Mode of action of ergonovine on isolated porcine coronary artery: the role of Ca2+

Coronary vasoconstrictor responses to ergonovine were examined in helical coronary arterial strips of young swine. Both ergonovine and serotonin (5-hydroxytryptamine) produced dose-dependent contractions of the strips. The distal region (less than 1.00 mm outer diameter) of the circumflex coronary artery was most sensitive to the responses of serotonin and ergonovine. Methysergide and nifedipine significantly depressed the contractions induced by ergonovine and serotonin. Atropine, propranolol, and the alpha 1 blocker, prazosin, did not antagonize ergonovine-induced contractions. The ergonovine response may depend entirely upon extracellular Ca2+ while the effect of serotonin may be mediated in part through the mobilization of intracellular Ca2+ stores. Increases in 45Ca2+ cellular contents occurred after ergonovine or serotonin and these increases were blocked by methysergide or nifedipine at concentrations which blocked mechanical responses to the agonist. It is concluded that the contractions of the porcine coronary artery produced by ergonovine and serotonin are as follows: (i) regionally sensitive; (ii) blocked by Ca2+ antagonists and therefore may utilize Ca2+ channels similar to those described in other excitable tissues; (iii) blocked by methysergide. These studies indicate that the major mechanism of ergonovine's action in the porcine coronary artery is through the activation of serotonin receptors on coronary arteries which are, in turn, linked to Ca2+ channels. However, this mechanism of action may be different in an intact animal.     

Evolution of feeding niches in New World monkeys.

The adaptive radiation of modern New World monkeys unfolded as the major lineages diversified within different dietary-adaptive zones predicated upon a fundamentally frugivorous habit. The broad outlines of this pattern can be seen in the fossil record, beginning in the early Miocene. Cebids are obligate frugivorous predators. The smallest forms (Cebuella, Callithrix) are specialized exudativores, and the largest (cebines) are seasonally flexible omnivores, feeding particularly on insects (Saimiri) or "hard" foods, such as pith and palm nuts (Cebus), when resources are scarce. The smaller-bodied atelids (Callicebus, Aotus) may use insects or leaves opportunistically, but pitheciins (saki-uakaris) specialize on seeds as their major protein source. The larger atelines (Alouatta, Brachyteles) depend on leaves or on ripe fruit (Ateles). Locomotion, body size, and dietary adaptations are linked: claws and small body size opened the canopy-subcanopy niche to callitrichines; climbing and hanging, the fine-branch setting to the atelines; large size and strength, semiprehensile tails, and grasping thumbs, the extractive insectivory of Cebus; deliberate quadrupedalism, the energy-saving transport of folivorous Alouatta. Body size increases and decreases occurred often and in parallel within guilds and lineages. Conventional dietary categories, particularly frugivory, are inadequate for organizing the behavioral and anatomical evidence pertinent to evolutionary adaptation. Related models of morphological evolution based on feeding frequencies tend to obfuscate the selective importance of "critical functions," responses to the biomechanically challenging components of diet that may be determined by a numerically small, or seasonal, dietary fraction. For fossils, body size is an unreliable indicator of diet in the absence of detailed morphological information. More attention needs to be given to developing techniques for identifying and quantifying mechanically significant aspects of dental form, the physical properties of primate foods, their mode of access, and the cycles of availability and nutritional value.     

Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.     

Analysis of human extrachromosomal DNA elements originating from different β-satellite subfamilies

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

Design considerations for two-stage enrichment clinical trials

When there is a predictive biomarker, enrichment can focus the clinical trial on a benefiting subpopulation. We describe a two-stage enrichment design, in which the first stage is designed to efficiently estimate a threshold and the second stage is a “phase III-like” trial on the enriched population. The goal of this paper is to explore design issues: sample size in Stages 1 and 2, and re-estimation of the Stage 2 sample size following Stage 1. By treating these as separate trials, we can gain insight into how the predictive nature of the biomarker specifically impacts the sample size. We also show that failure to adequately estimate the threshold can have disastrous consequences in the second stage. While any bivariate model could be used, we assume a continuous outcome and continuous biomarker, described by a bivariate normal model. The correlation coefficient between the outcome and biomarker is the key to understanding the behavior of the design, both for predictive and prognostic biomarkers. Through a series of simulations we illustrate the impact of model misspecification, consequences of poor threshold estimation, and requisite sample sizes that depend on the predictive nature of the biomarker. Such insight should be helpful in understanding and designing enrichment trials.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

Cell attachment to collagen: The ionic requirements

This study is concerned with three aspects of the ionic requirements for cell attachment to collagen; namely (a) the divalent, (b) monovalent cation specificities and (c) pH optima for cell interaction with collagen. The divalent cation requirement for cell attachment to collagen can be sufficed by Ca²⁺, Mg²⁺ and certain transition group metals; whereas Ba²⁺, Sr²⁺, and polyamines are inactive. The pH optimum for cell attachment in this system occurs in the physiological range. The monovalent cation requirement for cell attachment to collagen is satisfied by isotonic NaCl, KCl, LiCl, NH₄Cl, sucrose, and glucose. Pronounced inhibition of cell attachment occurs under both hypertonic and hypotonic conditions.