Oxygenation of Hexadecane in the Biosynthesis of Cyclic Glycolipids in Torulopsis Apicola

Biotransformation of [1-¹³C] labelled hexadecane, hexadecanol and hexadecanoic acid have been investigated using the yeast Torulopsis apicola. The yeast produces a microcrystalline mixture of two glycolipids, the lipophilic moiety of which consists of ω- or (ω-l)-hydroxylated hexadecanoic acid. Biosynthesis of these glycolipids takes place via hydroxylation of hexadecane, oxidation to hexadecanoic acid and ω or (ω-l)-hydroxylation of hexadecanoic acid. Feeding the cell cultures with a mixture of hexadecane and [1-¹³C] labelled hexadecane derivatives one observes ¹³C enrichment ratios which indicate that neither of the biohydroxylation or oxidation steps are rate limiting in the formation of the glycolipids, furthermore, two different monooxygenase systems appear to be involved in hydroxylation of hexadecane and hexadecanoic acid.     

Interactions between thiamine and large anions. Crystal structures of (H-thiamine)[Pt(SCN)4] · 3H2O and (H-thiamine)[Pt(SCN)6] · H2O

The reaction of thiamine with K₂Pt^IICl₄ and with Pt^IVCl₄ in the presence of excess NaSCN in aqueous solution gave thiamine salts, (H-thiamine)[Pt(SCN)₄] · 3H₂O (1) and (H-thiamine)[Pt(SCN)₆] · H₂O (2), respectively, structures of which have been determined by X-ray diffraction. The thiamine molecule adopts the usual F conformation in each salt. In 1, [Pt(SCN)₄]²⁻ ions act as large planar spacers in the crystal lattice and interact scarcely with thiamine, except for a hydrogen bonding with the terminal hydroxy O(5γ). Instead, water molecules form two types of host–guest-like interactions with the pyrimidine and the thiazolium moieties of a thiamine molecule, one being a C(2)–Hwaterpyrimidine bridge and the other being an N(4′)–Hwaterthiazolium bridge. In 2, despite the much larger ion size, octahedral [Pt(SCN)₆]²⁻ ions form a C(2)–Hanionpyrimidine bridge and an N(4′)–Hanionthiazolium bridge. An additional hydrogen bonding between the anion and the terminal O(5γ) of thiamine creates a hydrogen-bonded macrocyclic ring {thiaminium–[Pt(SCN)₆]²⁻}₂, a supramolecule.     

Determination of (-)-threo-chlorocitric acid in human plasma by gas chromatography—positive chemical-ionization mass spectrometry

A method is described for measuring (-)-threo-chlorocitric acid in human plasma. Plasma is acidified to pH 1 to minimize lactonization and a¹³C analogue of (-)-threo-chlorocitric acid is added as internal standard. The acidified plasma is then extracted with ethyl acetate containing 10% methanol. The ethyl acetate—methanol extract is back-extracted with acetate buffer (pH 5). This extract, following adjustment to pH 1, is reextracted with ethyl acetate. The residue after removal of the ethyl acetate is treated with ethereal diazomethane. The wet residue is reconstituted in ethyl acetate and a portion of this solution is analyzed by gas chromatography—chemical ionization mass spectrometry. The mass spectrometer is set to monitorm/z269 [MH⁺ of trimethylated (-)-threo-chlorocitric acid] andm/z270 [MH⁺ of trimethylated (-)-threo-[¹³C]chlorocitric acid] in the gas chromatographic effluent. Them/z/269 tom/z270 ion ratio in a sample containing an unknown amount of (-)-threo-chlorocitric acid is converted to an amount of compound using a calibration curve. The calibration curve is generated by analyzing control plasma spiked with various known amounts of (-)-threo-chlorocitric acid and a fixed amount of (-)-threo-[¹³C]chlorocitric acid. The limit of quantitation is 0.1–0.6 μg ml⁻¹, depending on the characteristics of the calibration curve generated with each set of samples. The precision (relative standard deviation) at a concentration of 2 μg ml⁻¹ is 3.3%.     

Effects of defined synthetic phospholipids on glycosylation processes. Modifications of membrane-bound N-acetylglucosaminyl-transferase and solubilized sialyl-transferase activities

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12–96-h incubations of hamster embryo cells with 4 μM [³H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites ( <0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [³H]B[e]P.     

