Fetal rat brain hemisphere tissue in nonadherent stationary organ culture

A simple organ culture system for brain tissue is described. Fragments of fetal rat brain hemisphere tissue are explanted to multiwell dishes base-coated with semisolid agar. In this system nonadherent organ culture can be performed for at least 50 days. Cell migration, biochemical and morphological differentiation and the formation of a layered architecture seem to mimic some of the phenomena occurring in the developing rat brain in vivo. The fragments may therefore be a useful organ culture model for nervous tissue.     

Ultrastructural investigation of fetal rat brain hemisphere tissue in nonadherent stationary organ culture

Fetal rat brain fragments grown in nonadherent stationary organ culture for 50 days have been investigated ultrastructurally. Synaptogenesis and myelin formation occurred at the same time as the corresponding time-dependent events in the developing brain in vivo. Intermediate junctions were observed between cellular processes lining a central cavity in the fragments and later associated with astrocytes at the surface. Gap junctions and tight junctions were also present. In some fragments cilia were observed in the central cavity. Subependymal basement membrane labyrinths were observed in all fragments after 10 days in culture. The ultrastructural characteristics and the tissue-like structure in general were preserved for at least 50 days in this tissue culture system. The brain fragments may therefore be a valuable supplement to existing culture methods for nervous tissue.     

Ultrastructural investigation of fetal rat brain hemisphere tissue in nonadherent stationary organ culture

Summary Fetal rat brain fragments grown in nonadherent stationary organ culture for 50 days have been investigated ultrastructurally. Synaptogenesis and myelin formation occurred at the same time as the corresponding time-dependent events in the developing brain in vivo. Intermediate junctions were observed between cellular processes lining a central cavity in the fragments and later associated with astrocytes at the surface. Gap junctions and tight junctions were also present. In some fragments cilia were observed in the central cavity. Subependymal basement membrane labyrinths were observed in all fragments after 10 days in culture. The ultrastructural characteristics and the tissue-like structure in general were preserved for at least 50 days in this tissue culture system. The brain fragments may therefore be a valuable supplement to existing culture methods for nervous tissue.Research fellow from the Norwegian Research Council for Sciences and Humanities. This work was supported by the Norwegian Cancer Society.     

Inhibition of plasminogen activator production in organ cultures by cycloheximide

Summary The production of tissue plasminogen activator (TPA) in rat tongue organ cultures is strongly inhibited by low concentrations of the protein synthesis inhibitor cycloheximide. TPA production is fully resumed after the removal of cycloheximide from the culture medium.This study was supported by a USPHS grant (HE-05050) from the National Heart and Lung Institute.     

Identification of organ-specific glycosylation of a membrane protein in two tissues using lectins

Since glycosylation of proteins is performed by the host cell, and variable sugar groupings can confer heterogeneity on the same polypeptide, we wished to see whether membrane proteins, in particular the ubiquitous transmembrane Na, K-ATPase, could be glycosylated differently in different organs. Using a highly sensitive enzyme-linked antibody detection system of bound digoxigenin-labelled lectins on nitrocellulose sheets containing electroblotted and subunits of kidney and brain Na,K-ATPase, isolated from various rat strains, in combination with isoform-specific immunoblots, we discovered that brain Na,K-ATPase was highly mannosylated in contrast to renal Na,K-ATPase. Thus, we describe the existence of organ-related glycoforms of an integral ubiquitous membrane protein, i.e. diversification of the same polypeptide by organ-typical sugars. At the same time, the presence of the same glycosylation pattern can make distinct protein isoforms occurring in a same organ more homogeneous. Such organ-related glycoforms may serve for tissue identification and as tissue-specific receptors.     

Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain

Summary The morphology and histochemistry of dissociated newborn rat brain was studied in tissue culture. Direct microscopy of developing cells, electron microscopy and the alkaline phosphatase activity were used to identify the capillary endothelial cells.Acknowledgments. We thank Dr J. Gripenberg for technical assistance. This research was supported by Finnish Cultural Foundation and carried out during the tenure of a fellowship provided for F. J. from the Finnish-Hungarian Cultural Exchange Program.     

Demonstration of testosterone secretion by testicular tissue of hypophysectomized boar as affected by HCG in organ culture]

Boar Leydig cells undergo a strong atrophy from 1 to 3 months after hypophysectomy but can be reactivated by the gonadotropin HCG in organ culture conditions. This reactivation which appeared at histological and ultrastructural level was evidenced by the capacity of testicular tissue to synthesize testosterone as judged by radioimmunoassay. Both synthesis in the tissue and release into the medium increased according the incubation time with HCG; the adjonction of 17 alpha-OH-pregneolone to culture medium led to increase the intra and extra-tissular concentration of testosterone.     

Role of glucocorticoids on the maturation of brush border enzymes in fetal rat gut endoderm

Heterospecific recombinants between fatal rat intestinal endoderm and chick mesenchyme, and also undissociated fetal rat intestine, were submitted to different hormonal environments. The present study shows that exogenously-supplied dexamethasone in organ culture, like endogenous hormones provided by the adult rat (grafting experiments) led to similar qualitative and quantitative results, i.e., a 9-fold stimulation of maltase and a precocious induction of sucrase activity in comparison with an hormonal conditions.     

