Perfusion-microcarrier cultivation of rCHO-cells in serum-free medium for production of human renin

A recombinant CHO cell line producing human prorenin was cultivated on microcarriers in serum-free medium. Best growth was obtained when the cells were cultivated on a collagen coated microcarrier (Cytodex 3) using a serum-free medium (SF-02) supplemented with fibronectin. It was possible to reduce the necessary concentration of fibronectin in the feed medium from 10 g/cm³ to 2 g/cm³ during perfusion cultures in a spinner reactor equipped with an UF-membrane. Also in this system, the prorenin concentration increased up to 8 times higher compared to that in a conventional repeated-batch culture. The cells grew in multilayers on the microcarriers during the perfusion culture. The specific prorenin productivity was not significantly affected by the cell growth rate, and the secretion of prorenin continued even after the cells had ceased to grow.     

β-Glucosidase biosynthesis inThermomonospora curvata

Summary -Glucosidase biosynthesis was induced inThermomonospora curvata during early exponential growth on cellobiose, cellulose and protein-extracted lucerne fibre in mineral salts minimal medium at 53°C. Only about 3% of the total culture -glucosidase was found in cell-free fluid when the actinomycete was grown on cellobiose or purified cellulose. A variety of non-lytic agents including detergents, salts, proteinase and electroporation failed to release the cellbound enzyme. However, cells grown on the fibre released more than 50% of their -glucosidase. The maximal amount of extracellular accumulation was dependent on the initial concentration of fibre in the medium. Thermal instability at 53°C was the major cause for low exracellular -glucosidase activity in cellobiose- and cellulose-grown cultures. In cultures grown on the fibre, the extracellular enzyme was stabilized against thermal denaturation by the composition of the cell-free fluid, but was degraded by transient proteinase activity. Proteolysis decreased the average beta-glucosidase specific activity from about 460mU/mg extracellular protein to about 80mU/mg within 1 day after the appearance of the proteinase.

---

Biosynthèse de -glucosidase chez Thermomonospora curvata

Résumé La biosynthèse de -glucosidase a été induite chezThermomonospora curvata pendant la phase exponentielle précoce de croissance sur cellobiose, sur cellulose et sur fibre de lucerne dont on avait extrait les protéines, en milieu minéral minimum à 53°C. A peine 3% de la -glucosidase totale dans la culture a été retrouvée dans le milieu acellulaire lorsque l'actinomyète a crû sur cellobiose ou sur cellulose purifiée. Une foule d'agents non-lytiques, comprenant des détergents, des sels, la protéinase et l'électroporation n'ont pas réussi à libérer l'enzyme liée aux cellules. Toutefois, les cellules développées sur la fibre ont libéré plus de 50% de leur -glucosidase. La quantité maximum d'accumulation extra-cellulaire a dépendu de la concentration initiale de fibre dans le milieu. L'instabilité thermique à 53°C a été la casue majeure des seuils faibles de -glucosidase extra-cellulaire dans les cultures sur cellobiose ou cellulose. Dans les cultures développées sur fibre, l'enzyme extra-cellulaire était stabilisée contre la dénaturation thermique par la composition du milieu acellulaire, mais elle était dégradée par l'activité transitoire de la protéinase. La protéolyse a décru l'activité spécifique moyenne de la -glucosidase d'environ 460 mU/mg de protéine extra-cellulaire à environ 80 mU/mg endéans la journée après l'apparition de la protéinase.

     

Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass

A comparison of different habitats, biogas plant, rumen fluid and sewage sludge, for cellulolytic organisms indicated sewage studge was the best source. Enrichment cultura gave a mixed culture which exhibited CMCase activity as well as extracellular Avicelase, xylanase, -glucosidase, -xylosidase activities and cell-bound -glucosidase, and -xylosidase production in a synthetic medium with eleven different cellulosic and lignocellulosic substrates. The activity of extracellular -glucosidase and -xylosidase production was significantly higher than endogenous activities. Hemicellulases were induced better than cellulases. The anzyme system was stable under aerobic conditions. Of the different lignocellulosic substrates, kallar grass was the best inducer of extracellular enzymes.

