20α-hydroxysteroid dehydrogenase activity in the rat corpus luteum; A quantitative cytochemical study

A quantitative cytochemical method for the demonstration of 20α-hydroxysteroid dehydrogenase activity (20α-HSD) in the regressing corpora lutea of the adult rat ovary is described. The method employs unfixed tissue sections and relies upon the oxidation of 20α-hydroxy-4-pregnen-3-one (20α-OH-P) with nitro blue tetrazolium as the hydrogen acceptor. The enzyme was dependent upon NADP⁺ for its activity and was inactive when 20β-hydroxy-4-pregnen-3-one (20β-OH-P) was used as a substrate. The apparent K_m values for 20α-OH-P and NADP⁺ were 3 × 10⁻⁴M and 2.5 × 10⁻⁵M respectively. Inhibition of 20α-HSD activity by steroids was demonstrable at pH 8. Androstenedione was by far the most potent inhibitor, followed by progesterone (the product of the enzyme activity) 17α-hydroxyprogesterone. Compound S and 20β-OH-P. At pH 6.8, a pH more favourable to the progesterone → 20α-OH-P reaction, only progesterone and 17α-hydroxyprogesterone were inhibitory. Testosterone was without demonstrable effect at either pH.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K^k. In the absence of exogenous IL-2 the clones require the presence of Ia⁺, Thy-1⁻ accessory cells and of Thy-1⁺, Lyt-1⁺2⁻ cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A_αA_β and/or E_αE_β had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2⁺ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E_αE_β when B10.A(4R) spleen cells, which do not express E_αE_β, or when B10.A(4R) accessory cells, which were reconstituted with (BALB/c X B10.A(4R)) F1 T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2K^d). These and previous results suggest that H-2K-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A_αA_β and E_αE_β molecules with syngeneic accessory cells. This interaction results in IL-2 production by the stimulator T cells and thus in the proliferation of the clones.     

A (1→3,1→4)-β-glucan-specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)-β-glucans

Monoclonal antibodies were raised against a (1→3,1→4)-β-glucan-bovine serum albumin (BSA) conjugate. One antibody (BG1) selected for further characterization, was specific for (1→3,1→4)-β-glucan, displaying no binding activity against a (1→3)-β-glucan-BSA conjugate and minimal binding against a cellopentaose-BSA conjugate. A range of oligosaccharides was prepared by enzymatic digestion of (1→3,1→4)-β-glucan, purified by size exclusion chromatography and characterized by ¹H-NMR and anion exchange chromatography. These (1→3,1→4)-β-oligoglucosides, together with (1→3)-β- and (1→4)-β-oligoglucosides were used to characterize the binding site of the monoclonal antibody (BG1) by competitive inhibition. The monoclonal antibody showed maximal binding to a heptasaccharide with the structure Glc(1→3) Glc(1→4) Glc(1→4) Glc(1→3) Glc(1→4) Glc(1→4) Glc and was determined to have an affinity constant of 3.8 × 10⁴ M⁻¹ for this oligoglucoside. The monoclonal antibody (BG1) has been used to develop a sensitive sandwich ELISA for the specific quantitation of (1→3,1→4)-β-glucans. The assay operates in the range 1–10 ng ml⁻¹ and shows no significant cross-reaction with tamarind xyloglucan, wheat endosperm arabinoxylan or carboxymethyl-pachyman ((1→3)-β-glucan). When used with a second-stage, rabbit anti-mouse gold conjugate and viewed under the electron microscope, the monoclonal antibody probe was found to bind strongly to the walls of the aleurone in thin sections of immature wheat (Triticum aestivum) cv. Millewa grains but not to the middle lamella region. A previously described specific anti-(1→3)-β-glucan antibody (Meikle et al., 1991) bound to discrete patches on the aleurone walls, believed to be plasmodesmata.     