Role of glucocorticoids on the maturation of brush border enzymes in fetal rat gut endoderm

Summary Heterospecific recombinants between fetal rat intestinal endoderm and chick mesenchyme, and also undissociated fetal rat intestine, were submitted to different hormonal environments. The present study shows that exogenouslysupplied dexamethasone in organ culture, like endogenous hormones provided by the adult rat (grafting experiments) led to similar qualitative and quantitative results, i.e., a 9-fold stimulation of maltase and a precocious induction of sucrase activity in comparison with anhormonal conditions.Supported by grant CRL 80 70 17 from the INSERM and by the CNRS. This work was presented in part at the XVth EDBO International Embryological Conference.     

Tissue-specific inhibition of embryonic neural cell proliferation by rat brain extract

Summary The effect of rat brain tissue extract on the proliferative activity of chicken embryonic neural tube and other tissues was studied. Only tissue-specific inhibitory action was found to be similar to substances of the chalone group.     

Solubility of the neurosecretory products of Crepidula fornicata Phil., a prosobranch mullusk]

Study of solubility of neurosecretory products in Crepidula fornicata shows that the presence of ethanol in the fixative is incompatible with good fixation. This character permits the choice of a more effective extraction technique. An experimental study in organ culture shows that the action of differentiation of brain appears in the extract obtained by this method.     

gamma-Aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for gamma-aminobutyric acid (GABA), a high affinity system (Km = 10.9 microM) and a low affinity system (Km = 1203 microM). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, beta-alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5 c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMV uptake systems were clearly different from the GABA transport systems present in brain tissue.     

Immobilized subunits can function as an affinity sorbent to purify oligomeric enzymes: The usefulness of hybridization on the solid support

Summary Immobilized dimers of yeast glyceraldehyde-3-phosphate dehydrogenase covalently bound to sepharose were shown to form hybrids with soluble dimers of the homologous enzymes present in crude tissue extracts (rat skeletal muscle, rat, rabbit and bovine hearts, rat liver, rat brain). Immobilized hybrid tetramers were then dissociated to form purified soluble enzymes.     

A biphasic effect of estradiol on serotonin metabolism in rat pineal organ cultures

Summary Physiological amounts of estradiol (1 nM) significantly increased serotonin, conversion to melatonin and 5-methoxytryptophol by rat pineal glands in organ culture whereas larger, pharmacological, doses (i.e. 1000 nM) impaired it significantly. The stimulatory but not the inhibitory effect of estradiol was blocked by the simultaneous addition of puromycin in the culture medium.This study was supported by grant No. 6638 from the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina.     

In vitro biosynthesis of beta-lipotropin in brain tissue]

We have recently developed methods to identify biosynthetized beta-endorphin and beta-lipotropin (beta-LPH) following incubation of Rat pars intermedia with radioactive amino acids. We used the same approach for rat brain tissue. In the striatum we found a peptide similar to beta-LPH while its identification in hypothalamus was less positive. This is the first demonstration of such biosynthesis and it could well be an important step in determining the biosynthetic patterns of cerebral endorphins and enkephalins.     

A radioisotopic method for the determination of S-adenosyl-L-homocysteine in tissues in the 10(-7) M range

S-Adenosyl-L-homocysteine is able to bind to brain membranes. We used this characteristic to measure the level of S-adenosyl-L-homocysteine in rat brain tissue. The method is rapid, at the same time very sensitive (down to 10(-7) M) and specific.     

Complement evasion by the Lyme disease spirocheteBorrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance

The effectiveness of complement-mediated killing ofBorrelia burgdorferi, the causative agent of Lyme disease, in the presence of host-derived tissues was studied. Second and high passage forms ofB. burgdorferi 297 isolate were grown in a LEW/N rat joint tissue co-culture system and in artificial BSK medium. Guinea pig complement and third week immune serum from hamsters with experimental Lyme disease were added to the cultures. Both high and low passage borrelia grown in BSK medium died and did not revive after 3 weeks incubation in BSK medium. However, 5–12% of tissue co-cultured borrelia survived the first complement-mediated lysis. Repeated re-growth and lysis cycles in tissue co-culture resulted in isolation of an 85% complement-resistant population ofB. burgdorferi. Joint tissue culture supernatant collected on the third day of tissue culture, and fibronectin (25 g/ml), also protected spirochetes from complement-mediated lysis in contrast to BSK or fresh co-culture medium. Complement-mediated lysis may not be an effective mechanism in eradication of borrelia, and the chronicity of Lyme disease may be due to resistance ofB. burgdorferi variants to host immune defense mechanisms in the presence of host-derived tissues.     

Métaplasie épidermoïde de l'épithélium mammaire humain,en culture organotypique à long-terme

Summary Squamous metaplasia of the mammary epithelium was observed in human breast tissue maintained in long-term organ culture. The phenomenon occurred only in the synthetic medium 199 with Earle's salts. Insulin and/or glucose enrichment enhanced its occurrence.     

Promotion of epithelial keratinization by N-methyl-N'-nitro-N-nitrosoguanidine in rat forestomach in organ culture.

The effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on epithelial differentiation of fetal rat forestomach was investigated in organ culture. When forestomach tissues removed from 16.5-day fetuses were treated with 5 microgram and 3 microgram of MNNG per ml for 1 h, epithelial keratinization was observed after 4 and 5 days, respectively, whereas it occurred after 6 days in control cultures. A clear dose-response relationship was found in the promotion of epithelial keratinization by MNNG.