---

Résumé La comparaison de différents habitats: digesteur méthanique, fluide du rumen ou boue de station d'épuration, pour leur contenu en organismes cellulolytiques, indiquent que la boue de station d'épuration est la meilleure source. Une culture par enrichissement a produit une culture mixte qui a exhibé aussi bien une activité CMCase que des activitiés extracellulaires avicelasique, xylanasique, -glucosidasique et -xylosidasique et qu'une production de -glucosidase et de -xylosidase liées à la cellule, dans un milieu synthétique et pour onze substrats cellulosiques et lignocellulosiques différents. L'activité de la -glucosidase extracellulaire et la production de -xylosidase sont significativement plus élevées que les activitiés endogènes. Les hemicellulases sont mieux induites que les cellulases. Le système enzymatique est stable dans des conditions aérobies. Parmi les divers substrats lignocellulosiques, l'herbe Kallar est le meilleur inducteur d'enzymes extracellufaires.

     

Autoradiographic study of RNA synthesis in isolated cells in culture from chick embryo spinal ganglia

Summary Dissociated cells from 9, 12 and 15 day-old chick embryo spinal ganglia were cultivated in presence of total embryo-extract, brain embryo extract, or total embryo extract supplemented with purified nerve growth factor (NGF). The cells were maintained during 4 days in Maximow assembly and during 1 month in Rose chamber. Neurons showed growth of nerve fibres. The non-neural cells evolved to spindle cells, Schwann cells, or fibroblasts.Ribonucleic acid (RNA) synthesis was followed with tritiated uridine by autoradiography. Some nerve cells showed tritiated uridine incorporation. The highest incorporations for short-term cultures were at 15 hours in presence of NGF, at 48 hours in presence of total or brain extract, and for long-term cultures at 8 days. These periods corresponded to the highest growing activity of the nerve fibres. After 4 days all the non-neural cells incorporated tritiated uridine.The tritiated uridine was first incorporated into the RNA of the nucleus and, afterwards was found also in the cytoplasm. The presence of brain extract or of NGF stimulates the incorporation of labelled uridine into RNA. No labelling was found in the nerve fibres, even after 4 hours incubation.Chargée de Recherche au C.N.R.S.This communication is a part of the Doctorat és-Sciences thesis, presented by Mrs. J. Treska-Ciesielski.With the technical assistance of Mrs. M. F. Knoetgen and A. Bieth.     

The release of sterile males into natural populations of the German cockroach Blattella Germanica

A pilot experiment in genetic control of the German cockroach was conducted aboard a small vessel based at Norfolk, Virginia. It utilized sterile (double translocation heterozygotes) whose sterility effects were due to embryonic lethality plus complete entrapment of any remaining viable embryos within the egg case. Initial infestations were reduced by insecticides prior to male release. Three releases of unmated were made at monthly intervals. Infested harborages, located in the course of insecticide application, were used as release sites. The experiment was terminated after 41/2 months. The results showed that released joined groups near release sites and that they competed well against wild type . Apparently neither they nor with which they mated moved far from these sites. Sterility effects differed in respect to specific sites and general areas. Population growth was retarded markedly in the galley, the area of heaviest initial infestation. Terminal infestation was heaviest in the mess deck, although the highest sterility among occurred at/near the mess-deck release site (only one mess deck-infested site was found prior to initation of the experiment). It is suggested that a slight increase in the number released would have suppressed/eliminated groups inhabiting galley harborages, but that site selection was the major problem in the mess deck. Analyses of nymphal age classes and mating types among led to a hypothesis that insecticide-induced dispersal of nymphs resulted in the infestation of many new mess-deck harborages. Population growth was unchecked at these sites because they were too far removed from the sterile male release sites.

