Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus

The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKR^kd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (C^ko) virus but had no effect on the growth of either wild-type (WT) or V knockout (V^ko) virus. Increased growth of the C^ko virus in PKR^kd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, V^ko, or especially C^ko virus caused significantly less apoptosis in PKR^kd cells than in PKR-sufficient cells. Although apoptosis induced by C^ko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of C^ko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.     

Fish infected with Sphaerospora spp. Thélohan (Myxosporea) from waters enzootic for proliferative kidney disease of salmonids

Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.     

Trypsin-sensitive neutralization site on VP1 of Theiler''s murine encephalomyelitis viruses.

We generated Theiler's murine encephalomyelitis virus mutants resistant to several neutralizing monoclonal antibodies (MAbs) having their epitopes near a trypsin cleavage site of VP1. Neutralization and Western blot (immunoblot) studies suggest that two of the MAbs have identical epitopes that partly overlap the epitope of a third MAb. Sequencing of RNA of the mutants localized the epitopes to a site near the carboxyl end of VP1. The limited diversity of nucleotide changes seen in the mutants and the immunodominance of the site suggest that the carboxyl end of VP1 may have an important function.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Über die Entwicklung der Arbeits- und Erregungsleitungsmuskulatur des Herzens von Ratte und Meerschweinchen

Zusammenfassung Die Chemodifferenzierung der Herzmuskulatur von Ratte (215 Tiere) und Meerschweinchen (180 Tiere) wird untersucht und zur Morphodifferenzierung und elektrophysiologisch nachweisbaren Funktionsentwicklung in Beziehung gesetzt. Ferner wird die Entwicklung der Arbeitsmuskulatur mit der des Reizleitungssystems verglichen. Prinzipiell verhält sich die Herzentwicklung bei Ratte und Meerschweinchen ähnlich, doch bestehen erhebliche Unterschiede im Terminplan der Entwicklung. Bei der Ratte erfolgen wesentliche Schritte der Herzentwicklung nach der Geburt, beim Meerschweinchen ist die Entwicklung etwa zur Zeit der Geburt abgeschlossen. In der frühen Embryonalzeit ist die Herzmuskulatur reich an Glykogen, aber arm an Enzymen des oxidativen Stoffwechsels (Bernsteinsäuredehydrogenase, Cytochromoxidase) und -Hydroxibuttersäuredehydrogenase. Kapillaren fehlen. Dann — bei der Ratte etwa ab 12. Embryonaltag — beginnt der Glykogenbestand der Arbeitsmuskulatur abzunehmen. Zunächst werden die subepikardial gelegenen Schichten betroffen. Von hier schreitet die Glykogenverminderung nach innen fort. In den glykogenarm gewordenen Zonen vermehrt sich der Bestand an Atmungsfermenten und Kapillaren. Die Chemodifferenzierung in der Kammermuskulatur erfolgt grundsätzlich von außen nach innen. Parallel hierzu verläuft die Strukturentwicklung der Herzmuskelzellen und es kommt zu elektrophysiologisch nachgewiesenen Änderungen in der Permeabilität der Herzmuskelzellmembranen. Abgeschlossen ist die Entwicklung bei der Ratte nach färberisch-lichtmikroskopischen Befunden in der Mitte der 2. Lebenswoche, nach histochemischen Befunden in der 4. Lebenswoche, nach Maßgabe elektrophysiologischer Beobachtungen nach dem 31. Lebenstag. Die Vorhofmuskulatur ist mit Ausnahme frühembryonaler Stadien stets reicher an Glykogen und ärmer an Atmungsfermenten als die Kammermuskulatur. — Das Reizleitungssystem durchläuft eine eigene Entwicklung. Ursprungsgewebe für das Atrioventricularsystem ist der atrioventriculäre Muskelring. Von hieraus wachsen spezifische Fasern in die Kammermuskulatur vor. Sie unterscheiden sich histochemisch von vornherein von der Arbeitsmuskulatur.

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Summary The present study deals with the chemodifferentiation of the cardiac muscle in rats (215 animals) and Guinea pigs (180 animals). The results obtained are compared with the morphological differentiation and the functional development, as determined by electrophysiological methods. Finally, the development of the cardiac muscle is compared with the development of the conducting system. Fundamentally the development as such is the same in rats and Guinea pigs; however, there are striking differences as far as the schedule for the development is concerned. In rats, important steps in the development of the heart take place after birth, where as in Guinea pigs the development is terminated at approximately the time of birth. In the early embryonic stages the cardiac muscle has a high content of glycogen, but it is poor in enzymes of the oxidative metabolism (succinate dehydrogenase, cytochrome oxidase) and -hydroxibutyricaciddehydrogenase. There are no capillaries. Approximately from the 12th embryonic day onwards a decrease of the glycogen content is observed in the cardiac muscle of rats. The first layers to undergo this change are the subepicardial layers. From there it progresses to the innermost layers. The activity of the respiratory enzymes and the number of the capillaries increases in the areas where the glycogen content is now relatively low. On principle the chemodifferentiation of the ventricular mucsle progresses always from the outermost to the innermost layer. The structural development of the cells of the cardiac muscle goes parallel with it; at the same time changes in the permeability of the membranes of cardiac muscle cells are demonstrable by electro-physiological methods. According to light microscopical findings the development in rats is terminated in the middle of the second week of life, according to histochemical findings in the 4th week of life, electrophysiologically after the 31st day of life. It seems to be a rule that apart from the early embryonic stages the auricular muscle is richer in glycogen and poorer in respiratory enzymes than the ventricular muscle. The conducting system has a separate pattern of development. The initial form of the atrioventricular system is the atrio-ventricular muscular ring. From there specific fibres penetrate into the ventricular musculature. Histochemically they are a priori different from the cardiac muscle.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Stipendiatin der Humboldt-Stiftung.

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Frau Prof. Berta Scharrer mit herzlichem Glückwunsch zum 60. Geburtstag gewidmet.