The biosynthesis of melanin-concentrating hormone in a fish.

Abstract This work investigated the biosynthesis of a neurohypophysial hormone, melanin-concentrating hormone (MCH), in the trout. Sephadex G-75 chromatography showed the presence of several large MCH-immunoreactive molecules in hypothalamic and pituitary gland extracts, with different retention times on high-performance liquid chromatography from the mature MCH(1-17). About 10% of the total MCH-immunoreactivity in the hypothalamus was attributable to large molecular weight forms but these contributed less than 1% to the immunoreactivity in the neurointermediate lobe. Both [(35) S]methionine and [(3) H]leucine were injected into the hypothalamus near the MCH perikarya (nucleus lateralis tuberis region) of anaesthetized fish, after which the fish were killed at intervals of up to 8 h post-injection and the basal hypothalami, pituitary pars distales and neurointermediate lobes were extracted in acid. MCH-related immunoprecipitates from these extracts were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis or by Sephadex G-50 chromatography. The results show the incorporation of radiolabel into 15.3 K and 11.3 K precursors within 0.75 h, and their conversion, via several smaller intermediates, to a molecule resembling MCH(1-17). The results are discussed in relation to the known cDNA sequence of salmon MCH. Labelled MCH first appeared in the neurointermediate lobe 4 h after injection, giving an estimated transit rate of 0.4 mm/h.

A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe.

Corpora lutea and follicles were taken from the ovaries of 12 ewes at intervals from the start of luteolysis until 3 days after ovulation. RIA analysis of the tissue oxytocin content showed that luteal oxytocin concentrations declined during luteolysis to reach basal values at about the time of the next ovulation. Oxytocin was first measurable in the walls of 3 out of 6 preovulatory follicles during the LH surge, with a small increase in concentration to 26.1 +/- 6.6 pg/mg before ovulation, and a further increase in the young corpus luteum to concentrations exceeding 1 ng/mg 2-3 days later. After the LH surge, oxytocin was also found in the follicular fluid at a concentration of 3.4 +/- 0.3 ng/ml. Using immunocytochemical techniques, oxytocin and neurophysin were first detected in the follicle wall immediately before ovulation, and were localized in the granulosa cells. After ovulation the stained cells initially formed strands which appeared to break down to clusters and then to individual cells as the corpus luteum matured. The immunocytochemical picture also suggested that neurophysin immunoreactivity increased within a few hours of ovulation but that processing to oxytocin may be delayed. Measurements of circulating oxytocin concentrations revealed a pulsatile release pattern throughout the follicular phase with the height of the pulses decreasing from 25 +/- 5 pg/ml during luteolysis to a minimum of 11 +/- 2 pg/ml during the LH surge.

Localization of chromatographically characterized oxytocin and arginine-vasopressin to sensory neurones in the rat.

Following treatment with colchicine 50-60% of all neurones in rat trigeminal and L5 spinal ganglia showed oxytocin (OXT)- and arginine-vasopressin (AVP)-like immunoreactivity. Further, OXT and AVP, together with their associated neurophysins, could be isolated from trigeminal ganglia by reversed-phase high-performance liquid chromatography followed by radioimmunoassay.

Biosynthesis of oxytocin in the corpus luteum.

In this report we demonstrate that ovine and bovine luteal cells synthesise oxytocin by way of a precursor protein similar to that found in the hypothalamus. Isolated ovine or bovine luteal cells were incubated for up to 12 h with [35S]cysteine. Neurophysin-Sepharose column separation and HPLC of cell extracts demonstrated the presence of [35S]oxytocin. Incorporation of [35S]cysteine was confirmed by performic acid oxidation. Immunoprecipitation of cell extract with anti-rat oxytocin-neurophysin followed by SDS-PAGE yielded 2 radioactive bands of 14 kDa and 11-12 kDa. Immunoprecipitation with anti-oxytocin yielded 1 band at 14 kDa. On SDS-PAGE the 14 kDa band had a similar mobility to rat-hypothalamic oxytocin precursor.

