Mycoplasma in African green monkey kidney cell cultures: biochemical detection and effects in virus-infected cells.

Among a number of techniques for the detection of mycoplasmal contamination in African green monkey kidney (AGMK) cell lines, the assay of uridine phosphorylase activity is unsuitable because of the presence of high levels of endogenous enzymatic activity. A thymidine phosphorylase test, however, based on the chromatographic analysis of radiolabeled thymidine breakdown, turned out to be a simple and sensitive mycoplasma detection method. We found, using the latter technique, that 0.22-micrometer-filtered virus inocula could still transfer mycoplasma unless treated with diethyl ether. The effect of mycoplasmal contamination on the synthesis of simian virus 40 and adenovirus in AGMK cells was negligible under the conditions used (no depletion of arginine). Incorporation of radioactive thymidine in viral macromolecules, however, was inhibited severely by the presence of mycoplasma.     

Transendothelial channels in fenestrated endometrial blood capillaries of rats

Summary Three types of transendothelial channels are described in the endothelium of blood capillaries in the endometrium of the rat. It is postulated that they may function as pores draining interstitial fluid to the venous blood.Supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek — Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Belgium)     

Oxidation of sulfur compounds and electron transport in Thiobacillus denitrificans

Summary Intact cells of Thiobacillus denitrificans catalyzed the oxidation of thiosulfate, sulfide and sulfite with nitrate or oxygen as the terminal acceptor. The anaerobic oxidation of thiosulfate, sulfide and sulfite was sensitive to the inhibitors of the flavoprotein system. Under aerobic conditions the oxidation of sulfide and sulfite was sensitive to these inhibitors but the thiosulfate oxidation was unaffected. Cyanide and azide inhibited the aerobic and anaerobic respiration when thiosulfate, sulfide or sulfite served as electron donors. The oxidation of thiosulfate by cell-free preparations was mediated by cytochromes of c, a and o-types. The cell-free extracts also catalyzed the oxidation of NADH and succinate, involving flavoproteins and b, c, a and o-type cytochromes. In addition, a cytochrome oxidase sensitive to cyanide and azide was also present.Non-Standard Abbreviations TTFA Thenoyltrifluoroacetone - HQNO 2-heptyl-4-hydroxyquonoline N-oxide Aspirant van het Nationaal Fonds voor Wetenschappelijk Onderzoek (Belgian National Science Foundation).     

Phagocytotic and iron-storing capacities of stromal cells in the rat endometrium

Summary Cytoplasmic pigment inclusions of rat endometrial stromal cells were studied by histology, histochemistry, fluorescence microscopy, electron microscopy and X-ray microprobe analysis. It is shown that a number of endometrial perivascular stromal cells contain numerous free cytoplasmic ferritin particles as well as hemosiderin vacuoles. The larger pigment inclusions reveal also positive PAS- and Schmorl reactions indicating that they contain polysaccharide and lipofuscin material, respectively. These pigmentstoring stromal cells also display acid phosphatase activity; they avidly phagocytose instillated latex particles. No pigment-storing cells occur within the surface or glandular epithelium, either in the basal endometrium or in the myometrium.It is demonstrated that the endometrial iron-storing cells function as iron depots; they take part in the phagocytosis and endocytosis of extracellular tissue components and therefore can be named phagocytes. Our data show that fibroblastoid endometrial stromal cells may differentiate into endometrial resident phagocytes, which ensure interstitial proteolysis and hence facilitate the drainage of extracellular fluid into the venous blood capillaries.Supported by a grant from the Nationaal Fonds voor Wetenschappelijk Onderzoek — Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Belgium.     

Electron microscopic immunocytochemical demonstration of separate vasotocinergic and mesotocinergic nerve fibres in the neural lobe of the amphibian hypophysis

Summary Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the electron microscopic level, it was demonstrated that the hormones of the posterior pituitary of Rana temporaria are located in separate vasotocinergic and mesotocinergic nerve fibres. This observation confirms the results of our previous immunocytochemical studies at the light microscopic level.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Cell population kinetics of zymogen and parietal cells in the stomach of mice

Summary With autoradiography after labelling with tritiated thymidine, the kinetics of zymogen and parietal cells were studied in the gastric mucosa of mice. After one intraperitoneal injection of the DNA precursor, zymogen cells in the DNA synthesis phase were clearly identified on autoradiograms, whereas no parietal cells were seen to synthesize DNA.In another group of mice, multiple injections were used in order to obtain a greater number of labelled cells. Following the latter procedure, analysis of grain count distributions over labelled zymogen cells and of labelling indices allowed detection of two subsequent zymogen cell divisions within an interval of approximately two months. This indicates that the cell turnover of zymogen cells is at least partly assured by their own mitotic activity.By contrast, parietal cells showed no evidence of cell division, but appeared to be derived through differentiation from other cells in the neck area of the gland. Analysis of spatial distribution of the labelled parietal cells in the glandular tube indicated that, in time, most newly formed parietal cells undergo a slow migration directed downwards to the bottom of the fundic glands.These results clearly show that the zymogen and the parietal cell population of the fundic glands have a different kinetic behaviour.This work was supported by a grant of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Adenosine phosphorylase activity in a mutant Hep-2 cell line contaminated withmycoplasma hyorhinis

