Biosynthesis of delta-7-cholesten-3-beta-ol, delta-5,7-cholestadien-3-beta-ol, and delta-5-cholesten-3-beta-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Single nucleotide polymorphisms in the FADS gene cluster are associated with delta-5 and delta-6 desaturase activities estimated by serum fatty acid ratios

Genetic variability in the FADS1-FADS2 gene cluster [encoding delta-5 (D5D) and delta-6 (D6D) desaturases] has been associated with plasma long-chain PUFA (LCPUFA) and lipid levels in adults. To better understand these relationships, we further characterized the association between FADS1-FADS2 genetic variability and D5D and D6D activities in adolescents. Thirteen single nucleotide polymorphisms (SNPs) were genotyped in 1,144 European adolescents (mean ± SD age: 14.7 ± 1.4 y). Serum phospholipid fatty acid levels were analyzed using gas chromatography. D5D and D6D activities were estimated from the C20:4n-6/C20:3n-6 and C20:3n-6/C18:2n-6 ratios, respectively. Minor alleles of nine SNPs were associated with higher 18:2n-6 levels (1.9E-18 ≤ P ≤ 6.1E-5), lower C20:4n-6 levels (7.1E-69 ≤ P ≤ 1.2E-12), and lower D5D activity (7.2E-44 ≤ P ≤ 4.4E-5). All haplotypes carrying the rs174546 minor allele were associated with lower D5D activity, suggesting that this SNP is in linkage disequilibrium with a functional SNP within FADS1. In contrast, only the rs968567 minor allele was associated with higher D6D activity (P = 1.5E-6). This finding agrees with an earlier in vitro study showing that the minor allele of rs968567 is associated with a higher FADS2 promoter activity. These results suggest that rare alleles of several SNPs in the FADS gene cluster are associated with higher D6D activity and lower D5D activity in European adolescents.     

Bioinformatics study of delta-12 fatty acid desaturase 2 (FAD2) gene in oilseeds

Fatty acid desaturases constitute a group of enzymes that introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids. In plants, seed-specific delta-12 fatty acid desaturase 2 (FAD2) is responsible for the high content of linoleic acid by inserting a double bond at the delta-12 (omega-6) position of oleic acid. In this study, sixteen FAD2 and FAD2-2 protein sequences from oilseeds were analyzed by computational tools including two databases of the NCBI and EXPASY and data management tools such as SignalP, TMHMM, Psort, ProtParam, TargetP, PLACE and PlantCARE. These services were used to predict the protein properties such as molecular mass, pI, signal peptide, transmembrane and conserved domains, secondary and spatial structures. The polypeptide sequences were aligned and a neighbour-joining tree was constructed using MEGA5.1 to elucidate phylogenetic relationships among FAD2 genes. Based on the phylogenetic analysis species with high similarity in FAD2 sequence grouped together. FAD2 proteins include highly conserved histidine-rich motifs (HECGHH, HRRHH and HV[A/C/T]HH) that are located by three to five transmembrane anchors. For further investigations Sesamum indicum FAD2 was selected and analyzed by bioinformatics tools. Analysis showed no N-terminal signal peptide for probable localization of FAD2 protein in cytoplasmic organelles such as chloroplast, mitochondria and Golgi. Instead the C-terminal signaling motif YNNKL, Y(K/N)NKF or YRNKI allows FAD2 protein to selectively bind to and embed in the endoplasmic reticulum. FAD2 promoter contains different cis-regulatory elements involve in the biotic and abiotic stresses response or control of gene expression specifically in seeds.     

Activation of delta- and kappa-opioid receptors by opioid peptides protects cardiomyocytes via KATP channels

To examine the receptor specificity and the mechanism of opioid peptide-induced protection, we examined freshly isolated adult rabbit cardiomyocytes subjected to simulated ischemia. Cell death as a function of time was assessed by trypan blue permeability. Dynorphin B (DynB) and Met5-enkephalin (ME) limitation of cell death (expressed as area under the curve) was sensitive to blockade by naltrindole (NTI, a delta-selective antagonist) and 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6,7-2',3'-indolomorphinan (GNTI dihydrochloride, a kappa-selective antagonist): 85.7 +/- 2.7 and 142.9 +/- 2.7 with DynB and DynB + NTI, respectively (P < 0.001), 94.1 +/- 4.2 and 164.5 +/- 7.3 with DynB and DynB + GNTI, respectively (P < 0.001), 111.9 +/- 7.0 and 192.1 +/- 6.4 with ME and ME + NTI, respectively (P < 0.001), and 120.2 +/- 4.3 and 170.0 +/- 3.3 with ME and ME + GNTI, respectively (P < 0.001). Blockade of ATP-sensitive K+ channels eliminated DynB- and ME-induced protection: 189.6 +/- 5.4 and 139.0 +/- 5.4 for control and ME, respectively (P < 0.001), and 210 +/- 5.9 and 195 +/- 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); 136.0 +/- 5.7 and 63.4 +/- 5.4 for control and ME, respectively (P < 0.001), and 144.6 +/- 4.5 and 114.6 +/- 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01); 189.6 +/- 5.4 and 139.0 +/- 5.4 for control and ME, respectively (P < 0.001), and 210 +/- 5.9 and 195 +/- 6.1 for 5-HD and ME + 5-HD, respectively (P < 0.001); and 136.0 +/- 5.7 and 63.4 +/- 5.4 for control and ME, respectively (P < 0.001), and 144.6 +/- 4.5 and 114.6 +/- 7.7 for HMR-1098 and ME + HMR-1098, respectively (P < 0.01). We conclude that opioid peptide-induced cardioprotection is mediated by delta- and kappa-receptors and involves sarcolemmal and mitochondrial ATP-sensitive K+ channels.     

The inactivation of ornithine transcarbamoylase by N delta-(N''-sulpho-diaminophosphinyl)-L-ornithine.

Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N delta-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N delta-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 10(6) M-1 X s-1 at 0 degree C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3 microM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine in vivo appears to be an important function in the physiology of the disease.