Content and composition of hopanoids in Zymomonas mobilis under various growth conditions.

By using a new method for quantification of the different hopanoid derivatives, a total hopanoid content of about 30 mg/g (dry cell weight) was observed in Zymomonas mobilis. This value is the highest reported for bacteria so far. The major hopanoids in Z. mobilis were the ether and glycosidic derivatives of tetrahydroxy-bacteriohopane, constituting about 41 and 49% of the total hopanoids. Tetrahydroxybacteriohopane itself, diplopterol, and hopene made up about 6, 3, and 1%, respectively. Only minor changes in hopanoid composition were observed with changes in growth conditions. Earlier reports on a correlation between hopanoid content and ethanol concentration in the medium could not be confirmed. Over a wide range of ethanol concentrations (5 to 60 g/liter), growth rates (0.08 to 0.25 h-1), and temperatures (25 to 37 degrees C), the molar ratio of hopanoids to phospholipids in the cells amounted to about 0.7. Only at growth rates of greater than 0.30 h-1 did the molar ratio increase to about 1.     

Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid‐producing bacteria

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C₃₅ extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild‐type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid‐producing bacteria.     

Hopanoids,like sterols,modulate dynamics,compaction, phase segregation and permeability of membranes

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.     

Oxidation of Fe(II) leads to increased C‐2 methylation of pentacyclic triterpenoids in the anoxygenic phototrophic bacterium Rhodopseudomonas palustris strain TIE‐1

Hopanoids are among the most widespread biomarkers of bacteria that are used as indicators for past and present bacterial activity. Our understanding of the production, function, and distribution of hopanoids in bacteria has improved greatly, partly due to genetic, culture‐independent studies. Culture‐based studies are important to determine hopanoid function and the environmental conditions under which these compounds are produced. This study compares the lipid inventory of Rhodopseudomonas palustris strain TIE‐1 under anoxic photoautotrophic conditions using either H₂ or Fe(II) as electron donor. The high amount to which adenosylhopane is produced irrespective of the used electron donor suggests a specific function of this compound rather than its exclusive role as an intermediate in bacteriohopanepolyol biosynthesis. C‐2 methylated hopanoids and tetrahymanol account for as much as 59% of the respective C‐2 methylated/non‐methylated homologs during growth with Fe(II) as electron donor, as compared with 24% C‐2 methylation for growth with H₂. This observation reveals that C‐2 methylated hopanoids have a specific function and are preferentially synthesized in response to elevated Fe(II) concentrations. The presence of C‐2 methylated pentacyclic triterpenoids has commonly been used as a biosignature for the interpretation of paleoenvironments. These new findings suggest that increased C‐2 methylation may indicate anoxic ferrous conditions, in addition to other environmental stressors that have been previously reported.     

Diversity of hopanoids and squalene-hopene cyclases across a tropical land-sea gradient

Bacterial hopanoids are ubiquitous in Earth surface environments. They hold promise as environmental and ecological biomarkers, if the phylogeny and physiological drivers of hopanoid biosynthesis can be linked with the distribution of hopanoids observed across a breadth of samples. Here we survey the diversity of hopanoid cyclases from a land‐sea gradient across the island of San Salvador, in the easternmost part of the Bahamas. The distribution of lipids was determined for the same sites, for the first time overlaying quantification of bacteriohopanepolyols with sqhC phylogeny. The results are similar to previous reports: environmental sqhCs average < 65% translated amino acid identity to their closest named relatives, and sequences from putative Proteobacteria dominate. Additionally, a new and apparently ubiquitous group of marine hopanoid producers is identified; it has no identifiable close relatives. The greatest diversity of hopanoid lipids occurs in soil, but hopanoids represent a minor fraction of total soil‐derived lipids. Marine samples contain fewer identifiable hopanoids, but they are more abundant as a fraction of the total extractable lipids. In soil, the dominant compounds are 35‐aminobacteriohopane‐32,33,34‐triol and adenosylhopane. In an upper estuarine sample, bacteriohopanetetrol and 32,35‐anhydrobacteriohopanetetrol dominate; while in lower estuarine and open marine samples, the most abundant are bacteriohopanetetrol and bacteriohopaneribonolactone. Cyclitol ethers are trace components in the soil, absent in the estuary, and of moderate abundance in the open marine setting, suggesting a dominant marine source. Conversely, aminotriol and aminotetrol decrease in abundance or disappear completely from land to ocean, while 2‐methyldiplopterol shows the opposite trend. Small quantities of 2‐methylbacteriohopanepolyols are detectable in all samples. The overall hopanoid distributions may correlate to the major phylogenetic families of hopanoid producers or to the environments in which they are found.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Hopanoids play a role in stress tolerance and nutrient storage in the cyanobacterium Nostoc punctiforme

