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Biomedical subjects

H R Lindner

Publications and source records attributed to H R Lindner.

At least 19 recordsLinked to original sources
PubMed1983

Development of chemiluminescence monitored immunoassays for steroid hormones.

Solid-phase chemiluminescence based immunoassay methods for the measurement of steroids and urinary steroid conjugates are described. The assays incorporate the following features: (i) a steroid-chemiluminescent marker conjugate serves as the labeled ligand; (ii) polyclonal or monoclonal antibodies covalently coupled to polymer beads are utilized as the specific immunoadsorbent; and (iii) a centrifugation step after the binding reaction is used to remove interferring luminescent compounds along with unbound steroid. These assays proved satisfactory with respect to sensitivity, precision and accuracy and offer significant advantages over existing methods in speed, safety and economy. Moreover, the use of monoclonal antibodies provides optimal specifity and rigorous standardization.

Animals↗
PubMed1983

Receptor-mediated internalization of LHRH antagonists by pituitary cells.

A fluorescently labeled antagonist of luteinizing hormone releasing hormone (LHRH), D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys6-(tetramethylrhodamine)-Leu-Arg-Pro-Gly-NH2, was prepared. This peptide retained high-affinity binding to the LHRH receptor of pituitary plasma membrane preparations. The analog was able to block LHRH-stimulated LH release from pituitaries incubated in vitro, and exhibited minor agonistic activity. This rhodamine-labeled antagonist was utilized for the microscopic visualization and localization of LHRH receptors in dispersed rat pituitary cells. The fluorescently labeled receptors were initially distributed uniformly on the cell surface. The hormone-receptor complexes were redistributed after incubation at 23 degrees C and formed clusters which subsequently became internalized (at 37 degrees C) into endocytic vesicles. Addition of LHRH (10(-6) M) abolished these processes, indicating specific binding sites for the rhodamine-labeled peptide to the gonadotrope cells. A quantitative comparison of temperature-dependent internalization by iodinated LHRH agonist and antagonist revealed that both analogs were internalized to a similar extent. These findings suggest that LHRH-receptor complex internalization is related to LHRH receptor regulation.

Animals↗
PubMed1983

Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin-releasing hormone analog on ovarian functions.

The paradoxical effects of gonadotropin-releasing hormone (GnRH) on the ovary have hitherto been believed to result from different regimens of administration; an acute treatment was shown to stimulate the ovary while chronic administration of the hormone inhibited LH-induced responses. In the present report we demonstrate that a single injection of a GnRH analog (D-Ala6)des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) is sufficient to obtain a significant inhibition (75%) of hCG-induced ovulation in PMSG-primed, either intact or hypophysectomized, immature rats. Inhibition of ovarian development, in terms of growth and ovulation, by multiple injections with GnRHa (2 micrograms/rat, twice daily for 3 days) could be obtained only upon administration of the hormone at early stages of follicular development, i.e. concomitantly with the PMSG injection. When administered after PMSG, GnRHa could not inhibit the ovary but rather induced ovulation by itself in the absence of hCG. A 12-24 h delay in initiation of GnRHa treatment triggered 65% of the rats to ovulate while a delay of 48 h resulted in 100% ovulation. Under both regimes of GnRHa administration, either the inhibitory or the stimulatory, the oocytes of the treated rats were induced to resume meiotic maturation. Since under the inhibitory regime ovulation did not occur, maturation was followed by a massive degeneration of the oocytes trapped within their follicles. These findings demonstrate that the follicular stage of development rather than the dose and/or duration of GnRHa administration determines whether GnRHa inhibits ovarian growth and ovulation, while the competence of the oocytes to respond to the GnRHa stimulus and mature is independent of hormonal priming.

Animals↗
PubMed1983

Follicle-stimulating hormone-induced prostaglandin E formation in isolated rat ovarian theca.

The aim of this study was to search for direct biochemical effects of highly purified FSH on isolated ovarian follicular theca in vitro. Granulosa cells (GC; approximately 1 x 10(5) cells per follicle) were flushed from isolated follicles of pro-oestrous rats. The remaining theca layer and the isolated GC were incubated with highly purified ovine FSH. Prostaglandin E (PGE) accumulation was measured by radioimmunoassay. Follicle-stimulating hormone induced a 15-fold increase in PGE accumulation over the basal level in the follicular theca, the stimulated rate exceeding threefold that observed in the GC fraction derived from the same follicle. Follicle-stimulating hormone caused no significant increase in cyclic AMP level or steroidogenesis in the theca layer, but was active on these parameters in the GC. In contrast, LH increased the accumulation of cyclic AMP, progesterone and testosterone, as well as of PGE, in follicular theca. Exogenous 8-bromo cyclic AMP or cyclic GMP also stimulated PGE production in follicular theca or GC, but FSH was without any effect on the level of endogenous cyclic GMP in GC or follicular theca. Antibodies to FSH prevented the effect of FSH (but not that of LH) on PGE formation by follicular theca and GC, while antibodies to the beta-subunit of LH blocked the effect of LH but not of FSH. We conclude that highly purified FSH has a stimulatory effect on PGE formation by the follicular theca.

Animals↗
PubMed1982

Preparation of monoclonal antibodies able to discriminate between testosterone and 5 alpha-dihydrotestosterone.

A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl) oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (Ka = 10(10) 1/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5 alpha-dihydrotestosterone (range 2-100%) and androstenedione (less than 0.1-4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.

Animals↗
PubMed1982

Inhibition by lithium of PGE1-sensitive adenylate cyclase in neuroblastoma X glioma hybrid cells: approach to the attenuation of the opiate withdrawal syndrome.

