Beta END Research Articles

Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary.

Proopiomelanocortin (POMC) is posttranslationally processed in the intermediate lobe of the pituitary to both N-terminally acetylated and nonacetylated forms of alpha MSH and beta-endorphin (beta END). N-Acetylation substantially modifies the physiological responses produced by both peptides, suggesting that the activity of the peptide acetyltransferase, which posttranslationally acetylates beta END and des-acetyl-alpha MSH, may play an important role in defining the biological activity of the secretory products of the intermediate pituitary lobe. The present results demonstrate that peptide acetyltransferase activity is induced by treating rats chronically with the dopamine receptor antagonist haloperidol. Haloperidol administration produced parallel and essentially equivalent increases in acetyltransferase activity, POMC mRNA levels, and the content and secretion of POMC-derived peptides in the neurointermediate pituitary. Time-course and dose-response studies further demonstrated that acetyltransferase activity covaried with POMC mRNA and peptide levels. Chronic treatment with the dopamine receptor agonist bromocriptine had the opposite effects; it lowered acetyltransferase activity, POMC mRNA levels, and alpha MSH and beta END immunoreactivities. Subcellular fractionation showed that acetyltransferase activity was localized in three subcellular compartments corresponding in density to secretory vesicles, rough endoplasmic reticulum and Golgi apparatus, and cytosol. Haloperidol treatment significantly increased the specific activity of the secretory vesicle-associated acetyltransferase without affecting the specific activity of the enzymes present in the endoplasmic reticulum or cytosol. Together, these data indicate that peptide acetyltransferase activity and POMC biosynthesis are coregulated.

Proopiomelanocortin-derived peptides in testicular interstitial fluid: characterization and changes in secretion after human chorionic gonadotropin or luteinizing hormone-releasing hormone analog treatment.

Recent studies indicate that proopiomelanocortin (POMC)-derived peptides are present in the testis and may be involved in the regulation of gonadal function, although their precise role still remains obscure. In the present study, we investigated in the rat whether testicular interstitial fluid (TIF), a functionally important compartment of the testis, contains POMC-derived peptides. Adult rats were used for all the experiments, and TIF was collected from each individual testis and analyzed for the presence of ACTH and beta-endorphin-like immunoreactivity (beta end-LI). Initial studies indicated that both ACTH and beta end-LI can be readily detected in TIF from intact rats, and that the concentrations of these peptides are severalfold higher than those in the peripheral plasma of the same animals. Similar studies in rats 3-5 days after hypophysectomy indicate that although ACTH and beta end-LI levels in plasma were undetectable, high levels of both peptides were measured in TIF. Moreover, serial dilution curves of TIF showed parallelism with the respective standard curves in the beta end and ACTH assays. Additional studies indicated that levels of POMC peptides in TIF were modified by treatments that affect the endocrine function of the testis. In that respect, hCG given sc to hypophysectomized rats increased testosterone levels in TIF and plasma, and similarly increased beta end-LI concentrations in TIF. A LHRH analog (LHRH-A; [D-Ala6,des-Gly10]LHRH-ethylamide) given sc to hypophysectomized rats resulted in increased testosterone production, but decreased beta end-LI in TIF, suggesting that the effects of hCG and LHRH-A on testicular beta end-LI secretion are not directly coupled with testosterone production. Prolonged treatment of intact rats with the LHRH-A for 2-10 days resulted in progressive declines in testicular weight and testosterone levels in TIF and in plasma, and a significant suppression of beta end-LI levels in TIF as early as 2 days after analog treatment. On the other hand, acute intratesticular injection of LHRH-A to intact rats (5 ng into the right testis) produced a short-lived increase in beta end-LI in TIF 2 h after injection, coincident with a temporary decrease in TIF volume. These results indicate that the POMC-derived peptides ACTH and beta end are present in TIF and are secreted locally into this compartment, supporting previous reports demonstrating the presence and local synthesis of POMC peptides in testicular tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural organization of the DR subregion of the human major histocompatibility complex.

Two clusters of overlapping cosmid and lambda phage clones comprising 205 kilobases (kb) have been isolated from the DR subregion of the human major histocompatibility complex from a DR4 haplotype. A single DR alpha and three DR beta genes were identified. In one cluster (135 kb), the DR alpha gene is 90 kb distant from the DR beta gene encoding a molecule that carries the MT3 serological specificity. In the second cluster (70 kb), the DR beta gene determining the DR4 specificity is located 22 kb apart from a DR beta pseudogene (DR beta psi). A 3- to 4-kb sequence located at the 5' end of the DR beta (MT3) gene is common to all three DR beta-chain genes. In addition, three more copies of this sequence are spaced between the DR alpha and the DR beta (MT3) genes in the first cluster and one of these, at least, is associated with a DR beta 1 exon, suggesting that additional genes could be encoded in this region and that multiple duplication events have led to its evolution.

Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli.

We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end). These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.

Polypeptide fragments of the third component of complement in urine of hereditary nephritis patients.

Pro-HNP, a urine protein isolated from hereditary nephritis patients, is derived from C3 and resembles the C3c domain. It contains disulfide-linked polypeptides of beta 75, alpha 40, and alpha 28. Plasmin degraded pro-HNP in vitro to HNP, which was also isolated from the urine of patients and which contained disulfide-linked polypeptides of beta 60, alpha 38, and alpha 26, and noncovalently bound polypeptide of beta 17. Amino terminal sequence analyses and amino acid compositions of the seven polypeptides isolated from pro-HNP and HNP show that beta 75 degrades to beta 60 and beta 17 (beta 17 locates at the amino end of beta 75), alpha 40 degrades to alpha 38 (both locate at the carboxyl end of the alpha-chain of C3), and alpha 28 degrades to alpha 26 (both are from the amino end of the alpha'-chain of C3b). These results confirm the enzymatic specificity of plasmin on pro-HNP. In HNP, the half-cystine contents of beta 60, alpha 38, alpha 26, and beta 17 were approximately 3, 12, 3, and 4, respectively. Partial reduction readily released alpha 40 from pro-HNP and alpha 38 from HNP. There were about five intra-chain disulfide bonds in alpha 40 or alpha 38; stepwise reduction of these intra-polypeptide bonds apparently accounted for multiple conformations of alpha 40 or alpha 38.

Rapid and specific radioimmunoassays for beta-endorphin and beta-lipotropin in affinity-purified human plasma.

Using rapid and reusable affinity columns, we established a method to measure beta-endorphin (beta END) and beta-lipotropin (beta LPH) separately in human plasma. One column contained antibodies against the N-terminal portion of beta LPH, and the other contained antibodies against beta END. The first column separated beta LPH from beta END and concentrated beta LPH as well, and the second separated beta LPH from concentrated beta END. Mean plasma levels (at 0830-0930 h) of beta END and beta LPH in 10 normal subjects were 1.1 +/- 0.1 (+/- SE) and 4.1 +/- 0.4 fmol/ml, respectively; both were markedly elevated in patients with ACTH/LPH hypersecretory states. The molar ratios of plasma levels of beta END and beta LPH were fairly constant in normal subjects (0.28 +/- 0.01), unchanged in patients with Cushing's disease and Cushing's disease after adrenalectomy, lower in patients with Addison's disease (P less than 0.001), higher in patients with chronic bone pain (P less than 0.001), and variable in patients with the ectopic ACTH syndrome. These data indicate that beta END and beta LPH can be measured separately in plasma by simple and reproducible procedures.

Beta endorphin selectively stimulates aldosterone secretion in hypophysectomized, nephrectomized dogs.

We examined the effects of synthetic human beta-endorphin (beta END) and a stable methionine (Met)-enkephalin analogue on aldosterone and cortisol secretion rates in anesthetized, hypophysectomized, and nephrectomized dogs and compared them to those of (1-39) ACTH. The circulation of the adrenal glands was completely isolated on the arterial and venous sides (Hilton Pouch). The peptides were infused to deliver 3 pmol/min into the aortic "pouch." Blood was collected from the vena caval pouch, which received blood only from the adrenal gland. Secretion rates of aldosterone and cortisol were calculated as the product of adrenal blood flow and venous steroid concentration. Duplicate steroid measurements were obtained during a control period, at 10, 30, and 50 min of peptide infusion and during a postcontrol period. BetaEND increased aldosterone secretion rate from 2.4 +/- 0.5 ng/min (mean +/- SEM) to 3.2 +/- 0.9 ng/ min at 10 min (N.S.), 8.2 +/- 2.5 ng/min (P less than 0.05) at 30 min and 11.0 +/- 3.7 ng/ min (P less than 0.05) at 50 min of infusion. Cortisol secretion rate was not affected by infusion of betaEND. Infusion of the stable Met-enkephalin analogue D-alanine2; Metphenylalanine4, Met(O)-enkephalin-ol or saline alone had no effect on aldosterone or cortisol secretion rates. ACTH infusion increased mean aldosterone secretion rate by approximately 215% and significantly stimulated cortisol secretion rate. These results indicate that beta END selectively stimulates aldosterone secretion with a potency similar to that of an equimolar dose of ACTH.