Synthesis, spectroscopy and electrochemistry of some polynuclear complexes of aminoethanethiol

Polynuclear S-bridged complexes of the general formula {[Co₂L₆]M}ⁿ⁺ with M = Co(III), Cd(II), Pb(II), Ni(II), Zn(II) and Hg(II) were prepared from [Co(2-aminoethanethiolate)₃] and the appropriate metal salt. Proton and ¹³C NMR spectra are consistent with structures previously proposed for these species with ¹³C chemical shifts dependent on the bridging metal ion. Electrochemical studies are consistent with a model in which an S-bonded ML₆ moiety (i.e., the bridging metal ion and the six aminoethanethiolate ligands) acts as a ‘dodecadentate’ ligand bonded to two Co³⁺ ions. Reduction of the terminal cobalt ions in these trinuclear complexes is observed in the range −0.75 to −1 V vs. SCE on mercury, gold or glassy carbon working electrodes. For complexes with relatively labile bridging ions, the electrode reaction is irreversible, presumably due to rapid decomposition of the labile cobalt(II) product. For the tricobalt(III) derivative, however, the electrode reaction is reversible consistent with other recent observations on cage or otherwise stereorestrictive ligand systems [1].     

Electrospray mass spectrometry of trans-[Ru(NO)Cl(dpaH)2] (dpaH=2,2′-dipyridylamine) and ‘caged NO’, [RuCl3(NO)(H2O)2]: loss of HCl and NO from positive ions versus NO and Cl from negative ions

The positive ion electrospray mass spectrometry (ESI-MS) of trans-[Ru(NO)Cl)(dpaH)₂]Cl₂ (dpaH=2,2′-dipyridylamine), obtained from the carrier solvent of H₂O–CH₃OH (50:50), revealed 1+ ions of the formulas [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ (m/z=508), [Ru^IIICl(dpaH)(dpa⁻)]⁺ (m/z=478), [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472), [Ru^III(dpa)₂]⁺ (m/z=442), originating from proton dissociation from the parent [Ru^II(NO⁺)Cl(dpaH)₂]²⁺ ion with subsequent loss of NO (17.4% of dissociative events) or loss of HCl (82.6% of dissociative events). Further loss of NO from the m/z=472 fragment yields the m/z=442 fragment. Thus, ionization of the NH moiety of dpaH is a significant factor in controlling the net ionic charge in the gas phase, and allowing preferential dissociation of HCl in the fragmentation processes. With NaCl added, an ion pair, {Na[Ru^II(NO)Cl(dpa)₂]}⁺ (m/z=530; 532), is detectable. All these positive mass peaks that contain Ru carry a signature ‘handprint’ of adjacent m/z peaks due to the isotopic distribution of ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru and ⁹⁶Ru mass centered around ¹⁰¹Ru for each fragment, and have been matched to the theoretical isotopic distribution for each set of peaks centered on the main isotope peak. When the starting complex is allowed to undergo aquation for two weeks in H₂O, loss of the axial Cl⁻ is shown by the approximately 77% attenuation of the [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ ion, being replaced by the [Ru^II(NO⁺)(H₂O)(dpa)₂]⁺ (m/z=490) as the most abundant high-mass species. Loss of H₂O is observed to form [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472). No positive ion mass spectral peaks were observed for RuCl₃(NO)(H₂O)₂, ‘caged NO’. Negative ions were observed by proton dissociation forming [Ru^II(NO)Cl₃(H₂O)(OH)]⁻ in the ionization chamber, detecting the parent 1− ion at m/z=274, followed by the loss of NO as the main dissociative pathway that produces [Ru^IIICl₃(H₂O)(OH)]⁻ (m/z=244). This species undergoes reductive elimination of a chlorine atom, forming [Ru^IICl₂(H₂O)(OH)]⁻ (m/z=208). The ease of the NO dissociation is increased for the negative ions, which should be more able to stabilize a Ru^III product upon NO loss.     

Investigations into the biosynthetic pathways for classical and ring B-unsaturated oestrogens in equine placental preparations and allantochorionic tissues

In on-going studies of ‘classical’ and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-²H₅]-testosterone and [16,16,17-²H₃]-5,7-androstadiene-3β,17β-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [²H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography–mass spectrometry (GC–MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell™ cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [²H₅]-Testosterone was converted into [²H₄]-oestradiol-17β, [²H₄]-oestrone and [²H₃]-6-dehydro-oestradiol-17 in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse.

