A tyrosine-based signal plays a critical role in the targeting and function of adenovirus RIDalpha protein

Early region 3 genes of human adenoviruses contribute to the virus life cycle by altering the trafficking of cellular proteins involved in adaptive immunity and inflammatory responses. The ability of early region 3 genes to target specific molecules suggests that they could be used to curtail pathological processes associated with these molecules and treat human disease. However, this approach requires genetic dissection of the multiple functions attributed to early region 3 genes. The purpose of this study was to determine the role of targeting on the ability of the early region 3-encoded protein RIDalpha to downregulate the EGF receptor. A fusion protein between the RIDalpha cytoplasmic tail and glutathione S-transferase was used to isolate clathrin-associated adaptor 1 and adaptor 2 protein complexes from mammalian cells. Deletion and site-directed mutagenesis studies showed that residues 71-AYLRH of RIDalpha are necessary for in vitro binding to both adaptor complexes and that Tyr72 has an important role in these interactions. In addition, RIDalpha containing a Y72A point mutation accumulates in the trans-Golgi network and fails to downregulate the EGF receptor when it is introduced into mammalian cells as a transgene. Altogether, our data suggest a model where RIDalpha is trafficked directly from the trans-Golgi network to an endosomal compartment, where it intercepts EGF receptors undergoing constitutive recycling to the plasma membrane and redirects them to lysosomes.     

Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

Clinician reviewers in birth defects surveillance programs: survey of the National Birth Defects Prevention Network

BACKGROUND: Some birth defects surveillance programs utilize a clinician reviewer ("Clinician") to assist the multidisciplinary staff in the process of case review, coding and classification. The untested assumption is that expertise in the evaluation of individuals with birth defects and genetic syndromes in clinical practice, usually clinical genetics, is useful in reviewing medical records. METHODS: We conducted an exploratory survey of the 50 functioning birth defects surveillance programs that participated in the National Birth Defects Prevention Network in 2004. The survey was mailed electronically to program coordinators and included 10 questions with check-off option replies. Open-ended comments were also solicited. RESULTS: Responses were received from 31 of 50 (62%) programs, 21 (68%) which used a Clinician. In addition to the 9 centers that identified themselves as participants in the National Birth Defects Prevention Study (NBDPS), there were 12 non-NBDPS programs using a Clinician, 2 of whom were not clinical geneticists (1 nurse genetic counselor and 1 primary pediatrician). A total of 86% (18/21) of Clinicians were employed part-time or as consultants; 1 was full-time, and 2 were volunteers. In addition to the core activities of classifying defects and reviewing cases to determine if a syndrome was present, over one-half of the Clinicians participated in education of birth defects surveillance programs staff, research, and program development. Most (86%; 18/21) Clinicians had been trained informally for their roles. Only 1 had received a formal performance review. CONCLUSIONS: Aside from the 9 centers in the NBDPS in which the position of Clinician is funded by the Centers for Disease Control and Prevention (CDC), about one-half of the remaining respondent birth defects surveillance programs used a Clinician. Future research is needed to determine why a birth defects surveillance program hires or refrains from using a Clinician, and whether a Clinician accomplishes the desired goals. This survey reveals a lack of formal training for the Clinicians in their roles in the surveillance programs, and a lack of quality monitors, which might be addressed in the future.     

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.     

Mechanisms and pathophysiological implications of sinusoidal endothelial cell gap formation following treatment with galactosamine/endotoxin in mice