Proopiolipomelanocortin peptides in normal pituitary, pituitary tumor, and plasma of normal and Cushing's horses.

Using RIAs for six regions within proopiolipomelanocortin (proOLMC), gel filtration, and electrophoresis, we studied pituitary peptides in a normal horse and one with Cushing's disease caused by a pars intermedia adenoma. Almost all immunoreactive (IR) ACTH (78%) was 4,500 mol wt (4.5K) ACTH in normal pars distalis, but it was almost 100% corticotropin-like intermediate lobe peptide (CLIP) in normal pars intermedia. alpha MSH and beta MSH were found mainly in pars intermedia: equal concentrations of the beta MSH precursors, beta-lipotropin (beta LPH) and gamma LPH, were found in pars distalis. Most IR-beta-endorphin (IR-beta END) was found as beta END in pars intermedia, but roughly equal concentrations of beta END and its precursor, beta LPH, were found in pars distalis. A 33K molecule containing IR-ACTH, IR-gamma 3MSH, and IR-beta END, presumed to be proOLMC, and a variety of 15-27K presumed biosynthetic intermediates were found in both normal pars distalis and pars intermedia. The pars intermedia adenoma causing Cushing's syndrome contained high IR-peptide concentrations. Several differences in precursors were noted, including the presence of three larger presumed precursors (38.5K, 47K, and 63K) that had both ACTH and beta END immunoreactivities and both deletions and additions of 15-27K intermediates. The Cushing's horse's plasma peptides reflected tumor concentrations; 4.5K ACTH was modestly elevated, but the concentrations of CLIP, alpha MSH, beta MSH, gamma LPH, and beta END were dramatically increased. About 20% of plasma IR-ACTH and 5% of IR-beta MSH and IR-beta END were found as high molecular weight forms. Normal processing of horse proOLMC appears to be similar to that in other species, but may be altered in pars intermedia tumors of horses with Cushing's disease, the plasma of which contains disproportionately increased concentrations of pars intermedia proOLMC peptides.

Evidence for independent secretion of beta-endorphin immunoreactivity from rat pars distalis in vivo.

The distinctive chromatographic patterns of beta-endorphin-like immunoreactivity (beta END-LI) released from rat pars distalis (PD) or pars intermedia in vitro as well as the selective inhibitory effects of dexamethasone on PD were used to determine which lobe secretes beta END-LI into plasma after clonidine administration in vivo. Gel chromatography (Sephadex G-50) indicates that cultured PD cells released two major immunoreactive species which coelute with beta-lipotropin (beta LPH) or beta END standards. Conversely, virtually all of the beta END-LI secreted by neurointermediate lobe [pars intermedia plus pars nervosa (NIL)] cells in vitro resembled beta END in size, while none was detected which cochromatographed with beta LPH. Incubation of PD cells with clonidine (10(-6) M) evoked a 2-fold increase in beta END-LI release, while the drug had no effect on beta END-LI released from cultured NIL cells. Dexamethasone (10(-7) M) inhibited the clinidine-induced release of beta END-LI from PD cells, whereas it did not influence the stimulated release of beta END-LI from NIL by isoproterenol (10(-6) M). In vivo administration of clonidine (0.5 mg/kg, ip, for 15 min) increased total plasma beta END-LI from 0.75 +/- 0.16 to 1.35 +/- 0.18 ng/ml; 85% of this rise corresponded to beta LPH as determined by gel chromatography. Prior administration of dexamethasone (60 micrograms/kg, ip, for 4 h) completely prevented the clonidine-induced release of beta END-LI in vivo. These results demonstrate that clonidine probably acts selectively on the PD in vivo to release pituitary beta END-LI, and further, that PD can release beta END-LI independently of the pars intermedia.

Human placental immunoreactive corticotropin, lipotropin, and beta-endorphin: evidence for a common precursor.