Summary Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor coformycin. Isopycnic separation of [³H]thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [¹⁴C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified asMycoplasma hyorhinis, a porcine mycoplasma. Following γ-irradiation (3000 rads) to block cellular mitosis, the mycoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma. Presented at the 25th Annual Meeting of the Tissue Culture Association, Philadelphia, Pa., June 1976. This work was supported by Public Health Service Grants DE 02731 from the National Institute of Dental Research and CA 16219 from the National Cancer Institute.     

Intrapulmonary neuro-epithelial bodies in newborn rabbits

Summary Neonatal rabbit neuro-epithelial bodies (NEB) were investigated under various experimental conditions with light microscopy, microspectrography, morphometry and electron microscopy. (1) Hypoxia causes a decreased amine fluorescence intensity and an increased secretory exocytosis of dense core vesicles (DCV). Otherwise the NEB appear structurally normal. (2) Hypercapnia also produces a decreased fluorescence and an increased exocytosis; ultrastructurally, however, the dense core of DCV fragmentizes. (3) Hyperoxia does not appear to affect significantly either fluorescence or exocytosis. (4) The uptake of biogenic amines such as 5-HTP and L-DOPA was demonstrated by fluorometry and electron microscopy. (5) Reserpine, on the other hand, provokes an amine depletion with a decrease of the NEB fluorescence and an ultrastructural palor of the DCV. (6) Intratracheally administered nicotine is accompanied by a decreased fluorescence and a distinct exocytosis of fragmented DCV.The reaction of NEB to hypoxia and hypercapnia suggests that these corpuscles could be intrapulmonary chemoreceptors (in addition to the classically known central and peripheral chemoreceptors), inducing a reflex reaction through the liberation of DCV at the corpuscular sensible nerve endings and via the CNS. In addition, they may subserve a local intrapulmonary effect by modulating directly the hypoxic and hypercapnic pulmonary vasoconstriction and thus the V/Q ratio. Acknowledgment. This study was supported by a grant from The Council for Tobacco Research, U.S.A., and the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. We thank M.R. Van Hamme, R. Renwart and K. Armee for technical, G. Pison and St. Ons for photographical and N. Tyberghien and G. Verbeeck for secretarial assistanceMartin Deleersnyder (deceased May 11, 1976) was Candidate of the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium     

Immunocytochemical demonstration of the hypothalamo-hypophysial vasotocinergic system of Lampetra fluviatilis

Summary With the use of the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level, it was shown that the preoptico-hypophysial neurosecretory system of the adult migrating Lampetra fluviatilis is a vasotocinergic system. It does not synthesize vasopressin. The results are entirely consistent with earlier chromatographic and pharmacological indications that it produces little or no oxytocin-like peptide hormone. In the adenohypophysis, immunoreactive neurohypophysial peptidergic fibres are absent.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Identification of separate vasopressin-neurophysin II and oxytocin-neurophysin I containing nerve fibres in the external region of the bovine median eminence

Summary Immuno-enzyme histochemical investigations showed the presence, in the external region of the bovine median eminence, of accumulations of vasopressin-neurophysin II-and oxytocin-neurophysin I-complexes. These two hormone-neurophysin complexes are located in separate fine varicose nerve fibres. The results strongly plead against an important role of tanycytes in the transport of vasopressin, oxytocin and neurophysins from the cerebrospinal fluid to the hypophysial portal blood-vessels.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek.     

An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Summary Autoradiographic detection of incorporation of tritiated thymidine into the cytoplasm of cultured mammalian cells has been evaluated as a test of contamination of the cultures by cell-associated microorganisms, which usually are mycoplasmas. Criteria which indicate the presence of cell-associated mycoplasmas have been established, and the reliability of the standardized autoradiographic method has been assessed by testing the same cultures by two colony formation methods of mycoplasmal detection. The autoradiographic method demonstrated cell-associated microorganisms in all cultures from which characteristic colonies were grown on mycoplasma agar. The autoradiographic method did not produce false positive results, and the outcome of this test was evident in 3 days as opposed to 7 to 14 days by agar culture methods. Some applications of the autoradiographic method are shown, and it is suggested that this method be employed for routine surveillance for mycoplasmal contamination in laboratories where facilities for frequent agar culture tests are not easily available. This research was supported by U.S. Public Health Service Grants CA 12351-02 and CA 12334-01 from the National Cancer Institute.     

