Recommendations for accelerating global action to prevent folic acid-preventable birth defects and other folate-deficiency diseases: meeting of experts on preventing folic acid-preventable neural tube defects

BACKGROUND: In April of 2003, The Micronutrient Initiative, in collaboration with several other organizations, convened a group of knowledgeable scientists and policy experts to discuss ways to accelerate the global pace at which countries implement effective and sustainable programs to prevent folic acid-preventable birth defects and other folate-deficiency diseases. Programs implemented to date by fewer than 40 countries have prevented only 10% of the estimated 240,000 annual cases of folic acid-preventable spina bifida and anencephaly. METHODS: Participants in this meeting summarized and presented scientific evidence showing that increased consumption of synthetic folic acid prevents a large proportion of spina bifida and anencephaly cases. They also reviewed related guidance and endorsement issued by national professional societies and advisory bodies as well as policies and programs implemented by some countries that have already demonstrated successful results in terms of reduced rates of neural tube defects and improved folate nutrition. CONCLUSIONS: The group formulated and discussed recommendations and strategies for increasing the pace of neural tube defect prevention globally. The recommendations and strategies are published here.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.     

Regulation of p70 S6 kinase by complex formation between the Rac guanine nucleotide exchange factor (Rac-GEF) Tiam1 and the scaffold spinophilin

Tiam1 is a ubiquitous guanine nucleotide exchange factor (GEF) that activates the Rac GTPase. We have shown previously that the N terminus of Tiam1 contributes to the signaling specificity of its downstream target Rac via association with IB2, a scaffold that promotes Rac activation of a p38 kinase cascade. Here we show that the N terminus of Tiam1 can influence Rac signaling specificity in a different way by interaction with spinophilin, a scaffold that binds to p70 S6 kinase, another protein regulated by Rac. In particular, spinophilin binding promotes the plasma membrane localization of Tiam1 and enhances the ability of Tiam1 to activate p70 S6 kinase. In contrast, spinophilin binding suppresses the ability of Tiam to activate Pak1, a different Rac effector. Finally, a mutant spinophilin that cannot bind to Tiam1 suppresses serum-induced p70 S6 kinase activation in cells, suggesting that a Tiam1/spinophilin complex contributes to p70 S6 kinase regulation by extracellular signals. These findings add to a growing body of evidence supporting the concept that some Rac-GEFs not only activate Rac GTPases but also participate in the selection of Rac effector by binding to particular scaffolds that complex with components of specific Rac effector pathways.     

Functional analysis of a divergent system II protein,Ccs1, involved in c-type cytochrome biogenesis

The Ccs1 gene, encoding a highly divergent novel component of a system II type c-type cytochrome biogenesis pathway, is encoded by the previously defined CCS1 locus in Chlamydomonas reinhardtii. phoA and lacZalpha bacterial topological reporters were used to deduce a topological model of the Synechocystis sp. 6803 Ccs1 homologue, CcsB. CcsB, and therefore by analogy Ccs1, possesses a large soluble lumenal domain at its C terminus that is tethered in the thylakoid membrane by three closely spaced transmembrane domains in the N-terminal portion of the protein. Molecular analysis of ccs1 alleles reveals that the entire C-terminal soluble domain is essential for Ccs1 function and that a stromal loop appears to be important in vivo, at least for maintenance of Ccs1. Site-directed mutational analysis reveals that a single histidine (His(274)) within the last transmembrane domain, preceding the large lumenal domain, is required for c-type cytochrome assembly, whereas an invariant cysteine residue (Cys(199)) is shown to be non-essential. Ccs1 is proposed to interact with other Ccs components based on its reduced accumulation in ccs2, ccs3, ccs4, and ccsA strains.     

Domains in tropoelastin that mediate elastin deposition in vitro and in vivo

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.     

Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.