Beta END Research Articles

Earthquake Early Warning ShakeAlert System: Testing and Certification Platform

Earthquake early warning systems provide warnings to end users of incoming moderate to strong ground shaking from earthquakes. An earthquake early warning system, ShakeAlert, is providing alerts to beta end users in the western United States, specifically California, Oregon, and Washington. An essential aspect of the earthquake early warning system is the development of a framework to test modifications to code to ensure functionality and assess performance. In 2016, a Testing and Certification Platform (TCP) was included in the development of the Production Prototype version of ShakeAlert. The purpose of the TCP is to evaluate the robustness of candidate code that is proposed for deployment on ShakeAlert Production Prototype servers. TCP consists of two main components: a real‐time in situ test that replicates the real‐time production system and an offline playback system to replay test suites. The real‐time tests of system performance assess code optimization and stability. The offline tests comprise a stress test of candidate code to assess if the code is production ready. The test suite includes over 120 events including local, regional, and teleseismic historic earthquakes, recentering and calibration events, and other anomalous and potentially problematic signals. Two assessments of alert performance are conducted. First, point‐source assessments are undertaken to compare magnitude, epicentral location, and origin time with the Advanced National Seismic System Comprehensive Catalog, as well as to evaluate alert latency. Second, we describe assessment of the quality of ground‐motion predictions at end‐user sites by comparing predicted shaking intensities to ShakeMaps for historic events and implement a threshold‐based approach that assesses how often end users initiate the appropriate action, based on their ground‐shaking threshold. TCP has been developed to be a convenient streamlined procedure for objectively testing algorithms, and it has been designed with flexibility to accommodate significant changes in development of new or modified system code. It is expected that the TCP will continue to evolve along with the ShakeAlert system, and the framework we describe here provides one example of how earthquake early warning systems can be evaluated.

Abstract A177: Mode of action of PM060184, a new interfacial microtubule inhibitor of marine origin.

Abstract PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total chemical synthesis and is under evaluation in Phase I clinical studies in patients with advanced cancer diseases. Recently published results indicate that members of the PM060184 family are interfacial microtubule inhibitors that bind with nanomolar affinity to unassembled αβ-tubulin dimers at the beta tubulin plus end (Pera et al. 2013, ACS Chem. Biol.) In an attempt to shed light into the mechanism responsible of its antitumor activity, we have studied the effects of PM060184 in tumor cells including microtubule network structure, centrosome modifications and type of cell death induced by this compound. PM060184 rapidly internalizes into the tumor cells and shows a high intracellular retention rate, reflecting the high avidity for its cellular target. In addition, the antitumor activity of the compound seems to be rather irreversible after short (30 min) exposure times. At the cellular level, PM060184 inhibits tubulin polymerization. Interestingly, PM060184 affects interphase cells, where the compound induces disorganisation and fragmentation of the microtubule network and disturbing cell migration. PM060184 also affects mitotic cells, inducing the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. All these effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. In summary, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A177. Citation Format: Marta Martínez-Diez, Juan Fernando Martínez-Leal, Luis F. Garcia-Fernandez, Carlos M. Galmarini. Mode of action of PM060184, a new interfacial microtubule inhibitor of marine origin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A177.

Positron source Ge‐68 through copper and bronze irradiated by carbon ions

AbstractThis communication reports on the formation of radioactive germanium isotope (G68) in copper (Cu) and bronze irradiated with Carbon ions (70MeV, 700 nA for 20 h) at the pelletron facility at TIFR, Mumbai. The irradiated targets were analyzed by gamma spectroscopy using HPGe detector and end point energy using GM counter. By analysis of gamma spectrum, it has been observed that Ge68 isotope along with some other isotopes like Se75 (119 days), Ge66 (2.26 days), Ga74 (8.12 minutes), As69 (15.2 minutes), Co60 (1925days), Zn65 (243days), Ga68 (67.7 minutes) were formed as estimated by PACE Code calculations. These results show that both Cu and bronze have contributed to Ge68 production but the irradiated bronze is found to form a good positron emitter in comparison to that of irradiated copper. The beta end point energy of Ge68 estimated by GM counter has been found to be 1.80 MeV. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

First Precision Mass Measurements of Refractory Fission Fragments

Atomic masses of 95-100Sr, 98-105Zr, and [corrected] 102-110Mo and have been measured with a precision of 10 keV employing a Penning trap setup at the IGISOL facility. Masses of 104,105Zr and 109,110Mo are measured for the first time. Our improved results indicate significant deviations from the previously published values deduced from beta end point measurements. The most neutron-rich studied isotopes are found to be significantly less bound (1 MeV) compared to the 2003 atomic mass evaluation. A strong correlation between nuclear deformation and the binding energy is observed in the two-neutron separation energy in all studied isotope chains.

