and after receiving CBT self-help delivered via a parent (total therapy time = five hours) by primary mental health workers (PMHWs). Therapy sessions were rated for treatment adherence and parents and PMHWs completed satisfaction questionnaires after treatment completion. Over 80% of therapy sessions were rated at a high level of treatment adherence. Parents and PMHWs reported high satisfaction with the treatment. Sixty-one percent of the children assessed no longer met the criteria for their primary anxiety disorder diagnosis following treatment, and 76% were rated as 'much'/'very much' improved on the Clinical Global Impression-Improvement (CGI-I) scale. There were significant reductions on parent and child report measures of anxiety symptoms, interference and depression. Preliminary exploration indicated that parental anxiety was associated with child treatment outcome. The findings suggest that guided CBT self-help represents a promising treatment for childhood anxiety in primary care.     

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.     

Ethical questions concerning research on human embryos, embryonic stem cells and chimeras

Research using human embryos and embryonic stem cells is viewed as important for various reasons. Apart from questions concerning legal regulations, numerous ethical objections are raised pertaining to the use of surplus embryos from reproductive medicine as well as the creation of embryos and stem cells through cloning. In the hopes of avoiding ethical problems, alternatives have been proposed including the extraction of egg cells from "dead" embryos derived from in vitro fertilization procedures, the extraction of pluripotent stem cells from blastocysts, technologies such as "altered nuclear transfer" (ANT) and "oocyte-assisted reprogramming" (ANT-OAR) as well as parthenogenesis. Initial ethical assessments show that certain questions pertaining to such strategies have remained unanswered. Furthermore, with the help of new or more differentiated biotechnological procedures, it is possible to create chimeras and hybrids in which human and non-human cells are combined. Human-animal chimeras, in which gametes or embryonic tissue have been mixed with embryonic or adult stem cells, demonstrate a different "quality" and "degree of penetration" from those produced in previous experiments. Not only does this have consequences regarding questions of patentability, this situation also raises fundamental questions concerning the human being's self image, the concept of person, identity and species and the moral rights and duties that are connected with such concepts. There is a need for legal regulation, on the national as well as the international level.     

Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome

Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.     

Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.     

Different polyketide folding modes converge to an identical molecular architecture

Metabolic diversity is being studied intensively by evolutionary biologists, but so far there has been no comparison of biosynthetic pathways leading to a particular secondary metabolite in both prokaryotes and eukaryotes. We have detected the bioactive anthraquinone chrysophanol, which serves as a chemical defense in diverse eukaryotic organisms, in a bacterial Nocardia strain, thereby permitting the first comparative biosynthetic study. Two basic modes of folding a polyketide chain to fused-ring aromatic structures have so far been described: mode F (referring to fungi) and mode S (from Streptomyces). We have demonstrated that in eukaryotes (fungi, higher plants and insects), chrysophanol is formed via folding mode F. In actinomycetes, by contrast, the cyclization follows mode S. Thus, chrysophanol is the first polyketide synthase product that is built up by more than one polyketide folding mode.