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.     

Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.     

Evidence for an inhibition of thyroid hormone effects during chronic treatment with amiodarone

The effects of chronic amiodarone treatment on several thyroid and cardiac function parameters were studied in 50 euthyroid patients with refractory ventricular arrhythmias, divided in responders and nonresponders according to their sensitivity to the antiarrhythmic action of the drug. No differences in the severity of cardiac disease and blood amiodarone concentrations were found in the two groups. Amiodarone induced a significant inhibition of peripheral T4 monodeiodination, more pronounced in responders compared to nonresponders. On the contrary, only in responsive patients, elevated basal and TRH-stimulated TSH levels were observed (despite serum T3 levels were not different from those in nonresponders) and the indirect indices of cardiac performance, particularly the systolic time intervals, fell in a range usually observed in the hypothyroid states. These findings suggest that amiodarone, besides the well-known inhibition of T4 to T3 conversion, also induces a partial resistance to the thyroid hormones, which is probably involved in the therapeutical effectiveness of the drug.     

Interactions between mutations affecting ribosome synthesis in Escherichia coli

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.     

A colorimetric method for measuring the surface area of aquatic plants

A procedure is described that allows the determination of the surface area of aquatic plants by dipping them in a surfactant (Liquinox) dye (acridine orange) mixture and measuring the dye retained colimetrically. The method was applied to Vallisneria sp., Elodea canadensis Michx, Potamogeton richardsonii (A. Benn.) Rydb. and Myriophyllum spicatum L. For each species good relationships (0.91 < r² < 0.99) between computed area and dye retention capacity were found.     

Cell entry by measles virus: long hybrid receptors uncouple binding from membrane fusion.

The pH-independent fusion of membranes induced by measles virus (MV) requires, in addition to the fusion-competent protein F, hemagglutinin (H), and on the target membrane, the virus receptor CD46. We constructed hybrid receptors composed of different numbers and combinations of the four CD46 short consensus repeat (SCR) domains, followed by immunoglobulin-like domains of another cell surface protein, CD4. Hybrid proteins containing SCRs I and II bound MV particles and conferred fusion competence to rodent cells. SCRs III and/or IV strengthened MV binding. Increasing the distance between the MV binding site and the transmembrane domain enhanced virus binding but reduced fusion efficiency. A hybrid protein predicted to be about 120 Angstroms (12 nm) longer than the standard receptor lost fusion support function and was dominant negative over a functional receptor. These data indicate that receptor protein length influences virus binding and determines fusion efficiency.     

Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor

Together with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on-line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 muM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (+/- 4.4%). Both the bi-enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. (c) 1993 John Wiley & Sons, Inc.