Bolometric bounds on the antineutrino mass.

High statistics calorimetric measurements of the beta spectrum of 187Re are being performed with arrays of silver perrhenate crystals operated at low temperature. After a substantial modification of the experimental setup, a new measurement with ten silver perrhenate microbolometers has been running since July 2002. The crystals have masses around 300 microg and their average FWHM energy resolution is of 28.3 eV at the beta end point. The Kurie plot collected during 4485 h x mg effective running time has an end-point energy of 2466.1+/-0.8(stat)+/-1.5(syst) eV, while the half lifetime of the decay is found to be 43.2+/-0.2(stat)+/-0.1(syst) Gy. These values are the most precise obtained so far for 187Re. The best fit value for m(2)(nu(e)) is 147+/-237(stat)+/-90(syst) eV(2), which corresponds to an upper limit for the electron antineutrino mass m(nu(e))< or =21.7 eV at 90% C.L.

High-resolution X-ray structure of isoaspartyl dipeptidase from Escherichia coli.

Isoaspartyl dipeptidase from Escherichia coli functions in protein degradation by catalyzing the hydrolysis of beta-L-isoaspartyl linkages in dipeptides. The best substrate for the enzyme reported thus far is iso-Asp-Leu. Here we report the X-ray analysis of the enzyme in its resting state and complexed with aspartate to 1.65 and 2.1 A resolution, respectively. The quaternary structure of the enzyme is octameric and can be aptly described as a tetramer of dimers. Each subunit folds into two distinct domains: the N-terminal region containing eight strands of mixed beta-sheet and the C-terminal motif that is dominated by a (beta,alpha)(8)-barrel. A binuclear zinc center is located in each subunit at the C-terminal end of the (beta,alpha)(8)-barrel. Ligands to the binuclear metal center include His 68, His 70, His 201, His 230, and Asp 285. The two zincs are bridged by a carboxylated lysine residue (Lys 162) and a solvent molecule, most likely a hydroxide ion. The product of the reaction, aspartate, binds to the enzyme by displacing the bridging solvent with its side chain functional group. From this investigation it is proposed that the reaction mechanism of the enzyme proceeds through a tetrahedral intermediate and that the bridging solvent attacks the re face of the carbonyl carbon of the scissile peptide bond. This structural analysis confirms the placement of isoaspartyl dipeptidase into the urease-related amidohydrolase superfamily.

Site actif de la glucocérébrosidase humaine : prédictions structurales et validations expérimentales

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase which cleaves the beta-glucosidic linkage of glucosylceramide, a normal intermediate in glycolipid metabolism. Glucocerebrosidase belongs to the clan GH-A of glycoside hydrolases, a large group of enzymes which function with retention of the anomeric configuration at the hydrolysis site. Accurate three-dimensional (3D) structure data for glucocerebrosidase should help to better understand the molecular bases of Gaucher disease. As such 3D structure data were not available, we used the two-dimensional hydrophobic cluster analysis (HCA) method to make structure predictions for the catalytic domains of clan GH-A glycoside hydrolases. We found that all the enzymes of clan GH-A may share a similar catalytic domain consisting of an (alpha/beta)8 barrel with the critical acid/base and nucleophile residues located at the C-terminal ends of strands beta 4 and beta 7, respectively. In the case of glucocerebrosidase, Glu 235 was predicted to be the putative acid/base catalyst whereas the nucleophile was located at Glu 340. Next, in order to obtain experimental evidence supporting these HCA-based predictions, we used retroviral vectors to express, in murine null cells, E235A and E340A mutant proteins, in which alanine residues unable to participate in the enzymatic reaction replace the presumed critical glutamic acid residues. Both mutants were found to be catalytically inactive although they were correctly folded/processed and sorted to the lysosome. Thus, Glu 235 and Glu 340 do indeed play key roles in the active site of human glucocerebrosidase as predicted by the HCA analysis. In a broader perspective, our work points out that bioinformatics approaches may be highly useful for generating structure-function predictions based on sequence-structure interrelationships, especially in the context of a rapid increase in protein sequence information through genome sequencing.

