Saccharin induces morphological changes and enhances prolactin production in GH4C1 cells

Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells (GH₄C₁ cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment of GH₄C₁ cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells. After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin on cell morphology and on PRL production are reversible in GH₄C₁ cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell morphology or PRL production. This investigation was supported by a Faculty Research Grant from Wheaton College     

Septal ultrastructure in Candida albicans.

Fine structural studies on the septa of Candida albicans in vitro (when leading a saprophytic existence) and the organism in its invasive form (as a pathogen in oral candidosis) have shown that in the former the septum exhibits a unique central perforation resembling an aggregate of fine canaliculi connecting one cell to the other. In the invasive form the septum is non-perforated and appears as a solid structure. Septal ultrastructure is well characterised in many pathogenic fungi. Our observations on Candida albicans do not resemble any previous studies carried out on other deutromycetous fungi.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Gamete structure and fertilization in the brown alga Hormosira banksii (Turner) Decaisne

Light microscope and fine structural studies of the gametes of Hormosira banksii show the antherozoids as typical Fucales biflagellate gametes with a posterior whiplash flagellum and an anterior flagellum with mastigonemes. The oospheres are enclosed in a plasma membrane, are highly vacuolate and contain abundant phenolic inclusions.

Antherozoids attach to the surface of the oospheres by their anterior flagellum at conjugation. Following fertilization Golgi derived vesicles underlying the plasma membrane decrease in electron density and an exterior surface coat appears outside this membrane. This may be a “fertilization barrier” which prevents further penetration by other male gametes.

Subsequently a fibrous layer appears which develops into a cell wall around the developing zygote. Histochemical studies show the cell wall of these young zygotes is probably made up of alginic acid. Fine structural and histochemical examination of the young zygotes suggest that whereas there is no preformed adhesive in the oosphere, large reserves of lipid and polysaccharide provide materials necessary for synthesis of wall material and the potential for adhesive production following fertilization.     

Effects of elevated soil solution Al concentrations on fine roots in a middle-aged Norway spruce (Picea abies (L.) Karst.) stand

Aluminium (Al), mobilized by acidic deposition, has been claimed to be a major threat to forest vitality. Fine root mortality, decreased root growth and reduced nutrient uptake have been observed in controlled laboratory experiments where roots of tree seedlings were exposed to elevated concentrations of Al. Yet, evidence for Al-induced root damage from forest stands is scarcely reported. Nevertheless, Al dissolved in soil water has received a key role in the critical load concept for forests. Here, we present effects of artificially elevated concentrations of Al in the soil solution on fine roots in a middle-aged stand of Norway spruce (Picea abies (L.) Karst.). Although the inorganic Al concentrations about 200 µM and Ca:Al ratio about 0.7 that were established in the soil solution within this experiment have been associated with reduction of root growth and root mortality for spruce seedlings in hydroponic studies, no acute damage on fine roots was observed. Three years of treatment did not cause visual root damage, nor were effects on fine root necromass observed. Fine root necromass made up about 10% of fine root biomass for all treatments. However, significantly lower molar Ca:Al and Mg:Al ratios in living and dead fine roots were found in the plots where Al concentrations were highest and ratios of Ca to Al in the soil solution were lowest. The lack of response on fine root biomass suggests that forest stands tolerate higher Al levels than results from laboratory experiments indicate. We conclude that effect studies in the laboratory have limited value for field conditions. The key role of Al toxicity, expressed as the Ca/Al ratio, in critical load calculations for forests may have to be reconsidered.     

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.     

Drosophila PRL-1 Is a Growth Inhibitor That Counteracts the Function of the Src Oncogene

Phosphatase of Regenerating Liver (PRL) family members have emerged as molecular markers that significantly correlate to the ability of many cancers to metastasize. However, contradictory cellular responses to PRL expression have been reported, including the inhibition of cell cycle progression. An obvious culprit for the discrepancy is the use of dozens of different cell lines, including many isolated from tumors or cultured cells selected for immortalization which may have missing or mutated modulators of PRL function. We created transgenic Drosophila to study the effects of PRL overexpression in a genetically controlled, organismal model. Our data support the paradigm that the normal cellular response to high levels of PRL is growth suppression and furthermore, that PRL can counter oncogenic activity of Src. The ability of PRL to inhibit growth under normal conditions is dependent on a CAAX motif that is required to localize PRL to the apical edge of the lateral membrane. However, PRL lacking the CAAX motif can still associate indiscriminately with the plasma membrane and retains its ability to inhibit Src function. We propose that PRL binds to other membrane-localized proteins that are effectors of Src or to Src itself. This first examination of PRL in a model organism demonstrates that PRL performs as a tumor suppressor and underscores the necessity of identifying the conditions that enable it to transform into an oncogene in cancer.     