Variations in oxytocin, vasopressin and neurophysin concentrations in the bovine ovary during the oestrous cycle and pregnancy.

Bovine ovaries were obtained from the abattoir and corpora lutea were classified as: (1) early luteal phase (approximately Days 1-4); (2) mid-luteal phase (Days 5-10); (3) late luteal phase (Days 11-17); (4) regressing (Days 18-20) and (5) pregnant (Days 90-230). In addition, preovulatory follicles and whole ovaries without luteal tissue were collected. Concentrations of oxytocin, vasopressin, bovine neurophysin I and progesterone were measured in each corpus luteum by radioimmunoassay. Progesterone and neurophysin I levels increased from Stage 1 to Stage 2, plateaued during Stage 3 and declined by Stage 4. Oxytocin and vasopressin concentrations increased from Stage 1 to Stage 2 but declined during Stage 3 and were low (oxytocin) or undetectable (vasopressin) in follicles, whole ovaries and pregnancy corpora lutea. Therefore the concentrations of both peptide hormones were maximal during the first half of the cycle and declined before those of progesterone. The high concentration of oxytocin within the corpus luteum coupled with the presence of bovine neurophysin I suggests that oxytocin is synthesized locally.

Identification of oxytocin and vasopressin in the testis and in adrenal tissue.

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.

A double-blind study of the effectiveness of a 3% erythromycin and 5% benzoyl peroxide combination in the treatment of acne vulgaris.

One hundred sixty-five subjects completed a 10-week, double-blind controlled study comparing the following: (1) a combination of 3% erythromycin and 5% benzoyl peroxide in a gel, (2) 5% benzoyl peroxide gel, (3) 3% erythromycin gel, and (4) the gel vehicle. The benzoyl peroxide gel and the erythromycin gel were superior to the control gel; however, the combination product was more effective than any of the others. This was true for both pustular and papular lesions, but the most dramatic effect was on combined inflammatory lesions, i.e., papules and pustules.

Analysis of the neurohypophyseal components accumulating in the supraoptic nucleus of the rat after injection of colchicine.

[35S]Cysteine has been injected into the supraoptic nuclei of normal rats and of animals given 7 micrograms colchicine into the cerebrospinal fluid to inhibit transport of neurosecretory granules. Analysis of extracts of the supraoptic nuclei 20 min or 6 h after isotope injections showed that (i) colchicine does not affect biosynthesis, i.e., incorporation of tracer into the common precursors of neurohypophyseal hormones and their associated neurophysins, and (ii) processing of precursors inside the arrested granules proceeds normally.

Characterization of oxytocin, vasopressin, and neurophysin from the bovine corpus luteum.

Acid extracts of corpora lutea collected from nonpregnant cows were found to contain oxytocin, arginine vasopressin, and neurophysin. The inhibition curves of the oxytocin and vasopressin extracts showed parallelism with the appropriate standard preparations in specific RIAs and eluted at the same position as the standards using high performance liquid chromatography (HPLC). The neurophysin extract showed parallelism in a bovine neurophysin I RIA and had a similar elution position to the standard on both Sephadex G-50 and HPLC. However, its immunoreactive profile on HPLC differed slightly from that obtained with hypophyseal bovine neurophysin I. In nonpregnant cows the oxytocin content (about 1 microgram g-1 wet wt of tissue) was three orders of magnitude greater than the vasopressin content. Levels of luteal oxytocin were considerably lower in pregnant animals. These results show that the bovine ovary is a rich source of neurohypophysial peptides and suggest that oxytocin biosynthesis may occur within the corpus luteum.

Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography.

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.

Effects of tunicamycin on the hypothalamo-neurohypophysial system of the rat.