Dosimetric penumbra effects in catheter-based intravascular brachytherapy using a centered photon or beta line source

Purpose: In catheter-based intravascular brachytherapy, either photon or beta emitters are often used in a linear arrangement so that blood vessels of 10–30 mm lengths can be treated. With a line source, the dose gradient in the radial direction and longitudinal direction depend on the type of radionuclides used in the treatment. The purpose of this study was to investigate the dose fall-off at the edges of a linear source in a blood vessel for different types of photon and beta emitters. Materials/Methods: Dose distributions were calculated on cylindrical blood vessels of various radii. Radioactive sources of 192Ir, 125I, 103Pd, 188Re, 32P, and 90Y/Sr were studied. All the sources were assumed to be in the form of a line. The dose rate at a point in space produced by a radioactive source was computed by integrating the point dose rate kernel of the corresponding radionuclide over the radioactive line. The point dose rate kernel was computed with Monte Carlo simulation of radiation transport. The edge effects were characterized with three newly defined quantities: longitudinal dose uniformity (LDU), effective coverage length (ECL), and margin length (ML). LDU was defined as the ratio of dose at a distance along the long axis of the vessel to the dose at center. ECL was defined as the length over which the LDU was greater than 0.95. ML was defined as half of the length difference between source length L and ECL, which is essentially the length segment at each edge that is covered by the source physical length but is being underdosed. Results: All beta emitters provided more uniform dose distributions and covered a larger portion of blood vessels longitudinally than photon emitters. Typical MLs were 2–3 mm for beta emitters and 4–6 mm for gamma emitters. As the radial depth of the point of interest increased, both the LDU and ECL decreased and ML increased. The ML increased from 2 to 3 mm for beta emitters and from 4 to 6 mm for photon emitters when the radial depth of the point of interest increased from 1.5 to 2.5 mm (typical proximal and distal media points for a 3-mm diameter lumen). The ML increased with increasing source length for all radionuclides. For beta emitters the ML increased initially from 1.5 mm to more than 2.5 mm as source length increased from 5 to 10 mm. When the source length was longer than 15 mm, the ML remains nearly constant, about 3 mm. For photon emitters, ML increased continuously from 1.5 mm to more than 6.0 mm, as source length increased from 5 to 50 mm. Conclusions: A formalism to quantify the dose uniformity along the length of a blood vessel undergoing catheter-based intravascular brachytherapy has been developed. This formalism was used to study the edge effects at the ends of several beta and photon sources. The results indicated that for a centered source the ML at each end due to penumbra effects was about 2 to 3 mm for beta emitters; about 4–6 mm for photon emitters. The ML increases as the radial depth of point of interest in the vessel increases. The ML increases also with increasing source length, especially for photon sources.

A High-Affinity Fluorenone-Based β2-Adrenergic Receptor Antagonist with a Photoactivatable Pharmacophore

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.

Characterization of carotenes in a combination of a C(18) HPLC column with isocratic elution and absorption spectra with a photodiode-array detector.

Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C(18) HPLC column with simple isocratic elution. The elution order of the carotenes was C(30), C(40),C(45) then C(50). The C(40) carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: phi, Psi, in then beta end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (phi and beta) influenced the absorption spectra and the non-conjugated end groups (Psi and in) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra.

The Subtilisin/Kexin Family of Precursor Convertases: Emphasis on PC1, PC2/7B2, POMC and the Novel Enzyme SKI‐1

Proopiomelanocortin (POMC) is a precursor to various, bioactive peptides including ACTH, beta LPH, alpha MSH, and beta endorphin (beta END). Processing of POMC at dibasic residues is tissue-specific and is performed by either PC1 alone (resulting in ACTH and beta LPH, anterior pituitary corticotrophes) or by a combination of PC1 and PC2 (yielding alpha MSH and beta END, pituitary neurointermediate lobe and hypothalamus). The PC2-specific binding protein 7B2 is intimately involved in the zymogen activation of proPC2 into PC2. Structure-function studies of these enzymes demonstrated the presence of N- and C-terminal domains, as well as specific amino acids within the catalytic segment that influence the degree of activity of each enzyme and the interaction of PC2 with 7B2. The tissue distribution, plasticity of expression, and the multiple precursors that are differentially cleaved by PC1 and/or PC2, predict a wide array of combinatorial activities of these convertases within the endocrine and neuroendocrine system. The phenotypic consequences of the absence of genetic expression of either PC1 or PC2 are now explored using knockout mice and in human patients suffering from obesity and diabetes.

