Identification of the mRNA coding for the ACTH-beta-lipotropin precursor in a human ectopic ACTH-producing tumor

The mRNA coding for the ACTH-β-lipotropin precursor from a human ectopic ACTH-producing thymic carcinoid was identified by blot hybridization analysis with the bovine cDNA as a probe. The mRNA from the tumor had the same length (approximately 1,100 nucleotides) as that from the human pituitary. An additional hybridization-positive RNA species of a larger size was found in the tumor. Cell-free translation of the mRNA from the tumor as well as from the pituitary yielded a product with an apparent molecular weight of 38,000 that was reactive both with antibody to ACTH and with antibody to β-endorphin.     

Pro-opiocortin peptides in rat cerebrospinal fluid

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae was subjected to gel filtration chromatography on Sephadex G-50. Fractions were monitored using radioimmunoassays for N-terminal pro-opiocortin (N-POC), gamma 3-melanotropin (gamma 3-MSH), C-terminal adrenocorticotropin (C-ACTH), alpha-endorphin, beta-endorphin, gamma-lipotropin (gamma-LPH) and alpha-MSH. Two peaks which corresponded in elution position to rat N-POC (1-74) and gamma 3-MSH were detected. The major C-ACTH-immunoreactive (IR) peak was found to correspond to 14k ACTH. While no alpha-endorphin immunoreactivity was detected in rat CSF, three beta-endorphin-IR peaks were identified in positions expected for beta-LPH, beta-endorphin (1-31) and beta-endorphin (1-27), as well as a major peak of activity with the elution characteristics and cross-reactivity of rat gamma-LPH. HPLC of the alpha-MSH-IR material in rat CSF revealed the presence of a major peak of immunoreactivity whose retention time did not correspond to the known oxidised and reduced forms of alpha-MSH and its desacetylated and diacetylated derivatives. The identity of this peak is unknown.     

Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum.

β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [¹²⁵I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At different time intervals the release of [¹²⁵I]-tyrosine or the change in immunoreactivity of the endorphins was determined. The cSPM preparation displayed both high aminopeptidase and endopeptidase activities. In contrast, human serum mainly contained aminopeptidase activity. The data suggest that functional endorphin metabolism may occur at the synaptosomal plasma membrane. These membranes may potentially be involved in the formation of behaviorally active endorphin fragments.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Isolation of corticotropin-β-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-β-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824–2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel eletrophoresis, using immunoprecipitated and electrophoretically purified [¹²⁵I]corticotropin-β-lipotropin common precursor as a marker. The preparation was judged homogenous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and β-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-β-lipotropin common precursor possessed specific activities of 116 μg of immunoreactive corticotropin and 210 μg of immunoreactive β-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Occurrence of a new melanocyte stimulating hormone in the salmon pituitary gland

A new melanocyte stimulating hormone has been identified in the pituitary of the teleost, $\text{Oncorhynchus keta}$ (chum salmon). The newly isolated MSH like peptide is a heptadeca peptide, which differs in size from salmon α-MSH (13 residues) and β-MSH I and II (17 residues each). The structural determination, however, revealed that it is similar to but distinct form α-MSH, with following amino acid sequence, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Ile-Gly-His-OH. This peptide, named α-MSH II is the third line of evidence in the salmon that the teleost pituitary gland secretes two different forms of processed hormones, for which the precursor molecules are coded on two separate genes.     

High pressure NMR studies of hemoproteins. The effect of pressure on the quaternary structure of hemoglobin

Proton NMR spectra for nitrosyl-, aquomet- and deoxy des-Arg(α141)-hemoglobin in H₂O were studied at high pressures up to 1400 atm with attention to the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds. For aquomethemoglboin, the T state marker signal at 6.4 ppm is insensitive to pressure while the R state marker signal at 6.0 ppm exhibits progressive upfield shift upon pressurization. For nitrosylhemoglobin, the T state signals at 9.6 and 6.5 ppm decrease their intensities upon pressurization while the R state marker signal at 6.0ppm remains unchanged. Pressure-induced spectral changes for some of exchangeable resonances are also encountered for deoxy des-Arg(α141)-hemoglobin while the R and T quaternary structural indicators at 6.0 and 9.4 ppm are insensitive to pressure. These pressure-induced spectral changes for these hemoglobin derivatives are significantly distinguished from those associated with the R-T transition induced by addition of IHP or by variatiuon of pH. It is therefore concluded that pressure induces subtle quaternary structural changes in these hemoglobin derivatives without causing the R-T transition.     

