Gail S. Martin

Mutations in Capillary Morphogenesis Gene-2 Result in the Allelic Disorders Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis

Juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH) are autosomal recessive syndromes of unknown etiology characterized by multiple, recurring subcutaneous tumors, gingival hypertrophy, joint contractures, osteolysis, and osteoporosis. Both are believed to be allelic disorders; ISH is distinguished from JHF by its more severe phenotype, which includes hyaline deposits in multiple organs, recurrent infections, and death within the first 2 years of life. Using the previously reported chromosome 4q21 JHF disease locus as a guide for candidate-gene identification, we identified and characterized JHF and ISH disease-causing mutations in the capillary morphogenesis factor-2 gene (CMG2). Although CMG2 encodes a protein upregulated in endothelial cells during capillary formation and was recently shown to function as an anthrax-toxin receptor, its physiologic role is unclear. Two ISH family-specific truncating mutations, E220X and the 1-bp insertion P357insC that results in translation of an out-of-frame stop codon, were generated by site-directed mutagenesis and were shown to delete the CMG-2 transmembrane and/or cytosolic domains, respectively. An ISH compound mutation, I189T, is predicted to create a novel and destabilizing internal cavity within the protein. The JHF family-specific homoallelic missense mutation G105D destabilizes a von Willebrand factor A extracellular domain alpha-helix, whereas the other mutation, L329R, occurs within the transmembrane domain of the protein. Finally, and possibly providing insight into the pathophysiology of these diseases, analysis of fibroblasts derived from patients with JHF or ISH suggests that CMG2 mutations abrogate normal cell interactions with the extracellular matrix.

Effect of palm oil, margarine, butter, and sunflower oil on the serum lipids and lipoproteins of normocholesterolemic middle-aged men☆

Abstract Twenty-nine healthy middle-aged men participated in a Latin square-designed study containing six dietary fats: butter; crude palm oil; hard margarine; refined palm oil; 80% refined palm oil + 20% sunflower oil blend; and sunflower oil. Each diet period was 6 weeks in duration followed by 6 weeks of habitual diet. Test fats were consumed in ice cream, milk, cookies, and as spreads and represented 50% of the total fat energy (38%) on all diets. Serum lipid responses to the high level of test fats in the diets were small relative to habitual diet values. Large changes in quantity and type of fatty acids consumed daily were not reflected in the fatty acid composition of the total serum lipids. Butter did not elevate total serum cholesterol or low density lipoprotein (LDL) cholesterol relative to habitual diet levels, but these values were significantly higher than sunflower oil-diet and palm oil-diet values. The sunflower oil diet produced the most dramatic changes: total serum cholesterol was reduced significantly relative to all diets except margarine, and apolipoprotein B values were the lowest of any diet. Unfortunately, the desirable high density lipoprotein (HDL) cholesterol and apolipoprotein A1 were also reduced on the sunflower oil diet. Diets containing either crude or refined palm oil did not elevate total serum cholesterol relative to habitual diet values or LDL cholesterol or apolipoprotein levels relative to any diet. Unexpectedly, the refined palm oil diet HDL-cholesterol and apolipoprotein A1 levels were the highest of all diets and significantly higher than sunflower oil diet values. The hard margarine diet, containing 26% trans fatty acids, reduced apolipoprotein B values relative to habitual diet levels, but HDL cholesterol was reduced significantly relative to the refined palm oil diet values. Comparison of the diets fatty acid compositions suggests the decrease in the HDL cholesterol on the hard margarine diet is attributable to the trans fatty acids. The data indicate that the trans fatty acids, produced during the partial hydrogenation of fats and oils, are not neutral and adversely affect serum lipid profile.

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-δ-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.

Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partrum. Vitamin K1 was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.

Gene and microRNA expression in p53-deficient day 8.5 mouse embryos.