On the basis of GC–MS characteristics (M⁺ m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M⁺). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [²H₄]-17β-dihydroequilin was also shown, and a biosynthetic pathway is proposed.

In static incubations of placental microsomal fractions, the 17β-dihydro forms of both equilin and equilenin were shown to be major metabolites of [²H₃]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17β-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17β-isomer of dihydroequilenin. Confirmation of the identity of 17β-dihydroequilin and 17β-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-deuterated steroids (ions at m/z 414 (17β-dihydroequilin) and 412 (17β-dihydroequilenin) (both as bis-trimethyl silyl ether derivatives) were observed. Biosynthetic pathways for formation of the ring B-unsaturated oestrogens from 5,7-androstadiene-3β,17β-diol are proposed.     

Activation of [C]chlorobiphenyls to protein-binding metabolites by rat liver microsomes

The irreversible binding of [¹⁴C]2,2′-di- and [¹⁴C]2,4,5,2′,4′,5′-hexachlorobiphenyl ([¹⁴C]DCB and [¹⁴C]HCB) to protein was studied in the presence of rat liver microsomes and a NADPH-generating system. Protein-bound radioactivity was found with [¹⁴C]DCB but not with [¹⁴C]HCB. The binding of ¹⁴C-metabolites was increased by pretreatment of the rats with phenobarbital or polychlorinated biphenyls. Protein binding was linear for 80 min. In contrast, monohydroxy-metabolites of DCB were formed and degraded within 40 min. Inhibition of secondary oxidation of DCB by scavening superoxide anions or by glucuronidation of the monophenols markedly decreased the protein binding. Addition of trichloropropene oxide or styrene oxide, both inhibitors of epoxide hydrase, did not significantly stimulate the binding. The results suggest that the majority of reactive metabolites of DCB arise from secondary metabolism, i.e., the subsequent oxidation of the phenolic metabolites. Arene oxides, the primary products, appear to play a minor role in the protein binding of DCB.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

Synthesis and characterization of trisodium salt of bis(thiosalicylato)aurate(I): Na3[Au(TSA)2]·5H2O

Sodium salt of a water-soluble, anionic, and monomeric 1:2 complex of Au(I) with a dianion of thiosalicylic acid TSA²⁻(Hin2TSA) = o-HS(C₆H₄)COOH) was first prepared and isolated as colorless needle crystals through a stoichiometric reaction of NaAuCl₄:H₂TSA:NaOH = 1:4:8 molar ratio in aqueous/EtOH solution. In this reaction, TSA²⁻ ligand has played a role of a reducing agent for the starting Au(III) ion and also of donor ligands coordinating to the reduced Au(I). This compound was characterized by complete elemental analyses, TG/DTA, FT-IR, 2D-NMR (¹H-¹H COSY, ¹H-¹³C HMBC, and ¹H-¹³C HMQC) spectroscopy, and the molmass measurement based on the cryoscopic method. It was shown that this complex was a monomeric species of Au(I) with a formula of Na₃[Au(TSA)₂]·5H₂O in the solid state, but not a polymeric species even in aqueous solution. A full assignment of seven carbon and four proton resonances in the coordinated TSA²⁻ ligand was achieved by the 2D ¹H-¹³C HMBC NMR technique.     

Effects of vesamicol on acetylcholine metabolism and synaptic transmission in the electric organ of Torpedo

Vesamicol [2-(4-phenylpiperidino)cyclohexanol, formerly AH5183] at a concentration of 10 μM reduced by 16–20% the amount of vesicle-bound ACh in intact pieces of Torpedo electric organ (isolated prisms). When [¹⁴C]acetate was applied to prisms in the presence of 10 μM vesamicol, vesicular translocation of newly synthesized [¹⁴C]ACh was inhibited by 40%. During short trains of field shocks given at 10 Hz to the tissue, vesamicol inhibited by 93% the release of [¹⁴C]ACh, but left the release of prestored ACh unaltered. In spite of these alterations, 10 μM vesamicol did not impair nerve-electroplaque transmission, even after prolonged electrical stimulation and during a recovery period. It is concluded that in the Torpedo electric organ the actions of vesamicol on ACh metabolism have apparently little or no effect on the efficiency of synaptic transmission.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Variation in δC values for the seagrass Thalassia testudinum and its relations to mangrove carbon

