Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

Structure fine du rein de l'Epinoche (Gasterosteus aculeatus L.) au cours de sa transformation muqueuse

Résumé Pendant la période de reproduction, les néphrons du rein de l'Epinoche mâle subissent d'importantes modifications de structure sous l'action des hormones sexuelles. Au niveau de chacun d'entre eux, se différencient deux segments distincts par leur fonction et par leur cytologie. Le segment urinaire, très court, est formé de cellules identiques à celles du jeune, qui remplissent leur fonction d'excrétion. Le segment glandulaire, plus volumineux, subit une transformation muqueuse et élabore une sécrétion qui sert à construire le nid. L'évolution de ces deux segments est étudiée au cours de la période de reproduction et les modifications cytologiques correspondantes sont décrites.

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Fine structure of the kidney of the three-spined-stickleback (Gasterosteus aculeate L.) during its mucous transformation

Summary Under the action of sexual hormones the nephrons of the kidney of the male three-spined-stickleback undergo considerable transformations during the breeding period. They differentiate into two segments which differ from one another in function and cytology. The cells of the urinary segment are identical to those of the young fish. They have an excretory function. The glandular segment undergoes a mucous transformation and synthesizes a secretion which is used for the building of the nest. The cytological transformations occuring at the level of these two segments during the breeding period are described with special attention to the mucous cells.

     

Ultrastructure de l'endomètre humain normal

Résumé L'étude du chorion cytogène de 15 endomètres humains normaux prélevés à divers moments du cycle menstruel a précisé d'importantes variations des vaisseaux et des cellules.Les vaisseaux subissent une maturation progressive: en phase proliferative moyenne, des pointes d'accroissement se forment à partir du réseau vasculaire profond activé; au milieu du cycle, elles se transforment en capillaires typiques; en phases sécrétoires moyenne et avancée, des péricytes migrent dans le chorion cytogène et se différencient en cellules choriales.Ces cellules choriales, dites fixes, ont une évolution biphasique au cours du cycle menstruel. Au milieu de chacune des phases, proliférative et surtout sécrétoire, les cellules choriales fixes, dites alors fibroblastoïdes, montrent une intense activité de synthèse. A ces périodes de synthèse succède une involution cellulaire, peu marquée en fin de phase proliferative, intense en phase sécrétoire avancée.Les cellules dites prédéciduales sont des cellules choriales fixes involuées et hyperhydratées; elles vont, en phase menstruelle, évoluer de plusieurs façons: la plupart d'entre elles régénèrent, certaines se nécrosent focalement ou totalement, d'autres font preuve d'activité macrophagique, en particulier collagénolytique.

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Ultrastructure of the normal human endometriumI. The stroma

Summary 15 human endometria during the normal menstrual cycle have been investigated. Important alterations of the vessels and the stroma cells occur.The vessels are the site of gradual maturation. In the mid proliferative phase, growing capillaries rise from the deep-seated vascular system. In the middle of the cycle, they change into typical capillaries. In the mid and late secretory phases, pericytes leave the walls of the capillaries and differentiate into stroma cells.These stroma cells undergo a biphasic cyclic evolution. The middle of the proliferative and particularly of the secretory phase is marked by an intensive synthetic activity of the stroma cells which are called, at this time, fibroblastoïd stroma cells. These two periods of synthesis are followed by cellular involution, mild in the proliferative, intense in the secretory phase.The so-called predecidual cells are hyperhydrated involuted stroma cells. In the menstrual phase they behave very differently of: the majority regenerates, some predecidual cells are the site of focal or total necrosis, others show a macrophagic activity which is conspicuous in some cells having a collagenolytic activity.

Nous remercions Monsieur le Professeur Gandar, Directeur de la Clinique Gynécologique et Obstétricale 1 de la Faculté de Médecine de Strasbourg de nous avoir confié, pour examen ultrastructural, le matériel nécessaire à cette étude.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Interferon inhibits the accumulation of glycerol phosphate dehydrogenase mRNA in oligodendrocytes and C6 cells

We investigated the effect of rat interferon-/ (IFN) on the expression of glycerol phosphate dehydrogenase (E.C.1.1.1.8; GPDH), in both C6 cells and pure cultures of oligodendrocytes. IFNs are naturally produced inhibitors of cell growth that can also affect differentiated cell functions. GPDH is a biochemical marker for oligodendrocytes and is known to be developmentally regulated and steroid inducible. GPDH activity is induced by hydrocortisone (HC) 3.5 fold in C6 cells and 5 fold in oligodendrocytes compared to untreated cultures. A pretreatment of these cells with 75 U/ml of rat IFN-/ resulted in an inhibition of the HC induction of GPDH enzymatic activity by 50% and 40% in C6 cells and oligodendrocytes respectively. We also found that IFN impaired the accumulation of GPDH mRNA in both cell types. These results demonstrate that IFNs are capable of modifying the cellular response to hormones in cells of neuroepithelial origin, and suggest the possibility that IFNs may be able to influence the development and function of the brain.Special issue dedicated to Dr. Paola S. Timiras     

Influence of cytosolic pH changes on the organisation of the endoplasmic reticulum in epidermal cells of onion bulb scales: Acidification by loading with weak organic acids

Summary The anastomosing ER system of epidermal cells of onion bulb scales is composed of three modifications: lamellar and tubular elements, located in the cell periphery, and long tubular stands located deeper in the cytoplasm. Cytoplasmic acidification of epidermal cells by loading with weak organic acids like acetic or propionic acid causes the decay of the lamellar elements and the disappearance of long tubular strands. Organelle movement is also inhibited. The effects depend on the pH of the incubation medium and on the administered acid concentration, and are characterized by a distinct lag phase of about 7 min. The induced ER changes are transient with adaptation starting after about 50min. Buffer components alone have little influence on the cellular ER organization within a pH-range of 4.0–8.0. However, the pH of the medium strongly affects the time course of the effects as well as recovery after omitting the administered acid. Both modulation and recovery occur more rapidly at neutral or slightly alkaline pH. Actin filaments, which play a major role in ER organization and organelle movement, are not affected by cytosolic acidification.Dedicated to the memory of Professor Oswald Kiermayer     

Molecular analysis of the extent of asymmetry in two asymmetric somatic hybrids of tomato

Summary Two somatic hybrid plants generated from a single fusion event between Lycopersicon esculentum and irradiated L. pennellii protoplasts have been analyzed at the molecular level. Over 30 loci have been analyzed using isozymes and RFLPs. All loci tested on chromosomes 2–10 were heterozygous, while those loci on chromosome 12 were homozygous L. pennellii in both somatic hybrids. In one of the somatic hybrids, 2850, loci on chromosome 1 were also homozygous L. pennellii. The other somatic hybrid, 28F5, was heterozygous at all chromosome 1 loci tested, but exhibited altered stoichiometry of parental bands as compared to the sexual hybrid. Loci on chromosome 2 from both somatic hybrids have altered stoichiometry, with L. pennellii alleles being four times more abundant than expected. Both somatic hybrids contain the L. esculentum chloroplast genome, while only L. pennellii polymorphisms have been detected in the mitochondrial genome.     

Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.