Tubulin stability and decay: mediation by two distinct classes of IKP104-binding sites.

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751-15759]. Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both alpha and beta subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the alpha and beta subunits of alphabeta tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl titer or the exposure of hydrophobic areas of this subtilisin-treated tubulin (alpha(s)beta(s)). Moreover, IKP104 lowers the sulfhydryl titer of alpha(s)beta(s) tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of alpha(s)beta(s) tubulin. The cleavage at the C-terminal ends of both alpha and beta modulates the effect of IKP104 on the beta subunit, but not on the alpha subunit. Fluorometric binding data analysis suggests that IKP104 binds to the alpha(s)beta(s) tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for alpha and beta and the fluorometric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of alpha and beta and the effect of the high-affinity site is restricted largely to the alpha subunit, while the low-affinity-site binding is modulated by the C-terminal domain of beta. It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.

High plasma proopiomelanocortin in aggressive adrenocorticotropin-secreting tumors.

A specific propiomelanocortin (POMC) immunoradiometric assay was developed using antibodies directed against ACTH and beta-endorphin (beta end). Partially purified standard POMC was prepared from the human small cell lung carcinoma cell line DMS-79 culture medium. Ten units (U) POMC had the same displacement ability as one pg beta end in a C-terminal beta end radioimmunoassay and thus were close if not equal to 10 pg POMC. This POMC assay was used to investigate patients with ACTH-dependent Cushing's syndrome. Plasma POMC was undetectable (< 60 U/mL) in 17 normal controls and in 4 patients with Addison's disease (concomitant ACTH plasma levels between 362 and 1058 pg/mL). Forty-two patients with Cushing's disease were studied, either before (n = 25) or after (n = 17) bilateral adrenalectomy: 7 patients with highly invasive macroadenomas had high POMC plasma levels, between 240 and 4200 U/ml (concomitant ACTH plasma levels between 77 and 5730 pg/mL); 35 patients, including one with an invasive macroadenoma, had undetectable POMC plasma levels (concomitant ACTH plasma levels between 31 and 2820 pg/mL). Among 20 patients with histologically proven ectopic ACTH syndrome, 16 had high POMC plasma levels, between 80 and 8000 U/mL (concomitant ACTH plasma levels between 45 and 9265 pg/mL); all those tumors were malignant, and the highest POMC/ACTH plasma levels ratios (taken as an index of altered POMC processing) were observed in the 3 patients with small cell carcinomas of the lung; in one of these patients, ACTH and POMC plasma levels both decreased during the course of chemotherapy, in parallel with the reduction of the tumoral mass. Four patients with ectopic ACTH syndrome had undetectable POMC plasma levels (concomitant ACTH plasma levels between 78 and 335 pg/mL): they were all typical bronchial carcinoids. These data show that high POMC plasma level is neither specific for nor constant in ectopic ACTH syndrome. Rather it should be considered as a marker of tumor aggressivity, in pituitary- and non-pituitary tumors. Its diagnostic help appears limited for the most frequent cause of occult ectopic ACTH syndrome, the typical bronchial carcinoids.

The carotenoid 7,8-dihydro-psi end group can be cyclized by the lycopene cyclases from the bacterium Erwinia uredovora and the higher plant Capsicum annuum.

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc-crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8-dihydro-beta-carotene, via monocyclic beta-zeacarotene. zeta-Carotene was also found to be cyclized to bicyclic 7,8,7',8'-tetrahydro-beta-carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8-dihydro-psi end group to a 7,8-dihydro-beta end group, in addition to the usual cyclization of the psi end group to the beta end group. Furthermore, beta-carotene hydroxylase from Erwinia (CrtZ) was able to add a hydroxyl group to the 7,8-dihydro-beta end group and the beta end group.

Beta-Endorphin enhances phagocytosis of latex particles in mouse peritoneal macrophages.

The effects of beta-endorphin (beta End) on phagocytosis in peritoneal macrophages were examined by using flow cytometry (FCM). Beta End enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), alpha-endorphin (alpha End), gamma-endorphin (gamma End), alpha End (18-31) and beta End (28-31) had no such activity. Beta End (1-27) and beta End (6-31) enhanced phagocytosis less effectively than beta End did. Naloxone did not inhibit the enhancement of phagocytosis induced by beta End. Unstimulated control phagocytosis was partially suppressed in Ca2(+)-free EGTA-containing solution and even in this solution beta End enhanced phagocytosis. However, the enhancement was suppressed in the solution containing BAPTA-AM. The present study showed that beta End enhanced extracellular Ca2+ ([Ca2+]o)-dependent and -independent phagocytosis and that the enhancement is largely dependent on intracellular Ca2+ ([Ca2+]i). These results support the contention that beta End is one of the mediators that modulates the immune system.