---

Résumé Une expérience pilote dans la lutte génétique contre Blattella germanica L. a été réalisée sur un petit navire encré à Norfolk, Virginie. La léthalité embryonnaire des mâles stériles utilisés (hétérozygotes à double translocation) était associée au piégeage des quelques embryons viables dans les oothèques. Les pullulations initiales avaient été réduites par des insecticides avant le lâcher des mâles. Des mâles vierges ont été lâchés à trois reprises à un mois d'intervalle. Les abris contaminés, localisés au cours des traitements insecticides, ont été utilisés comme lieux de lâcher. L'expérience s'est achevée au bout de quatre mois et demi. Les résultats montrent que les mâles introduits rejoignent les autres blattes près des lieux de libération et qu'ils ne sont pas défavorisés dans leur compétition avec les autres mâles. Ni eux, ni les femelles avec lesquelles ils se sont accouplés, ne s'éloignent apparemment de ces emplacements. Les effets de stérilisation varient en fonction des sites spécifiques et des zones plus vastes. La croissance de la population est significativement retardée dans la cuisine, zone où l'infestation initiale était la plus élevée. L'infestation finale la plus importante est dans le pont du mess, bien que la stérilité des femelles la plus élevée s'observe à (ou près) du lieu de libération au pont du mess (seulement un lieu d'infestation avait été observé sur le pont du mess avant le début de l'expérience). On pense qu'une légère augmentation de l'effectif libéré aurait supprimé (ou éliminé) les groupes occupant des abris dans la cuisine, mais que la sélection des sites était le principal problème pour le pont du mess. L'analyse des classes d'âges des larves et des catégories de femelles accouplées laisse supposer que la dispersion des larves provoquée par l'insecticide a entrainé la contamination de beaucoup de nouveaux abris dans le pont du mess. La croissance de la population n'a pas été contenue dans ces sites parce qu'ils étaient trop éloignés des lieux de lâcher des mâles stériles.

     

Le collagène du byssus de Mytilus edulis L.

Résumé L'autoradiographie révèle, au niveau du pied, une incorporation massive et sélective de la ³H-Proline dans la glande blanche de Mytilus edulis. Cette étude a permis de suivre le processus qui mène de la synthèse de la sécrétion dans la partie basale des cellules jusqu'a son émission dans le sillon pédieux où elle participe à la formation du filament. La collagénase détruit la presque totalité du marquage, attestant ainsi la nature collagénique du produit sécrété. Les autres glandes pédieuses ainsi que la glande du byssus proprement dite, située à la base du pied, montrent une incorporation très faible, sans commune mesure avec celle de la glande blanche. Ceci démontre de façon définitive que le collagène présent dans le filament prend naissance dans cette glande et justifie la dénomination de glande du collagène. Des contrôles réalisés dans différentes régions (bords du manteau, manteau, branchies) montrent que l'injection du précurseur dans le bord palléal constitue une méthode satisfaisante pour marquer de façon relativement rapide et différentielle le collagène de la glande.

---

The collagen of the byssus in Mytilus edulis L.II. Autoradiographic study on the incorporation of ³H-Proline

Summary Autoradiographic studies reveal a strong specific incorporation of ³H-Proline in the white gland in the foot of Mytilus edulis. The author could trace the radioactive secretory product from its synthesis in the basal part of the cells down to its outflow into the pedial groove where it takes part in the formation of the filament. Purified collagenase takes out radioactivity from the sections. This observation confirms the collagenous nature of the secretion.The other foot-glands as well as the main byssus gland located at the base of the foot show but a very weak labelling not comparable with that of the white gland. This clearly evidences that the collagen occuring in the filament originates from the latter. The white gland may be properly called: collagen gland.Control sections through different parts of the body (mantle-edge, mantle, gills) confirm that our injection technique of the precursor into the palleal margin is a suitable method for a rather quick and specific labelling of the glandular collagen.

Cette note fait partie d'un travail pour l'obtention d'une thèse de doctorat.     

Présence de collagène dans le proventricule des Syllidiens (Annélides Polychètes)

Résumé L'auteur décrit en particulier à l'aide du microscope électronique les différentes couches constituant la paroi du proventricule de Syllidiens (Annélides Polychètes) en insistant spécialement sur celles (zones sous-épithéliales) situées de part et d'autre de la couche des muscles radiaires caractéristique de cet organe. II est montré que, contrairement à ce qu'il était admis jusqu'à présent, les zones sous-épithéliales sont constituées par des fibres de collagène noyées dans un matériel granuleux vraisemblablement de nature mucopolysaccharidique. D'autre part, un système d'attache particulier entre zones sous-épithéliales et musculature radiaire est mis en évidence.Les fibres de collagène de périodicité 560 Å constituent, autour de la musculature radiaire, un réseau à mailles rectangulaires hautement organisé. Dans l'assise la plus interne de ce réseau, les fibres sont allongées parallèlement à l'axe antéro-postérieur du Ver; dans l'assise la plus externe, elles sont disposées circulairement dans des plans transversaux.La signification de la présence de ces fibres de collagène (les seules existant dans tout le corps des Syllis étudiés) est évoquée. Les problèmes de leur origine et du rôle possible de cellules différenciées (musculaires) dans la synthèse de ce collagène sont analysés.

