Individual quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human milk

Extraction, lipid-reduction, and chromatographic methods suitable for the resolution and subsequent quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25-hydroxy-vitamin D3 from human milk are described. This procedure utilizes a methanol:methylene chloride extraction, precipitation of unwanted lipids with cold methanol and ether, backwash with alkaline buffer, silica Sep-Pak preparative chromatography, normal- and reverse-phase high-performance liquid chromatography with final quantitation of the antirachitic sterols by competitive protein binding assay. The described assay was used to determine these antirachitic sterols in milk from women receiving various supplements of vitamin D or undergoing ultraviolet phototherapy.     

Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.     

Isolation and identification of 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2: new metabolites of 1,25-dihydroxyvitamin D2 produced in rat kidney

Three new metabolites of vitamin D2 were produced in vitro by perfusing isolated rat kidneys with 1,25-dihydroxyvitamin D2. They were isolated and purified from the kidney perfusate by the techniques of methanol-methylene chloride lipid extraction and high-performance liquid chromatography. By means of ultraviolet absorption spectrophotometry, mass spectrometry, and specific chemical reactions, the metabolites were identified as 1,24,25-trihydroxyvitamin D2, 1,24,25,28-tetrahydroxyvitamin D2, and 1,24,25,26-tetrahydroxyvitamin D2. Both 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2 were also produced when a kidney was perfused with 1,24,25-trihydroxyvitamin D2. Thus, it becomes clear that 1,25-dihydroxyvitamin D2 is first hydroxylated at C-24 to form 1,24,25-trihydroxyvitamin D2, which is then further hydroxylated at C-28 and C-26 to form 1,24,25,28-tetrahydroxyvitamin D2 and 1,24,25,26-tetrahydroxyvitamin D2, respectively. From several recent studies, it has been well established that 1,25-dihydroxyvitamin D3 is converted into various further metabolites in the kidney as a result of chemical reactions such as C-23, C-24, and C-26 hydroxylations, C-24 ketonization, and C-23:C-26 lactonization. From our study it is obvious that 1,25-dihydroxyvitamin D2 does not undergo all of the aforementioned chemical reactions except C-24 and C-26 hydroxylations. Also, our study indicates that C-28 hydroxylation plays a significant role in the further metabolism of 1,25-dihydroxyvitamin D2. Thus, for the first time, we describe a novel further metabolic pathway for 1,25-dihydroxyvitamin D2 in a mammalian kidney.     

Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

Polypeptides of the synaptic membrane antigens D1, D2, and D3

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.     

Molecular cloning and characterization of three novel cytochrome P450 2D isoforms, CYP2D20, CYP2D27, and CYP2D28 in the Syrian hamster (Mesocricetus auratus)

We cloned three novel cytochrome P450 (CYP) 2D cDNAs in the Syrian hamster (Mesocricetus auratus). Each clone contained an open reading frame of 1500 nucleotides encoding a protein of 500 amino acids. The deduced amino acid sequences of these had high identities with those of the other CYP2D members, therefore, the clones were assigned as CYP2D20, CYP2D27, and CYP2D28. Northern blot analysis showed that the CYP2D27 mRNA was expressed in liver, but not in kidney, small intestine, and brain, while the CYP2D20 and CYP2D28 mRNAs were not detected in these tissues examined. The expression of CYP2D27 mRNA in liver did not show sex difference and was not induced by either 3-methylcholanthrene or phenobarbital treatment. We characterized the enzyme activities of recombinant CYP2D27 expressed in COS-7 cells. The CYP2D27 protein had the bufuralol 1'-hydroxylase and debrisoquine 4-hydroxylase activities that are specific to the CYP2D subfamily.     

Dopamine D1, D2, D3 Receptors,Vesicular Monoamine Transporter Type-2 (VMAT2) and Dopamine Transporter (DAT) Densities in Aged Human Brain

