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Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.

On the basis of 90 cerebral AVMs, the authors study clinical signs which show the AVM, before a dramatic bleeding. Such clinical signs are: benign subarachnoid hemorrhage or intracranial hypertension and, only for lobar AVMs, migraine, epileptic seizures, progressive neurological deficit. In such cases a misdiagnosis is avoided by CT Scan with contrast. In the second part, the authors show that the AVMs surgical removal gives better results than AVMs natural history studied over a 20 years period. Ten AVMs observed in deep coma died. Two surgical deaths are only observed out of 73 AVMs surgical removals. Out of 44 lobar AVMs totally removed, 37 show good results and seven disabilities. Out of 19 deep AVMs, 13 were totally removed and six partially. Such deep AVMs, especially AVMs of the corpus callosum or of lateral and third ventricles choroid plexus give excellent surgical results. The authors conclude that surgical removal is the safer treatment for the majority of AVMs (Acta neurol. belg., 1985, 85, 65-81).

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.

Cytochrome b5 has been visualized by an immunoferritin labeling in a Golgi preparation. When the Golgi apparatus was first disrupted before labeling, 60% of the profiles were clearly labeled with ferritin. This proportion of labeled vesicles was too high to be explained by contamination from other membranes. When the Golgi structure was maintained during the experimental process, obviously some small vesicles located at the periphery of the Golgi complex were labeled and at least one saccule per Golgi apparatus was labeled but only at the edges. These conclusions were sustained by a quantitative analysis which showed a low but constant labeling of Golgi saccules and tubules and a very high labeling of some small profiles. These results confirm the presence of cytochrome b5 in the Golgi apparatus but stress its heterogeneous distribution; they also sustain the idea of a possible membrane specific shuttle between the ER and the Golgi apparatus by a recycling process suggested by the presence of highly labeled small vesicles found in close vicinity with the cisternae.

Glucose 6-phosphate dehydrogenase has been found to be altered in ageing human fibroblasts in culture. In this article, we have studied the effects of incubation conditions on glucose 6-phosphate dehydrogenase present in isolated cells or homogenates from young and old cells. We show that incubation at 4 degrees C and pH 7.4 induced the appearance of a heat-labile enzyme; this was not the case at pH 6.5. Also NADP+, when present, protects the enzyme from being modified. The kinetics of the modification indicates that the altered form of the enzyme is an intermediary stage between the active and inactive forms. We now have a model system in which the appearance of an altered enzyme can be induced in only a few hours; this model will be used in our next paper to study the mechanism of this alteration.

Products of the major histocompatibility complex (H-2) are important in allograph rejection. In view of the close relationship between mother and foetus, we can consider the latter as an allograph which is however not rejected by an immunological reaction. We studied the presence of H-2 antigens on embryo membranes at the time of implantation, by immunochemical labeling using gold particles coupled with protein A. Results showed that the expression of H-2 antigens is different before and after implantation. It seems that after implantation, H-2 antigens disappear from trophoblastic membranes. This could explain the absence of immunological reaction of the mother against the foetus.

A two-stage chemostat was used to study removal of cadmium by microorganisms in continuous culture. The medium was contaminated with 0.8 mg of Cd per liter. At 20 degrees C, most of the microbial biomass formed aggregates which settled in the second stage of the chemostat. Effluent was free of bacteria. Up to 80% of the metal contained in the inlet flux was removed by the biomass, with 20% remaining in solution. At 10 degrees C and with a shorter retention time, flocculation was poorer and metal removal by settling biomass did not exceed 35%.

The bacterial communities of three aquatic systems were analyzed in order to compare the influence of heavy metals. The first system was a sedimentation pond in a zinc-copper factory. The second was the bank of the Belgian river Meuse covered by the mossPlatyhypnidium riparioides (Hedw.) Dix. contaminated with heavy metals. The third was the bank of the same river covered by the same uncontaminated moss.The study was focused mainly on cadmium.The reciprocal averaging method showed that some bacterial strains could develop in very high concentrations of cadmium, but their physiological characteristics were not the same as those of the sensitive strains. In addition, the characteristics of the resistant strains depended on the environment. Correlation between resistance to heavy metals and to antibiotics was observed but was not the same in all communities. The density of resistant strains was roughly related to the level of toxicity in the environment.

A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages. The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions. The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.

Esophageal dilatations under topical pharyngeal anesthesia with the Eder-Puestow's metallic olives system were performed 101 times on 30 patients presenting peptic strictures. This method uses first a metallic wire introduced into the esophagus and the stomach under endoscopic control. Then, the wire allows to guide dilatating olives through very tight strictures. Functional results were excellent 28 times out of 30; two patients only required repeated dilatations because of uncomplete treatment (stenosis on esojejunal anastomosis). Finally, surgical procedure was never indicated. The use of Eder-Puestow's material together with fiberoptic esophagoscope reduces the hazard of peroral bougienage: no complication occured in our series.