---

Presence of collagen in the proventriculus of Syllids

Summary The layers of the wall of the proventriculus of Syllids (Annelids Polychetes) were studied, mainly with the aid of the electron microscope. The areas on both sides of the radial musculature (subepithelial zones) were given special attention. Contrary to previous views, it was shown that the subepithelial zones contain collagen fibrils within a granular material probably of mucopolysaccharide nature. Furthermore, a system of special attachment devices between subepithelial zones and radial musculature was demonstrated.The collagen fibrils (periodicity 560 Å) form a highly organized network with rectangular meshes around the radial musculature. In the innermost layer of this network, the fibers run parallel to the antero-posterior axis of the worm; in the outermost layer, they run circularly.The significance of the presence of these collagen fibers, the only in existence in the entire organism studied, is discussed. The problem of their origin, and the possible role played by differentiated (muscular) cells in the synthesis of this collagen are analyzed.

     

The behavior of retinal tissue in vitro,light and electron microscopic observations

Summary Retinae from two day old rats were used in this study and the cultures were handled according to standard methods used in this laboratory. In the first few days of cultivation an abundant outgrowth of nerve fibers into the cell-free medium was observed. These fibers later degenerated and by the beginning of the second week they had completely disappeared. In the living cultures, differentiating ganglion cells, bipolar and horizontal neurons could be seen in the main explant in association with various types of glial cells. Rod cells became arranged as epithelial sheets or as clusters of cells which often formed rosettes. The nuclei of these sensory cells possessed a characteristic chromatin pattern by which they always could be differentiated from other cells in the cultures. Cytoplasmic extensions that developed from the free surface of the sensory rod cells were observed within a week following explantation. A limiting membrane separated these extensions from the nucleated part of the rod cells. Morphologic details of the different neuronal cell types could be demonstrated in cultures by Bodian's silver impregnation technique.With the electron microscope, retinal development in culture was observed and compared to the development of the retina of the intact eye. Cilia developed from processes extending from the rod cell free surface. These processes were the rod cell inner segments in which many mitochondria were seen. At the bases of these segments terminal bars developed forming the outer limiting membrane. In the area of the terminal bars microvillous extensions projected between the rod cell inner segments. After twelve days in vitro a bulb-like enlargement containing a lamellar membrane system developed at the end of the cilium. This bulb-like enlargement was a beginning of the rod cell outer segment. The lamellar system did not acquire the symmetry or precise organization during cultivation that was observed in the retina of the intact eye. The distinguishing characteristics of individual neuronal cell types seen in cultivated retinae were the same as those described for their counterparts in the retina in situ, but regular plexiform layers failed to develop. Likewise, there were no indications of typical synapses in the neuropils of the cultures. There were many processes containing vesicles similar to those in presynaptic endings and mitochondria but membrane thickenings were not apparent.The results indicate that the retina cultivated in vitro does not behave as an organized entity. The component cells dissociated more and more with time, and developmental differentiation was observed only at the cellular level.Supported by USPHS Grants 5R01NB03114-06 and 5T01GM00459 from the National Institutes of Health, Bethesda, Maryland.Sincere appreciation is expressed to Mrs. Eleanor Morris for management of the cultures, and to Mr. E. E. Pitsinger, Jr. for his photographic assistance.     