The dopamine D₁, D₂, D₃ receptors, vesicular monoamine transporter type-2 (VMAT2), and dopamine transporter (DAT) densities were measured in 11 aged human brains (aged 77–107.8, mean: 91 years) by quantitative autoradiography. The density of D₁ receptors, VMAT2, and DAT was measured using [³H]{"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, [³H]dihydrotetrabenazine, and [³H]WIN35428, respectively. The density of D₂ and D₃ receptors was calculated using the D₃-preferring radioligand, [³H]WC-10 and the D₂-preferring radioligand [³H]raclopride using a mathematical model developed previously by our group. Dopamine D₁, D₂, and D₃ receptors are extensively distributed throughout striatum; the highest density of D₃ receptors occurred in the nucleus accumbens (NAc). The density of the DAT is 10–20-fold lower than that of VMAT2 in striatal regions. Dopamine D₃ receptor density exceeded D₂ receptor densities in extrastriatal regions, and thalamus contained a high level of D₃ receptors with negligible D₂ receptors. The density of dopamine D₁ linearly correlated with D₃ receptor density in the thalamus. The density of the DAT was negligible in the extrastriatal regions whereas the VMAT2 was expressed in moderate density. D₃ receptor and VMAT2 densities were in similar level between the aged human and aged rhesus brain samples, whereas aged human brain samples had lower range of densities of D₁ and D₂ receptors and DAT compared with the aged rhesus monkey brain. The differential density of D₃ and D₂ receptors in human brain will be useful in the interpretation of PET imaging studies in human subjects with existing radiotracers, and assist in the validation of newer PET radiotracers having a higher selectivity for dopamine D₂ or D₃ receptors.     

Electrophoretic mobility of N- and C-terminal monoferric fragments of bovine transferrin phenotypes AA,D1D1, D2D2, and EE,and N-terminal amino acid sequences

Iron-saturated bovine transferrins A, D1, D2, and E were cleaved by trypsin yielding monoferric fragments. The N-terminal fragments (F) of transferrins A and D2 had identical mobility in cellulose acetate electrophoresis, that of transferrin D1 a slower mobility, and that of E a still slower mobility. The C-terminal fragments (S) gave multiple bands which were essentially identical in the case of transferrins A, D1, and E, but of slower mobility in the case of transferrin D2. All four variants had identical N-terminal amino acid sequences. The electrophoretic mobility of the C-terminal fragments was reduced by neuraminidase treatment, but the N-terminal fragments were unaffected. The four transferrin variants therefore appear to be made up from three electrophoretically distinguishable N-terminal halves and two C-terminal halves. The feature responsible for the electrophoretic double banding of homozygous bovine asialotransferrins is consistently associated with the C-terminal half of the molecule.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Protein kinase D1 and D2 are involved in chemokine release induced by toll-like receptors 2, 4, and 5

The protein kinase D (PKD) family consists of three serine-threonine kinases involved in cellular proliferation, motility, and apoptosis. We previously reported that human toll-like receptor 5 (TLR5) contains a consensus PKD phosphorylation site. Flagellin stimulation of cells activated PKD1, and inhibition of PKD1 reduced flagellin-induced interleukin-8 (IL-8) production in epithelial cells. In the current work, we examined PKD1 and PKD2 involvement downstream of TLR5, TLR4 and TLR2. We found that inhibition of either kinase with shRNA reduced IL-8 and CCL20 release due to TLR4 and TLR2 agonists to a similar extent as previously reported for TLR5. PKD1 and PKD2 inhibition reduced NF-κB activity but not MAPK activation. These results demonstrate that both PKD1 and PKD2 are required for inflammatory responses following TLR2, TLR4, or TLR5 activation, although PKD1 is more strongly involved. These kinases likely act downstream of the TLRs themselves to facilitate NF-κB activation but not MAP kinase phosphorylation.     

Localization of the antigens D1, D2 and D3 in the rat brain synaptic membrane

The inside-outside localization of the nervous system specific membrane proteins D1, D2 and D3 was investigated by a immunoabsorption technique. It was found that D1 was located at least partly on the outside of the synaptic membrane in contrast to D3 which was inside on the membrane, facing the cytoplasm. The protein D2 was outside on the synaptic membrane, and it was found very accessible to the antiserum. It is speculated that D2 might be involved in the axonal-dendritic recognition process during synaptogenesis.     

Relationships among the O-Antigen Gene Clusters of Salmonella enterica Groups B, D1, D2, and D3

The O antigen is an important cell wall antigen of gram-negative bacteria, and the genes responsible for its biosynthesis are located in a gene cluster. We have cloned and sequenced the DNA segment unique to the O-antigen gene cluster of Salmonella enterica group D3. This segment includes a novel O-antigen polymerase gene (wzy_D3). The polymerase gives α(1→6) linkages but has no detectable sequence similarity to that of group D2, which confers the same linkage. We find the remnant of a D3-like wzy gene in the O-antigen gene clusters of groups D1 and B and suggest that this is the original wzy gene of these O-antigen gene clusters.     