Endocrine,paracrine and autocrine actions of prolactin on immune cells

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.     

Regulated expression of the prolactin gene in rat pituitary tumor cells

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.     

Changes in the morphology and synthetic activity of cultured rat tail tendon

Summary Isolated single fascicles from tail tendons of young rats were freed of epitenon cells and cultured in vitro for up to 7 days. The tissue remained viable, as judged by the structural integrity of cell organelles and the ability to synthesize DNA and glycosaminoglycans (GAG). The rate of DNA synthesis peaked after 2 days in culture and decreased slowly thereafter. Concomitantly, an increase in cell number was noted at the periphery of the fascicle. GAG production also increased during culture, sulphated GAG being increased proportionately more than hyaluronic acid. Dermatan sulphate was the predominant sulphated GAG in freshly isolated fascicles, but in cultured tissue, the newly synthesized sulphated GAG was more sensitive to degradation by chondroitinase AC and had an increased electrophoretic mobility. Fine structural changes were observed in cultured tissues such as the retraction of cell processes. rounding up of cell bodies and the appearance of gaps between collagen fibrils. Cultured tenocytes also frequently contained apparently phagocytized collagen fibrils which were not seen in freshly isolated fascicles, and this appearance was suggestive of collagen degradation occurring in vitro, although no change in the total hydroxyproline content was noted. The data show that when individual fascicles are cultured in vitro they undergo a process of matrix remodelling which has features in common with events occurring in vivo when tendons have been surgically manipulated.     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

Nine Years of Irrigation Cause Vegetation and Fine Root Shifts in a Water-Limited Pine Forest

Scots pines (Pinus sylvestris L.) in the inner-Alpine dry valleys of Switzerland have suffered from increased mortality during the past decades, which has been caused by longer and more frequent dry periods. In addition, a proceeding replacement of Scots pines by pubescent oaks (Quercus pubescens Willd.) has been observed. In 2003, an irrigation experiment was performed to track changes by reducing drought pressure on the natural pine forest. After nine years of irrigation, we observed major adaptations in the vegetation and shifts in Scots pine fine root abundance and structure. Irrigation permitted new plant species to assemble and promote canopy closure with a subsequent loss of herb and moss coverage. Fine root dry weight increased under irrigation and fine roots had a tendency to elongate. Structural composition of fine roots remained unaffected by irrigation, expressing preserved proportions of cellulose, lignin and phenolic substances. A shift to a more negative δ¹³C signal in the fine root C indicates an increased photosynthetic activity in irrigated pine trees. Using radiocarbon (¹⁴C) measurement, a reduced mean age of the fine roots in irrigated plots was revealed. The reason for this is either an increase in newly produced fine roots, supported by the increase in fine root biomass, or a reduced lifespan of fine roots which corresponds to an enhanced turnover rate. Overall, the responses belowground to irrigation are less conspicuous than the more rapid adaptations aboveground. Lagged and conservative adaptations of tree roots with decadal lifespans are challenging to detect, hence demanding for long-term surveys. Investigations concerning fine root turnover rate and degradation processes under a changing climate are crucial for a complete understanding of C cycling.     

Heat shock induced ultrastructural alterations in Stylonychia mytilus cells

Fine structural observations on heat shocked cells of S. mytilus reveal that cell organelles undergo structural alterations. Mitochondria show distorted shapes with disorganized cristae and vacuolar spaces. Pulse heat shock results in dilated rough endoplasmic reticulum, abundant polysomes as well as smooth endoplasmic reticulum. Heat shocked cells show membrane bound bodies containing osmiophilic cores. In macronuclei, dense chromatin breaks up into discrete bodies accompanied by the appearance of bundles of fine filaments and clustering of nuclear pores. The most prominent changes are noticed in nucleoli. Within 15 min of heat shock, nucleoli show hypertrophy and fine fibrillar zone which gradually replaces the granular zone by 120 min giving the nucleoli ring shaped configuration. In S phase cells, macronuclei show the arrested replication band in which the diffused zone (the site of DNA replication) is absent.     