Galα1→4Gal-glycans are expressed on myofibrillar associated proteins

There is evidence that glycans carrying terminal galactose residues are differently expressed in the sarcoplasm of different muscle fiber types. In this study monoclonal antibodies directed against P blood group antigens P^k: Galα1–4Galβ1–4Glcβ- and P₁: Galα1–4Galβ1–4GlcNAcβ- were used to detect terminal α-galactosylated glycoconjugates on muscle proteins. Electrotransfer of proteins, extracted from human masseter and biceps muscles, to nitrocellulose after polyacrylamide gel electrophoresis (PAGE) and incubation with anti-P^k (CD77) consistently showed two bands with apparent molecular weights of 66 kDa and 64 kDa. In fresh frozen muscle sections from some humans there was endothelial reaction with anti-CD77 in capillaries, venules and veins but not in arterioles and arteries. In muscle samples from other humans there was no staining of endothelial cells. Formalin-fixed human muscle displayed a CD77 reaction with highest accumulation of reaction product at the periphery of the fibers. This may be explained by the presence of P^k glycoconjugates on intermediate filaments in muscle fibers. In preparations of cat masseter muscle proteins the antibodies against P₁P^k antigens reacted with a 170 kDa and a 55 kDa band while in preparations of cat biceps brachii only a 55 kDa band was reactive. The specificities of the antibodies were investigated by fluorescence-activated cell sorter (FACS), α- and β-galactosidase digestion and inhibitory sugars. This study indicates that glycans carrying Galα1–4Galβ1- epitopes are expressed on myofibrillar associated proteins.     

Isolation of antibodies of improved specificity after fractionation on Antigen-Sepharose affinity columns of antisera to bovine serum albumin conjugates of C-3- and C-7-linked (O-carboxymethyl)oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

Rabbit antisera to bovine serum albumin (BSA) conjugates of 3-(O-carboxymethyl)oximino-, 7-(O-carboxymethyl)oximino- and 7β-hemi-succinamido derivatives of 5α-dihydrotestosterone (DHT) were applied to four affinity columns bearing respectively these three antigens and a fourth 3β-hemisuccinamido-5α-androstan-17β-ol-BSA antigen as ligands.The antibodies retained on the columns were totally desorbed by an excess of DHT, but in DHT-bound form, whereas 1M mh₄oh and electrophoretic elution allowed a recovery of 60% of the retained antibodies in unbound form. The antibody fractions (40%) remaining on the columns after NH₄OH or electrophoretic elution were totally recovered by addition of DHT following the electrophoretic elution only. All the DHT-bound fractions were dissociated by dialysis but with a 70% loss of binding activity.The association constants for DHT of most of the antibody fractions were similar to those of the crude antisera (Ka ~ 10¹⁰M^?1), with the exception of the antibodies recovered from the antibody fractions resistant to electrophoretic elution which had higher affinities (Ka ~ 2.0 to 30 × 10¹⁰M^?1).The specificity charts of the antisera were in some cases considerably modified after fractionation, according to the choice of the ligand employed in the affinity columns as well as of the elution methods. The lowest cross-reactions with testosterone were observed after elution with 1M NH₄OH (17–20%) or electrophoresis (23–25%) of the anti-7-(O-carboxymethyl)oximino-DHT antisera fractions retained on 3β-hemisuccinamido-5α-androstan-17β-ol-BSA-Sepharose columns.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

The synthesis and hydrolysis of a series of deoxyfluoro-d-glucopyranosyl phosphates

The synthesis of all four deoxyfluoro-α-d-glucopyranosyl phosphates is described. Rate constants for their acid-catalyzed hydrolysis were determined, and fluorine substitution was shown to have a significant effect in lowering the rate, particularly when the substitution is adjacent to the anomeric center. Relative rate-constants measured in m HClO₄ at 25° are 60.30:1.00:7.05:3.97:16.5 for α-d-glucopyranosyl phosphate and the 2-, 3-, 4- and 6-deoxyfluoro derivatives, respectively. The hydrolysis of 2-deoxy-2-fluoro-α-d-glucopyranosyl phosphate was studied in more detail, and an activation entropy and enthalpy of 4.1 e.u. (m reactant) and 113.5 kJ.mol⁻¹, respectively, were determined for hydrolysis in m HClO₄ at 60° The pH dependence of its hydrolysis was investigated, and rate constants for hydrolysis of the monoanion (k_M = 1.88 × 10⁻⁶ s⁻¹) and neutral (k_N = 6.23 × 10⁻⁵ s⁻¹) species were thus extracted. Hydrolysis of the monoanion is not significantly affected by fluorine substitution, as expected. The ability or inability of several mechanistically distinct enzymes to utilize these fluorinated substrates is rationalized in the light of these findings.     