PROTMAP2D: visualization, comparison and analysis of 2D maps of protein structure

MOTIVATION: Protein structure comparison is a fundamental problem in structural biology and bioinformatics. Two-dimensional maps of distances between residues in the structure contain sufficient information to restore the 3D representation, while maps of contacts reveal characteristic patterns of interactions between secondary and super-secondary structures and are very attractive for visual analysis. The overlap of 2D maps of two structures can be easily calculated, providing a sensitive measure of protein structure similarity. PROTMAP2D is a software tool for calculation of contact and distance maps based on user-defined criteria, quantitative comparison of pairs or series of contact maps (e.g. alternative models of the same protein, model versus native structure, different trajectories from molecular dynamics simulations, etc.) and visualization of the results. AVAILABILITY: PROTMAP2D for Windows / Linux / MacOSX is freely available for academic users from http://genesilico.pl/protmap2d.htm     

Treatment with 50000 IU Vitamin D2 Every Other Week and Effect on Serum 25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and Total 25-Hydroxyvitamin D in Aclinical Setting

ObjectiveTo examine the effect of 50 000 IU-vitamin D₂ supplementation in a clinical setting on serum total 25-hydroxyvitamin D (25[OH]D), 25-hydroxyvitamin D₂ (25[OH]D₂), and 25-hydroxyvitamin D₃ (25[OH]D₃).MethodsThis retrospective cohort study was performed in an urban tertiary referral hospital in Boston, Massachusetts. Patients who had been prescribed 50 000 IU vitamin D₂ repletion and maintenance programs were identified through a search of our electronic medical record. Baseline and follow-up total serum 25(OH)D, 25(OH)D₂, and 25(OH)D₃ levels were compared.ResultsWe examined the medical records of 48 patients who had been prescribed 50 000 IU vitamin D₂ in our clinic. Mean ± standard deviation baseline total 25(OH) D was 31.0 ± 10.6 ng/mL and rose to 48.3 ± 13.4 ng/mL after treatment (P <.001). 25(OH)D₂ increased from 4.2 ± 4.3 ng/mL to 34.6 ± 12.3 ng/mL after treatment (P <.001), for an average of 158 days (range, 35-735 days). Serum 25(OH)D3 decreased from 26.8 ± 10.8 ng/mL to 13.7 ± 7.9 ng/mL (P <.001).ConclusionsFifty thousand IU vitamin D₂ repletion and maintenance therapy substantially increases total 25(OH)D and 25(OH)D2 despite a decrease in serum 25(OH)D3. This treatment program is an appropriate and effective strategy to treat and prevent vitamin D deficiency.(Endocr Pract. 2012;18:399-402)     

Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer’s disease, patients with Parkinson’s disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296C > T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of “mutation patterns” for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4 ± 0.9 kYBP. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gabriela Alexe and Noriyuki Fuku are joint first authors.     

Functional characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D): RNA binding affinity and selectivity

The protein tristetraprolin (TTP, also known as NUP475 and TIS11) is a nonclassical zinc finger protein that is involved in regulating the inflammatory response. Specifically, TTP binds to AU-rich sequence elements located at the 3'-untranslated region of cytokine mRNAs forming a complex that is degraded by the exosome. The nucleic acid binding region of TTP is comprised of two CysX(8)CysX(5)CysX(3)His domains that are activated in the presence of zinc. A two-domain construct of TTP (TTP-2D) has been cloned and overexpressed in E. coli. TTP-2D picks up visible red coloration from the expression media, unless it is expressed under iron-restricted conditions. The iron-binding properties of TTP-2D and the effect of iron substitution on RNA recognition have been investigated. Both Fe(II) and Fe(III) bind to TTP-2D and a full titration of Fe(III) with TTP-2D revealed that this metal ion binds with micromolar affinity. Upon reconstitution of TTP-2D with either Fe(II) or Fe(III), the protein recognizes a canonical RNA-binding sequence, UUUAUUUAUUU, with nanomolar affinity. Substitution of a single adenine or both adenines results in a decreased affinity of TTP-2D for the RNA molecule, demonstrating that both Fe(II)-TTP-2D and Fe(III)-TTP-2D selectively recognize a physiologically relevant RNA sequence. The relative affinities of Fe(II)-TTP-2D and Fe(III)-TTP-2D for the series of RNA sequences mirror those observed for Zn(II)-TTP-2D and suggest that iron is a viable substitute for zinc in this protein.