Three‐dimensional structure prediction of the NAD binding site of proton‐pumping transhydrogenase from Escherichia coli

A three-dimensional structure of the NAD site of Escherichia coli transhydrogenase has been predicted. The model is based on analysis of conserved residues among the transhydrogenases from five different sources, homologies with enzymes using NAD as cofactors or substrates, hydrophilicity profiles, and secondary structure predictions. The present model supports the hypothesis that there is one binding site, located relatively close to the N-terminus of the alpha-subunit. The proposed structure spans residues alpha 145 to alpha 287, and it includes five beta-strands and five alpha-helices oriented in a typical open twisted alpha/beta conformation. The amino acid sequence following the GXGXXG dinucleotide binding consensus sequence (residues alpha 172 to alpha 177) correlates exactly to a typical fingerprint region for ADP binding beta alpha beta folds in dinucleotide binding enzymes. In the model, aspartic acid alpha 195 forms hydrogen bonds to one or both hydroxyl groups on the adenosine ribose sugar moiety. Threonine alpha 196 and alanine alpha 256, located at the end of beta B and beta D, respectively, create a hydrophobic sandwich with the adenine part of NAD buried inside. The nicotinamide part is located in a hydrophobic cleft between alpha A and beta E. Mutagenesis work has been carried out in order to test the predicted model and to determine whether residues within this domain are important for proton pumping directly. All data support the predicted structure, and no residue crucial for proton pumping was detected. Since no three-dimensional structure of transhydrogenase has been solved, a well based tertiary structure prediction is of great value for further experimental design in trying to elucidate the mechanism of the energy-linked proton pump.

Domains of rat interleukin 1 beta involved in type I receptor binding.

In the present study the inhibitory effects of a panel of 21 monoclonal antibodies (moabs) to rat interleukin 1 beta (rIL-1 beta) on the binding of 125I-labeled rIL-1 beta to murine type I IL-1 receptors on EL4 cells were investigated. Furthermore, the epitopes of these moabs were determined by the use of the pepscan technique, and these epitopes were visualized on a three-dimensional model of rIL-1 beta. Some moabs (SILK 3, 4, 5, 6, and 22) inhibited receptor binding of radioiodinated rIL-1 beta at concentrations that are similar to the dissociation constant values of antibody-rIL-1 beta binding. Another group of moabs (SILK 7, 11, 20, 21, and 23) also inhibited receptor binding but only at concentrations that are 10-150 times higher than their dissociation constants. A large group of moabs did not affect receptor binding in the concentration range tested, and two moabs enhanced the binding of rIL-1 beta to type I receptors. The result of pepscan analysis shows that the moabs bound to one or more of the amino acid sequences 35-49, 66-85, 78-97, 106-124, and 123-143 of mature rIL-1 beta. Modeling of rIL-1 beta shows that the binding domains of SILK 4, 5, 6, and 22 (sequence 123-143) is located at the closed end of the molecule, indicating that this part of rIL-1 beta harbors domains that are crucial for type I receptor binding. The binding domain of SILK 3 (sequence 66-85) is also located at this end of the molecule. In contrast, the binding domains of SILK 7, 11, 20, 21, and 23 (sequence 78-97) are located at the open end of the molecule, which is at the same face as the amino- and carboxy-terminals. The binding domain of SILK 16 (sequence 106-124) is positioned at the center of the molecule. It is concluded that the closed end of rIL-1 beta contains sequences that are crucial for its binding to type I receptors on murine EL4 cells. Because of the high concentrations of antibodies to residues 78-97 of rIL-1 beta that are needed to interfere with receptor binding, the importance of these domains in binding to type I receptors remains uncertain.