Electron microscopic observation of sera from 59 subjects(HBs Ag-negative controls, HBs Ag-positive asymptomatic blood donors, and HBs Ag-positive patients with acute viral hepatitis, chronic persistant or active hepatitis and alcohol-induced liver damage) was capable of specifying the morphology of the HB virus (spherical particles, rod-shaped particles and Dane particles) and of correlating the aspect of the observed viral material with the nature and evolution of the liver damage. The viral particles are observed in small amounts in asymptomatic blood donors and in patients with acute viral hepatitis, but are present in very significant quantities in chronic hepatitis. Dane particles and viral particle aggregates are more numerous in chronic active hepatitis than in chronic persistant hepatitis. The detection of HBeAg in the serum, especially found in chronic active hepatitis, correlates with the presence of Dane particles. In most cases, the observed viral particle aggregates do not seem to be associated with an immunological process.

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

Yeast alcohol dehydrogenase was successfully immobilized on tresyl-chloride-activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%. Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.

Fibroblasts from hamster embryos cultivated in vitro present the typical ageing process of other fibroblastic lines, but they also suddenly give rise to giant non dividing cells which could be considered to represent terminally differentiated cells [36]. We investigated the latter mechanism, first by showing that microtubules in these cells depolymerized from the centrosome and not from the cell periphery as in other cells; secondly we analysed the structure of the centrosome on serial sections and found a diminished pericentriolar material; finally time lapse sequence studies of cell division confirmed that this process sometimes aborts giving rise to these giant polyploid cells. As a consequence, what first appeared as a differentiation process is in fact the result of an environmental deterioration which probably reaches a critical level thus creating a catastrophic consequence for the cell.

When the aflatoxin-producing strain of Aspergillus flavus, nr 14409 developed in mixed cultures with non toxigenic strains, in solid or liquid media, it produced less aflatoxin than in pure culture. Investigations in view to elucidate the process promoting the toxin disappearance showed that the mycelium could immobilize a large amount of aflatoxin by adsorption on its walls.

This study had for objective to detect the psychological morbidity of 176 non-consulting primiparas in the region of Liege using both the PSE and a sociological questionnaire. A high incidence of anxio-depressive manifestations was recorded in the month following birth (almost 27% of the cases) and their persistence at a lower rate in the 8th and 18th month. We were finally able to point out a certain number of risk factors: socioeconomic and familial conditions, somatic factors (difficult pregnancy and birth), recent psychological trauma or persistent conflict.

The appearance of a heat-labile glucose 6-phosphate dehydrogenase fraction can be induced by incubation of human fibroblasts at 4 degrees C and pH 7.4. Using this system we first studied the effects of pH and NADP+ on the reversibility of the alteration. In young cells, the induced alteration of glucose 6-phosphate dehydrogenase disappeared if the pH of the medium was lowered to 6.5 or if NADP+ was added. In old cells, the disappearance of the natural heat-labile glucose 6-phosphate dehydrogenase was possible only if both pH 6.5 and NADP+ were present. Secondly, we studied the effects of the alteration and its reversibility on the equilibrium between the enzyme subunits; we showed that alteration and inactivation are linked to a dissociation of the enzyme subunits into inactive monomers. We also proposed a model where the heat-labile enzyme is a transient form between the active dimer and inactive monomer. Our results are in perfect agreement with the theories of post-translational modifications now proposed to explain the presence of the altered enzymes in old cells.

In order to facilitate the homogenization of lung tissue it was previously incubated with collagenase during 30 minutes. Morphological observations were performed in order to ascertain the cell integrity. The enzymatically digested tissue was homogenized in a 0.25 M sucrose solution containing 1 mM EDTA, 3 mM imidazole (pH.7.3) and supplemented with 1 mM imipramine in order to stabilize the mitochondria, which otherwise might contaminate the microsomal fraction. The homogenate was then centrifuged and subdivided into four fractions which were analyzed for their content in protein and for the activities of so-called marker enzymes. The cytochrome P450 level was measured in both control and 3-methylcholanthrene preparations. The activities and the kinetic parameters of lung benzpyrene hydroxylase and aldrin epoxidase were measured using the lung microsomal fractions from control and previously 3-methylcholanthrene treated rats; 3-methylcholanthrene pretreatment modified the catalytiac properties of both enzymes.

Microtubules and microfilaments were investigated in hamster lung fibroblasts, during their in vitro life-span. These cells show a senescence process characterized by a drastic phenotypic change, resulting in two phenotypes: the type 1 cells, characteristic of young cultures and the type 2 cells appearing progressively with culture passages. Microtubules and microfilaments were observed at the TEM and also visualized by the unlabelled peroxidase-anti-peroxidase method. Moreover, the susceptibility of microtubules to nocodazole was tested in type 1 and 2 cells. We could not provide evidence for a different susceptibility to the drug. However the depolymerization wave occurred centripetally in type 1 cells whilst centrifugally in type 2 cells. These observations are discussed in relationship with the early arrest of division growth of the type 2 differentiated cells.