Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland.

The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M(r) range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [125I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (Ka of 4.7 +/- 0.2 x 10(9) M-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M(r) 24,000. The functional status of PRL- and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.

Identification of luteinizing hormone-like proteins in the ovine pineal gland.

A chemical analysis was instigated to investigate the identity of the luteinizing hormone (LH)-like immunoactivity present in ovine pineal protein homogenates. Isolation of pineal LH-like material was accomplished using a 0.1 M ammonium sulphate (pH 4.0) extraction followed by anion-exchange chromatography. The resulting 3.0 M ammonium sulphate precipitate containing 70% of the LH-like immunoactivity was refractionated by cation-exchange and Sephadex G-100 chromatography. Analysis of the pattern of recovered LH-like immunoactivity in the Sephadex G-100 eluate indicated the presence of molecular weight (MW) less than 60,000 besides MW 21,000 species of LH-like proteins. Bioactivity was tested in the rat Leydig cell steroidogenesis assay. In terms of steroid production, the activity was associated with the MW 21,000 LH-like proteins only. Further purification by CM-Sephadex chromatography and gel permeation HPLC was conducted in order to determine whether the physicochemical properties of pineal LH-like material represented endogenous LH, synthesized and released by the ovine pituitary. It is concluded by a variety of means, including polyacrylamide gel electrophoresis, and amino acid and carbohydrate analyses, that at least two molecular forms of immunoactive LH-like proteins occur in ovine pineal tissue. The MW 21,000 forms showed much similarity with ovine adenohypophyseal LH or with a complex mixture of its subunits. These observations contribute to the understanding of endocrine-endocrine transducing events that may occur in this organ.

Partial purification of a polypeptide extract derived from ovine pineal that suppresses the growth of human melanoma cells in vitro.

A combination of gelfiltration and reverse-phase high performance liquid chromatography with postcolumn antitumour assay has been developed. A melatonin insensitive human melanoma cell strain was used to guide the purification of the antitumour effect of an ovine pineal aqueous extract (MW 1,000 to 10,000) that possessed the ability to decrease the hypophysiotropic activity of rat and mice hypothalami in vitro. This allows a specific identification of a pineal factor (MW 2,000 to 6,000) that inhibits the growth of human melanoma cells at a dose of 0.47 mg/ml medium. It was shown that the activity of this pineal compound differs from structures known to be present in the pineal, such as melatonin, pteridines, and beta-carbolines. There appears to be evidence for a peptidic nature of this pineal antitumour factor.

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay.

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.

Characterization of a neurohypophyseal hormone-like activity isolated from ovine pineal glands.

The milk-ejecting response of lactating mouse mammary gland tissue to ovine pineal extracts indicated the presence of a neurohormone-like bioactivity in this tissue. After successive fractionation on gel permeation chromatography and reversed-phase liquid chromatography (HPLC) in conjunction with radioimmunoassays (RIA), it was demonstrated that the milk-ejection response to ovine pineal components with an Mr less than 1,000 corresponded to a biologically active peptide sequence that probably differs from that of arginine vasopressin, arginine vasotocin, and oxytocin and from peptides with a COOH-terminal Pro-Arg-Gly-amide ending. Gel permeation chromatography in formic acid appeared also to indicate the presence of a noncovalent interaction of the neurohormone-like bioactivity with proteins (Mr greater than 25,000) of the pineal.

Studies on a high molecular weight luteinizing hormone release stimulating factor of the ovine pineal gland.

Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high Mr (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in short-term culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226 +/- 23 micrograms LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high Mr forms, slightly heavier than authentic pituitary LH (Mr 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.

Partial purification of (a) low molecular weight ovine pineal compound(s) with an inhibiting effect on the growth of human melanoma cells in vitro.

An in vitro human melanoma cell assay was used to work up the partial purification of (a) low molecular weight (MW) substance(s) from aqueous extracts of ovine pineal tissue shown to contain a growth-inhibiting activity. A combination of paper chromatography, ion-exchange and reverse-phase high performance liquid chromatography with post-column antitumor assay has been developed. This allows a specific identification of an ovine pineal factor (MW less than 500) which inhibits the growth of human melanoma cells in vitro. The substance was partially purified to about 1,000 times as compared to the IC100-value of the starting material (retentate 5). The growth inhibition of human melanoma cells in culture was complete at a dose of 0.1 microgram/ml of purified pineal factor(s). It was demonstrated that the activity of this pineal compound differs from some substances known to be present in the pineal, such as melatonin, serotonin, peridines and beta-carbolines. The activity was not destroyed by treatment with proteolytic enzymes.

Purification and characterization of DSIP-like material from ovine pineal glands: possible peptide-protein interaction.

