The cell coat of the developing olfactory epithelium in the chick

Summary During development of the olfactory epithelium in the chick embryo, the cell coat is revealed by treatment with Ruthenium red. On day 4 of incubation the developing sensory epithelium displays a thicker apical and basal cell coat than the neighbouring head ectoderm. The lateral cell coat is of equal thickness in both epithelia. The apical cell coat of the olfactory epithelium increases in thickness from day 4 to day 19 of embryonic life, finally attaining a thickness of about 55 nm.This paper is dedicated to Dr. A.J. ZamoraPortions of this paper were taken from the thesis by the author in fulfilment of the requirement for the degree of Dr. rer. nat. at the University of Essen Supported by the Deutsche Forschungsgemeinschaft (SFB114)     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium

Embryonic chick (7–9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionicf strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7–9-day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio in newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanined by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium.

Embryonic chick (7-9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionic strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7--9 day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio of newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanied by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Effects of prostaglandins on cyclic AMP levels in isolated cells from developing chick limbs

Effects of prostaglandins (PGs) on accumulation of cyclic AMP (cAMP) in the presence of a phosphodiesterase inhibitor were investigated in cells isolated from avian limb buds at various stages of development. Cells were responsive to PGE2 at the earliest stage investigated (stage 20-21) which was well in advance of specific cytodifferentiation of limb tissues. At three later stages (24-25; 26-28; 30-32), the responsiveness of cells isolated from the developing skeletal anlagen of the limb progressively increased coincident with the differentiation and maturation of the cartilage phenotype. Cells isolated from stage 26-28 cartilage rods were responsive also to prostacyclin (PGI2); however, the response produced was only about 50% of the response to an equivalent concentration of PGE2. Cells were not responsive to either PGF2 alpha or 6-keto PGF1 alpha, at concentrations of 30-33 micrograms/ml demonstrating a degree of specificity for PGE2 and PGI2. In the absence of the phosphodiesterase inhibitor, PGE2 increased cAMP accumulation two-fold over the controls and produced a concentration-dependent response between 0.3-30 micrograms/ml. The results demonstrate that PGs are capable of modulating cAMP levels of undifferentiated limb mesenchymal cells as well as embryonic cartilage cells and suggest a role for these compounds in limb chondrogenesis.     

The effect of inhibitors upon intracellular cyclic AMP levels and chick myoblast differentiation.

The small free-living nematode Caenorhabditis elegans is usually found as a hermaphrodite, but occasionally true males appear in the population. This study provides an account of gonadogenesis in the normal male and in a mutant that is a temperature-sensitive sex transformer.Male and hermaphrodite gonads develop from morphologically identical primordia. The small primordial gonad lies on the ventral side of the worm in the coelomic cavity. The gonadial primordium contains four nuclei at parturition. As this primordium develops in a hermaphrodite, it produces a double-armed, mirror symmetrical gonad that produces first sperm and then eggs. In the male, however, this primordium develops into an asymmetrical structure composed of a ventrally located testis, a loop region, a seminal vesicle, and a vas deferens. The male gonad presents a linear sequence of nuclei in successive stages of spermatogenesis beginning with a mitotic region in the testis, followed by clearly distinguishable stages of meiosis throughout the loop region to the seminal vesicle.A temperature-sensitive sex transformer mutant, tsB202, has been isolated. tsB202 carries an autosomal recessive mutation in linkage group II that at restrictive temperature transforms an XX hermaphrodite into a phenotypic male, complete with a normal male gonad and vestigial external genitalia. These transformed males are classified as pseudomales because they do not exhibit mating behavior. Temperature shift experiments have determined the specific temporal sequences of gonadogenesis, oogenesis, and spermatogenesis. Proper manipulation of the temperature regimen causes the production of intersexes. In one intersex, a male gonad complete with sperm, seminal vesicle, and vas deferens also contains oocytes. In another intersex produced by the complementary temperature shift, a hermaphrodite-shaped gonad develops that produces only sperm and no oocytes.     

