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.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.     

Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates

The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.     

Tyrphostin A23 inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the AP-2 adaptor complex

Several intracellular membrane trafficking events are mediated by tyrosine-containing motifs within the cytosolic domains of integral membrane proteins. Many such motifs conform to the consensus YXXPhi, where Phi represents a bulky hydrophobic residue. This motif interacts with the medium chain (mu) subunits of adaptor complexes that link the cytosolic domains of integral membrane proteins to the clathrin coat involved in vesicle formation. The YXXPhi motif is similar to motifs in which the tyrosine residue is phosphorylated by tyrosine kinases. Tyrphostins (structural analogs of tyrosine) are inhibitors of tyrosine kinases and function by binding to the active sites of the enzymes. We previously showed that, in vitro and in yeast two-hybrid interaction assays, some tyrphostins can inhibit the interaction between YXXPhi motifs and the mu2 subunit of the AP-2 adaptor complex (Crump, C., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073-28077). A23 is such a tyrphostin. We now show that molecular modeling of tyrphostin A23 into the tyrosine-binding pocket in mu2 provides a structural explanation for A23 being able to inhibit the interaction between YXXPhi motifs and mu2. Furthermore, we show that A23 inhibited the internalization of (125)I-transferrin in Heb7a cells without having any discernible effect on the morphology of compartments of the endocytic pathway. Control tyrphostins, active as inhibitors of tyrosine kinase activity, but incapable of inhibiting the YXXPhi motif/mu2 interaction, did not inhibit endocytosis. These data are consistent with A23 inhibition of the YXXPhi motif/mu2 interaction in intact cells and with the possibility that different tyrphostins may be used to inhibit specific membrane trafficking events in eukaryotic cells.     

Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.     

Stage-specific expression of Smad2 and Smad3 during folliculogenesis

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.     

Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint

Deletion of the Saccharomyces cerevisiae TOP3 gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2 content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391-8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Delta strains. We show that top3Delta mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion, top3Delta strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Delta mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.     

The gamma-tubulin gene family in humans

Despite the central role of gamma-tubulin in the organization of the microtubule cytoskeleton, the gamma-tubulin gene family in humans has not been characterized. We now report the identification of a second expressed human gamma-tubulin gene (TUBG2) and a gamma-tubulin pseudogene (TUBG1P) in addition to the previously identified gamma-tubulin gene (TUBG1). Evidence from Southern hybridizations suggests that there are probably no additional gamma-tubulin sequences in the human genome. TUBG1 and TUBG2 are within 20 kb of each other in region q21 of chromosome 17, and TUBG1P is on chromosome 7. The proteins encoded by TUBG1 and TUBG2 share 97.3% amino acid identity, and the two genes are coexpressed in a variety of tissues. Previous studies of gamma-tubulin in human tissues and cell lines have been based on the tacit assumption that a single gamma-tubulin (the gamma-tubulin encoded by TUBG1) was present. While this assumption is not correct, the similarity of the products of TUBG1 and TUBG2 suggests that results of previous immunolocalization and immunoprecipitation studies in human cells and tissues are likely to be valid. In addition, any pharmacological agents that target one human gamma-tubulin are likely to target both.     

Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.     

Soil Methane Sink Capacity Response to a Long-Term Wildfire Chronosequence in Northern Sweden

Boreal forests occupy nearly one fifth of the terrestrial land surface and are recognised as globally important regulators of carbon (C) cycling and greenhouse gas emissions. Carbon sequestration processes in these forests include assimilation of CO₂ into biomass and subsequently into soil organic matter, and soil microbial oxidation of methane (CH₄). In this study we explored how ecosystem retrogression, which drives vegetation change, regulates the important process of soil CH₄ oxidation in boreal forests. We measured soil CH₄ oxidation processes on a group of 30 forested islands in northern Sweden differing greatly in fire history, and collectively representing a retrogressive chronosequence, spanning 5000 years. Across these islands the build-up of soil organic matter was observed to increase with time since fire disturbance, with a significant correlation between greater humus depth and increased net soil CH₄ oxidation rates. We suggest that this increase in net CH₄ oxidation rates, in the absence of disturbance, results as deeper humus stores accumulate and provide niches for methanotrophs to thrive. By using this gradient we have discovered important regulatory controls on the stability of soil CH₄ oxidation processes that could not have not been explored through shorter-term experiments. Our findings indicate that in the absence of human interventions such as fire suppression, and with increased wildfire frequency, the globally important boreal CH₄ sink could be diminished.