Hormonal action in human cervix—II specific progestogen binding proteins in human cervix

Progesterone binding activity has been detected in cytosols prepared from human cervical tissue. Cytosols showed high affinity (K_a, 0.2–1 nM⁻¹) for progesterone and synthetic progestogens but not for corticosteroids or other steroids (norethindrone, medroxyprogesterone acetate > chlormadinone acetate, progesterone > 5α-pregnane-3,20-dione > norethisterone acetate > 17α-hydroxyprogesterone > cortisol, estradiol). Addition of 49 nM cortisol to the assay buffer permitted measurement of high affinity progesterone binding sites in those cytosols with high concentrations of CBG-like contaminants. The hormone dissociated from the complex with a dissociation rate constant of 6.9 × 10⁻⁵s⁻¹ (0°C, 18% glycerol).Binding sites exhibited similar K_a values throughout the cervix and were found in highest concentration in the region encompassing the columnar epithelium when expressed per mg protein and per g. The concentrations of sites per mg DNA were similar in the columnar epithelium and in the stroma but higher than in the region of the squamous epithelium.Endometrial tissue exhibited higher concentrations of progesterone binding sites than did the corresponding cervix. In both the endometrium and cervix, the mean concentrations expressed per mg protein were significantly higher for proliferative phase than for secretory phase tissues. The corresponding differences in concentration per mg DNA were significant only for cervical cytosols.     

Effects of cyclic AMP and of 2-mercapto-1-(β-4-pyridethyl)benzimidazole on composition of steroids secreted by a testicular tumour cell line

We studied the effect of cyclic AMP (cAMP) on steroidogenesis in a mouse Leydig cell tumor line (1–10). known to secrete exclusively progesterone (P) and 20α-dihydroprogesterone (20α-H₂P). Radioimmunoassays that distinguish these two steroids were used. Total steroidogenesis was stimulated by cAMP in a dose-dependent manner over the range tested (10⁻⁶-10⁻³ M). Up to 2 × 10⁻⁵ M cAMP, progesterone constituted 11–13% of the secreted progestins: at higher concentrations of cAMP (10⁻⁴-10⁻³ M), the P/(P + 20α-H₂P) ratio progressively increased (37% at 10⁻³ M), but the incremental progestin secretion consisted of 50% progesterone throughout this range. The change in progestin profile occurred within less than 45 min. 2-Mercapto-1-(β-4-pyridethyl)benzimidazole (MPB) reduced basal steroidogenesis, progesterone secretion being more severely affected than that of 20α-H₂P. MPB inhibited cell growth and noncompetitively inhibited cAMP-dependent protein kinase activity in the cytosol of 1–10 cells. In a faster-growing variant of 1–10. higher concentrations of exogenous cAMP were required to exert similar effects on steroidogenesis. and MPB was less effective in suppressing cell growth. The possibility is discussed that cAMP may accelerate an active process of progesterone release, thus minimizing the intracellular exposure of the hormone to 20α-hydroxysteroid dehydrogenase, and that MPB antagonizes cAMP at a site influencing both steroid synthesis and release.     

Identity of aliphatic L- -hydroxyacid oxidase and glycolate oxidase from rat livers

l-α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K_m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K_m values are as follows; glycolate, 2.4 × 10^?4m; l-α-hydroxyisocaproate, 1.26 × 10^?3; glyoxylate, 1.41 × 10^?4m; and FMN, 1.13 × 10^?6m. K_m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.     