The concentrations and molecular sizes of immunoreactive corticotropin (ACTH), lipotropin (LPH, beta LPH plus gamma LPH), gamma LPH, and beta-endorphin (beta END) were determined in human placental extracts. Serial dilutions of a water extract of placenta generated competitive binding curves parallel with that of the standard in each assay. The concentrations of ACTH, LPH, gamma LPH, and beta END were 3.3, 0.8, 0.7, and 1.1 ng/g wet weight of tissue, respectively. A partially purified extract applied to a Sephadex G-50 column contained high Mr components with ACTH, LPH, gamma LPH, and beta END immunoreactivities. The extract was applied to an immune affinity chromatography column consisting of affinity-purified (1-24)ACTH antiserum covalently bound to agarose. The material that adsorbed to the column and eluted with buffer containing sodium dodecyl sulfate had ACTH, LPH, and beta END immunoreactivities, indicating that there was a component or components containing antigenic determinants for all of these peptides. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the affinity-purified placental extract revealed at least two high Mr components (Mr approximately 48,000 and 36,000) with all three immunoreactivities. These data suggest, but do not prove, that the placenta synthesizes ACTH, the LPHs, and beta END from a common precursor molecule.

IMMUNOREACTIVE ENDORPHINS, LIPOTROPINS AND CORTICOTROPINS IN A HUMAN NONPITUITARY TUMOR: EVIDENCE FOR A COMMON PRECURSOR

The immunoreactive (RIA), ACTH, LPH, alpha endorphin (alpha End) and beta End in an extract of a human pancreatic islet cell carcinoma causing the ectopic ACTH syndrome were assayed after gel exclusion chromatography. In addition to "big", "intermediate" and "little" ACTHs, beta lipotropin (beta LPH) and gamma LPH, the eluate fractions contained RIA-alpha End and -beta End. The major RIA-beta End component appeared to be h beta LPH, which has beta End as its carboxy-terminus, but significant concentrations of a component that coeluted with synthetic p beta End were also found. Small amounts of RIA-alpha End were found in two peaks, one that may have represented 1-76 h beta LPH, with alpha End as its carboxy-terminus, and one probably representing alpha End itself. RIA-ACTH, -LPH and -beta End were found in the void volume fractions. Thus, a human nonpituitary tumor that produced ACTH and LPHs also produced endorphins, all perhaps deriving from a common precursor.

Electron microscopic characterization of DNAs of non-defective deletion mutants of bacteriophage Mu

We have discovered that the Mu prophage in Escherichia coli strain DK445, a λ cIts857 Sam7 lac5: :Mu cI + dilysogen (in which Mu is integrated in the lacZ gene of the p lac5 prophage in the E. coli chromosome) contains a 2600 base-pair long insertion of unknown nature. The DNAs of seven independent phage Mu isolates recovered from induced DK445 have been studied by electron microscopic heteroduplex methods. All were found to contain either a deletion or a substitution in the G and/or beta regions of the chromosome. All seven mutants have growth rate, burst size and plaque morphology indistinguishable from wild-type Mu. Thus, they are the first non-defective deletion mutants of Mu. The regions deleted in these mutants are non-essential. They span the right-hand 1.3 kilobases of the G segment in the lytic orientation and the entire beta region except the rightmost 0.15 kilobase on the S (right) end. The mechanism which generates these Mu deletion mutants with high efficiency ( 7 7 ) is not known. The lengths of the bacterial DNA on the S end vary according to the amount of DNA inserted into or deleted from the Mu genome. Therefore, the model of headful encapsidation of Mu DNA from the c (left) end (Bukhari & Taylor, 1975) is substantiated. None of the mutants has lost the right-hand end of beta, as would have been anticipated by straight-forward headful packaging from the c end of prophage with a sizeable insertion. Therefore, the right-hand end of beta, one of its normal attachment sites, is probably essential for Mu viability. The identification of a Mu mutant with a substitution in beta which is incapable of promoting inversion of the G segment suggests that part of the G segment inversion system might be located on the left portion of the beta region. The G segment inverts normally in the Mu prophage in DK445. Yet, all seven deletion mutants have the G segment arranged in the lytic orientation. These observations suggest that the orientation of Mu G DNA may affect the phage viability and that there may be essential functions encoded within the left-hand 1.7 kilobases of the G segment in the lytic orientation.