Sur l'ultrastructure des ≪corps de call et d'exner≫ dans l'ovaire du lapin

Résumé On a effectué une étude ultrastructurale sur les corps de Call et d'Exner. Ceux-ci se montrent constitués, sous leur aspect le plus typique, par une couronne de cellules de la granulosa disposées autour d'une cavité pleine d'un liquide semblable au liquor folliculi .Les cellules, de forme irrégulière, accolées dans leur partie proche du corps et écartées dans celle éloignée, déterminent des fentes dans lesquelles s'insinuent des groupes d'autres cellules voisines; la membrane cytoplasmique, parfois présente sous forme de microvilloités, parfois incisée, montre de fréquentes interruptions; le cytoplasme riche en ribosomes libres et adhérant aux parois du réticule endoplasmique (ergastoplasme), possède en outre un appareil de Golgi modérément développé et de nombreuses mitochondries; le noyau, très volumineux, présente à l'intérieur un nucléole grossièrement réticulé.Le corps de Call et d'Exner proprement dit est, au contraire, constitué par une cavité unitaire d'aspect à peu près sphérique d'environ 15–30 de diamètre, pleine d'un liquide dont les caractéristiques sont comparables à celles du liquor folliculi; dans cette cavité on voit un fin réseau à mailles irrégulières, tandis que sa surface montre une mince zone de condensation.L'aspect ultrastructural des cellules disposées autour des corps de Call et d'Exner est caractérisé par des cellules en élaboration, ce qui laisse penser qu'une partie de cette élaboration peut être versée à l'intérieur du corps, en contribuant ainsi à l'augmentation de son volume.En ce qui concerne, enfin, le réticule que l'on voit à l'intérieur du corps il est vraisemblable qu'il soit du à des phénomènes de condensation et consécutif à un état physicochimique particulier du liquide endocavitaire.

---

Summary The so-called Call-Exner bodies have been found to be formed, in their most typical shape, by a ring of granulosa cells enclosing a cavity filled with a fluid comparable to the liquor folliculi. The cells, irregular in shape, lying side by side in their portions closer to the body and far apart in their distal portions leave open spaces which are occupied by prolongations of adjacent cells. The cytoplasmic membrane, sometimes forming mierovilli, sometimes incisions, presents a discontinuous aspect; the cytoplasm is very rich in ribosomes which can be free or sticking to the walls of the endoplasmic reticulum (ergastoplasm). It has a fairly developed Golgi's apparatus and numerous mitochondria, sometimes open. The very voluminous nucleus shows internally a roughly reticulated nucleolus.The Call-Exner body, on the other hand, is a unitary cavity approximately spherical in shape, having about a 15–30 micron diameter filled with a fluid characteristically similar to the liquor folliculi, which shows internally a fine reticulation of irregular meshwork, while a thin condensation zone can bee seen peripherically.The ultrastructural aspect of the cells radiating from the Call-Exner bodies is typical of cells in formation. Therefore we cannot exclude the possibility that a portion of the substances elaborated by the cells may be thrown into the center of the body, thus contributing to its increase in volume. The mesh-like structure visible inside the body may be due to condensation phenomena following a particular physico-chemichal change in the endocavitary luid.

     

Sur l'existence de cellules de type ≪corticotrope≫ dans la pars intermedia de l'hypophyse du rat

Résumé Nos observations démontrent l'existence de deux types de cellules glandulaires dans la pars intermedia de l'hypophyse du rat: des cellules propres, à M.S.H., dont les grains de sécrétion sont détruits par la fixation osmiée et relativement bien conservés par la fixation au glutaraldéhyde; des cellules à petits grains denses, osmio-résistants, qui s'apparentent morphologiquement aux éléments corticotropes de l'antéhypophyse. L'existence bien connue d'A.C.T.H. ou, plus précisément, d'un facteur A.C.T.H.-like dans le lobe neuro-intermédiaire nous fait considérer comme très vraisemblable la nature corticotrope de ce deuxième type cellulaire de la pars intermedia.

---

On the existence of cells of the Corticotrophic type in the intermediate lobe of the rat hypophysisAn electronmicroscopic study

Summary Two types of glandular cells have been shown to occur in the intermediate lobe of the rat hypophysis: 1) M.S.H. producing cells whose secretory granules are destroyed by osmium fixation and relatively well preserved by glutaraldehyde fixation, 2) cells with small dense osmium resistant granules being morphologically similar to the corticotrophic cells of the anterior lobe. The well known occurrence of A.C.T.H. or, more precisely, of an A.C.T.H.-like factor in the neuro-intermediate lobe makes the corticotrophic nature of this second cell type very likely.

Travail dédié à la mémoire de Nicole Granboulan.     

