Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Summary Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium.Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15–30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.     

Endocytosis of cationized ferritin in human resting peripheral blood by T-lymphocytes

Summary We have examined the binding and internalization of cationized ferritin in T-lymphocytes of human peripheral blood, as a model for resting cells. After 30 min of incubation only 8% of endocytotic vesicles contain cationized ferritin. T-cells internalie the equivalent of their entire surface area in approximately 54 h, a longer time than is required by non-resting cells such as PHA-stimulated human lymphocytes. These tracer experiments suggest that the endocytosis of cationized ferritin by T-lymphocytes follows a lysosome pathway similar to that described for other cell types.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Localization and partial characterization of a rat ovarian granulose cell protein with a monoclonal antibody

Summry— Hybridoma cell lines were obtained from mouse splenocytes sensitized to granulosa cells collected from rat ovaries after gonadotropin stimulation. A monoclonal antibody (5G5) was obtained which reacted with granulosa cells and showed a positive reaction with serum-free conditioned medium containing granulosa cell secreted proteins. Immoblotting of the conditioned medium and light- and electron-microscopic immunocytochemistry of rat ovary show that mAb 5G5 is directed against a 59-kDa protein which is located on the plasma membrane of granulosa cells. Furthermore, the immunoreactivity of the granulosa cells depends both on the degree of follicle development and on the position of the granulosa cells within the follicles. Strong immunoreactivity was observed in the innermost granulosa cell layers, close to the oocyte and the antral cavity. The results obtained show that mAb 5G5 is a useful marker of a 59-kDa granulosa cell protein which might be of importance for the follicle and the occyte maturation.     

Unique iron binding and oxidation properties of human mitochondrial ferritin: a comparative analysis with Human H-chain ferritin

Ferritins are ubiquitous iron mineralizing and storage proteins that play an important role in iron homeostasis. Although excess iron is stored in the cytoplasm, most of the metabolically active iron is processed in the mitochondria of the cell. Little is known about how these organelles regulate iron homeostasis and toxicity. The recently discovered human mitochondrial ferritin (MtF), unlike other mammalian ferritins, is a homopolymer of 24 subunits that has a high degree of sequence homology with human H-chain ferritin (HuHF). Parallel experiments with MtF and HuHF reported here reveal striking differences in their iron oxidation and hydrolysis chemistry despite their similar diFe ferroxidase centers. In contrast to HuHF, MtF does not regenerate its ferroxidase activity after oxidation of its initial complement of Fe(II) and generally has considerably slower ferroxidation and mineralization activities as well. MtF exhibits sigmoidal kinetics of mineralization more characteristic of an L-chain than an H-chain ferritin. Site-directed mutagenesis reveals that serine 144, a residue situated near the ferroxidase center in MtF but absent from HuHF, is one player in this impairment of activity. Additionally only one-half of the 24 ferroxidase centers of MtF are functional, further contributing to its lower activity. Stopped-flow absorption spectrometry of Fe(II) oxidation by O(2) in MtF shows the formation of a transient diiron(III) mu-peroxo species (lambda(max) = 650 nm) as observed in HuHF. Also, as for HuHF, minimal hydroxyl radical is produced during the oxidative deposition of iron in MtF using O(2) as the oxidant. However, the 2Fe(II) + H(2)O(2) detoxification reaction found in HuHF does not occur in MtF. The structural differences and the physiological implications of the unique iron oxidation properties of MtF are discussed in light of these results.     

Progesterone maintains the status of granulosa cells and slows follicle development partly through PGRMC1

Progesterone receptor membrane component 1 (PGRMC1) mediates antimitotic and antiapoptotic actions of progesterone in granulosa cells, which indicates that PGRMC1 may play a key role in maintaining the status of granulosa cells. The current study investigated the effects of progesterone on intracellular signaling involved in differentiation, follicle development, inflammatory responses, and antioxidation, and determined the role of PGRMC1 in these processes. Our results demonstrated that progesterone slowed follicle development and inhibited p-ERK1/2, p-p38, caspase-3, p-NF-κB, and p-IκB-α signals involved in differentiation, steroidogenesis, and inflammatory responses in granulosa cells. Progesterone inhibited the steroidogenic acute regulatory protein and the cholesterol side-chain cleavage enzyme and decreased pregnenolone production. A PGRMC1 inhibitor and a PGRMC1 small interfering RNA ablated these inhibitory effects of progesterone. Interfering with PGRMC1 functions also decreased cellular antioxidative effects induced by an oxidant. These results suggest that PGRMC1 might play a critical role in maintaining the status of granulosa cells and balancing follicle numbers.     