Hopanes are abundant in ancient sedimentary rocks at discrete intervals in Earth history, yet interpreting their significance in the geologic record is complicated by our incomplete knowledge of what their progenitors, hopanoids, do in modern cells. To date, few studies have addressed the breadth of diversity of physiological functions of these lipids and whether those functions are conserved across the hopanoid‐producing bacterial phyla. Here, we generated mutants in the filamentous cyanobacterium, Nostoc punctiforme, that are unable to make all hopanoids (shc) or 2‐methylhopanoids (hpnP). While the absence of hopanoids impedes growth of vegetative cells at high temperature, the shc mutant grows faster at low temperature. This finding is consistent with hopanoids acting as membrane rigidifiers, a function shared by other hopanoid‐producing phyla. Apart from impacting fitness under temperature stress, hopanoids are dispensable for vegetative cells under other stress conditions. However, hopanoids are required for stress tolerance in akinetes, a resting survival cell type. While 2‐methylated hopanoids do not appear to contribute to any stress phenotype, total hopanoids and to a lesser extent 2‐methylhopanoids were found to promote the formation of cyanophycin granules in akinetes. Finally, although hopanoids support symbiotic interactions between Alphaproteobacteria and plants, they do not appear to facilitate symbiosis between N. punctiforme and the hornwort Anthoceros punctatus. Collectively, these findings support interpreting hopanes as general environmental stress biomarkers. If hopanoid‐mediated enhancement of nitrogen‐rich storage products turns out to be a conserved phenomenon in other organisms, a better understanding of this relationship may help us parse the enrichment of 2‐methylhopanes in the rock record during episodes of disrupted nutrient cycling.     

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D₀) acrylamide and deuterated (D₃) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8 M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10^-5 less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.     

Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell‐signaling pathways. Despite this, high‐throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO₂)‐based 3‐D LC (strong cationic exchange/TiO₂/C18)‐MS³‐linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low‐abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho‐biomarkers including plectin‐1(phopho‐Ser‐4253) and alpha‐HS‐glycoprotein (phospho‐Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO₂‐based online‐3‐D LC‐MS³‐linear ion trap system with high‐throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.     

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization

Ammuxetine (AMT), a novel chiral antidepressant candidate compound, exhibits better antidepression effects than duloxetine in different animal models. In this article, a chiral derivatization method, combined with online solid phase extraction (online SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), was developed for the chiral separation of AMT enantiomers after administration of racemic AMT to dogs. The derivatization reaction employed 2,3,4,6‐tetra‐O‐acetyl‐b‐glucopyr‐anosyl isothiocyanate (GITC) as a precolumn chiral derivatization reagent. A SPE column Retain PEP Javelin (10 × 2.1 mm) was used to remove proteins and other impurities in plasma samples. The enantiomeric derivatives were separated on a ZORBAX SB‐C₁₈ column (50 × 2.1 mm × 3.5 μm) with an isocratic elution procedure. The selected multiple reaction monitoring mode of the positive ion was performed and the parent to the product transitions m/z 681.0/543.1 and m/z 687.4/543.1 were used to measure the derivatives of AMT and duloxetine (internal standard) with electrospray ionization. The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The method was applied to a pharmacokinetics study of AMT racemate in dogs. The results suggested that the pharmacokinetic of AMT enantiomers might be stereoselective in dogs.     