The effect of Solcoseryl on explant cultures of the hippocampus

Explants of hippocampus from fetal rats were cultivated in Maximow chambers in semisynthetic medium up to 12 days in vitro. The cultures were fixed Bouin, slided 15 micron, coloured with Klüver-Barrera and some morphological parameters were tested. 1. The nerve fiber index increased by influence of 1% Solcoseryl in relation to control cultures, which growed in minimal medium. An essential stimulation was observed by application of placentar serum and embryonal extract into the culture medium. 2. Die decrease of the number of neurons and glial cells per unit of area and a small decrease of the area of neuron nuclei was discussed in relation to the effect of the pharmacon Solcoseryl on O2- consumption. 3. Solcoseryl (firm Solco AG, Base) is an extract of calf blood. It can not substitute other tissue extracts.     

Effects of cyclic AMP and Sephadex fractions of chick embryo extract on cloned retinal pigmented epithelium in tissue culture

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10^-4 M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.     

High cyclic AMP levels in young chick embryonic hearts.

Acid phosphatase (Acph) activities and protein content were measured in developing ovaries of adult flies. Acph and protein increased approximately logarithmically for the first 2 days of adult life and then plateaued at about 80 and 35 times, respectively, the levels present at eclosion. The specific activity of Acph was constant for the first 15 hr and then increased by a factor of three over the next 2 days. Analysis of staged follicles showed that the specific activity of Acph starts to increase at stage 10. Ovaries from homozygotes for Acph-1ⁿ⁴, a null activity mutant, showed constant low specific activity, indicating that this gene codes for the major ovarian Acph. Ovarian transplantations between Acph-1ⁿ⁴ and wild type showed that Acph is made by the ovary. Ovaries from isolated abdomens failed to increase in Acph activity or protein, but treating isolated abdomens with ZR-515, a juvenile hormone analog, caused nearly normal levels to be attained. Ovaries of the female sterile mutant ap⁴ failed to develop Acph activity unless they were implanted into a normal host or treated with ZR-515. Ovaries from the female sterile mutant fs(3)L3 developed no increase in Acph activity even when treated with ZR-515. The results demonstrate that the activity of a genetically localized enzyme is controlled by a chemically defined hormone in a genetically favorable higher eucaryote.     

The induction of ovalbumin and conalbumin mRNA by estrogen and progesterone in chick oviduct explant cultures

Estrogen pretreated chick oviduct tissue can be restimulated in vitro by physiological concentrations of estrogen and progesterone. The rates of synthesis of the major egg white proteins, ovalbumin and conalbumin, as well as the cellular levels of their respective mRNAs, increase after characteristic lag periods; this confirms previously reported results in vivo and demonstrates that both the lag phenomena and the mRNA induction are a function of the direct interaction of steroids with oviduct cells.The antagonistic action of progesterone on an estrogen-mediated induction of conalbumin mRNA also occurs in vitro, and the kinetics of this response are examined. Progesterone terminates the estradiol-induced accumulation of conalbumin mRNA within 30 min after addition to the medium; progesterone alone or in combination with estrogen, however, is capable of inducing conalbumin mRNA after a 4 hr lag period. The temporary nature of this antagonism and the fact that it does not occur with ovalbumin induction indicate the complexity of the oviduct's response to steroids.The role of protein synthesis in the induction of both ovalbumin and conalbumin was examined by including protein synthesis inhibitors in the culture medium. Puromycin, cycloheximide, emetine, pactamycin and high salt all block the induction of both ovalbumin and conalbumin mRNA when added together with either estrogen or progesterone. The effect of puromycin is reversible. After the drug is removed from the medium, the mRNA accumulation begins with the same characteristic lag period seen when no inhibitors are added. When given 2 hr after estrogen, puromycin stops the accumulation of conalbumin mRNA within 30 min, whereas cycloheximide and emetine allow the mRNA to accumulate for another 2 hr before causing complete inhibition. There is no effect of protein synthesis inhibitors on the number of estrogen receptors localized in the nucleus. The data suggest a direct link between protein synthesis and the steroid-induced accumulation of specific mRNAs in this system.     