Carbon isotope ratios (¹³C/¹²C) were measured for the leaves of the seagrass Thalassia testudinum Banks ex König and carbonates of shells collected at the seagrass beds from seven sites along the coast of southern Florida, U.S.A. The δ¹³C values of seagrass leaves ranged from −7.3 to −16.3‰ among different study sites, with a significantly lower mean value for seagrass leaves from those sites near mangrove forests (−12.8 ± 1.1‰) than those far from mangrove forests (−8.3 ± 0.9‰; P < 0.05). Furthermore, seagrass leaves from a shallow water area had significantly lower δ¹³C values than those found in a deep water area (P < 0.01). There was no significant variation in δ¹³C values between young and mature leaves (P = 0.59) or between the tip and base of a leaf blade (P = 0.46). Carbonates of shells also showed a significantly lower mean δ¹³C value in the mangrove areas (−2.3 ± 0.6‰) than in the non-mangrove areas (0.6 ± 0.3‰; P <0.025). In addition, the δ¹³C values of seagrass leaves were significantly correlated with those of shell carbonates (δ¹³C seagrass leaf = −9.1 + 1.3δ¹³C shell carbonate (R² = 0.83, P < 0.01)). These results indicated that the input of carbon dioxide from the mineralization of mangrove detritus caused the variation in carbon isotope ratios of seagrass leaves among different sites in this study.     

Specificity and the reliability of quantitation in the analysis of neurotransmitters

The unique advantages of selected ion monitoring, when using gas chromatography-mass spectrometry (GC-MS) as an analytical method for neurotransmitters are presented, using two examples: (1) paired quantitations of GABA from 11 brain regions, using different ion ratios of native and deuterated GABA: (a) m/z 190/194 and (b) m/z 204/210. Of 44 data pairs of GABA concentration calculated using these ratios, only 2 differed by > 10%, 23 differed by <5%, 15 differed by <2.0% and 3 were identical. In no case was the difference statistically significant. (2) The analysis of dopamine in the developing nervous system of rat. Based on its GC-MS characteristics, a dopamine-like compound was detected in the central nervous system (CNS) of rat, in high concentration, during embryonic and neonatal life. It co-eluted with dopamine, and generated two (m/z 387,415) of the three (m/z 387,415,428) ions normally monitored from authentic dopamine. The analysis of this compound by GC alone, with an electron capture detector, using the preparative procedure described, would have lead to the false, positive conclusion of high concentrations of dopamine in the CNS of rat during fetal and neonatal life.     

Mechanism of chain initiation by dextransucrase: Absence of terminal ‘sucrose’ linkage in newly synthesized dextran chains

Dextran was synthesized using dextransucrase from Streptococus sanguis 10558 and (F)-[¹⁴C]sucrose as substrate to test the possibility that sucrose may be the initial acceptor for glucose. If sucrose is the initial acceptor, then dextran chains should have [¹⁴C] fructose in a terminal ‘sucrose’ linkage which can be cleaved under mild conditions. Although incorporation of [¹⁴C]fructose into dextran was observed, the label was not released by mild hydrolysis, indicating that sucrose is not the initiator for dextran synthesis. Incorporation of [¹⁴C]fructose into dextran might represent its ability to act as an acceptor, as suggested by the isolation of leucrose as a by-product in the reaction.     

Transport in C4 mesophyll chloroplasts. Characterization of the pyruvate carrier

1. Evidence is presented for high rates of carrier-mediated uptake of pyruvate into the stroma of intact mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis, but not the chloroplasts of the C₃ plant Spinacea oleracea. Uptake of pyruvate in the dark with the C₄ mesophyll chloroplasts was followed using two techniques: uptake of [¹⁴C]pyruvate as determined by silicon oil centrifugal filtration and uptake as indicated by absorbance changes at 535 nm (shrinkage/swelling) after addition of 0.1 M pyruvate salts.

2. Uptake of the pyruvate anion by an electrogenic carrier is suggested to be the major mode of transport. Chloroplast swelling was observed in potassium pyruvate plus valinomycin and uptake of [¹⁴C]pyruvate was inhibited by membrane-permeant anions. Valinomycin reduced uptake in the absence of external potassium and the inhibition could be reversed by addition of external potassium.