Adenosine phosphorylase activity in a mutant HEp-2 cell line contaminated with Mycoplasm hyorhinis.

Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor conformycin. Isopycnic separation of [3H] thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [14C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified as Mycoplasma hyorhinis, a porcine mycoplasma. Following gamma-irradiation (3000 rads) to block cellular mitosis, the mucoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma.     

Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The activated hypothalamic magnocellular neurosecretory system and the one neuron -one neurohypophysial hormone concept

Summary The activated hypothalamic magnocellular neurosecretory system of the rat was studied in tissue sections, double stained with the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. The results indicate that in animals with an activated hypothalamic magnocellular neuroendocrine system, as well as in normal animals, vasopressin and oxytocin are exclusively synthesized in separate vasopressinergic and oxytocinergic neurons.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

An ultramicrochemical test for mycoplasmal contamination of cultured cells.

A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-14C]adenine and ribose-1-phosphate to [8-14C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosine-phosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contamination in which most of the mycoplasma particles are attached to cell membranes.     

Cytidine monophosphate as substrate for the electron microscopic visualization of alkaline phosphatase activity

Summary Since in histochemical reactions the liver alkaline phosphatase discloses only low activity with respect to beta-glycerophosphate, the authors have replaced the latter by cytidine monophosphate. With this substrate they obtained much better results: greater sensibility permitting to reduce the incubation time, and much more precise localization of the enzyme activity, at the ultrastructural level. Details of the method are given with a discussion of its specifity. The method was applied on liver tissue of normal albino rats and animals of the same strain with ligated common bile duct.This study was supported by a grant from the Fonds voor Wetenschappelijk Geneeskundig Onderzoek.     

Interference in hybrid clone selection caused by Mycoplasma hyorhinis infection

Mycoplasma hyorhinis strains were isolated from Chinese hamster DON cells which lacked the ability to produce hybrid colonies in HAT medium. The mycoplasma isolates were virtually devoid of HGPRT activity in vivo and in vitro in the presence of excess co-enzyme, phosphoribosylpyrophosphate. Deliberate infection of mycoplasma-free cells caused no alterations in the HGPRT^? and TK^? phenotypes of the cells. Heterokaryon formation with infected cells was normal and the failure to produce hybrid colonies resulted from depletion, by nucleoside phosphorylase activity, of exogenous thymidine required for rescue of hybrid cells in HAT medium. Increasing the thymidine concentration and repeatedly replenishing HAT medium permitted hybrid clone formation.     

Immunocytochemical localization of somatostatin in the brain of the lizard,Ctenosauria pectinata

Summary The brain of the lizard, Ctenosauria pectinata, was studied light microscopically using an immunocytochemical staining method that is specific for neurohypophysial hormones and somatostatin. It was shown that the telencephalon and particularly the diencephalon contain somatostatin-producing perikarya, while somatostatinergic fibers occur in the entire brain. Similar to the situation in other vertebrates, somatostatin neurons in Ctenosauria pectinata form a population distinct from the neurohypophysial hormone-producing neurons. The small-sized somatostatin neurons were found in the cortex and the hypopallium of the telencephalon, and in two distinct clusters in the diencephalon: (1) ventral from, and partially overlapping with, the classical neurosecretory para ventricular nucleus; and (2) in the region of the infundibular (tuberal) nucleus. Somatostatin fibers were found among the classical neurosecretory fibers of the supraoptico-paraventricular system (tract, median eminence, neural lobe), near to and within the epiphysis, in the septum, in the vicinity of the tectum opticum and the cerebellum, and in the tegmentum.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species

Summary The distribution of somatostatin (SRIF) — and corticotropin-releasing factor (CRF)-like — immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek and the CNRS     

Mycoplasma contamination greatly enhances the apparent transport and concentrative accumulation of formycin B by mammalian cell culture

S49 mouse leukemia cells exhibit both equilibrative and Na(+)-dependent, concentrative formycin B transport. The latter represents only a minor nucleoside transport component and is detectable only when equilibrative nucleoside transport is inhibited by dipyridamole or another transport inhibitor. Thus in uncontaminated S49 cells formycin B accumulated only to slightly above the intracellular-extracellular equilibrium level. In contrast, in suspensions of S49 cells contaminated with mycoplasma, formycin B accumulated in the intracellular water space in unmodified form to 40-50-times the extracellular concentration in a dipyridamole-independent manner during 90 min of incubation at 37 degrees C. The mycoplasma active formycin B transport system was inhibited by all nucleosides tested, including thymidine and deoxycytidine, which are not substrates for the concentrative nucleoside transporter of S49 cells. Mycoplasma contamination was detected by the presence of cell-associated adenosine phosphorylase activity.