Neutrophil extravasation from sinusoids is a critical step for acute inflammatory tissue injury. However, the role of sinusoidal endothelial cells (SECs) in this process remains unclear. Matrix metalloproteinases (MMPs) have been shown to involve gap formation in SECs in several liver diseases. Therefore, the present study examined SEC modifications elicited by galactosamine (Gal)/endotoxin (ET). Treatment of male C3Heb/FeJ mice with Gal/ET or Gal/TNF caused the formation of numerous gaps in SECs at 4 h when no neutrophil extravasation occurred. Six hours after Gal/ET or Gal/TNF treatment, blood elements started to penetrate to the extrasinusoidal space through large gaps. Treatment with ET alone caused sinusoidal neutrophil accumulation but no gap formation, neutrophil extravasation, or hemorrhage. Gal/ET treatment increased hepatic MMP-2 and MMP-9 mRNA expression (6.7- and 11-fold, respectively). Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg), minimized gap formation after Gal/ET and Gal/TNF treatment. The MMP inhibitor reduced injury only in the Gal/ET model mainly due to reduced TNF formation. The MMP inhibitor attenuated sinusoidal neutrophil accumulation at 6 h but failed to attenuate Gal/TNF-induced liver injury at 7 h due to excessive apoptosis. These results suggest that Gal/ET or Gal/TNF activates MMPs, which are responsible for SEC gap formation. Although the initial appearance of gap formation is independent of neutrophils, the gaps allow initial contact of neutrophils with damaged hepatocytes. In addition, MMP activation promotes neutrophil accumulation in sinusoids.     

Two motifs with different function regulate the anterograde transport of the adiponectin receptor 1

The anterograde trafficking of GPCR has been described as a tightly controlled process involving specific amino acid sequences that mediate the receptor transport. In this study, we investigated whether the cell surface delivery of the adiponectin receptor 1, a newly identified class of heptahelix receptors different from G protein-coupled receptors, is regulated. Sequential N-terminal deletion revealed that the export of the AdipoR1 from the endoplasmic reticulum (ER) is controlled by distinct parts of the receptor N-terminus. Strong evidence is provided that the ER exit is mediated by two specific sequences, a F(X)(3)F(X)(3)F and a D(X)(3)LL motif. Disruption of these motifs led to a substantial accumulation of the AdipoR1 in the ER. Mutation of similar motifs in the AdipoR1 C-terminus did not result in aberrant receptor localization, suggesting that these motifs are sequence and position specific to the AdipoR1 N-terminus. Further analysis of the regulation mechanism identified an interaction with the chaperone BiP and additionally, strong evidence is provided that both motifs exert different biological function in the AdipoR1 ER export. In conclusion, our data demonstrate that the receptor transport shares similar ER exit motifs although AdipoR are structurally different from GPCR. However, since even two specific sequences are identified, the anterograde trafficking of the AdipoR1 seems to be regulated in a more complex manner.     

Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA₂₀ and GA₈, in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.Semidwarfism is a valuable trait in many crop species and agricultural environments. In cereal crops, it can result in decreased lodging, increased yield, and improved harvest index (Dalrymple, 1985; Hedden, 2003). Therefore, it was a critical foundation of the “Green Revolution” that resulted in large improvements of yield in wheat (Triticum aestivum) and rice (Oryza sativa; Hargrove and Cabanilla, 1979; Perovic et al., 2008). Semidwarfism has had substantial benefits for fruit tree production, where it enables earlier fruit bearing, higher yields, and easier harvests in orchards (Battisini and Battisini, 2005). Semidwarf woody species are also extensively used in ornamental horticulture, where they allow more compact forms to be fit into small areas around homes and on streets and reduce the need for pruning to avoid interference with structures and transmission lines (Busov et al., 2003).Although against the current orthodoxy of forest tree breeding, where height growth is emphasized, semidwarfism might also have benefits for wood and biomass production (Bradshaw and Strauss, 2001). Such trees could be useful if they were less prone to wind throw due to their shorter, stockier forms and expected greater allocation to roots. Reduced stature could also result in less bending and slanting of trunks in the face of wind and gravity on hillslopes and thus reduce the extent of reaction wood formation, which degrades the performance and value of solid wood and pulp products. Reduced height and increased allocation of growth to roots might enhance stress tolerance, soil nutrient uptake, bioremediation, and carbon sequestration.Semidwarfism can be achieved by the modification of several types of genes and physiological mechanisms, but the most prevalent and advanced forms in agriculture affect GAs or their signaling (for review, see Busov et al., 2008). GAs are endogenous plant hormones that influence several aspects of plant growth and development, including seed germination, leaf expansion, shoot growth, cell division, flower induction, and fruit development (Sun and Gubler, 2004; Fleet and Sun, 2005; Swain and Singh, 2005). With respect to shoot growth, the most obvious effect of GA is its promotion of stem elongation by stimulating both cell elongation and division (Marth et al., 1956). GA modification also has significant effects on plant biochemistry, changing the amounts and distribution of a wide variety of metabolites in shoots and roots (Rossetto et al., 2003; Chen et al., 2004; Busov et al., 2006).Little is known about how semidwarfism affects belowground growth. GA has been shown to play a controlling role in lateral root development (Gou et al., 2010), and GA and ethylene synergistically promote both the initiation and growth of adventitious roots (Osmont et al., 2007). In tomato (Solanum lycopersicum), isogenic GA-deficient mutants (gib) allocate more biomass to roots compared with shoots (Nagel et al., 2001). In poplar (Populus spp.), semidwarf transgenic plants grown in vitro had a lower shoot-to-root ratio, which was at least partly due to proliferation of lateral roots (Busov et al., 2006; Gou et al., 2010).As a domestication trait, semidwarfism has been proposed as a means for mitigating the spread of transgenic plants within and outside of crop environments (Al-Ahmad et al., 2005). The genetic dominance of most semidwarfism transgenes would cause reduced height growth in transgene-containing progeny, reducing their ability to compete for light. Moreover, because of the close linkage of the semidwarfism genes to other genes that were cointroduced on the same plasmid, they would also powerfully retard their spread or introgression, even in cases where the linked transgene would, on their own, impart a selective advantage. However, there have been very few plant species where this concept has been explicitly tested (Al-Ahmad and Gressel, 2006; Gressel and Valverde, 2009), and we know of no examples in woody or perennial plants.To study the effects of semidwarfism genes in a woody plant grown under field conditions, we inserted a number of dominant GA-modifying transgenes into hybrid poplar (Populus tremula × Populus alba), the widely recognized model woody plant for genomics and biotechnology (Herschbach and Kopriva, 2002; Brunner et al., 2004a; Tuskan et al., 2004). Most of the genes studied were overexpressed forms of GA 2-oxidase, GA-Insensitive (GAI), or Repressor of GAI-Like (RGL), all known to cause semidwarfism in other plant species. GA 2-oxidase is a major GA catabolic enzyme in plants, and GAI and RGL are negative regulators of the GA signal transduction pathway (Appleford et al., 2007; Busov et al., 2008). The transgenic trees were first analyzed in the greenhouse (Busov et al., 2006) and then assayed for their effect on height growth in a 2-year field trial (Zawaski et al., 2011), from which we selected 10 transgenic events that grew at approximately three-quarters the rate of wild-type trees. The goal was to select semidwarf trees whose phenotype was not so severe as to be irrelevant to possible crop uses but strong enough to give a clear phenotype in a field study. In this study, we analyzed changes in a number of morphological, physiological, and growth traits and investigated the prospect for semidwarfism to be used as a mitigation tool to reduce the frequency of spread of transgenic and exotic species.     

A dysregulated endocannabinoid-eicosanoid network supports pathogenesis in a mouse model of Alzheimer's disease

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Highlights? Monoacylglycerol lipase (MAGL) is a major contributor to brain arachidonic acid pools ? MAGL mutation decreases neuroinflammation and amyloid β in Alzheimer's disease mouse ? MAGL blockade recapitulates brain prostaglandin and cytokine-lowering effects ? MAGL inhibitors may be a next-generation strategy for combating Alzheimer's disease     

Protein trafficking rates assessed by quantum dot quenching with bromocresol green

Quantum dots are bright, photostable fluorophores used extensively to investigate biological processes. In this study, we report that bromocresol green (BCG) at low micromolar concentrations rapidly, efficiently and reversibly quenches the fluorescence of commercial quantum dots having a wide range of functionalities. The broad utility of BCG quenching of quantum dots in cell biology is showed in quantitative assays of trafficking of the β(2) -adrenergic receptor (β(2) AR) and the cystic fibrosis transmembrane conductance regulator (CFTR).