Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.     

FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis

Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23(-/-) or Klotho(-/-) knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.     

Effectiveness and safety of a long-acting,once-daily,two-phase release formulation of methylphenidate (Ritalin<Superscript>®</Superscript> LA) in school children under daily practice conditions

Long-acting (LA) preparations of methylphenidate allow for once-daily dosing; however, pharmacokinetics may vary and depend on food intake. The objective was to evaluate effectiveness of a two-phase release formulation (Ritalin^® LA) under daily practice conditions. This was a prospective, multicenter, observational study in Germany. Eligibility and dosing were determined by the physician based on the drug label. Outcomes included changes over 3 months of treatment in assessments of effect duration, clinical global impression (CGI), and quality of life (ILK). In 101 sites, 262 patients (197 boys, 63 girls, and two unknown) with a mean age of 10.9 years were enrolled; 50 were treated for the first time; 212 switched medication to Ritalin^® LA. After 3 months, CGI improved in 59.4 % of patients, and well-being overall was rated as good by 61.0 % of parents and 63.7 % of children. Based on parents’ assessment, the proportion of children suffering from strong disease burden decreased from 40.7 to 15.1 %. In 123 insufficient responders to previous ADHD medications, benefit from Ritalin^® LA was above average and effect duration was significantly prolonged as compared to pretreatment. Overall, 28 patients (10.7 %) had treatment-related adverse events with one case being serious; 23 patients (8.8 %) discontinued therapy, 7 (2.7 %) due to poor treatment response; and 212 patients (81 %) continued treatment beyond the study. In line with clinical trial data, Ritalin^® LA provides significant benefit also under routine practice conditions.     

Tobacco smoke exposure before, during, and after pregnancy and risk of overweight at age 6

Maternal smoking during pregnancy has been associated with overweight and obesity in childhood and is strongly correlated with children's tobacco smoke exposure before and after pregnancy. We investigated the independent association of tobacco smoke exposure at various pre- and postnatal periods and overweight at age 6. A total of 1,954 children attending the 2001-2002 school entrance health examination in the city of Aachen, Germany, were included into this study. Height and weight were measured, BMI was calculated. Tobacco smoke exposure at various periods, other lifestyle and sociodemographic factors were ascertained by questionnaire. Multiple logistic regression models were used to assess the association between tobacco smoke exposure and overweight. Prevalence of overweight was 8.9%. Significant positive associations were found with maternal smoking before and during pregnancy and during the first and sixth year of life. When all smoking periods were included into one logistic model simultaneously, secondhand smoke exposure after birth remained positively associated with overweight at age 6 at either one of the two time periods (first year only: odds ratio (OR) (95% confidence interval (CI)): 2.94 (1.30-6.67), sixth year only: 2.57 (1.64-4.04), respectively) or at both (4.43 (2.24-8.76)). Exposure to tobacco smoke during the first years of life appears to be a key risk factor for development of childhood overweight.     

Methyl or ethyl makes the difference: Synthesis and solid-state structure of 9,10-dialkyl-9,10-dihydro-9,10-digallaanthracenes

The donor-free 9,10-dialkyl-9,10-dihydro-9,10-digallaanthracenes 1 (alkyl: methyl) and 2 (alkyl: ethyl) were prepared by reaction of 1,2-di(chloromercurio)benzene with the corresponding trialkylgallium and isolated as colourless air- and moisture sensitive crystalline compounds. In the solid-state structure of 1, two slightly different monomers 1A and 1B are found, which form a dimer 1A?1B held together by “medium” strong gallium arene π-interactions. Further weak π-interactions between 1A and 1A and 1B and 1B constitute a one-dimensional coordination polymer containing strands of the composition [?(1A?1B)?(1B?1A)?]_n. In contrast, compound 2 crystallizes in the form of distinct molecular units without any further intermolecular π-interactions. The molecular units possess D_2d symmetry and are built by strong π-interactions between two digallaanthracene monomers. Two symmetrical aryl group bridges between two gallium atoms are observed for the first time in the subunits of 2. By addition of a Lewis-base (THF, Pyridine) to 2, a monomeric planar digallaanthracene framework is restored, as proven by an X-ray crystal structure analysis of 2 · 2Py. The different structures of 1 and 2 are explained on the basis of steric effects.     

Transfer and Expression of Lithoautotrophy and Denitrification in a Host Lacking These Metabolic Activities

The conjugative 450-kilobase-pair megaplasmid pHG1 from Alcaligenes eutrophus H16 was transferred to the herbicide-degrading soil bacterium A. eutrophus JMP134. This transfer was achieved by means of RP4 mobilization and a Tn5-Mob insertion provided in trans on the megaplasmid replicon. Although kanamycin-resistant transconjugants also occurred with other gram-negative species such as Rhizobium, Agrobacterium, and thiobacteria, A. eutrophus JMP134 was the only recipient which stably maintained the megaplasmid. pHG1-containing transconjugants derived from JMP134 expressed all metabolic functions associated with the plasmid: the ability to oxidize hydrogen through catalysis of two hydrogenases, to assimilate carbon dioxide via the Calvin cycle pathway, and to grow with nitrate anaerobically. All of these metabolic activities were absent in the original strain JMP134.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.