I. Ebels

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 reversephase high performance liquid chromatography with post-column antitumor assay has been developed. This allows a specific identification of an ovine pineal factor (MW<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 (retentate5). The growth inhibition of human melanoma cells in culture was complete at a dose of 0.1 μg/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 β-carbolines. The activity was not destroyed by treatment with proteolytic enzymes.

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.

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

Abstract: 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 Mr 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 × 109 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 Mr 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.

Partial Purification of a Polypeptide Extract Derived From Ovine Pineal That Suppresses the Growth of Human Melanoma Cells In Vitro

A combination of gel filtration 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 pined 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 β‐carbolines. There appears to be evidence for a peptidic nature of this pineal antitumour factor.

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 < 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 > 25,000) of the pineal.

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

Abstract: 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) < 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 physicochemi‐cal 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 M W 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.

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≤ 1,000 DSIP‐like material. This free DSIP‐LI form co‐eluted with the synthetic DSIP nonapeptide from μBondapak 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≥ 10,000) of the ovine pineal gland.

Pteridines in the Pineal and Effects of These Substances on the Indole Metabolism of This Organ

Pteridines are very light sensitive substances. The first compounds of this class were isolated as a yellow pigment from the wings of the brimstone butterfly by Wieland and Schopf (1925) and as the white pigment from the wings of the cabbage butterfly by Schopf and Wieland (1926). These pigments were named xanthopterin and leucopterin indicating the colour and source of the compounds. The real chemical structure of these substances was not solved until 1940 by Purrmann. The structure of a third insect pigment, isoxanthopterin was also elucidated by Purrmann in 1941. The well-known structure folic acid, and its derivatives which play a key role in metabolism, contains also a pteridine ring. For a survey of the most important literature in the pteridine field till 1969, see Blakly’s “Biochemistry of folic acid and related pteridines”.

The influence of light of different wavelengths on the methylating capacity of the pineal gland of male golden hamsters in relation to reproduction.

In the present experiments the influence of light of different wavelengths on pineal indole metabolism in relation to reproduction was studied. Therefore, during autumn and winter male golden hamsters were kept under natural conditions but for the sunlight which was filtered exposing the hamsters to either normal (control), red or blue light. During the gradually shortening photoperiod at the start of the experiments under normal light conditions, a marked decrease of FSH and LH plasma content as well as testicular weight was found, indicating the onset of gonadal atrophy. During this period a high synthesis of 5-methoxytryptophan (MW) and 5-methoxytryptamine (MT) was determined. The synthesis of other 5-methoxyindoles (MI) was low, while O-acetyl-5-methoxytryptophol (aML) synthesis even markedly decreased. Red and blue light did not cause significant changes in MI synthesis. As long as MT synthesis is high (under blue light), there is no increase in FSH content and testes weight is still decreasing. This influence of blue light confirms the putative antigonadotropic properties of MT. The increase of FSH content at week 9 was the first indication that recrudescence had started. At week 19, this recrudescence was also manifested in the increasing testes weight. The synthesis of melatonin (aMT), 5-methoxytryptophol (ML), 5-methoxyindole-3-acetic acid (MA) and aML increased whereas the production of MT decreased. Blue light exposure caused a significantly higher increase of synthesis of ML, MA, aML and, not-significantly, of aMT, whereas red light caused a significantly lower synthesis of MA. It was concluded that MT, a putative antigonadotropic, and aML, a putative counter-antigonadotropic, are probably important pineal compounds that transduce the photoperiodic messages, which cause either gonadal atrophy or recrudescence. The effect of blue light on indole metabolism and the reproductive cycle was more clear than that of red light. From the present results of blue light on indole metabolism, it was suggested that blue light delayed gonadal atrophy and stimulated gonadal growth, compared to red light. An opposite effect of red light was less obvious.

The Influence of Some Pteridines on the Circadian and Seasonal Rhythmicity of Hydroxyindole-O-Methyltransferase (HIOMT) in the Pineal Gland of 42-days-old Male Wistar Rats

ABSTRACT The pineal of 42 days old male Wistar rats was studied with regard to the influence of some pteridines on HIOMT activity in different seasons of the year. The circadian rhythmicity was influenced by pteridines, when the maximal HIOMT activity was measured at 24.00 oclock (in June and in the period from half September till the beginning of October). There was no or only a small influence detectable, when the maximal HIOMT activity was determined at 04.00 oclock (in April and in the last half of October). These alterations are probably of physiological importance.