What do prisoners eat? Nutrient intakes and food practices in a high secure prison

There are limited studies on the adequacy of prisoner diet and food practices, yet understanding these are important to inform food provision and assure duty of care for this group. The aim of this research was to assess the dietary intakes of prisoners to inform food and nutrition policy in this setting. This research used a cross-sectional design with convenience sampling in a 945 bed male high secure prison. Multiple methods were used to assess food available at the group level, including verification of food portion, quality, and practices. A pictorial tool supported the diet history method. Of 276 eligible prisoners, 120 dietary interviews were conducted and verified against prison records, with 106 deemed plausible. The results showed the planned food to be nutritionally adequate, with the exception of vitamin D for older males and long chain fatty acids, with sodium above Upper Limits. The Australian Dietary Targets for chronic disease risk were not achieved. High energy intakes were reported with median 13.8MJ (SE 0.3MJ). Probability estimates of inadequate intake varied with age groups: magnesium 8% (>30 years), 2.9% (<30 years); calcium 6.0% (>70 years), 1.5% (<70 years); folate 3.5%; zinc and iodine 2.7%; and vitamin A 2.3%. Nutrient intakes were greatly impacted by self-funded snacks. Results suggest nutrient intakes nutritionally favourable when compared to males in the community. This study highlights the complexity of food provision in the prison environment, and also poses questions for population level dietary guidance in delivering appropriate nutrients within energy limits.

Dihydrofolate reductase from soybean seedlings: A fluorescence and circular dichroism study

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of −181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Genetic and epigenetic variants in the MTHFR gene are not associated with non-Hodgkin lymphoma

The methylenetetrahydrofolate reductase (MTHFR) gene codes for the MTHFR enzyme which plays a key role in the pathway of folate and methionine metabolism. Polymorphisms of genes in this pathway affect its regulation and have been linked to lymphoma. In this study we examined whether we could detect an association between two common non-synonomous MTHFR polymorphisms, 677C>T (rs1801133) and 1298A>C (rs1801131), and susceptibility to non-Hodgkin lymphoma (NHL) in an Australian case-control cohort. We found no significant differences between genotype or allele frequencies for either polymorphisms between lymphoma cases and controls. We also explored whether epigenetic modification of MTHFR, specifically DNA methylation of a CpG island in the MTHFR promoter region, is associated with NHL using blood samples from patients. No difference in methylation levels was detected between the case and control samples suggesting that although hypermethylation of MTHFR has been reported in tumour tissues, particularly in the diffuse large B-cell lymphoma subtype of NHL, methylation of this MTHFR promoter CpG island is not a suitable epigenetic biomarker for NHL diagnosis or prognosis in peripheral blood samples. Further studies into epigenetic variants could focus on genes that are robustly associated with NHL susceptibility.

Clinical relevance of MTHFR, eNOS, ACE, and ApoE gene polymorphisms and serum vitamin profile among Malay patients with ischemic stroke

Background The purpose of this study was threefold. First, it was to determine the relationship between serum vitamin profiles and ischemic stroke. The second purpose was to investigate the association of methylenetetrahydrofolate reductase (MTHFR), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE), and apolipoprotein-E (ApoE) gene polymorphisms with ischemic stroke and further correlate with serum vitamin profiles among ischemic stroke patients. The third purpose of the study was to highlight the interaction of MTHFR and eNOS haplotypes with serum vitamin profiles and ischemic stroke risks. Methods Polymorphisms of these genes were analyzed in age-, sex-, and ethnicity-matched case–controls (n = 594); serum vitamin profiles were determined using immunoassays. Results The MTHFR 677C>T, 1298A>C, eNOS intron 4a/b, and ApoE polymorphisms were significantly associated with the increased risk of ischemic stroke. Elevated serum homocysteine and vitamin B12 levels were associated with MTHFR 677C>T and eNOS intron 4a/b polymorphisms. The ApoE and eNOS −786T>C polymorphisms were associated with increased serum vitamin B12 levels. However, none of the polymorphisms influenced serum folate levels except for the MTHFR 1298A>C. Different patterns of MTHFR and eNOS haplotypes tend to affect serum vitamin profiles to different degrees, which contribute to either different susceptibility risk or protective effect on ischemic stroke. Overall, increased levels of serum homocysteine and vitamin B12 levels were associated with higher risk of ischemic stroke in the investigated population. Conclusions The present study suggests that the genotypes and haplotypes of MTHFR 677C>T and eNOS intron 4a/b polymorphisms are potential serum biomarkers in the pathophysiological processes of ischemic stroke, by modulating homocysteine and vitamin B12 levels.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India