Exposure of neuroblastoma X glioma hybrid cells to LiCl (Li+) for 48 h significantly reduces prostaglandin E1 (PGE1)-dependent cyclic adenosine monophosphate (AMP) accumulation. When applied simultaneously with morphine for the same period, Li+ inhibits the excessive accumulation of cyclic AMP in response to PGE1 characteristically exhibited by these cells upon opiate withdrawal. Likewise, Li+ inhibits, in vitro, PGE1-dependent cyclic AMP accumulation in tissue slices from rat corpus striatum. Prophylactic administration of Li+ in the diet attenuates the withdrawal symptoms in morphine-addicted rats. These findings support clinical reports that suggest that prophylactic treatment with Li+ attenuates the severity of the opiate withdrawal syndrome.

Adenylyl Cyclase Inhibitors↗
PubMed1982

A novel method for localization of gonadotropin releasing hormone receptors.

Two 125I-labeled analogs of GnRH, [acidobenzoyl-D-Lys6]GnRH (I) and [D-Lys6]GnRH (II) were used for the localization of GnRH receptors in pituitary cells. The analogs exhibited high binding affinity to pituitary membrane preparations and after photoactivation (analog I) or cross-linking with glutaraldehyde (analog II), these analogs are bound covalently to pituitary cells. The distribution of the labeled hormones by light and electron microscopic autoradiography indicated that after exposure of pituitary cells to 125I-labeled hormones at 4 C (90 min), most of the labeled hormones were associated with the cell surface membrane, while at 37 degrees C (30 min) most of the cell-bound labeled hormones were internalized.

Animals↗
PubMed1982

Measurement of plasma estradiol-17 beta by solid-phase chemiluminescence immunoassay.

We describe a simple, solid-phase chemiluminescence immunoassay for the measurement of estradiol-17 beta in extracts of peripheral venous plasma. The method has similar sensitivity, specificity, precision, and accuracy to a conventional radioimmunoassay with a tritiated antigen. An immunoglobulin G fraction od monoclonal antibodies to estradiol-6-carboyxmethyl oxime-bovine serum albumin is passively adsorbed onto the walls of polypropylene tubes. The labeled antigen is estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol. After the binding reaction (1 h at 22 degrees C), the solution is removed by aspiration (400 microliters). Sodium hydroxide (5 mol/L, 300 microliters) is added and the mixture incubated for 30 min at 37 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. The light yield is inversely proportional to the concentration of estradiol in the standard or sample.

Adult↗
PubMed1981

Elevation of apparent membrane viscosity in ovarian granulosa cells by follicle-stimulating hormone.

Continued exposure of cultured granulosa cells to follicle-stimulating hormone (FSH) induced: (i) a rise in apparent membrane microviscosity, as reflected by an increase in fluorescence polarization of the lipid-soluble probe, 1,6-diphenyl-1,3,5,-hexatriene; and (ii) a progressive decline in the cyclic AMP response to renewed challenge with the same hormone. Both changes were reduced or prevented by pretreatment of the cells with oleic or linoleic acid, agents which reduce membrane viscosity, but not by elaidic or palmitic acid which increase the rigidity of membrane lipids. Other agents that inhibited FSH-induced changes in membrane fluidity (gonadotropin-releasing hormone, actinomycin D and cycloheximide) also prevented desensitization to FSH. Cyclic AMP and cyclic GMP derivatives did not mimic the effects of FSH on apparent membrane viscosity or desensitization. Changes in membrane fluidity are unlikely to be the sole cause of desensitization since (i) pretreatment of the cells with fatty acids that increase lipid viscosity did not induce desensitization to FSH, and (ii) desensitization of granulosa cells to lutropin and prostaglandin E2 by exposure to the homologous hormone was not attended by increased membrane viscosity. The experiments described provide the first example of a hormonally induced increase in the target cell apparent membrane viscosity.

Animals↗
PubMed1981

Possible involvement of macrophages in embryo--maternal relationships during ovum implantation in the rat.

Distribution of phagocytic cells in the rat endometrium during the estrous cycle and early gestation was examined by histological, electron microscopic, and histochemical techniques. The results show that numerous macrophages emerge around the nidus shortly after the onset of ovum implantation. Such macrophages, however, were not present within the decidua, suggesting that this tissue may be a protective barrier against the migration of phagocytic cells towards the implants. Approximately 48 hours after the onset of implantation, the number of endometrial macrophages decreased dramatically.

Animals↗
PubMed1981

Development of a solid-phase chemiluminescence immunoassay for plasma progesterone.

A solid-phase immunoassay procedure for the determination of progesterone in human plasma is described, which utilizes chemiluminescence as the end-point. A progesterone-isoluminol conjugate serves as the chemiluminescent marker. An IgG fraction of antiserum to progesterone-11 alpha-hemisuccinate bovine serum albumin is passively adsorbed to the walls of Lumacuvettes P polystyrene test tubes. After the binding reaction, the cuvettes are washed. The light yield of the bound conjugate upon oxidation with a H2O2-microperoxidase system at pH 13 decreased with increasing free progesterone concentration in a log-linear manner over the range 15-1000 pg/tube. The chemiluminescence immunoassay is comparable to radioimmunoassay with regard to sensitivity, specificity, precision and accuracy. The assay offers the advantages of speed and ease of automation. In addition, this method does not involve the use of radioisotopes or of a centrifugation step.

Animals↗
PubMed1981

Use of monoclonal antibodies to pregnanediol-3 alpha-glucuronide for the development of a solid phase chemiluminescence immunoassay.

Monoclonal antibodies to pregnanediol-3 alpha-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specificity and affinity to pregnanediol-3 alpha-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3 alpha-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r = 0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.

Animals↗