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.     

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.     

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.     

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.     

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.     

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.     

Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

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).     

The evolution of pro-opiomelanocortin: looking for the invertebrate fingerprints

The presence and role of the pro-opiomelanocortin (POMC) gene and encoded peptides in invertebrates are here summarized and discussed. Some of the POMC-derived peptides show a significant similarity regarding their functions, suggesting their appearance before the split of protostomian–deuterostomian lineages and their maintenance during evolution. The basic mechanisms that govern the exchange of information between cells are usually well conserved, and this could have also been for POMC-derived peptides, that are mainly involved in fundamental functions such as immune and neuroendocrine responses. However, the presence and functions that POMC-derived peptides exhibit in taxonomically distant models, are not always reflected by the expected gene homology, leaving the problem of POMC evolution in invertebrates in need of additional study.     

Short hybrid peptides incorporating β- and γ-amino acids as antimicrobial agents

The peptides containing β- and γ-amino acids, LA-Lys(Z)-PEA, P1; LA-Lys(Z)-β^3,3-Ac₆c-PEA, P2; LA-Orn(Z)-β^3,3-Ac₆c-PEA, P3; LA-Lys(Z)-Gpn-PEA, P4; LA-Orn(Z)-Gpn-PEA, P5; LA-Lys(Z)-γ⁴-Phe-PEA, P6, LA-γ⁴-Leu-Lys(Z)-PEA, P7 and LA-β^3,3-Pip(Ac)-Lys(Z)-PEA, P8 were synthesized, characterized and evaluated against Gram-positive and Gram-negative bacteria. Among all, peptides P2, P3, P4 and P5 exhibited potent activity (MIC 6.25 μM) against S. aureus MTCC 737 and P. aeruginosa MTCC 424. In order to understand the efficacy of peptides and mechanism of action, time kill kinetics and fluorescence microscopic studies were performed against S. aureus and P. aeruginosa for the peptides P2, P3, P4 and P5. P4 took half time to show the bactericidal effect on P. aeruginosa and S. aureus in comparison to P2 at their 2x MICs. Fluorescence microscopic studies suggested that peptides P2 and P4 both killed the bacteria via membrane disruption. Further, P4 exhibited lowest haemolytic activity among active peptides and negligible cytotoxic activity against human cancer cell lines A-549, PC-3 and HCT-116 at its MIC.     

Changes in muscle crossbridges when β,γ-imido-ATP binds to myosin

We have studied the binding of β,γ-imido-adenosine-5′-triphosphate to glycerol-extracted insect flight and rabbit back muscle fibres. The binding was at relatively high affinity, of the same quantity as that of other nucleotides, and was inhibited by the presence of ATP. We concluded that imido-ATP bound, without hydrolysis, at the enzymic site of myosin. The mechanical effects of imido-ATP on the glycerol-extracted fibres were measured: concentrations sufficient to bind to myosin caused a small increase in the length of the rigor muscle for a given tension without alteration in the shape of the length-tension diagram. The magnitude of the length change paralleled the binding curve of imido-ATP to the fibre. We concluded that binding caused some change in myosin without its detachment from actin. Electron microscopy and X-ray diffraction studies of glycerol-extracted flight muscle fibres showed an increase in the angle of attachment of myosin to actin when imido-ATP was added. The results are discussed in relation to current concepts of force generation in active muscle.     

Stereoselective Synthesis of (2R, 3S)-2-Benzyl-2-hydroxy-3- (3,4-methylenedioxybenzyl)-γ-butyrolactone from L-(+)-Arabinose

As a model experiment for the stereoselective synthesis of optically active cis-α,β-dibenzyl-α-hydroxy-γ-butyrolactone, (2R, 3S)-2-benzyl-2-hydroxy-3-(3,4-methylenedioxybenzyl)-γ-butyrolactone (3) was stereoselectively synthesized from L-(+)-arabinose.     

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.     

β-胡萝卜素抑制MCF-7细胞生长上调过氧化酶体增殖物激活受体γ（PPARγ）表达

为了研究过氧化酶体增殖物激活受体γ（PPARγ）表达在β 胡萝卜素影响乳腺癌MCF 7细胞活力中所起的作用,采用MTT法测定细胞活力、Western 印迹检测细胞中PPARγ的蛋白质水平,用RT-PCR从mRNA水平检测细胞内PPARγ、P21^WAF1/CIP1、COX-2和P27表达.研究发现,β 胡萝卜素显著抑制人乳腺癌细胞株MCF-7细胞的生长,β-胡萝卜素对细胞生长的抑制作用呈现出时间和计量依赖关系;β-胡萝卜素能够呈现时间效应地从mRNA和蛋白质水平显著上调PPARγ的表达,β-胡萝卜素能够通过PPARγ调节P21^WAF1/CIP1和COX-2mRNA水平;PPARγ的抑制剂GW9662和抗氧化剂还原型谷胱甘肽（GSH）都能部分阻止由β-胡萝卜素引起的细胞活力下降.研究结果提示,激活PPARγ途径和调制细胞氧化状态是β 胡萝卜素对乳腺癌细胞MCF-7的生长抑制效应原因之一.     

Isolation,characterization and opiate activity of β-endorphin from human pituitary glands

The isolation of a 31-amino acid peptide from human pituitary glands has been described. Its amino acid sequence has been proposed to be identical to the sequence of the carboxyl-terminal 31 amino acids of human β-lipotropin. The peptide, designated as β_h-endorphin, possesses significant opiate activity.     

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.     

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.