Background & objectives: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677C -> T and 1298 A -> C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C -> T and 1298 A -> C and risk of colorectal cancer (CRC).Methods: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). Results: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74,25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A -> C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45(95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18(95% CI 0.52-2.64, P =0.697).Interpretation & conclusion: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C -> T and 1298 A -> C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A -> C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

Methylenetetrahydrofolate reductase gene polymorphisms and risk of acute lymphoblastic leukemia in children

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA synthesis, DNA repair and DNA methylation. Genetic polymorphisms of this enzyme have been shown to impact several diseases, including cancer. Leukemias are malignancies arising from rapidly proliferating hematopoietic cells having great requirement of DNA synthesis. This case-control study was undertaken to analyze the association of the MTHFR gene polymorphisms 677 C"T and 1298 A"C and the risk of acute lymphoblastic leukemia in children. Materials and Methods: Eighty-six patients aged below 15 years with a confirmed diagnosis of acute lymphoblastic leukemia (ALL) and 99 matched controls were taken for this study. Analysis of the polymorphisms was done using the polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP) method. Results: Frequency of MTHFR 677 CC and CT were 85.9% and 14.1% in the controls, and 84.9% and 15.1% in the cases. The 'T' allele frequency was 7% and 7.5% in cases and controls respectively. The frequency of MTHFR 1298 AA, AC, and CC were 28.3%, 55.6% and 16.1% for controls and 23.3%, 59.3% and 17.4% for cases respectively. The 'C' allele frequency for 1298 A→C was 43.9% and 47% respectively for controls and cases. The odds ratio (OR) for C677T was 1.08 (95% CI 0.48- 2.45, p = 0.851) and OR for A1298C was 1.29(95% CI 0.65-2.29, p = 0.46) and OR for 1298 CC was 1.31 (95% CI 0.53-3.26, p =0.56). The OR for the combined heterozygous status (677 CT and 1298 AC) was 1.94 (95% CI 0.58 -6.52, p = 0.286). Conclusion: The prevalence of 'T' allele for 677 MTHFR polymorphism was low in the population studied. There was no association between MTHFR 677 C→T and 1298 A→C gene polymorphisms and risk of ALL, which may be due to the small sample size.

Regulation of monkey liver serine hydroxymethyltransferase by nicotinamide nucleotide

The positive homotropic binding of tetrahydrofolate to monkey liver serine hydroxymethyltransferase was abolished on preincubating the enzyme with NADH and NADPH. NAD+ was a negative heterotropic effector, whereas NADP+ was without effect. The allosteric effects of nicotinamide nucleotides on the serine hydroxymethyltransferase, reported for the first time, lead to a better understanding of the regulation of the metabolic interconversion of folate coenzymes.

The role of lysine-256 in the structure and function of sheep liver recombinant serine hydroxymethyltransferase

The active site lysine residue, K256, involved in Schiffs base linkage with pyridoxal-5'-phosphate (PEP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H(4)folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H(4)folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.

``Clickable'', Trifunctional Magnetite Nanoparticles and Their Chemoselective Biofunctionalization

A multifunctional iron oxide based nanoformulation for combined cancer-targeted therapy and multimodal imaging has been meticulously designed and synthesized using a chemoselective ligation approach. Novel superparamagnetic magnetite nanoparticles simultaneously functionalized with amine, carboxyl, and azide groups were fabricated through a sequence of stoichiometrically controllable partial succinylation and Cu (II) catalyzed diazo transfer on the reactive amine termini of 2-aminoethylphosphonate grafted magnetite nanoparticles (MNPs). Functional moieties associated with MNP surface were chemoselectively conjugated with rhodamine B isothiocyanate (RITC), propargyl folate (FA), and paclitaxel (PTX) via tandem nucleophic addition of amine to isothithiocyanates, Cu (I) catalyzed azide-alkyne click chemistry and carbodiimide-promoted esterification. An extensive in vitro study established that the bioactives chemoselectively appended to the magnetite core bequeathed multifunctionality to the nanoparticles without any loss of activity of the functional molecules. Multifunctional nanoparticles, developed in the course of the study, could selectively target and induce apoptosis to folate-receptor (FR) overexpressing cancer cells with enhanced efficacy as compared to the free drug. In addition, the dual optical and magnetic properties of the synthesized nanoparticles aided in the real-time tracking of their intracellular pathways also as apoptotic events through dual fluorescence and MR-based imaging.

Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5V-phosphate (PLP)-dependent enzyme catalyzes thetetrahydrofolate (H4-folate)- dependent retro-aldol cleavage of serine to form 5,10-methylene H4-folate and glycine. The structure–function relationship of SHMT wasstudied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an ‘open’ to ‘closed’ conformation rather than proton abstraction from the hydroxylgroup of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate.The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s)complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

Orthogonal biofunctionalization of magnetic nanoparticles via ``clickable'' poly(ethylene glycol) silanes: a ``universal ligand'' strategy to design stealth and target-specific nanocarriers

The present work demonstrates a novel strategy to synthesize orthogonally bio-engineered magnetonanohybrids (MNPs) through the design of versatile, biocompatible linkers whose structure includes: (i) a robust anchor to bind with metal-oxide surfaces; (ii) tailored surface groups to act as spacers and (iii) a general method to implement orthogonal functionalizations of the substrate via ``click chemistry''. Ligands that possess the synthetic generality of features (i)-(iii) are categorized as ``universal ligands''. Herein, we report the synthesis of a novel, azido-terminated poly(ethylene glycol) (PEG) silane that can easily self-assemble on MNPs through hetero-condensation between surface hydroxyl groups and the silane end of the ligand, and simultaneously provide multiple clickable sites for high density, chemoselective bio-conjugation. To establish the universal-ligand-strategy, we clicked alkyl-functionalized folate onto the surface of PEGylated MNPs. By further integrating a near-infrared fluorescent (NIRF) marker (Alexa-Fluor 647) with MNPs, we demonstrated their folate-receptor mediated internalization inside cancer cells and subsequent translocation into lysosomes and mitochondria. Ex vivo NIRF imaging established that the azido-PEG-silane developed in course of the study can effectively reduce the sequestration of MNPs by macrophage organs (viz. liver and spleen). These folate-PEG-MNPs were not only stealth and noncytotoxic but their dual optical and magnetic properties aided in tracking their whereabouts through combined magnetic resonance and optical imaging. Together, these results provided a strong motivation for the future use of the ``universal ligand'' strategy towards development of ``smart'' nanohybrids for theragnostic applications.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli

Most organisms possess bifunctional FolD 5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrandrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (Eco FolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (Cpe FoID) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of Cpe FoID is about five times more efficient than that of Eco FolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is similar to 10 times more efficient than that of Eco FolD. Kinetic parameters for Cpe Fhs were also determined for utilization of all of its substrates. Both Cpe FoID and Cpe FchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of Cpe FoID and Cpe FchA is also necessary to rescue an E coli folD deletion strain (harbouring Cpe Fhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

功能化乳酸类聚合物的生物应用研究

乳酸类聚合物具有广泛的生物学和医学应用潜力，但是乳酸类聚酯主链缺少活性位点限制了其应用的范围。用聚乙二醇与聚乳酸共聚进行改性，可以提高担载水溶性药物的效率和控释能力；氨基酸改性再偶联生物分子可以实现乳酸类聚合物的生物功能化和生物智能化的应用。本论文以功能化乳酸类聚合物为研究对象，按照聚乳酸生物功能由低级到高级的顺序分别考察了乳酸类聚合物作为载药纤维、表面活性材料、蛋白质固载和纯化材料、以及靶向药物载体等在一些生物领域的应用，并获得结果如下：1） 聚乙二醇改性的聚乳酸嵌段共聚物纺丝担载阿霉素，具有体内和体外的长效缓释作用；2）以biotin/PLL—PLA—PEG制备的高分子涂层，具有良好的特异性固载生物分子；3）将biotin/PLL—PLA—PEG与PLGA共混制备生物活性纤维，则特异性的固载蛋白质；4）用聚半胱氨酸改性聚乳酸合成PCys—PLA，再和PLGA共混制备纤维，再表面偶联还原型谷胱甘肽，则可以捕获谷胱甘肽转移酶；5）将folate/PLL—PLA—PEG制备成胶束，则具备了叶酸受体介导的生物智能化靶向送药的功能，尤其是在动物肿瘤模型的试验中表现了良好的叶酸受体靶向性。