Hypoxia inverts the negative chronotropic response to norepinephrine in normoxia in cultured neonatal rat cardiac myocytes: role of the α1 adrenergic signal transduction system

In the present study, we investigated the effect of hypoxia on the chronotropic response to norepinephrine (NE) of cultured neonatal rat ventricular myocytes. We measured beating of myocytes with the Fotonic sensor^TM, using a newly developed method for a noncontact displacement measurement. The beating rate counted with the sensor had a high correlation coefficient with that counted visually under a microscope (r = 0.997, P < 0.01). NE concentrations of 10⁻⁸–10⁻⁴ M caused negative chronotropy dose dependently in the presence of 5×10⁻⁷ M propranolol. NE-induced chronotropy was completely antagonized by 10⁻⁶ M prazosin. Three hours hypoxia decreased the spontaneous beating rate 40% (P < 0.01). Negative chronotropy induced by 10⁻⁴ M NE in normoxia was inverted to positive and was antagonized by prazosin. Hypoxia increased the basal level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) to 190% (P < 0.01), while NE-stimulated Ins(1,4,5)P₃ production was significantly suppressed. Immunoblotting analysis of G protein subunits demonstrated no quantitative changes in Giα, Gqα, Goα and G_βcommon subunits in hypoxia. In a saturation binding assay with [³H]prazosin, K_d values were increased to 152% by hypoxia (P < 0.05) without significant change in B_max. Basal activity of low K_m-GTPase was increased to 122% by hypoxia (P < 0.05). These results suggest that the hypoxia-induced increase in low-K_m GTPase activity, which could stimulate phospholipase C by an activated α_GTP subunit of G protein and consequently induce receptor-independent increase in Ins(1,4,5)P₃, may be responsible for the inversion of the NE-induced negative chronotropic response in normoxia.     

Pulse radiolytic study of α-tocopherol radical mechanisms in ethanolic solution

Pulse radiolytic studies of α-tocopherol (αTH) oxidation-reduction processes were carried out with low doses (5 Gy) of high-energy electrons in O₂₋, N₂₋, and air-saturated ethanolic solutions. Depending on the concentration of oxygen in solution, two different radicals, A· and B·, were observed. The first, A·, was obtained under N₂ and results from aTH reaction with solvated electron (k_aTH+csolv⁻ = 3.4 × 10⁸ mol⁻¹ liter s⁻¹) and with H₃C-ĊH-OH, (R·) (k_{aTH + R·} = 5 × 10⁵ mol⁻¹ liter s⁻¹). B·, observed under O₂, is produced by αTH reaction with RO₂ peroxyl radicals (k_aTH + RO₂^. = 9.5 × 10⁴ mol⁻¹ liter s⁻¹).     

17-Hydroxyprogesterone metabolism in the monkey fetal adrenal: C17–20Lyase and 21-hydroxylase activities

C^17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey ($\text{Macaca mulatta}$) fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [¹⁴C]17-hydroxyprogesterone to [¹⁴C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, K_m and V_max. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10^?3 μmoles NADPH oxidized/min. × mg^?1 protein with an apparent K_m of 3.6 × 10^?7 M and a V_max of 2.2 × 10^?3 μmoles/min. × mg^?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis.     

Mode of action of endo-(1→3)-β-d-glucanases from marine molluscs on the laminarin from Laminaria cichorioides: The structure and the inhibitory effect of the resulting (1→3;1→6)-β-d-gluco-oligosaccharides

The oligosaccharides released by the action of endo-(1→3)-β-d-glucanases from the marine molluscs Chlamys albidus (laminarinase Lo) and Spisula sachalinensis (laminarinase LIV) on Laminaria laminarin have been studied. For laminarinase Lo, the branched products were shown to be 6²-β-d-glucopyranosyl-laminaribiose and 6³- and 6²-β-d-glucopyranosyl-laminaritrioses by methylation analysis and ¹³C-n.m.r. spectroscopy. It is suggested that one or two (1→3) linkages adjacent to (1→6) branch-points result in resistance to enzymic attack. 6³-β-d-Glucopyranosyl-laminaritriose inhibited laminarinases Lo and LIV (I₅₀ 1.2 × 10⁻³m and 1.5 × 10⁻³m, respectively).     

Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute δ¹³C values of 5α-androstanetrione (5α-AT) and 5β-androstanetrione (5β-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5α-androstanedione (5α-AD) and 5β-androstanedione (5β-AD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean −3SD of δ¹³C depletion values from the controls were −1.46, −1.98, −1.78 and −2.42 for 5β-AT-5β-AD, 5α-AT-5β-AD, 5β-AT-5α-AD and 5α-AT-5α-AD, respectively, indicating −3‰ as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, δ¹³C depletion values peaked around −5‰ and −9‰ after oral and intramuscular administration of HC, respectively, and around −6‰ after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites.     

Identification by capillary gas chromatography-mass spectrometry of eleven non-ecdysteroid steroids in the haemolymph of larvae of Sarcophaga bullata

$\text{O-}\text{Pentafluorobenzyloxime}$ (OPFB)-heptafluorobutyrylester (HFB) derivatives and $\text{OPFB}\text{-O-}\text{methyloxime}$ (MO)-trimethylsilylether (TMS) derivatives of non-ecdysteroid steroids were prepared from haemolymph extracts of last instar larvae of the fleshfly Sarcophaga bullata. Using a negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS) technique the following steroids could be identified: progesterone, testosterone, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, androst-5-ene-3β,17β-diol, androstenedione, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, 17α-hydroxyprogesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxyprogesterone. Although the technique is very sensitive, estrogens could not be detected. These results suggest an active metabolism of progesterone and testosterone.     

Different signaling pathway between sphingosine-1-phosphate and lysophosphatidic acid in Xenopus oocytes: Functional coupling of the sphingosine-1-phosphate receptor to PLC-xβ in Xenopus oocytes

In Xenopus oocytes, both sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) activate Ca²⁺-dependent oscillatory Cl⁻ currents by acting through membrane-bound receptors. External application of 50 μM S1P elicited a long-lasting oscillatory current that continued over 30 min from the beginning of oscillation, with 300 nA (n = 11) as a usual maximum peak of current, whereas 1-μM LPA treatment showed only transiently oscillating but more vigorous current responses, with 2,800 nA (n = 18) as a maximum peak amplitude. Both phospholipid-induced Ca²⁺-dependent Cl⁻ currents were observed in the absence of extracellular Ca²⁺, were blocked by intracellular injection of the Ca²⁺ chelator, EGTA, and could not be elicited by treatment with thapsigargin, an inhibitor of endoplasmic reticulum (ER) Ca²⁺ ATPase. Intracellular Ca²⁺ release appeared to be from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store, because Cl⁻ currents were blocked by heparin injection. Pretreatment with the aminosteroid, U-73122, an inhibitor of G protein-mediated phospholipase C (PLC) activation, to oocytes inhibited the current responses evoked both by S1P and LPA. However, when they were injected with 10 ng of antisense oligonucleotide (AS-ODN) against Xenopus phospholipase C (PLC-xβ), oocytes could not respond to S1P application, whereas they responded normally to LPA, indicating that the S1P signaling pathway goes through PLC-xβ, whereas LPA signaling goes through another unknown PLC. To determine the types of G proteins involved, we introduced AS-ODNs against four types of G-protein α subunits that were identified in Xenopus laevis; G_qα, G₁₁α, G₀α, and G_i1α. Among AS-ODNs against the Gαs tested, AS-G_qα and AS-G_i1α to S1P and AS-G_qα and AS-G₁₁α to LPA specifically reduced current responses, respectively, to about 20–30% of controls. These results demonstrate that LPA and S1P, although they have similar structural features, release intracellular Ca²⁺ from the IP₃-sensitive pool, use different components in their signal transduction pathways in Xenopus oocytes. J. Cell. Physiol. 176:412–423, 1998. © 1998 Wiley-Liss, Inc.     

Localization and pharmacological characterization of nicotinic-cholinergic binding sites in cockroach brain using α- and neuronal bungarotoxin

[¹²⁵I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (K_d = 1.09 nM) having a B_max of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a k_obs = 4.37 × 10⁻³s⁻¹. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10⁻⁵ and 9.94 × 10⁻⁵s⁻¹. The K_ds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.