Ultrastructure of the developing gizzard epithelium in the chick embryo

Summary The fine structure of the gizzard epithelium is described at different stages in the development of the chick embryo. The elaborate apical processes, a characteristic feature, take part in secretion at thirteen days, but do not seem to have this function at nine and ten days. The formation of glands begins at thirteen days but the adult fine structure of the gland cells is not attained until hatching. The distinct surface layer present between thirteen and seventeen days may have a protective function. Acknowledgement. The author is grateful for research facilities provided by Professor G. M. Wyburn, Anatomy Department, The University of Glasgow.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Effect of cyclic AMP on retrogression of the Mullerian ducts in chick embryos]

Theophyllin and puromycine, inhibitors of the enzyme phosphodiesterase and AMPc are all able to inhibit the retrogression of mullerian ducts in the female chick embryo, grafted with an embryonic testis. We can think that these results are explained by an inhibitory action of AMPc on the mechanisms responsible for the mullerian retrogression. So the chick embryo reacts similarly as do the mammalian embryo.     

Effects of dibutyryl cyclic AMP on tyrosine uptake and metabolism in neuroblastoma cultures

A series of thin-layer Chromatographic (TLC) systems were employed to study the effects of dibutyryl cyclic AMP (db-cAMP) on the metabolism of ³H-tyrosine in neuroblastoma cultures. The neuroblastoma monolayer cultures incubated with radiolabelled tyrosine synthesized di-hydroxyphenylalanine (DOPA), dopamine (DA), and norepinephrine (NE), in confirmation of previous reports identifying these compounds in neuroblastoma cultures. In addition, we found evidence suggesting the presence of metabolites of DA and NE, that is, homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) together with 3-methoxy-4-hydroxymandelic acid (VMA). When these cultures were grown in the presence of db-cAMP for 3 days, tyrosine uptake was increased with a proportional increase in tyrosine hydroxylation. This effect persisted in the absence of db-cAMP, but it was not apparent with only 90 min exposure to db-cAMP. Suspension cultures showed the same baseline level of tyrosine uptake as did monolayer cultures, but the uptake in suspension cultures failed to increase with db-cAMP treatment. It is suggested that the db-cAMP induced differentiation of the neuroblastoma cells in monolayer cultures was associated with induction of a tyrosine uptake system.     

The effect of cyclic AMP on the growth of Toxoplasma gondii in vitro

To assess the role of cAMP on the growth and proliferation of Toxoplasma in HL-60 cells we tested the effect of exogenous cAMP and cAMP analogues to the co-culture system of Toxoplasma and HL-60 cells. cAMP, dbcAMP, and br-cAMP stimulated the growth of Toxoplasma at a specific concentration, i.e., 10(0) mM, 10(0) mM, and 10(-1) mM, respectively. There were differences in growth induction kinetics and in the rate of promotion. These results were further verified by treating the co-culture with adenylate cyclase activator, pNHppG, cAMP phosphodiesterase activators, imidazole and A23187, and cAMP phosphodiesterase inhibitors, IBMX, compound 48/80, and theophylline, separately. When the cytosolic cAMP levels increased by the reagents mentioned above, Toxoplasma in the cytoplasm of HL-60 cells stimulated to proliferate more rapidly with concentration-dependent modes compared to the control, and vice versa. It is suggested that some mechanisms are activated by the high levels of cAMP in the cytoplasm, which result in the stimulation of Toxoplasma proliferation.     

Fine structure of the developing gastric epithelium of the chick

The fine structure of the developing gizzard of the chick embryo has been studied to define the sequence of events in cytodifferentiation of the epithelium and to look for morphological evidence of epithelio-mesenchymal interaction. During the fourth day of incubation epithelial cells begin to form mucous secretory granules, later massive glycogen deposits appear, and finally by day 8 numerous cell processes have formed. Tissue was prepared by a number of methods to stain material associated with cell surfaces. At the time induction is presumbably occurring such stainable material is abundant. Epithelial and mesenchymal tissue components when cultured transfilter show no inductive effects and stainable cell surface material is greatly reduced near the epithelium.