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10⁻⁵M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

The uptake of foreign ferritin by macrophages in the spleen, trunk kidney and liver of platy

Melano-macrophage centres in the spleen and liver of platy Xiphophorus maculatus contain iron and may recycle iron compounds. The sinusoidal tissue in the platy spleen and trunk kidney can take up much foreign ferritin, and may clear the circulation for waste and foreign macromolecules and particles.     

Synthesis,G-quadruplexes DNA binding,and photocytotoxicity of novel cationic expanded porphyrins

Intensive reports allowed the conclusion that molecules with extended aromatic surfaces always do good jobs in the DNA interactions. Inspired by the previous successful researches, herein, we designed a series of cationic porphyrins with expanded planar substituents, and evaluated their binding behaviors to G-quadruplex DNA using the combination of surface-enhanced raman, circular dichroism, absorption spectroscopy and fluorescence resonance energy transfer melting assays. Asymmetrical tetracationic porphyrin with one phenyl-4-N-methyl-4-pyridyl group and three N-methyl-4-pyridyl groups exhibit the best G4-DNA binding affinities among all the designed compounds, suggesting that the bulk of the substituents should be matched to the width of the grooves they putatively lie in. Theoretical calculations applying the density functional theory have been carried out and explain the binding properties of these porphyrins reasonably. Meanwhile, these porphyrins were proved to be potential photochemotherapeutic agents since they have photocytotoxic activities against both myeloma cell (Ag8.653) and gliomas cell (U251) lines.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

Uptake and intracellular fate of gelonin,a ribosome-inactivating protein,in rat liver

Gelonin, a type 1 ribosome-inactivating protein, has been used as toxin conjugate for several therapeutic purposes. We have investigated the endocytosis of gelonin by rat liver in vivo. Subcellular distribution of [125I]gelonin was established after differential and isopycnic centrifugation. Fractions were analyzed for acid-soluble and acid-precipitable radioactivity. Results show that gelonin is rapidly cleared from the blood and within 15min reaches a peak (25% of total injected) in the liver. With time, radioactivity associated with the liver markedly decreases. Two important observations are made: (a) Radioactivity associated with all fractions, at any time point, is greater than 80% acid precipitable. (b) Even at 5min, a significant amount of intact gelonin is present in the cytosolic fraction. Our work suggests that, though gelonin is rapidly cleared from the blood, there are still intact molecules that have entered the cytosol where they could exert their toxic effect.     

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.     

Membrane binding and uptake of diflubenzuron in a cell line from Manduca sexta (L.)

The binding and accumulation of the chitin synthesis inhibitor diflubenzuron (DFB) by a cell line derived from embryonic tissue of the tobacco hornworm, Manduca sexta (L.), was analyzed. A rapid and reversible binding to viable and nonviable cells suspended in the culture medium was observed at soluble concentrations of DFB for short exposure periods. Scatchard analysis gave no indication of a saturable uptake mechanism. The DFB-binding capacity of intact cells was found to be similar to that of a crude membrane preparation (70,000g pellet); however, plasma membrane-enriched fractions bound almost three times as much DFB as the homogenate. Repetitive shorttime incubations (up to 3 h) of suspended cells with DFB resulted in a stepwise intracellular accumulation of DFB. Treatment of growing cells with DFB at high concentrations (50 μM) of DFB for longer periods (up to 7 days) resulted in elevated intracellular accumulation of DFB, which exceeded the binding capacity of the cell membranes and the aqueous solubility of DFB. These results indicate that the intracellular crystals detected by transmission electron microscopy are precipitated DFB. No metabolites or other chemically modified products of intracellular DFB were detected by high pressure liquid chromatography (HPLC) after a 7-day incubation.     

Involvement of intracellular caveolin‐1 distribution in the suppression of antigen‐induced mast cell activation by cationic liposomes

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross‐linking of high‐affinity immunoglobulin E receptor in a time‐ and dose‐dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the inhibition of store‐operated Ca²⁺ entry. Further, we revealed that the mechanism underlying an impaired [Ca²⁺]_i increase is the inhibition of the activation of the phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Yet, how cationic liposomes inhibit the PI3K‐Akt pathway is still unclear. Here, we focused on caveolin‐1, a major component of caveolae, which is reported to be involved in the activation of the PI3K‐Akt pathway in various cell lines. In this study, we showed that caveolin‐1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin‐1 to the plasma membrane and the colocalization of caveolin‐1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.