Intracisternal injections of tunicamycin, an inhibitor of glycosylation, decreased the incorporation of [35S]cysteine into the neurophysins in the rat neurohypophysis. Histochemical and immunocytochemical studies showed that there was no concomitant decrease in the amount of secretory product in the perikarya of the hypothalamo-neurohypophysial neurones. Indeed there was an increase, although this was not associated with neurosecretory granules as judged electron-microscopically. Tunicamycin led to the formation of so-called "colloid droplets" which were immunopositive and of which the ultrastructural correlates appeared to be product-filled dilatations of the rough endoplasmic reticulum. The observations are interpreted to suggest that glycosylation plays a rôle in the packaging of secretory material in the hypothalamo-neurohypophysial system.

Isolation and properties of a tryptophan-containing peptide from bovine and turkey brains.

A tryptophan-containing peptide has been isolated from bovine cerebral hemispheres. A similar peptide has also been isolated from the turkey brain. Isoelectric focusing on polyacrylamide gels shows the peptides to be essentially homogeneous. Both peptides consist of 67 amino acids with isoleucine at the COOH-terminus. The NH2-terminal residues cannot be detected by end-group analysis. Both peptides exhibit 45--50% alpha-helix as revealed by circular dichroism spectra.

beta-Endorphin: stability, clearance behavior, and entry into the central nervous system after intravenous injection of the tritiated peptide in rats and rabbits.

Rabbits and rats were given intravenous injections of tritiated human beta-endorphin. The levels of beta-endorphin were followed by the decrease in radioactivity in the plasma of rats or rabbits and by the increase in radioactivity in the cerebrospinal fluid of the rabbit. The results were identical with the tritium label on either tyrosine-1 or -27. The plasma distribution times were 2 and 5 min in the rat and rabbit, respectively, with a later clearance time of approximately 1-8 hr. In the rat, approximately 50% of the radioactivity in the plasma was found to be intact human beta-endorphin 45 min after injection. Radioactivity appeared in the cerebrospinal fluid of the rabbit within 30 sec after injection and reached a plateau in approximately 60-90 min after injection. Approximately 75% of the radioactivity in the cerebrospinal fluid of the rabbit was intact human beta-endorphin. In the brain hemispheres of the rat and the rabbit, the only significant radiolabeled product was found to be radioactive tyrosine. Moreover, rat plasma levels of beta-endorphin decreased dramatically after hypophysectomy, which only slightly lowered the levels in the brain. It appears that beta-endorphin, upon entry into the plasma, is either not significantly taken up into the brain or is broken down with extreme rapidity upon entry into the brain, although it apparently does enter the cerebrospinal fluid.

Isolation and characterization of beta-endorphin-like peptides from bovine brains.

Four beta-endorphin-like peptides from bovine brain extracts have been identified by their behavior in CM-cellulose chromatography, gel filtration, high-performance liquid chromatography, and radioimmunoassay. Two of them have been isolated in sufficient quantity for amino acid analysis, radioimmunoassay, and radioreceptor assay. One peptide has an amino acid composition nearly identical to that of beta-endorphin with 56% of the radioimmunoreactivity and 2.3% of the potency in the radioreceptor assay of beta-endorphin. The amino acid content of the other beta-endorphin-like peptide is very different from that of bovine beta-endorphin but it has 47% of the radioimmunoreactivity and 1% of the potency in the radioreceptor assay of beta-endorphin.

Characterization of turkey myelin basic protein isolated by a simple procedure.

Highly purified basic proteins have been isolated from bovine and turkey brains by a novel method employing acid-acetone extraction. The final product gave a single band on polyacrylamide gel electrophoresis at pH 4.3 and in the presence of sodium dodecyl sulfate. Both proteins have arginine at the COOH-terminus while the NH2-terminal residue cannot be detected and is probably blocked. A higher ratio of histidine to lysine and a greater proportion of serine and valine was found for the turkey compared with the bovine protein. Both proteins contain one tryptophan and two methionine residues. However, it was found from cyanogen bromide treatment that there is a marked difference in the location of one of the methionine residues, while the tryptophan-containing peptides liberated after trypsin digestion have different mobilities on peptide maps. When dissolved in water these proteins give a typical random coil curve from circular dichroism (CD), whereas in 80% methyl alcohol they assume a 25% alpha-helix.