Localization and distribution of hopanoids in membrane systems of the cyanobacterium Synechocystis PCC 6714

Intracellular localization of triterpenic membrane stabilizers of the hopane series is described for the first time for a cyanobacterium. In Synechocystis PCC 6714, a bacteriohopanetetrol derivative (main compound) and diplopterol were detected in cell wall (CW) and thylakoid membrane (TM). Both hopanoids were enriched 4.5-fold and 9.0-fold in CW and outer membrane (OM) fractions, respectively, compared to TMs.     

Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.     

Draft Genome Sequence of Rhodomicrobium udaipurense JA643T with Special Reference to Hopanoid Biosynthesis

Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643^T isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100^T and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group containing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643^T will be useful in understanding the functional features of hopanoids in this bacterium.     

Synthesis,Chemical Characterization,DNA Binding,Antioxidant, Antibacterial,and Antifungal Activities of Ferrocence Incorporated Selenoureas

Ferrocene‐incorporated selenoureas 1‐(4‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P4Me), 1‐(3‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P3Me), and 1‐(2‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P2Me) were synthesized and characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, atomic absorption spectroscopy, CHNS, and single‐crystal X‐ray diffraction. DNA interaction of the compounds was investigated with cyclic voltammetry, UV–visible spectroscopy, and viscometry, which is a prerequisite for anticancer agents. Drug‐DNA binding constant was found to vary in the sequence: K_P4Me (4.9000 × 10⁴ M^?1) > K_P2Me (2.318 × 10⁴ M^?1) > K_P3Me (1.296 × 10⁴ M^?1). Antioxidant (1,1‐diphenyl‐2‐picrylhydrazyl), antifungal (against Faussarium solani and Helmentosporium sativum), and antibacterial (against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) activities have also been reported in addition.     

Determination of the residues of 50 anabolic hormones in muscle,milk and liver by very-high-pressure liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific method for determination of the residues of 50 anabolic hormones in muscle (pork, beef, shrimp), milk and pig liver was developed. Analytes were separated and acquired by liquid chromatography coupled with an electrospray ionization tandem mass spectrometer (LC–ESI–MS/MS). Target compounds were simultaneously extracted with methanol after enzyme hydrolysis, and purified using a graphitized carbon-black solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. Limits of quantification were 0.04–2.0 μg kg^?1; average recoveries were 76.9–121.3%; and the relative standard deviation was 2.4–21.2%. This method has been successfully applied in real samples.     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Lipid remodeling in Rhodopseudomonas palustris TIE‐1 upon loss of hopanoids and hopanoid methylation

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade‐off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α‐proteobacterium Rhodopseudomonas palustris TIE‐1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several‐fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro‐organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.     

Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor,modulating mammary epithelial cell growth

The vitamin D₃ receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D₃ synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D₃‐induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α‐hydroxylase, 1α‐OHase), the enzyme that generates the biologically active VDR ligand, 1α,25‐dihydroxyvitamin D₃ (1,25D₃), and CYP24 (24‐hydroxylase, 24‐OHase), a VDR‐1,25D₃ induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25‐hydroxyvitamin D₃ (25D₃) to the active ligand, 1,25D₃, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co‐cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D₃ or 1,25D₃ compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D₃ to 1,25D₃, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D₃‐induced growth regulation of ductal epithelium. J. Cell. Biochem. 112: 3393–3405, 2011. © 2011 Wiley Periodicals, Inc.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D₂ receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [³H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [³H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [³H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [³H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D₂ receptor and should prove useful in studies on functional reconstitution of the receptor.