Genetic polymorphisms in carnitine palmitoyltransferase 1A gene are associated with variation in body composition and fasting lipid traits in Yup'ik Eskimos

Variants of carnitine palmitoyltransferase 1A (CPT1A), a key hepatic lipid oxidation enzyme, may influence how fatty acid oxidation contributes to obesity and metabolic outcomes. CPT1A is regulated by diet, suggesting interactions between gene variants and diet may influence outcomes. The objective of this study was to test the association of CPT1A variants with body composition and lipids, mediated by consumption of polyunsaturated fatty acids (PUFA). Obesity phenotypes and fasting lipids were measured in a cross-sectional sample of Yup'ik Eskimo individuals (n = 1141) from the Center of Alaska Native Health Research (CANHR) study. Twenty-eight tagging CPT1A SNPs were evaluated with outcomes of interest in regression models accounting for family structure. Several CPT1A polymorphisms were associated with HDL-cholesterol and obesity phenotypes. The P479L (rs80356779) variant was associated with all obesity-related traits and fasting HDL-cholesterol. Interestingly, the association of P479L with HDL-cholesterol was still significant after correcting for body mass index (BMI), percentage body fat (PBF), or waist circumference (WC). Our findings are consistent with the hypothesis that the L479 allele of the CPT1A P479L variant confers a selective advantage that is both cardioprotective (through increased HDL-cholesterol) and associated with reduced adiposity.     

Haplotype homozygosity and derived alleles in the human genome

Haplotype-based techniques are being used to estimate the relative age of alleles--particularly in screening loci for signals of recent positive selection--but does this approach capture even coarse age differences? Using simulations and empirical data from the International HapMap Project, we show that a simple pairwise metric of haplotype homozygosity gives significantly higher mean values for human single-nucleotide-polymorphism alleles that appear to be derived than for those that appear to be ancestral, as determined by comparison with the chimpanzee genome. Our results support the use of haplotype-based techniques, such as extended haplotypic homozygosity, to assess the age of alleles.     

Clinically failed eggs as a source of normal human embryo stem cells

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.     

Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines

The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.     

Structural and Kinetic Characterization of 4-Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolase,a Protocatechuate Degradation Enzyme Evolutionarily Convergent with the HpaI and DmpG Pyruvate Aldolases

4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate α-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k_cat/K_m and k_cat for the enzyme is consistent with a single deprotonation with pK_a values of 8.0 ± 0.1 and 7.0 ± 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered α-β-β-α sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199′. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.     

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) m. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.