β-Endorphin: Complete primary structure is required for full analgesic activity

The synthesis of β_h-endorphin-(1–30) has been accomplished by the solid-phase procedure and its analgesic potency was assayed by the tail-flick method. Results showed that the synthetic analog had only 56% activity of the parent molecule. Thus, the complete sequence of 31 amino acid residues in β_h-EP is required for full analgesic activity.     

Des-acetyl MSH and γ-MSH act as partial agonists to α-MSH on the Anolis melanophore

The biological activities of α-MSH des-acetyl MSH, γ-MSH and LPH_37–58 were compared using the Anolis rate method of bioassay. Dose-response data showed LPH_37–58 to be equipotent with α-MSH, but des-acetyl MSH and γ-MSH were found to be much less active. The effect of LPH_37–58 was additive to that of α-MSH, indicating that LPH_37–58 is a full agonist of α-MSH. The lower potency peptides des-acetyl MSH and γ-MSH reduced the effect of α-MSH and are, therefore, partial agonists of α-MSH. The action of MSH peptides in vivo may be modulated by interaction with agonists.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Isolation and amino acid composition of two somatostatin-like peptides from ovine hypothalamus: somatostatin-28 and somatostatin-25

Two $\text{b}$-type cytochrome components, differing in their mid-point redox potentials as well as their reactivities toward CO, are demonstrated in the membrane particle of $\text{Azotobacter}\text{vinelandii}$ by potentiometric titration. The low redox component is revealed to be CO-reactive while the higher redox component is not. Accordingly, the later cytochrome is believed not to be directly involved in the reduction of oxygen while the former component is the cytochrome $\text{o}$ of $\text{Azotobacter}$.     

Regional heterogeneity in the ratio of α-MSH:β-endorphin in rat brain

The molar ratio of α-MSH:β-endorphin varies markedly among discrete microdissected regions of rat brain ranging from 0.57 in the median eminence to 2.74 in the lateral septum. This finding demonstrates that α-MSH and β-endorphin (β-END) are not uniformly distributed in a 1:1 molar ratio in rat brain as one might predict based on the consideration that the two peptides are synthesized in equimolar amounts as part of a common precursor molecule, pro-opiomelanocortin. The data indicate instead that the concentrations of α-MSH and β-END, the two predominant peptides expressed by opiomelantropinergic neurons, are independently regulated in rat brain. The heterogeneity of α-MSH:β-END ratios suggests that the regulation of α-MSH and β-END is regionally specific and may impart functional selectivity to the multisecretory opiomelanotropinergic neuronal system.     

Characterization of a plasma factor having opiate and immunoactivity like beta-endorphin.

A factor from human plasma having opiate-like activity was characterized in the present study. In addition to its ability to inhibit the binding of [³H]-methionine-enkephalin to opiate receptors, it also cross-reacted with two β-endorphin specific anti-sera. Compared with β-endorphin, the plasma factor had a shorter action on inhibiting the contraction of the guinea pig ileum. By gel-filtration chromatography, the size of this factor was intermediate between that of β-endorphin and methionine-enkephalin.     

ω-ethyl, ω-ethenyl and ω-ethynyl-α-alkylidene-γ-lactones from Clinostemon mahuba

The trunk wood of Clinostemon mahuba contains eight (3R)-2-alkylidene-3-hydroxy-4-methylenebutanolides, seven (3R,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides and seven (3S,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides distinguished by the alkylidene side chains with respect to their E- or Z-geometry, ethenyl, ethynyl or ethyl terminals and lengths (C₁₆ or C₁₈).     

Dimeric DOTA-α-Melanocyte-Stimulating Hormone Analogs: Synthesis and In Vivo Characteristics of Radiopeptides with High In Vitro Activity

Dimeric analogs of α-melanocyte-stimulating hormone (α-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for α-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled α-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [¹¹¹In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([¹¹¹In]DOTA)-amide, and 0.36 for [¹¹¹In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [¹¹¹In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled α-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Configuration at C-24 of sterols from the marine phanerogames Posidonia oceanica and Cymodocea nodosa

Sterols were extracted from two marine phanerogames, Posidonia oceanica and Cymodocea nodosa. The two plants contain 24α-ethyl sterols, while the 24α-methyl sterols are accompanied by 24β-epimers. The most abundant components are sitosterol, cholesterol and stigmasterol.