Proopiomelanocortin-Derived Peptides

Cloning of the cDNA of POMC mRNA led to the characterization of the ACTH precursor and established the molecular links between ACTH, beta LPH and gamma LPH, and beta end. Further previously unidentified POMC-derived peptides were also unravelled, including the N-terminal fragment and the JP. POMC is a precursor polypeptide that contains the amino acid sequences of numerous small polypeptide products. It contains eight pairs of basic amino acids and one sequence of four basic amino acids, which are the sites of cleavage for the recently identified prohormone convertases (PC1 and PC2), which have different specificities. PC1 but not PC2 is found in corticotroph cells of the anterior pituitary and cleaves POMC to generate NT, JP, ACTH, beta LPH and small amounts of gamma LPH and beta end. In the melanotroph cells of the intermediate pituitary, both PC1 and PC2 are present. The proteolysis of POMC is thus more extensive, resulting in a series of smaller peptides including gamma MSHs, CLIP, alpha MSH, beta MSH, beta end, and beta end. Equimolar amounts (because they are all products of the same precursor) of the POMC peptides are released into the blood by exocytosis from corticotroph cells of the anterior pituitary. No physiological role has been definitively identified for any of these products other than ACTH. POMC peptides have been assayed in human blood by specific radioimmunoassay (RIA). In most circumstances, the concentrations of all these peptides are perfectly correlated. Therefore, for practical reasons, LPH RIA is widely used as an indicator of overall corticotroph function. In rare cases, the correlation between the POMC peptides is disrupted by a general disorder selectively modifying the metabolism of the different members of the family as in chronic renal failure, or in tumors where POMC processing is abnormal. Examples include pituitary macroadenomas, some silent corticotroph adenomas, and most frequently non-pituitary tumors responsible for ectopic ACTH syndrome. These pathologies can lead to the formation and secretion into blood of CLIP and beta MSH, or alternatively POMC may be underprocessed and secreted intact. An elegant approach, specific IRMA, has been developed to directly recognize unprocessed POMC in blood.

Low external K+ regulates Na,K-ATPase alpha 1 and beta 1 gene expression in rat cardiac myocytes.

Incubation of mammalian cells in medium containing low K+ is often associated with an increase in Na,K-ATPase activity and content. In the present studies, we investigated the effect of low K+ on the regulation of Na,K-ATPase alpha 1 and beta 1 gene expression in primary cultures of neonatal rat cardiac myocytes. Northern blot hybridization analysis indicated that exposure of cardiocytes to a medium containing 0.3 mmol/L K+ for 72 h resulted in 1.8- and 3.3-fold increases in the abundance of alpha 1 and beta 1 mRNAs, respectively, compared with control cells exposed to 5.4 mmol/L K+. To investigate the molecular mechanism of the low K effect on beta 1 mRNA content, we constructed chimeric genes with a full-length and deleted portion of the 5' end of the rat beta 1 gene linked to the firefly luciferase gene. Transient transfection experiments using these constructs indicated that beta 1 gene sequences between -102 and +151 base pairs (bp) are required for low K(+)-induced trans-activation of reporter gene expression. Taken together, these results indicate that low K+ induction of beta 1 mRNA abundance in neonatal rat cardiac myocytes is mediated by regulatory DNA sequences in close proximity to the site of transcription initiation of the beta 1 gene.

Junctional diversity in the absence of N regions. Neonatal T cell receptor beta chain junctional sequences are more heterogeneous than neonatal T cell receptor gamma delta or IgH junctions.

Early in ontogeny, Ig, TCR-alpha beta, and TCR-gamma delta lack N regions. In addition, Ig and TCR-gamma delta junctions preferentially occur at regions of short sequence homology, thus limiting junctional diversity for these neonatal lymphocyte populations. Here, we analyze the extent of heterogeneity in TCR-beta chain junctions made early in ontogeny. DNA and cDNA from fetal/neonatal thymocytes were amplified by polymerase chain reaction, and the V-D and D-J junctions from these randomly generated sequences were analyzed. The D-J junctions were very heterogeneous, and displayed little evidence of homology-directed recombination. The V beta 8-D and V beta 5-D junctions that we analyzed each had a particular junctional sequence that was created at the site of a two-nucleotide homology, but in each case that sequence only comprised 10 to 17% of the total sequences. This junctional heterogeneity of N region lacking TCR-beta chains can be partially explained by a relative paucity of homologies at the appropriate locations near the coding ends, particularly at the D-J junction, but other factors such as the sequence surrounding the homology may also contribute. Thus, TCR-beta chains have extensive junctional heterogeneity early in ontogeny before N regions begin to be added. Since TCR-alpha beta CDR3 plays a major role in binding antigenic peptides, this junctional heterogeneity is likely to be advantageous for establishing a diverse TCR repertoire. We suggest that the sequences of the coding ends of the TCR-alpha beta have been selected through evolution to avoid the restricted junctional diversity resulting from homology-directed recombination.