The nonapeptide delta-sleep-inducing peptide (DSIP) has been isolated from venous blood of rabbits induced to sleep. Numerous reports have described sleep as well as extra-sleep effects. Radiochemical and immunochemical data suggest a relationship of DSIP with the pineal gland supported by interactions of this peptide with pineal functions such as the serotonin N-acetyltransferase activity. In order to demonstrate the natural occurrence of DSIP-like material associated with high Mr proteins in the ovine pineal, organs were water-extracted and fractionated by ultrafiltration and gel filtration. Radioimmunoassay (RIA) for DSIP-like fragments of the fractions revealed considerable amounts of pineal DSIP-like immunoreactivity (DSIP-LI) apparently existing in small as well as large molecular forms. Acidification of large DSIP-LI forms resulted in the elution from Sephadex G-50 of Mr less than or equal to 1,000 DSIP-like material. This free DSIP-LI form coeluted with the synthetic DSIP nonapeptide from microBondapak C18 on high-performance liquid chromatography. The results, therefore, appear to indicate the presence of a (biospecific) noncovalent intermolecular interaction of DSIP (1-9) with proteins (Mr greater than or equal to 10,000) of the ovine pineal gland.

Growth-inhibiting effect of crude pineal extracts on human melanoma cells in vitro is different from that of known synthetic pineal substances.

The effect was studied of a number of synthetic indoleamines, pteridines, beta-carbolines, of AVT and of crude extracts from rat and ovine pineal glands on human melanoma cells in vitro. The identified pineal substances as well as some of their analogues showed an inhibitory effect only at non-physiologically high concentrations. However, crude pineal extracts were more active than the synthetic pineal substances tested. They contain a compound which may have a tumor-inhibiting potency comparable to that of methotrexate but a different mechanism of action.

Lethal mutations induced in Drosophila melanogaster by direct or indirect action of pteridines.

The experiments described in this paper show that synthetic pteridines, especially biopterin and pterin, injected directly into Drosophila melanogaster induce recessive lethals. On the contrary, D-neopterin seems to have little effect. A mutagenic effect has previously been shown for an extract of Pieris brassicae in diapause, treated with these pteridines and tetrahydrofolic acid (FH4). It appears that chromosome II is more sensitive to these mutagenic treatments than chromosome X.

Biological activity of the alkaloids of Erythroxylum coca and Erythroxylum novogranatense.

The cultivated Erythroxylum varieties E. coca var. coca, E. coca var. ipadu, E. novogranatense var. novogranatense and E. novogranatense var. truxillense contain 18 alkaloids, identified so far, belonging to the tropanes, pyrrolidines and pyridines, with cocaine as the main alkaloid. The biological activity of the following alkaloids has been reported in the literature: cocaine, cinnamoylcocaine, benzoylecgonine, methylecgonine, pseudotropine, benzoyltropine, tropacocaine, alpha- and beta-truxilline, hygrine, cuscohygrine and nicotine. The biological activity of cocaine and nicotine is not reviewed here, because it is discussed elsewhere in the literature. Hardly anything is known about the biological activity of the other alkaloids present in the four varieties mentioned. The biosynthesis of the coca alkaloids has been outlined.

A model experiment in the study of cocaine base smoking. Isolation of methyl 4-(3-pyridyl) butyrate from cocaine pyrolysate.

Cocaine base was pyrolysed at 600 degrees C in a nitrogen atmosphere and methyl 4-(3-pyridyl) butyrate was isolated as one of the main components from the cocaine pyrolysate. The structure of the compound was determined by spectral means as well as by comparison with a synthetic sample.

Comparison of some peptidic and proteic ovine pineal fractions with a bovine pineal E5 fraction.

Using rather simple and mild extraction and separation methods, three ovine pineal fractions (XM 300 R-PP7.2' and PP7.2 S) were obtained, which contain peptidic/proteic substances and which show fluorescence characteristics of indoles. The ovine fractions were compared with the bovine pineal E-5 fraction. The ovine fractions are chemically sensitive to normal laboratory light and stable in red light (lambda greater than 600 nm). Immunologically, these fractions and the bovine E 5 fraction are stable. From the results of radioimmunological experiments it was concluded that the bovine pineal E 5 fraction as well as the ovine pineal fraction XM 300 R-PP7.2 and PP7.2S may contain (a) peptide(s) ending by the same carboxy terminal tripeptide Pro-Arg-Gly(NH2).

Papaver bracteatum Lindl.: quantitative extraction and determination of thebaine.

An interlaboratory procedure for the quantitative extraction and analysis of thebaine from different tissues of Papaver bracteatum Lindl. is presented. Each step was evalutated for the yield of thebaine by use of 1-3H-thebaine and GLC. The method of drying and milling of tissue and the size of resultant particles were important factors in the quantitative recovery of thebaine.