Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

The influence of level of infection of rats with Nippostrongylus brasiliensis on the haematology and phospholipase activity and mast cell numbers in the small intestine and colon

The haematology and phospholipase activity and mast cell numbers of the small intestine and colon of rats was studied 10 days after infection with various numbers of larvae of N. brasiliensis. A significant reduction in the RBC occurred after infections with 200 and 5000 larvae but not with 1000 larvae. Hb was significantly reduced after infection with 200 larvae and increases in the MCV and MCH indicated the development of a macrocytic anaemia. Reticulocyte count was increased at all levels of infection except after 200 larvae. WBC was increased at all levels of infection except in the 5000 larvae group. Lymphocytes were significantly increased in all groups except those infected with 5000 larvae. Neutrophils increased only at the lower levels of infection. The most marked changes occurred in eosinophil numbers, significant increases occurring with increasing levels of infection. However, after infection with 5000 larvae the numbers were significantly lower than after infection with 200 or 1000 larvae. Phospholipase activity, which is believed to be related to tissue eosinophil levels, was significantly increased at all infection levels in the proximal small intestine. Significant increases in the distal ileum and colon occurred mainly after infection with 1000 and 5000 larvae. Mast cell numbers did not change significantly at any infection level. It is suggested that the pathology observed, here in the form of anaemia, is multifactorial in origin and is largely a function of the immune response, the development and expression of which is dependent on the level of infection, with suppression of immune damage occurring at the high levels of infection when pathogenesis may involve a direct effect of the worms.     

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.     

Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

A possible phagocytic role for folliculo-stellate cells of anterior pituitary following estrogen withdrawal from primed male rats

Summary Ultrastructural changes suggesting a phagocytic role for the nongranular folliculo-stellate cells of the anterior pituitary are investigated in estrogen-primed male rats after withdrawal of estrogen. Morphological changes in mammotropes following the removal of a subcutaneous estradiol-containing Silastic implant include the formation of intracellular lipid bodies. These lipid bodies appear to be associated with enhanced estrogen-dependent prolactin secretion in mammotropes. Seven and 24 h after estrogen withdrawal intracellular lipid within mammotropes seems to be released into the intercellular space. Seventy-two h after estrogen withdrawal, lipid droplets are almost entirely cleared from mammotropes while folliculo-stellate cells become packed with lipid globules. Folliculo-stellate cells also undergo dramatic hypertrophy 7 and 24 h after the removal of E2-containing implants. Extensive intercellular junctions including zonulae adhaerentes, desmosomes, and putative gap junctions are formed. Intercellular junctions delineate extravascular channels into which numerous microvilli project. Folliculo-stellate cells appear capable of accumulating many lipid droplets, presumably related to mammotrope metabolism. What appear to be large secondary lysosomes as well as the lipid droplets are observed within folliculostellate cells; lipid, therefore, may be degraded through a lysosomal pathway in folliculo-stellate cells.     

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.