Cytogenetic analysis using fluorescence in situ hybridization (FISH) to evaluate occupational exposure to carcinogens

Chromosomal aberrations determined by conventional method or fluorescence in situ hybridization (FISH) technique with whole chromosome painting are used as biomarkers of effect. Groups occupationally exposed to 1,3-butadiene (BD), acrylonitrile, ethyl benzene and benzene in petrochemical industry, and carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) from ambient air were followed by conventional method and FISH painting for chromosomes # 1 and # 4, in total 383 subjects, including controls. No effect was observed by either method with exposure to 1,3-butadiene < 1mg/m(3) and acrylonitrile < 0.3mg/m(3). Ethyl benzene and benzene exposure significantly increased chromosomal aberrations by both methods, which decreased after the implementation of preventive measures. The genomic frequency of translocations by FISH calculated as FG/100 was significantly increased in city policemen versus control group exposed to c-PAHs from ambient air (1.72+/-1.57 versus 1.25+/-1.11, P<0.05). The method of FISH with whole chromosome painting seems to be more sensitive to detect chromosomal injury by occupational exposure to carcinogens than conventional method.     

P2X7 receptor expression after ischemia in the cerebral cortex of rats

Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.     

Constitutive overactivation of protein kinase C in guinea pig brain increases alpha-secretory APP processing without decreasing beta-amyloid generation

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and beta-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased alpha-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP alpha-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of beta-amyloid peptides 1-40 or 1-42 as measured by ELISA, suggesting that only the alpha-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.     

Increased neuronal and glial expression of protein kinase C isoforms in neocortex of transgenic Tg2576 mice with amyloid pathology

We investigated the influence of five- to sevenfold neuronal overexpression of the Swedish mutation of human APP695 (APPsw) in the transgenic mouse strain Tg2576 on neocortical protein kinase C (PKC) expression and subcellular distribution. Using specific antibodies to PKC alpha, PKC beta, PKC gamma, PKC epsilon and PKC zeta isoforms for Western blot analysis, we observed increased immunoreactivity for PKC alpha and PKC gamma isoforms in crude tissue homogenates from the neocortex of 16-month-old APPsw mice as compared with nontransgenic littermates, which was not present in 6 month-old Tg2576 mice. We also observed elevated levels of PKC alpha, PKC beta, PKC gamma and PKC zeta in membrane fractions and reduced concentrations of PKC alpha and PKC gamma in cytosolic fractions of aged Tg2576 mice, indicating that these PKC isoforms are in their activated state. In young, 6-month-old Tg2576 mice, however, the increase in membrane-bound PKC isoforms and concomitant decrease in cytosolic PKC isoforms was much less pronounced, demonstrating the age-dependent nature of alterations in PKC isoforms. Immunocytochemistry of brain sections supported these findings and revealed increased neuronal labelling for PKC alpha, PKC gamma and PKC lambda isoforms in neocortex of 16-month-old APPsw mice compared with nontransgenic littermates, with the increase being strongest for PKC gamma and PKC lambda isoforms. Additionally, PKC gamma and to a lesser extent PKC lambda isoforms were induced in reactive astrocytes in proximity to amyloid plaques. Our data indicate that neuronal overexpression of APPsw causes a dynamic change in neuronal expression and activation of multiple PKC isoforms known to be regulators of proteolytic amyloid precursor protein (APP) processing (PKC alpha) and of neuronal survival (PKC lambda and PKC zeta). The induction of the PKC gamma and PKC lambda isoforms in reactive astrocytes surrounding amyloid plaques might be required for astrocyte activation and astrocytic cytokine expression in response to amyloid plaque formation.     

Contractile properties of papillary muscle from young cardiomyopathic hamsters: effects of isoprenaline

Papillary muscles isolated from the left ventricle of 17-20 day old Syrian hamsters with genetic cardiomyopathy (BIO 14.6 strain) were examined for their mechanophysiological response to the beta adrenoceptor agonist isoprenaline. The indices of isometric twitch for both myopathic and aged matched normal (BIO RB strain) hamster papillary muscles were statistically the same before exposure to catecholamine. Isoprenaline, however, elicited an exaggerated degree of positive inotropism in myopathic papillary muscles. In addition, the characteristic relaxing effect of beta adrenoceptor agonists (abbreviation of twitch duration) was virtually absent in myopathic muscles. These results suggest that under conditions resembling sympathetic stimulation sarcolemma and perhaps sarcoplasmic reticulum become inadequate regulators of calcium, predisposing young myopathic heart cells to accrue damaging concentrations of sarcoplasmic calcium.     

Chronisch rezidivierender Lupus erythematodes visceralis mit akut tödlicher zentralnervöser Exacerbation

Zusammenfassung 1. Bei einem seit fast 9 Jahren an Lupus erythematodes visceralis leidenden 26jährigen Mann trat der Tod wenige Tage nach erstmaligem Auftreten zentralnervöser Krankheitssymptome ein. Die Autopsie ergab eine akute Meningomyeloencephalitis mit Schwerpunkt der Veränderungen im Rückenmark und im Tuber cinereum. Verschiedene feingewebliche Besonderheiten charakterisieren den Prozeß und trennen ihn von allen bisher bekannten Encephalomyelitiden ab.2. Hierher gehören regressive Zellkernveränderungen der entzündlichen Elemente im Sinne der Pyknose und Karyorrhexis sowie eigenartige Cytoplasmaeinschlüsse, die als Ausdruck des L.e.-Phänomens aufgefaßt werden.3. Hierher gehören ferner die Zeichen schwerer Permeabilitätsstörungen und deren Folgen, wie Verquellungen der Gefäßwände, Plasmaextravasate, Diapedesisblutungen, ödembedingte Nekrosen, und die gliös-mesenchymalen Reaktionen darauf.4. Subtile Veränderungen, vor allem endarteriitische Prozesse der Meningealgefäße, weisen darauf hin, daß schon früher zentralnervöse Krankheitsschübe klinisch unbemerkt abgelaufen sind.5. Obwohl das klinische Bild seit Jahren an der Diagnose keinen Zweifel ließ, fehlen am sonstigen Autopsiebefund jene Veränderungen, die als typisch für den L.e.v. gelten: Keine Endokarditis Libman-Sacks, keine deutlichen Drahtschlingengefäße in den Nieren, keine Hämatoxylinkörper, hingegen Granulome in Leber und Lunge und eine diffuse, vorwiegend lymphocytäre Hyperplasie der Lymphknoten.Mit 8 TextabbildungenHerrn Professor Berthold Ostertag mit herzlichem Gruß und Glückwunsch zum 28. Februar 1965.Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

An effective morphometric method for electron microscopic studies on papillary muscles

Summary Morphometry was performed on the left ventricular posterior papillary muscles of seven Wistar rats. The volume densities of myocardial cells, interstitial space, myocardial nuclei, sarcoplasm, mitochondria, myofibrils, ground substance and T tubules, and the surface densities of myocardial cells, mitochondrial membranes and T tubules, were calculated. Though only 1 ultrathin section per animal was evaluated the low standard errors of the means indicate that the method described here will be adequate in most experimental studies. Due to the anisotropy of the surfaces within myocardial cells, the papillary muscles were cut at an angle of 32.4 to their longitudinal axis. This angle is derived from an equation published by Whitehouse (1974). The procedure to correct the loss of cristal membrane images from oblique sectioning is discussed.This investigation was supported by the Sonderforschungsbereich 90 of the Deutsche Forschungsgemeinschaft. The skillful assistance of Zlata Antoni, Harald Derks and Peter Rieger are gratefully acknowledged.