非洲爪蛙分泌型叶酸结合蛋白及NKX6.3在早期胚胎神经发育中的功能研究

叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此叶酸是B族维生素的一员，参与体内一系列重要的生命过程包括DNA，氨基酸的合成，调控细胞周期，参与一碳单位供体循环，调节DNA,蛋白质甲基化等。叶酸的许多功能都和叶酸结合蛋白有关，体内有多种跨膜形式的叶酸结合蛋白，比如Folbp1，RFC，HCP等。以前的研究表明这些不同的叶酸结合蛋白具有不同的功能。分泌型叶酸结合蛋白是另外一类叶酸结合蛋白，在人类，小鼠，猪中都有序列报道，但是其功能却知之甚少。 我们在非洲爪蛙中鉴定出一个全新的分泌型叶酸结合蛋白并命名为Secreted Folate Binding Protein(sFBP)。在胚胎和转染细胞系中我们都证明该蛋白是分泌性的，表面等离子共振实验发现sFBP能够结合叶酸。在胚胎早期这个基因表达于粘液腺和神经板区域，神经管闭合后在神经管、粘液腺、眼睛，头部以及鳃弓都有表达。特异morpholino 阻断sFBP翻译后发现粘液腺发育异常，神经管闭合缺陷，前后体轴聚集延伸运动受到抑制，尾芽期胚胎表现出体轴缩短，无眼，小头或无头的表型。进一步研究发现显微注射sFBP morpholino 的胚胎神经板区域细胞发生凋亡，中胚层和神经外胚层的一系列粘附分子表达异常，神经细胞的正常分化也受到抑制。通过显微移植实验我们还发现抑制sFBP的翻译后，神经嵴细胞的正常分化和迁移都受到抑制。但是，显微注射叶酸及其类似物或者显微注射甲基供体S-腺苷甲硫氨酸或者亮氨酸甲基转移酶都不能挽救阻断sFBP造成的表形，由此提示sFBP可能不是通过叶酸传统的参与营养合成或者甲基化的途径发挥作用。我们发现注射sFBP morpholino可以抑制Islet-1mRNA和蛋白质的表达，Islet-1的表达区域与sFBP类似。共同注射Islet-1 mRNA和sFBP morpholino可以极大的挽救sFBP morpholino的表型。最后通过morpholino特异阻断Islet-1的表达后，我们发现其表现出与sFBP morpholino类似的粘液腺发育缺陷，神经板细胞凋亡，小头无眼的表形。由此我们认为sFBP结合叶酸后可能通过细胞膜上的受体传递信号，并且Islet-1可能在sFBP的下游发挥作用。 神经嵴是脊椎动物特有的一群多潜能干细胞，产生于表皮和神经板的边界，在原肠运动之后这群细胞通过表皮间充值转换从神经管背侧迁移到不同的区域，分化成不同的细胞类型，包括外周神经系统，色素细胞，软骨等。神经嵴的发生是一个多步骤多基因参与的精细调控过程。目前理论认为最初由一些分泌性信号分子又叫形态生成素比如BMP,Wnt，FGF，Notch等通过不同浓度梯度的相互作用调节一组在表皮和神经板边界的转录因子（Msx、Pax3/7、Zic1、Dlx3/5等）的表达，即边界决定。这些边界决定因子进一步在预定形成神经嵴的区域激活神经嵴特化基因比如Slug/Snail、FoxD3、Twist、Sox9/10的表达完成神经嵴的特化（Specification）。 Nkx6.3是Nkx6家族的一个转录因子，RT-PCR显示其呈现母源性表达。特异抗体显示Nkx6.3蛋白第9期在整个胚胎都表达，大部分蛋白集中在细胞核，有少部分蛋白定位于细胞膜上；神经板时期主要定位于神经嵴区域的细胞膜上。过表达Nkx6.3会影响细胞粘连分子的表达，由此干扰正常的胚胎原肠运动和Activin诱导的动物帽聚集延伸运动。显微注射Nkx6.3特异morpholino阻断其蛋白表达会抑制神经嵴的marker基因Wnt8，Fgf8，Pax3，Msx1，Zic1，FoxD3，Slug的转录，阻碍神经嵴的发育。在动物帽中单独注射Nkx6.3可以在mRNA水平上诱导Wnt8、Fgf8另一方面抑制BMP4的表达进而诱导神经嵴基因Pax3，Zic1，Slug的表达。报告基因实验也显示Nkx6.3能够激活Wnt信号而在动物帽中抑制BMP信号。Nkx6.3蛋白功能域分析发现其EH1结构域（domain）参与对Wnt8信号的激活，而EH1结构域和HD结构域之间的连接区域（linker domain）参与对FGF的激活和对BMP的抑制。进一步在动物帽和胚胎中分析发现Nkx6.3对Wnt8的激活依赖于FGF家族受体信号但是不依赖于Fgf8。有趣的是4细胞时期过表达Nkx6.3促进Fgf8和Wnt8 mRNA表达，但是抑制边界决定基因Msx1、Pax3和神经嵴特化基因Slug的转录。在32细胞时期显微注射Nkx6.3可以在内源神经嵴发生区域抑制Slug的表达，而异位却诱导Slug的mRNA。我们发现与动物帽中对BMP的调节不同，在胚胎中，过表达Nkx6.3会强烈的激活Smad1蛋白在细胞核中的表达即BMP信号被激活，高的BMP信号会抑制神经嵴的发生。另外我们发现过表达Nkx6.3在胚胎中抑制Dlx5而在动物帽中却不影响Dlx5的表达水平，Morpholino阻断Dlx5会抑制Msx1、Pax3和Slug的表达。BMP信号和Dlx5在动物帽和在整体胚胎中对Nkx6.3的不同响应可以一定程度上解释过表达Nkx6.3在2个系统中对神经嵴基因Slug相反的影响结果。