Initiation and growth characteristics of suspension cultures of Calendula officinalis cells

Callus cultures of marigold (Calendula officinalis L.) were induced on Murashige and Skoog medium with different concentrations of auxin (dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA) and cytokinin (kinetin or 6-(,-dimethylallylamino)purine (2iP). Of all hormone combinations used in the medium, two were the most efficient in promoting callus development: 1.81 M (0.4 mg l^–1) 2,4-D and 1.85 M (0.4 mg l^–1) kinetin (0.4d–0.4k culture) or 0.45 M (0.1 mg l^–1) 2,4-D and 2.02 M (0.5 mg l^–1) 2iP (0.1d–0.5p culture). These combinations were selected to induce cell suspension cultures. The suspension cultures were maintained under light or dark conditions. The light stimulated cell aggregation in the cultures. In both cultures cells were undifferentiated under darkness, whereas in the light, rhyzogenesis was observed in 0.1d–0.5p culture. The cell growth and protein and oleanolic acid contents were determined. Initially, biomass production was similar under light and dark conditions, but after 7–8 months from the induction the cell growth was reduced by approximately 30% in the light, whereas the cell growth of the cultures maintained under darkness did not reveal any changes. The presence of oleanolic acid was detected in the suspension cultures kept in darkness. This compound reached two quantitative peaks: in the lag and stationary phases –- beyond the active growth phase of the culture cycle and its concentration was several times higher in 0.1d–0.5p culture than that in 0.4d–0.4k culture. It was for the first time that callus and suspension cultures were induced from the marigold plant.     

Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds.     

Role respectif des males et des femelles dans la formation des sexués néoténiques chezCalotermes flavicollis

Résumé ChezCalotermes flavicollis, la formation des sexués néoténiques est plus facile, ou plus rapide, dans le sexe femelle que dans le sexe mâle.Les sexués femelles montrent un pouvoir inhibiteur à l'égard des individus femelles; les sexués mâles inhibent les mâles de façon moins complète; la stabilisation complète peut être obtenue dans les élevages unisexués, formés uniquement de mâles ou de femelles.La régulation du nombre des néoténiques ne se fait pas de la même façon dans les élevages et dans les élevages . Dans les premiers, 2 néoténiques subsistent, quelquefois 3; dans les seconds ne persiste qu'un seul néoténique , rarement deux.Ces résultats mettent en lumière le rôle différent joué par les mâles et les femelles dans les sociétés de Termites.     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.     

Rabbit articular chondrocytes in alginate gel: characterisation of immobilized preparations and potential applications

Summary Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After long-term immobilization, the cells still exhibited metabolic activities, patterns of division, synthesis and secretion of extracellular matrix macromolecules such as type II collagen and proteoglycans. After 38 days, immobilized rabbit articular chondrocytes predominantly expressed type II but not type I collagen. Thus, they maintained their cartilage phenotype. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. On the basis of these results, long-duration storage and large-scale production of extracellular matrix components are being investigated.Some of the results were presented at the international congress Physiology of immobilized cells, December 10–13, 1989, Wageningen, The Netherlands Correspondence to: M. Lièvremont     

Über das Wachstum der Pankreaszellen von Säugetieren als Monolayer Cultures

Zusammenfassung Aus Zellsuspensionen von Pankreasgewebe jugendlicher Ratten und Meerschweinchen sowie menschlicher Feten züchteten wir Monolayer Cultures großer fibroblastoider Zellen. Diese färben sich mit Aldehydfuchsin ebenso wie B-Zellen der Langerhansschen Inseln an; mit Pseudoisocyanin geben sie eine positive Reaktion auf Insulin. Mit Hilfe einer immunologischen Methode konnten wir sowohl in der gesammelten Nährlösung als auch in den Zellen nach Ausbildung eines Zellrasens Insulin (=IRI=immuno reactive insulin) feststellen.

---

Monolayer cultures of pancreatic tissueI. Methods of culture, morphology, insulin content of tissues and culture medium

Summary Monolayer cultures of large fibroblastoid cells could be cultivated from cell suspensions of pancreatic tissue of juvenile rats and guinea pigs as well as human fetuses. These cells are stained with aldehyde fuchsin exactly like the B-cells of the islets of Langerhans; with pseudo-isocyanine their reaction is positive to insulin. It is possible by means of an immunological method to determine insulin (=IRI=immuno reactive insulin) in the collected nutrient medium as well as in the cells after formation a complete monolayer.

