Fertility control in female rats by bovine pineal gland extracts.

Extracts of bovine pineal glands were partially purified by gel-filtration after initial extraction by an aqueous methanol-isobutanol method or with acetic acid. The extracts were tested for their ability to block pregnancy in young mature rats. The isobutanol extract yielded one fraction which was inhibitory to pregnancy when injected for one estrous cycle. Two distinct fractions from the acetic acid extract, each of which partially blocked compensatory ovarian hypertrophy, were also found to be contraceptive. The compounds responsible for these actions in either of the extracts are thought to be small polypeptides which are probably inhibitory to LH secretion.     

Inhibition by styrylpyridines of purified rat and bovine brain choline acetyltransferase

Nine styrylpyridine analogs were tested as inhibitors of choline acetyltransferase which had been highly purified from rat cerebrum and bovine caudate nuclei. In general, concentrations required to achieve 50% inhibiion (I₅₀ values) were in the micromolar range. For some analogs, I₅₀ values were similar to those obtained previously by other investigators who used less purified enzyme preparations. With certain analogs, however, the measured values of I₅₀ changed as the transferase became more purified, which may indicate the presence in the extract of other molecules which can interact with the enzyme. The methods used in purification of the enzyme suggest that the molecule which modifies the activity of CAT is probably a protein. The mode of inhibition by naphthylvinylpyridinium was found to be uncompetitive with respect to both choline and acetyl coenzyme A for both the rat and bovine transferases.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E- and prostaglandin F-like material (304 ± 20 and 582 ± 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10⁻⁴–10⁻⁶M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of ³H-prostaglandin E₂ (³H-PGE₂) and ³H-prostaglandin F_2α (³H-PGF_2α) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca⁺⁺ concentration in medium up to 2 mM, was heat-labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal ³H-PG binding was found in the 27000 × g pellet. Scatchard analysis of ³H-PGE₂ binding revealed the presence of a single population of binding sites with a K_d= 1.2 nM and a binding site concentration of 1–2 pmoles/g protein. A single population of binding sites for ³H-PGF_2α was also detected with a K_d= 1.7 nM and a similar binding site concentration. Non-radioactive PGE₁ and PGE₂ were almost equally effective to compete for ³H-PGE₂ binding sites (ED₅₀= 5 and 2 nM, respectively). Unlabeled PGF₂ was relatively ineffective to compete for ³H-PGE₂ binding (ED₅₀>1000 nM) but displaced effectively ³H-PGF_2α binding (ED₅₀= 1.2 nM).     

Arginine vasotocin mRNA revealed by in situ hybridization in bovine pineal gland cells

Arginine vasopressin (AVP) is the main antidiuretic hormone in mammals and arginine vasotocin (AVT) in submammalian vertebrates. The possibility that the genetic material encoding AVT is maintained in mammals is controversial. In this study, we investigated by radioactive in situ hybridization the possible presence of the mRNA encoding AVP and AVT, and using immunocytochemistry the presence of structures immunoreactive for AVP and AVT in the bovine pineal gland. In situ hybridization was performed by use of ³⁵S-labelled oligoprobes. Immunocytochemistry was performed using specific polyclonal rabbit antibodies and the avidin-biotin-complex method. In situ hybridization revealed positive signals for both AVP mRNA and AVT mRNA in a few cells scattered throughout the pineal body. Immunocytochemistry revealed thin AVP-immunoreactive fibres in the pineal stalk and the pineal gland. It also revealed staining of several AVT-immunoreactive nerve fibres in both the pineal stalk and the gland. In addition, polyhedral, neuron-like cell bodies from which two to three processes emerged were also AVT-immunoreactive. Thus, our investigation shows the presence of AVP/AVT-immunoreactive cellular structures in the bovine pineal gland. Our data further show the presence of mRNAs encoding both AVT and AVP. We therefore suggest that AVT mRNA is translated into an AVT-like peptide in the bovine pineal.     

Production of highly purified hydroxytyrosol from Olea europaea leaf extract biotransformed by hyperthermophilic beta-glycosidase

A large amount of highly purified hydroxytyrosol (91-94% in weight) is obtained in short time by a simple biotransformation of Olea europaea leaf extract by a partially purified hyperthermophilic beta-glycosidase immobilized on chitosan support. The biotransformation conditions have been modulated for increasing the hydroxytyrosol yield, whilst chitosan and chitin matrices are used as adsorbent materials in liquid phase hydroxytyrosol extraction from the biotransformed mixtures. Natural and non-toxic hydroxytyrosol has been by this way produced from a vegetal source, and this compound appeared for the first time highly purified by natural and biocompatible safe biopolymers in comparison to previous results. Moreover, the GC analyses have displayed that the eluates from a two-step bioreactor have qualitative composition very similar to that of the extra-virgin olive oil polar fraction. The proposed bioreactor could also find application in the utilization of olive mill waste waters (OMWW), medium rich in large amounts of oleuropein, which can be converted in pharmacologically active compounds.     

Studies on the properties of muscarinic cholinergic receptor sites in bovine pineal gland

1. Using the tritiated muscarinic receptor antagonist, quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic cholinergic receptors have been identified and characterized in the pineal glands of cow and swamp buffalo. 2. At 25 degrees C, the specific binding reached equilibrium within 60 min and remained constant for an additional two hours. Furthermore, the specific binding was saturable, reversible and tissue dependent in nature. 3. The kinetic analyses of muscarinic cholinergic receptor sites revealed KD values of 0.423 +/- 0.01 nM and 0.218 +/- 0.01 nM, and Bmax values of 69.75 +/- 20.91 fmol/mg protein and 74.19 +/- 32.73 fmol/mg protein for the cow's- and the swamp buffalo's pineal glands, respectively. 4. The presence of muscarinic cholinergic receptor sites originating from cholinergic innervation of the pineal gland is suggested.     

Purification of leukocytosis-promoting substance from bovine parotid gland extract

A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 · 10⁴, and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.