Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.     

Chemical modification of chalcone isomerase by mercurials and tetrathionate. Evidence for a single cysteine residue in the active site

Chalcone isomerase from soybean is inactivated by stoichiometric amounts of p-mercuribenzoate or HgCl2. Spectral titration of the enzyme with p-mercuribenzoate indicates that a single thiol group is modified. Treatment of modified enzyme with KCN or thiols results in a complete restoration of enzyme activity demonstrating that the inactivation is not due to irreversible protein denaturation. A product of the enzymatic reaction, naringenin, provides complete kinetic protection against inactivation by both mercurials. The binding constant (33 microM) for naringenin determined from the concentration dependence of the protection agrees with the inhibition constant (34 microM) for naringenin as a competitive inhibitor of the catalytic reaction. This agreement demonstrates that the observed kinetic protection results from the specific binding of naringenin to the active site. Incubation of native chalcone isomerase with sodium tetrathionate (0.1 M) results in a slow time-dependent loss of enzymatic activity. The inactivation of chalcone isomerase by tetrathionate and N-ethylmaleimide becomes very rapid in the presence of 6 M urea, indicating that the native tertiary structure is responsible for the low reactivity of the enzymatic thiol. The stoichiometric modification of reduced and denatured chalcone isomerase by [3H] N-ethylmaleimide indicates that the enzyme contains only a single cysteine residue and does not contain any disulfides. The evidence presented suggests that the only half-cystine residue in chalcone isomerase is located in the active site and thereby provides the first clue to the location of the active site in chalcone isomerase.     

Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions.

The soluble P450 isolated from Bacillus megaterium (the product of the CYP 102 gene) (P450BM-3) is a catalytically self-sufficient fatty acid hydroxylase which converts lauric, myristic, and palmitic acids to omega-1, omega-2, and omega-3 hydroxy analogs. The percentage distribution of the regioisomers depends on the substrate chain length. Lauric and myristic acids were preferentially metabolized to their omega-1 hydroxy counterparts while no hydroxylation occurred when capric acid was used as the substrate. Palmitic acid, when present at concentrations greater than the concentration of oxygen in the reaction medium (greater than 250 microM), was hydroxylated to its omega-1, omega-2, and omega-3 hydroxy analogs, with the percentage distribution of the regioisomers being 21:44:35, respectively. No omega hydroxylation of any of the fatty acids was detected. When the concentration of palmitic acid was less than the concentration of oxygen in the reaction mixture, it was noted that a number of additional products were formed. Under these conditions, unlike lauric and myristic acids, it was observed that palmitic acid was first converted to its monohydroxy isomers which were subsequently metabolized to a mixture of 14-ketohexadecanoic, 15-ketohexadecanoic, 13-hydroxy-14-ketohexadecanoic, 14-hydroxy-15-ketohexadecanoic, and 13,14-dihydroxyhexadecanoic acids with a relative distribution of 8:2:40:30:20, respectively. Thus, P450BM-3 is able not only to monohydroxylate a variety of fatty acids but also to further metabolize some of these primary metabolites to secondary and tertiary products. The present paper characterizes the products formed during the sequential hydroxylation of palmitic acid and proposes reaction pathways to explain these results.     

Arachidonic acid epoxidation: epoxyeicosatrienoic acids are endogenous constituents of rat liver

Epoxyeicosatrienoic acids have been isolated and purified from the livers of male rats. They were identified by gas chromatography-mass spectrometric techniques. These results expand the list of in vivo-produced eicosanoids. Their documented in vitro biological activities suggest a role for them in cell and tissue homeostasis.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Prediction of stenosis behaviour in artery by neural network and multiple linear regressions

Biomechanics and Modeling in Mechanobiology - Blood flow analysis in the artery is a paramount study in the field of arterial stenosis evaluation. Studies conducted so far have reported the...     

Bacopasaponins with cytotoxic activity against human breast cancer cells in vitro

Molecular Biology Reports - Globally, breast cancer is a serious concern that exhibits a persistent rise in its incidence and related mortality even after significant advancement in the field of...     

Genetic variants associated with breast cancer risk for Ashkenazi Jewish women with strong family histories but no identifiable BRCA1/2 mutation

The ability to establish genetic risk models is critical for early identification and optimal treatment of breast cancer. For such a model to gain clinical utility, more variants must be identified beyond those discovered in previous genome-wide association studies (GWAS). This is especially true for women at high risk because of family history, but without BRCA1/2 mutations. This study incorporates three datasets in a GWAS analysis of women with Ashkenazi Jewish (AJ) homogeneous ancestry. Two independent discovery cohorts comprised 239 and 238 AJ women with invasive breast cancer or preinvasive ductal carcinoma in situ and strong family histories of breast cancer, but lacking the three BRCA1/2 founder mutations, along with 294 and 230 AJ controls, respectively. An independent, third cohort of 203 AJ cases with familial breast cancer history and 263 healthy controls of AJ women was used for validation. A total of 19 SNPs were identified as associated with familial breast cancer risk in AJ women. Among these SNPs, 13 were identified from a panel of 109 discovery SNPs, including an FGFR2 haplotype. In addition, six previously identified breast cancer GWAS SNPs were confirmed in this population. Seven of the 19 markers were significant in a multivariate predictive model of familial breast cancer in AJ women, three novel SNPs [rs17663555(5q13.2), rs566164(6q21), and rs11075884(16q22.2)], the FGFR2 haplotype, and three previously published SNPs [rs13387042(2q35), rs2046210(ESR1), and rs3112612(TOX3)], yielding moderate predictive power with an area under the curve (AUC) of the ROC (receiver-operator characteristic curve) of 0.74. Population-specific genetic variants in addition to variants shared with populations of European ancestry may improve breast cancer risk prediction among AJ women from high-risk families without founder BRCA1/2 mutations.