Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.     

PHENOBARBITAL-INDUCED SYNTHESIS OF THE MICROSOMAL DRUG-METABOLIZING ENZYME SYSTEM AND ITS RELATIONSHIP TO THE PROLIFERATION OF ENDOPLASMIC MEMBRANES : A Morphological and Biochemical Study

Liver microsomes, isolated from rats which had been treated with phenobarbital in vivo, were found to exhibit increased activities of oxidative demethylation and TPNH-cytochrome c reductase and an increased amount of CO-binding pigment. Simultaneous administration of actinomycin D or puromycin abolished the phenobarbital-induced enzyme synthesis. Increased rate of P_i³² incorporation into microsomal phospholipid was the first sign of phenobarbital stimulation and appeared 3 hours after a single injection of this drug. Microsomes were divided into smooth-surfaced and rough-surfaced vesicle fractions. The fraction consisting of smooth-surfaced vesicles exhibited the greatest increase in protein content and oxidative demethylation activity after phenobarbital administration in vivo. Ultrastructural studies revealed that drug treatment also gave rise to proliferation of the endoplasmic reticulum in the hepatic parenchymal cells, first noticed after two phenobarbital injections. The phenobarbital-induced synthesis of the metabolizing enzymes is discussed with special reference to the relationship to the stimulated synthesis of the endoplasmic membranes.     

Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.     

Induced defence reaction in Scots pine following stem attacks by Tomicus piniperda

The induced defence reaction of Scots pine Pinus sylvestris to stem attacks by the common pine shoot beetle, Tomicus piniperda (Col, Scolytidae), was studied by inducing natural attacks on young Scots pine trees of different vigour Pruned trees were more heavily attacked by T piniperda than unpruned ones Vigorous trees successfully contained the attacking beetles within resinous lesions, whereas less vigorous trees failed to defend themselves The content of resin acids increased dramatically in the lesions on all trees, and the resin acid composition differed somewhat between some treatments Starch accumulation in the inner bark of the main stem was lower in pruned trees than in unpruned ones, and starch reserves were depleted in the most heavily attacked trees All but one of the unpruned trees survived the attack, whereas most of the severely pruned ones died following attack by beetles at densities exceeding c 300 egg galleries m⁻²     

Distribution of insulin binding sites on Leydig cells of rat testes using insulin-coated gold particles

Summary The distribution of insulin binding sites in Leydig cells dispersed with collagenase from rat testes was studied using insulin-coated gold particles as an electron opaque ligand. Using electron microscope is convenient to distinguish Leydig cells among a variety of cells in crude preparations by their ultrastructural characteristics. Leydig cells were shown to possess insulin-binding sites on their plasma membranes. Initial binding sites of insulin were located to the microvillous surfaces. Following binding, receptor-ligand complexes seemed to move to the intermicrovillous plasma membrane, then to be internalized. Two modes of the internalization were confirmed. Most of the receptor-ligand complexes on Leydig cells appeared to be internalized via large, uncoated plasma membrane invaginations, while the remainder became internalized via small pits into vesicles. The receptor-ligand complexes were subsequently transferred to large subsurface vacuoles with electron-lucent lumens believed to correspond to endosomes. The reason why IGCs on the postendosomal pathway moving toward lysosomes was also discussed.     

Fungal entomopathogens in a tritrophic context

Variation in plant quality has an important impact on insect growth and development and there is considerable evidence that plants can also influence an insect’s natural enemies. Here we discuss the potential for plant-mediated effects on fungal entomopathogens. Fungi differ from other insect pathogens in that they infect an insect directly through its cuticle. This means that they are particularly vulnerable to changes in microclimate and properties of the insect cuticle. Potential direct and indirect mechanisms for plant-mediated effects on fungal entomopathogens are discussed. It is clear from these studies that fungal entomopathogens could be affected by plant volatiles and plant surface chemistry. Plant secondary chemicals can also inhibit fungal growth, potentially protecting the insect herbivore. However, the site of action and the mechanism behind these effects in plant-based studies is not always clear. The implications for biocontrol using fungal entomopathogens are discussed.