C14-N14Mass Difference and Mass Excesses of Some Light Nuclides

Mass excesses of the nuclides ${\mathrm{H}}^{1}$, ${\mathrm{D}}^{2}$, ${\mathrm{C}}^{12}$, ${\mathrm{C}}^{14}$, ${\mathrm{N}}^{14}$, ${\mathrm{O}}^{18}$, and ${\mathrm{Ar}}^{40}$ were determined by measurement of 18 parent molecule ion doublets with the mass synchrometer. The ${\mathrm{C}}^{14}$-${\mathrm{N}}^{14}$ difference, 156.44\ifmmode\pm\else\textpm\fi{}0.29 kev, was obtained from measurements on the doublets [${\mathrm{C}}^{14}$${\mathrm{O}}^{18}$-${\mathrm{O}}_{2}$], [${\mathrm{N}}^{14}$${\mathrm{O}}^{18}$-${\mathrm{O}}_{2}$], [${\mathrm{C}}^{14}$${\mathrm{H}}_{4}$-${\mathrm{H}}_{2}$O], [${\mathrm{C}}^{14}$C${\mathrm{H}}_{2}$CO], and [${\mathrm{N}}_{2}$${\mathrm{H}}_{4}$-${\mathrm{O}}_{2}$] and other doublets which give some of those above. This is in excellent agreement with the value of 156\ifmmode\pm\else\textpm\fi{}1 kev obtained from determination of the ${\mathrm{C}}^{14}$ beta end point. The consistency of these data supports the 1 kev upper limit for the neutrino rest mass derived from the ${\mathrm{H}}^{3}$-${\mathrm{He}}^{3}$ mass difference and the ${\mathrm{H}}^{3}$ beta end point, and also supports the argument that mass synchrometer data are essentially free of systematic errors of the magnitude required to account for discrepancies between nuclear reaction and mass spectrographic mass excesses of ${\mathrm{C}}^{12}$ and ${\mathrm{O}}^{18}$. Mass excesses of the light nuclides determined were slightly higher than recently published mass synchrometer data, but agreement was generally within 1 \ensuremath{\mu}MU.

Experimental Limit of the Neutrino Rest Mass

An empirical determination of the neutrino rest mass can be obtained from the H/sup 3/-He/sup 3/ mass difference and the H/sup 3/ BETA end-point energy. If the experimental lower limit of the true BETA end point is subtracted from the H/sup 3/-He/sup 3/ mass difference, the result is an empirical for the neutrino rest mass. This upper limit is independent of any theory of beta decay and reenergy (W.D.M.)

Radioactive decay of Eu154

The radioactive decay of Eu154 (t 1/2=16y), obtained from thermal neutron irradiations of 95% enriched Eu153, has been studied by using the Seigbahn-Slatis intermediate image spectrometer, a thin lens magnetic spectrometer and a coincidence scintillation spectrometer. The following gamma-rays (energy in kev.) have been identified as belonging to this isotope: 123, 248, 585, 717, 759, 875, 998, 1007, 1285, 1600. Beta end points of Eu154 were measured at 275 (20%), 590 (45%), 890 (23%) and 1860 (12%). The K-conversion coefficient of 123 kev. gamma-ray has been experimentally determined to be ∼0·5. On the basis of gamma-gamma and beta-gamma coincidences, the decay scheme of Eu154 has been deduced: the resulting level-structure of Gd154 conforms well with the predictions of the unified nuclear model.

Decay of Europium-154

The gamma rays and electrons of fission-product europium (${\mathrm{Eu}}^{153}$, ${\mathrm{Eu}}^{154}$, and ${\mathrm{Eu}}^{155}$) have been studied, and the results obtained for ${\mathrm{Eu}}^{154}$ are presented. Gamma rays belonging to this isotope have been identified at 0.1229 Mev (35%), 0.2480 Mev, ${0.593}_{3}$ Mev (4%), ${0.694}_{1}$ Mev (\ensuremath{\le}3.5%), ${0.705}_{8}$ Mev (\ensuremath{\le}3.5%), ${0.724}_{9}$ Mev (21%), ${0.758}_{9}$ Mev (\ensuremath{\le}3.5%), ${0.875}_{3}$ Mev (13%), ${0.998}_{2}$ Mev (14%), 1.007 Mev (17%), 1.277 Mev (42%), and \ensuremath{\sim}1.6 Mev (\ensuremath{\sim}3%). Beta end points of ${\mathrm{Eu}}^{154}$ were measured at 1.85, 0.87, 0.59, and 0.25 Mev. Multipolarities for most of the above gamma-ray transitions have been suggested on the basis of the measured $K$-shell conversion coefficients. The decay scheme of ${\mathrm{Eu}}^{154}$ has been deduced, and, in most respects, the resulting levels of ${\mathrm{Gd}}^{154}$ conform well with the Bohr-Mottelson unified nuclear model and with other even-even nuclei in the strong-coupling regions.