Cytology of Spore Formation in Clostridium perfringens

The sequential morphological events in spore formation by Clostridium perfringens type D were observed in Ellner's medium where 80 to 100% of the cells formed spores. Gross structural changes were studied with the light microscope under phase-contrast, and in fixed cells by the use of both nigrosin and Giemsa preparations. Fine structure was examined with the electron microscope in both thin sections and frozen-etched preparations. During the first 3 hr of incubation, the original rod-shaped cells became ellipsoid to ovoid in shape; by 5 to 6 hr, subterminal spores had developed within these enlarged cells. The fine structural sequence was in most respects identical to that in other Bacillaceae, although some stages were illustrated with particular clarity. A unique feature was the development of a convoluted, membranous exosporium which adhered to the outer surface of the two coats and had an unusual fine structure resembling a rectangular array of subunits.     

Dopamine inhibition of prolactin release but not synthesis in the male Syrian hamster: in vitro studies

The hypothalamic mechanisms controlling prolactin (PRL) cell function in the male Syrian hamster are unclear. Equally unclear is the role of dopamine (DA) in regulating lactotrophic cell activity in long photoperiod-exposed hamsters particularly with respect to PRL synthesis and release. The synthesis of PRL, as measured by the incorporation of 3H-leucine into newly synthesized PRL, by anterior pituitary glands from male hamsters is linear over a five h incubation period. Approximately two-fold more 3H-PRL remained in the pituitary glands than in the medium by the end of the incubation period. The incubation of hamster hemipituitaries with DA at concentrations of either 5 X 10(-7) M or 5 X 10(-5) M, resulted in a 77% to 83% inhibition of the release of immunoreactive PRL into the medium as compared with controls. Similarly, the release of 3H-PRL into the medium was inhibited by 71% to 76% as compared with controls; however, the synthesis of PRL was virtually the same among the experimental and control groups. These results suggest that DA may be an important regulator of short-term PRL release but not synthesis in the long photoperiod-exposed male hamster.     

Spermiogenesis in the anthozoan Aiptasia pallida

Fine structural aspects of spermiogenesis in the anthozoan Aiptasia pallida have been examined. Nuclear condensation is similar to that reported for other anthozoans. The middle piece is formed by fusion of a few small mitochondria and is doughnut-shaped. A well-developed centriolar complex is attached to the nuclear envelope by fine filamentous material and is surrounded by the mitochondrial collar. Electron-dense vesicles, which correspond to 'proacrosomal vesicles', are scattered in the peripheral cytoplasm at the nuclear-mitochondrial junction. Cytochemical procedures indicate that these vesicles contain carbohydrates and basic proteins, suggesting they may be acrosomal-like.     

Studies on the possible role of protein kinase C in the prolactin regulation of cell replication in Nb2 node lymphoma cells

The results of several recent studies have indicated that protein kinase C (PKC) may be involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The PKC activator 12-O-tetradeconylphorbol-13-acetate (TPA) at certain concentrations has been shown to potentiate the mitogenic effect of PRL, whereas at higher concentrations, TPA inhibits the PRL response. Several inhibitors of PKC have also been shown to impair the PRL stimulation of metabolic process in the Nb2 cells. These studies provide further evidence for the likely involvement of PKC in the PRL stimulation of mitogenesis in the Nb2 cells. A transient, time-dependent accumulation of PKC in the particulate fraction of the Nb2 cells is observed in response to PRL. TPA is also shown to elicit a similar effect, albeit at a much earlier time and with a greater magnitude. On long-term exposure (3 days), high concentrations of TPA down-regulate the PKC enzyme; this down-regulation likely accounts for the inhibitory effect of high concentrations of TPA on the PRL stimulation of cell division. In further studies, the PKC inhibitors H-7 and gossypol were shown to inhibit the PRL stimulation of cell division in a concentration-dependent fashion.     

Spindle cell fragments in focal nodular hyperplasia of the liver. A case report

BACKGROUND: There are only few reports on the fine needle aspiration cytology (FNAC) findings of focal nodular hyperplasia (FNH) of the liver. CASE: A 30-year-old woman who had undergone surgery for a leiomyosarcoma of the calf, was found to have a hepatic mass five years later on imaging during routine follow-up. Fine needle aspiration was performed to rule out metastasis. Cytology revealed a few fragments of bland-looking spindle cells in a metachromatic stroma along with benign hepatocytes and bile duct cells. It was interpreted as "consistent with metastasis of leiomyosarcoma." The excised mass showed histologic features typical of FNH. CONCLUSION: Spindle cell fragments have not been previously observed in the FNAC of FNH. These fragments probably represent the muscular wall of the abnormal blood vessels of FNH. If smooth muscle fragment is seen accompanying benign hepatocytes and bile duct cells, one should consider the diagnosis of FNH in the needle aspirate.