Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Special secretory cells in the soybean seed coat

Summary The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a cone, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.This paper is dedicated to the memory of my parents, Joseph and Theresa Yaklich, who by their example taught me the value of work and the enjoyment of simple things.     

Enzymatic methylation of DNA and poly(dG-dC) X poly(dG-dC) modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide

Both the initial velocity and the overall methylation of Ac-4HAQO modified DNA by a calf brain DNA (cytosine-5-)-methyltransferase are increased as compared to native DNA. The affinity of the modified DNA for the enzyme decreases as a function of the extent of the modification. Heat-denatured, single-stranded DNA shows exactly the opposite results: the more it is modified, the less it is methylated. The poly(dG-dC) X poly(dG-dC) modified by 4NQO is as well methylated as the non-modified one. The carcinogen may induce a tertiary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity and cellular differentiation.     

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.     

Precocene causes male determination in the aphid Myzus persicae

When adult apterous viviparous females of Myzus persicae, reared in short night conditions at 21–23°C, are treated with the precocene analogue 6-methoxy-7-ethoxy-2,2-dimethylchromene, they deposit males towards the end of their reproductive lives. The first-born normal daughters of the treated females also deposit some males at various times in their reproductive lives. Karyotypic analysis was used to investigate the sequence of male and female embryos in the ovarioles of precociously metamorphosed aphids. The experiments support the hypothesis (Mittler et al., 1979) that juvenile hormone level controls the sex determination process in aphids. Since male aphids have an XO sex chromosome constitution, this implies that juvenile hormone level influences the behaviour of the X-chromosomes at or before the single maturation division of the egg. At this division one X-chromosome is eliminated from eggs which will develop as males. Aphids provide the first example of a specific endocrine influence on chromosome behaviour in sex determination.     

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.

     

Active Oxygen Contributes to the Major Part of Chromosomal Aberrations in V79 Chinese Hamster Cells Exposed to N-Hydroxy-2-Naphthylamine

N-hydroxy-2-naphthylamine (NOH-2NA). an active metabolite of human occupational bladder carcinogens, induced, in V79 Chinese hamster cells. chromosomal aberrations which were suppressed in the presence of catalase and/or superoxide dismutase. The induction of the aberrations was more efficient in a more basic pH in parallel with the generation of hydrogen peroxide from NOH-2NA. The possible role of the oxidation product of NOH-2NA in the induction of the aberrations is discussed.     

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.