3. Uptake of pyruvic acid (or a pyruvate ⁻/OH⁻ antiport) is ruled unlikely since [¹⁴C]pyruvate uptake was relatively independent of the pH gradient across the envelope and addition of pyruvate to chloroplasts did not result in an alkalization of the medium. The low rate of swelling observed in ammonium pyruvate may be due to non-mediated permeation of pyruvic acid, which is possible only at high pyruvate concentrations.

4. The concentration of pyruvate in the stroma increased with external concentration over the range tested (up to 40 mM) but the concentration ratio (internal/external) was always less than one. The steady-state concentration of [¹⁴C]pyruvate in the stroma was dependent on the ionic strength of the medium, with saturation at roughly I = 0.04 M, while accumulation of the membrane-permeant cation tetraphenylmethylphosphonium decreased with increasing ionic strength. This suggests that ionic strength modifies a membrane potential (inside negative) across the envelope and that pyruvate uptake responds to the magnitude and direction of that potential (−80 mV at low ionic strength).

5. Chloride and inorganic phosphate were potent inhibitors of [¹⁴C]pyruvate uptake. Of the sulfhydryl reagents tested, N-ethylmaleimide was not inhibitory while mersalyl completely blocked [¹⁴C]pyruvate uptake and swelling in potassium pyruvate plus valinomycin. Pyruvate uptake, as measured by valinomycin induced swelling in potassium pyruvate, was highly temperature sensitive, with an energy of activation of 39 kcal/mol above 9 °C.

6. Phenylpyruvate, -ketoisovalerate, -ketoisocaproate, -cyano-4-hydroxycinnamic acid and -cyanocinnamic acid inhibited [¹⁴C]pyruvate but not [¹⁴C]-acetate uptake in the dark and also reduced pyruvate metabolism by the chloroplasts in the light.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Studies on Bacillus stearothermophilus. Part 1. Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene-3,9,20-trione.

When Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, was incubated with exogenous progesterone for 24 h, three monohydroxylated metabolites were produced. 20-Hydroxyprogesterone was the major metabolite produced in 60.8 relative percentage yield. The other two monohydroxylated metabolites were identified as 6β-hydroxyprogesterone and the rare 6-hydroxyprogesterone in 21.0 and 13.6 relative percentage yields, respectively. A new metabolite 9,10-seco-4-pregnene-3,9,20-trione was isolated in 3.7 relative percentage yield. All metabolites were purified by preparative TLC and HPLC followed by their identification using ¹H, ¹³C NMR and other spectroscopic data.     

In vivo release of dopamine during perfusion of neurotensin in substantia nigra of the unrestrained rat

The functional effect of neurotensin on the kinetics of dopamine (DA) release in the substantia nigra of the freely moving rat was investigated. After guide tubes for push-pull perfusion were implanted stereotaxically just above the substantia nigra, endogenous stores of DA in this structure were labelled by micro-injection of 0.02–0.05 μCi of [¹⁴C]-DA. Then an artificial cerebrospinal fluid (CSF) was perfused within the site at a rate of 20 μl/min at successive 5 min intervals. Neurotensin added to the CSF perfusate in concentrations of 0.05–0.1 μg/μl evoked an immediate, Ca⁺⁺ dependent release of DA from sites directly within the substantia nigra or a delayed efflux when the peptide was perfused at the edge of this structure. Neurotensin failed to affect the pattern of release of this monoamine at sites which were not within the substantia nigra. Further, the body temperature of the rat also was not altered by neurotensin at any of the sites of perfusions. A relatively inactive analogue of the peptide, [D-Arg]⁹ neurotensin, was essentially without effect on DA activity. In double isotope experiments in which the substantia nigra of the rat was labelled with both [³H]-5-HT and [¹⁴C]-DA, the perfusion with neurotensin failed to affect 5-HT efflux while the release of DA was enhanced. Chromatographic analysis of the metabolites of DA in samples of push-pull perfusates revealed that neurotensin enhanced significantly the level of DOPAC and HVA. Overall, these results demonstrate that in the unrestrained rat neurotensin acts selectively within the substantia nigra to alter the presynaptic, Ca⁺⁺ dependent release of DA. It is suggested that the mechanism by which the tri-decapeptide functions within this brainstem structure is through its modulation of nigral dopaminergic neurons.     

Ion trap MS/MS of intact testosterone and epitestosterone conjugates--adducts, fragile ions and the advantages of derivatisation

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG + NH₄]⁺, [EG + NH₄]⁺ and [EG + H]⁺ were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG + H]⁺ but not [TG + H]⁺ is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.