BACKGROUND Neural tube defects (NTDs) are one of the most common human birth defects, with a prevalence of approximately 1 in 1000 live births in the United States. In animal studies, deletion of p53 leads to a significant increase in embryos that exhibit exencephaly. Whereas several studies have closely investigated the morphologic changes of p53-deficient embryos, no study has reported the molecular-level alteration in p53-deficient embryos. Here we attempt to identify genes and microRNAs (miRNAs) modified by deletion of p53 in day 8.5 mouse embryos. METHODS Mouse embryos from p53 heterozygous crosses were collected, genotyped, and embryos of similar genotype (+/+; +/-; -/-) were pooled. RNA from the pooled samples was isolated to determine mRNA and miRNA expression levels using Whole Genome Bioarrays and Low Density Arrays, respectively. RESULTS In p53 -/- embryos, 388 genes showed statistically significant alteration in gene expression of more than twofold compared to p53 +/+ embryos. Expression of p53 and well known p53 target genes, such as p21 and cyclin G1, were significantly down-regulated in p53 -/- embryos. In contrast, expression of other p53 target genes, such as Mdm2, Noxa, and Puma, were unchanged. We also identified six genes (Csk, Itga3, Jarid2, Prkaca, Rarg, and Sall4), known to cause NTDs when deleted, that are also down-regulated in p53 -/- embryos. Finally, five miRNAs (mir-1, mir-30e-3p, mir-142-3p, mir-301, and mir-331) also showed statistically significant alterations in expression levels in p53 -/- embryos compared to p53 +/+ embryos. Combined analysis of the experimental data using stepwise regression model and two publicly available algorithms identified putative target genes of these miRNAs. CONCLUSIONS Our data have identified genes and miRNAs that may be involved in the mechanisms underlining NTDs and begin to define the developmental role of p53 in the etiology of NTDs.

The roles of p53 and p21 in normal development and hyperthermia‐induced malformations

BACKGROUND Hyperthermia (HS) is a well-studied teratogen that induces serious malformations, including neural tube defects. Our previous studies have shown that HS induces apoptosis by activating the mitochondrial apoptotic pathway. Prior to activation of the mitochondrial apoptotic pathway, HS also activates p53 and its target genes. In the present study, we determine whether p53 and/or p21 play a role as teratogen suppressors or inducers of HS-induced malformations. METHODS Pregnant mice carrying all three p53 or p21 genotype embryos were exposed to HS on day 8.5. Subsequently, fetuses were collected on day 15.5, and genotyped. In addition to genotype, we also determined the number of resorptions and dead fetuses as well as the number and types of external malformations. RESULTS In the absence of HS exposure, fetuses exhibiting exencephaly and spina bifida were observed in approximately 11% of p53 -/- fetuses, whereas no malformations were observed among p21 -/- fetuses. Exposure to HS resulted in an increase in exencephaly and polydactyly in fetuses of all three p53 genotypes. However, the incidence of these malformations was statistically significantly higher in p53 -/- compared to p53 +/- and p53 +/+ fetuses. Exencephaly was the only malformation observed in p21 fetuses exposed to HS, with an approximately 2-fold increase among p21 +/- and a 3-fold increase among p21 -/- compared to p21 +/+ fetuses. CONCLUSIONS Our study confirms that p53 plays a role in normal development and has shown, for the first time that p53 and p21 function to suppress HS-induced malformations.

Absorption and metabolism of vitamin K in Swiss 3T3 mouse fibroblasts--a model system for study of vitamin K absorption and metabolism.

The vitamin K cycle previously described in liver has been demonstrated in Swiss 3T3 mouse fibroblasts. Vitamin K epoxide and gamma-carboxyglutamic acid were isolated from the cells and chemically characterized. Menaquinone (MK4) is also metabolized to its epoxide and vitamin K epoxide is reduced to vitamin K in these cells. Thus Swiss 3T3 mouse fibroblasts provide a useful model system for the study of vitamin K metabolism. Possible functions of the vitamin K-dependent protein(s) in fibroblasts are discussed.