     

Fragmenta mycologica

Summary Castellani's water culture method for microscopic fungi was re-examined and confirmed in its every detail. It is an ideal method for, at least, the smaller culture collection, in order to avoid continuous, short term subculturing. Benedek's suggestion for establishment of a mycotheca preserved in chlorallactophenol on the analogy of the herbarium of phanerogamists, may fill a long felt gap in mycological laboratories.

---

Zusammenfassung Castellani's Wasserkultur-methode für mikroskopische Pilze wurde einer Kontrolluntersuchung unterzogen. Sie konnte in all ihren Einzelheiten bestätigt werden. Sie ist eine ideale Methode, wenigstens für kleine Kultursammlungen, um eine fortgesetzte, kurz-fristige Überimpfung zu vermeiden. Benedek's Vorschlag für die Einrichtung einer Mykotheka in Chlorallactophenol in Analogie mit dem Herbarium für Blütenpflanzen, mag eine lang gefühlte Lücke im mykologischen Laboratorium beseitigen.

---

Résumé La méthode deCastellani, la culture en d'eau des champignons microscopiques, a été ré-examinée. Elle pouvait être confirmée en tous les détails. C'est une méthode idéale, au moins, pour la collection de cultures de petite dimension, pour éviter les transferts continus et d'intervalle courte.La suggestion deBenedek pour établir une mycothèque en chlorallactophénol, par analogie avec l' herbier pour les phanérogames peut remplir une lacune, existante pour long temps, aux laboratoires mycologiques.

     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

The aerial migration of the Six-Spotted Leafhopper and the spread of the virus disease Aster Yellows

Source areas for Six-Spotted leafhopper migrations into Manitoba are determined by wind trajectory studies and are found to be mainly in South Dakota, Nebraska and Kansas. There is a high enough frequency of southerly winds for the insects to migrate into Manitoba every spring. Attempts to correlate general weather conditions, both in southern Manitoba and in the source areas,with lettuce crop damage in Manitoba, were inconclusive.This leads to the conclusion that the intensity of aster yellows infestation in Manitoba depends on the environment experienced by the lettuce crop. Favourable meteorological conditions occurring for a few days only, at just the right time, can produce heavy infection in any year.

---

Zusammenfassung Nach Flugbahnstudien sind South Dakota, Nebraska und Kansas die Ursprungsgebiete der Heuschrecke MACROSTELES FASCIFRONS (Stal),(der wichtigste Wirt des Aster Yellow Virus, das zu schweren Schäden bei Salatpflanzen führt)bei den Wanderungen nach Manitoba. Die Häufigkeit von Südwinden ist gross genug, so dass die Insekten jeden Frühling nach Manitoba einwandern können.Eine Beziehung zwischen den allgemeinen Wetterbedingungen in den Ursprungsgebieten und Manitoba und den Schäden in Salatpflanzungen in Manitoba war nachweisbar.Dies führt zu dem Schluss, dass die Heftigkeit von Aster Yellow Virus Plagen in Manitoba von den klimatischen Bedingungen in den Salatpflanzungen abhängt. Einige Tage mit günstigen Wetterbedingungen können, wenn sie zur richtigen Zeit eintreten, in jedem beliebigen Jahr zu schweren Infektionen der Pflanzen führen.

---

Resume D'après l'étude des trajectoires des vents, le Dakota du Sud, le Nebraska et le Kansas sont les régions d'origine des sauterelles MACROSTELES FASCIFRONS (Stal.), c'est à dire du principal vecteur du virus Aster Yellow qui provoque de gros dégâts dans les cultures de salades. Les vents du sud sont suffisament fréquents pour que les insectes soient entraînés chaque printemps jusque dans le Manitoba. On peut en outre prouver qu'il existe une relation étroite entre les conditions météorologiques régnant aussi bien dans les régions d'origine qu'au Manitoba d'une part et les dégâts dans les cultures de salades de ce dernier état d'autre part. On en conclut que l'intensité des infections par Aster Yellow dépend des conditions météorologiques dans les cultures. Quelques journées présentant des conditions météorologiques favorables peuvent, si elles se manifestent en temps opportun, provoquer des infections dévastatrices et cela chaque année.

---

---

An Honors Mathematics and Physics student at the University of Manitoba who was employed as a Student Assistant during the summer of 1964 with Meteorological Service of Canada.     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.