Effects of phorbol esters on endothelial cell microfilaments: Laser scanning confocal microscopy and quantitative morphometry of dose dependent changes

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Production of trichothecene and non-trichothecene mycotoxins by Fusarium species isolated from maize in Minnesota

Eighty-two cultures of Fusarium species isolated in 1986 from moldy maize in Minnesota were each cultured on rice for 4 weeks and found to produce the following mycotoxins: F. graminearum isolates, deoxynivalenol (DON, 4–225 g/g), 3-acetyldeoxynivalenol (3-ADON, 2–4g/g), 15-acetyldeoxynivalenol (15-ADON, 1–35 g/g) and zearalenone (ZEA, 5–4350 g/g); F. moniliforme, fusarin C (detectable amounts to 1000 g/g); F. mòniliforme, F. oxysporum, F. proliferatum and F. subglutinans isolates, moniliformin (15–6775 g/g); F. moniliforme, F. proliferatum, and F. subglutinans isolates, fusaric acid (detectable amounts). Other mycotoxins screened for in each rice sample and not detected were T-2 toxin, HT-2 toxin, neosolaniol, T-2 tetraol, nivalenol, fusarenon-X, scirpenols, alpha and beta trans-zearalenols, wortmannin, and fusarochromanone. The rat feeding bioassay indicated that other, unidentified toxins may be present.     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Les régulateurs de croissance d'insectes (RCI), mimiques de l'hormone juvénile,en tant que moyen de lutte morphogénétique et ovicide contre les tordeuses des vergers

Résumé La métamorphose des insectes est régie par un équilibre hormonal complexe dans lequel l'hormone juvénile (HJ) joue un rôle important. Au dernier stade larvaire, la teneur en HJ est particulièrement faible dans le corps de l'insecte. Si un régulateur de croissance d'insectes (RCI)-un mimétique de l'HJ-est appliqué à ce moment-là, la mue nymphale est pertubée provoquant des déformations morphogénétiques caractéristiques. La teneur en HJ est également très faible dans les ufs fraîchement pondus. Les traitements aux RCI peuvent par conséquent perturber le développement embryonnaire de certaines espèces et produire ainsi un effet ovicide. Depuis quelques années deux RCI-le fenoxycarb et le CGA 45 128-ont été testés pour leur activité morphogénétique sur le dernier stade larvaire de quelques ravageurs tels qu'Adoxophyes orana F.v.R., ainsi que pour leur activité ovicide sur les ufs frais de Cydia pomonella L. et Grapholita funebrana Tr. Après quelques années d'expérimentation et de commercialisation des RCI dans les vergers européens, il s'avère que l'utilisation de ces produits peu toxiques, sélectifs et peu nocifs pour la faune utile, constitue une amélioration considérable pour l'aménagement de la lutte intégrée.

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Insect growth regulators (IGR), mimics of juvenile hormone, as morphological and ovicidal means of control against orchard tortricids

Summary Metamorphosis is regulated by a complex hormonal balance in which juvenile hormone (JH) plays an important part. At the last larval instar the content of JH is particularly low in the insect body. If an insect growth regulator (IGR) — a mimic of JH-is applied at this time, the pupal moult may be disturbed with the characteristic morphogenetical deformations. The JH content is also very low in freshly laid eggs. Therefore IGR treatments may disturb the embryonic development of some species and produce an ovicidal activity. During a few years two IGR-fenoxycarb and CGA 45128-were evaluated for their morphogenetical effect on the last larval instar of Adoxophyes orana F.v.R. and their ovicidal effect on freshly laid eggs of Cydia pomonella L. and Grapholita funebrana Tr. After a few years of experimentation with both compounds and of commercialisation of fenoxycarb in European orchards, IGR confirmed to present a considerable improvement in integrated pest management due to selectivity, and low mammal toxicity.

     

Effect of high-intensity endurance training on isokinetic muscle power

The purpose of this study was to determine the effects of high-intensity endurance training on isokinetic muscle power. Six male students majoring in physical-education participated in high intensity endurance training on a cycle ergometer at 90% of maximal oxygen uptake (VO2max) for 7 weeks. The duration of the daily exercise session was set so that the energy expenditure equalled 42 kJ.kg-1 of lean body mass. Peak knee extension power was measured at six different speeds (30 degrees, 60 degrees, 120 degrees, 180 degrees, 240 degrees, and 300 degrees.s-1) with an isokinetic dynamometer. After training, VO2max increased significantly from mean values of 51.2 ml.kg-1.min-1, SD 6.5 to 56.3 ml.kg-1.min-1, SD 5.3 (P less than 0.05). Isokinetic peak power at the lower test speeds (30 degrees, 60 degrees and 120 degrees.s-1) increased significantly (P less than 0.05). However, no significant differences in muscle peak power were found at the faster velocities of 180 degrees, 240 degrees, and 300 degrees.s-1. The percentage improvement was dependent on the initial muscle peak power of each subject and the training stimulus (intensity of cycle ergometer exercise).     

Immunoreactivity to the pancreatic polypeptide family in the nervous system of the adult human blood fluke,Schistosoma mansoni

Summary The presence and distribution of neuropeptides belonging to the pancreatic polypeptide family have been demonstrated by an indirect immunofluorescence technique in the nervous systems of adult male and female Schistosoma mansoni. Seven antisera of differing regional specificity to pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) were employed on both whole-mount and cryostat-sectioned material. Positive immunoreactivity (IR) was obtained with all antisera except an N-terminally-directed antiserum to NPY. In the CNS, immunoreactivity was restricted to cell bodies and nerve fibres in the anterior ganglia, central commissure and dorsal and ventral nerve cords of both sexes, whereas, in the PNS, positive-IR was present in the plexuses innervating the subtegumental musculature and the oral and ventral suckers. Intense immunoreactivity was observed in a plexus of nerve fibres and cell bodies in the lining of the gynaecophoric canal and in fine nerve fibres innervating the dorsal tubercles of the male. In contrast, in the female, strong immunoreactivity was evident in nerve plexuses innervating the lining of the ovovitelline duct and in the wall of the ootype, but most notably in a cluster of cells in the region of Mehlis' gland. Results suggest that molecules with C-terminal homology to the PP-family are present in S. mansoni. These peptides would appear to be important regulatory molecules in the parasite's nervous system and may play a role in the control of egg production.