Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.     

The effect of propylbenzilylcholine mustard on contraction and radioligand binding parameters of muscarinic receptors in guinea pig ileum

The receptor occupancy-biological effect relationship for muscarinic receptors in guinea pig ileal smooth muscle has been studied by comparison of radioligand binding and contractile response. Muscarinic receptors in homogenates of ileal smooth muscle were labeled with [3H]-1-Quinuclidinyl benzilate. Treatment with propylbenzilylcholine mustard (PrBCM), to inactivate irreversibly muscarinic receptors, caused a large dose dependent rightward shift of the dose-response curve to three agonistic furtrethonium derivatives with a concomitant decrease in maximal response. Using those data, the fraction of receptors remaining unoccupied (q-values) and "true affinity constants" (-log KA-values) were calculated. Exposure to 20 or 60 nM PrBCM for 15 minutes resulted in a 39% and a 61% reduction in specific [3H]-1-Quinuclidinyl benzilate binding sites respectively to be compared with a 62% and a 85% decrease expected from calculated q-values. KA-values for the methyl and ethyl derivative agreed well with the dissociation constants for the high affinity agonist sites determined from displacement of [3H-]-1-Quinuclidinyl benzilate. The KA-value for the propylfurtrethonium corresponds to the low affinity agonist dissociation constant. The fraction of receptors in the high affinity agonist state differs considerably for the three furtrethonium derivatives investigated. Neither the fraction of receptors in the high affinity agonist state, nor the ratio of dissociation constants for these states is affected by the alkylation of 85% of the functional muscarinic receptors. The inactivation of components of the effector system by PrBCM seems unlikely.     

β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [¹²⁵I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K_d=5.3±0.9 pmol/l and R_T=78±7fmol/g tissue). The β₁-selective antagonists atenolol and LK 203-030 inhibited specific [¹²⁵I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β₂-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK_H- and pK_L- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD₂-value of 6.63±0.19.     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Release of dormancy during after-ripening of Agrostemma githago seeds

Freshly harvested seeds of Agrostemma githago L. do not germinate when they are imbibed at 20°C. The block is located in the embryo and is relased by dry storage at 20°C (after-ripening). Freshly harvested seeds complete only a small part of the processes that occur in after-ripened seeds during the lag phase prior to germination (radicle protrusion). After-ripening removed the block on lag phase processes much faster than the block on germination. This was shown both by direct determinations of the completion of lag phase processes and by measurements of the rate of axial protein synthesis, which approximately doubles when seeds are progressing through the lag phase. It is concluded that the percentage germination does not adequately reflect the extent to which the dormancy mechanism has been overcome.     

Plant responses to H2S and SO2 fumigation. I. Effects on growth, transpiration and sulfur content of spinach

Exposure of spinach (Spinacia oleracea L. cv. Monosa) to 0.25 μl l_?1 H₂S reduced the relative growth rate by 26, 47 and 60% at 15, 18 and 25°C, respectively. Shoot to root ratio decreased in plants fumigated at 18 and 25°C. Growth of spinach was not affected by a 2-week exposure to 0.10 or 0.25 μl l_?1 SO₂. Both H₂S and SO₂ fumigation increased the content of sulfhydryl compounds and sulfate. A 2-week exposure to 0.25 μl l_?1 H₂S resulted in an increase in sulfhydryl and sulfate content of 250 to 450% and 63 to 248% in the shoots, respectively, depending on growth temperature. Exposure to 0.15 and 0.30 μl l_?1 H₂S at 20°C for 2 weeks resulted in a 46% increase in sulfate content of the shoots at 0.30 μl l_?1 and no detectable increase at 0.15 μl l_?1 H₂S; the sulfate content of the roots increased by 195 and 145% at 0.15 and 0.30 μl l_?1 H₂S, respectively. Fumigation with 0.25 μl l_?1 SO₂ at 20°C for 2 weeks resulted in an increase in sulfhydryl content and sulfate content in the shoots of 285% and 300 to 1100%. H₂S fumigation during the 12 h light period or only during the dark period resulted in identical growth reduction and accumulation of sulfhydryl compounds; they were about 50 and 67% of those observed in continuously exposed plants. H₂S- and SO₂-exposed plants showed an increased transpiration rate, which was mainly caused by an increased dark-period transpiration. No effect of H₂S and SO₂ on the water uptake of the plants and the osmotic potential of the leaves was detected. Plants fumigated with 0.25 μl l_?1 H₂S for 2 weeks were smaller and differed morphologically from the control plants by slightly more abaxially curved leaf margins. Cross sections of the leaves showed smaller cells at the margins and smaller and fewer air spaces. The increased transpiration in the H₂S-exposed plants is discussed in relation to the observed morphological changes.     

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A₅ (GA₅) as 1- and 1,2-[³H]GA₅ (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [³H]GA-like metabolites were separated from the highly H₂O-soluble [³H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted → isocratic eluted reversed-phase C₁₈ high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 × expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [³H] GA₅ were identified as GA₁, GA₃, and GA₆ by GC-SIM. The major highly water soluble metabolite of [³H]GA₅ at all levels of substrate GA₅ had chromatographic characteristics similar to authentic GA₁-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [³H]GA₅ feeds (2 × expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA₁, GA₃, GA₅ methyl ester, GA₆, GA₂₂, GA₂₉ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA₁, GA₃, GA₅, and GA₈ (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA₆ and GA₂₉ would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA₅. Increasing substrate GA₅ levels increased the proportion of metabolites with HPLC Rts similar to GA₁, GA₆, and especially GA₁ glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA₃, GA₃-glucosyl ester, and a postulated conjugate of GA₆. There was evidence that high doses of substrate GA₅ induced new metabolites which often, but not always, differed from GA₁, GA₃, and GA₆ in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M⁺ and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.