Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases.

Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17beta-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.

Activation of multiple signal transduction pathways by glucocorticoids: protection of ovarian follicular cells against apoptosis.

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFalpha-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1-5min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell-cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.

Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.

Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures. These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter). They are formed in response to UV radiation of cultures spread on high agar concentrations. Here we present an anatomical analysis of the stalks. Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core. This outer layer is physically separated from the core and contains many dead cells. The outer layer may form a protective shell for the core cells. Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells. We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization. We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology. Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures. Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects. The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.

Stimulation of myelin gene expression in vitro and of sciatic nerve remyelination by interleukin-6 receptor-interleukin-6 chimera.

Induction of myelin gene expression denotes the last stage of differentiation of myelinating glial cells. Following peripheral nerve transection, Schwann cells (SC) lose myelin gene expression and proliferate, resembling premyelinating embryonic SC (eSC). We show that a fusion protein of the soluble interleukin-6 receptor to interleukin-6 (IL6RIL6), a potent activator of the gp130 signaling receptor, is an inducer of MBP and Po gene products in rat E18 embryonic dorsal root ganglia (DRG) 3 day cultures. Cells whose growth is dependent on the IL6RIL6 chimera were isolated from DRG. These cells (designated CH cells) express Krox-20, as do promyelinating and myelinating SC (mSC). IL6RIL6 induces Po and MBP in CH cells and their cocultures with neurons. In addition, IL6RIL6 leads to a disappearance of Pax-3, a marker of eSC and nonmyelinating Schwann cells (nmSC). Glial fibrillary acidic protein, present in nmSC, is not significantly induced by IL6RIL6. The CH cells acquire glial morphology when exposed to IL6RIL6 and cover axons in cocultures. In a sciatic nerve-derived SC line, IL6RIL6 also induces Po and triggers a rapid attachment along axons. In vivo administration of IL6RIL6 intraperitoneally to rats after sciatic nerve transection and resuture increases 4-fold the number of myelinated nerve fibers (MF) measured on day 12, 2.5-5 mm distal to the suture. The stimulation by IL6RIL6 treatment is highest (7.1-fold) at the more distant 5 mm site, and the thickness of myelin sheaths is increased. Compared to known SC growth factors, the gp130 activator IL6RIL6 appears to combine both in vitro mitogenic effects and promotion of myelin gene expression.

Shear forces promote lymphocyte migration across vascular endothelium bearing apical chemokines.

Leukocyte transendothelial migration (TEM) is thought to be a chemotactic process controlled by chemokine gradients across the endothelium. Using cytokine-activated human umbilical vascular endothelial cells (HUVECs) as a model of inflamed endothelium, we have shown that apical endothelial chemokines can trigger robust peripheral blood lymphocyte (PBL) migration across endothelial cells. Lymphocyte TEM was promoted by physiological shear stress applied continuously to migrating lymphocytes. Lymphocyte integrins, intact actin cytoskeleton and G(i) protein-mediated chemokine signaling, but not a chemotactic gradient, were mandatory for TEM. PBL TEM did not require intracellular free calcium or intact phosphatidyl inositol kinase activity in migrating lymphocytes. Thus, lymphocyte TEM is promoted by fluid shear-induced mechanical signals coupled to G(i) protein signals at apical endothelial zones.

Expression and immunolocalization of the 14-3-3 protein of Schistosoma mansoni.

The 14-3-3 protein is a key player in signal transduction processes in various species. We have previously cloned and expressed the 14-3-3 of Schistosoma mansoni. Using the purified protein we have now raised antibodies against it. A highly specific, affinity-purified antibody preparation was employed for the localization of the 14-3-3 protein in the parasite, by both immunohistochemistry and immunoelectron microscopy. The results demonstrate wide distribution of this protein. It was observed in the female excretory system, the nephridia as well as in the genital systems of both sexes, namely in the vitelline gland of female and in the testis of the male. It is also present in the parenchyma and muscle of both male and female worms. Immunoelectron microscopy demonstrated the presence of immunogold-labelled protein in the tegument, subtegument, muscle, parenchyma and in the female reproductive system, in both the cytoplasm and nucleus of vitelline cells, and oocytes. The possible role of the 14-3-3 protein in the genital organs is discussed.

Hypomyelination and increased activity of voltage-gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon.

Protein tyrosine phosphatase epsilon (PTP epsilon) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTP epsilon exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed- rectifier, voltage-gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells. PTP epsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate-trapping mutant of PTP epsilon, and PTP epsilon profoundly reduces Src- or Fyn-stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTP epsilon antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury.

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express alpha2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.

A mutant of Synechococcus PCC 7942 impaired in HCO3- uptake.

An inactivation library was used to isolate high-CO2-requiring mutants of Synechococcus PCC 7942. One of them, mutant IL-7, is composed of elongated cells, some 5-15 times longer than the wild-type. IL-7 is impaired in the ability to accumulate inorganic carbon within the cells due to a lesion in HCO3- transport. Consequently, the apparent photosynthetic affinity for external inorganic carbon was about 50-100-fold lower than in the wild-type. Analysis of the genomic region modified in IL-7 demonstrated that the inactivating fragment was composed of two genomically unrelated fragments which were ligated together during the formation of the inactivation library. One of the fragments originated from a known genomic region, rbcLS, encoding ribulose 1,5-bisphosphate carboxylase/oxygenase and the other showed high homology to mutS encoding a DNA mismatch repair protein. We suggest that the primary lesion in IL-7 was in mutS and not in rbcLS, and that the phenotype of IL-7 resulted from secondary random mutations. We were unable to identify the spontaneous mutation(s) due to low transformability of IL-7. Our finding that two unrelated fragments ligated together points to possible mistakes in the identification of the function of putative genes with the aid of an inactivation library.

Cross-excitation in dorsal root ganglia does not depend on close cell-to-cell apposition.

About 90% of neurons in dorsal root ganglia (DRGs) of rats 2-5 weeks of age are depolarized and excited by impulse activity in neighboring neurons that share the same DRG. Synaptic contacts are extremely rare in DRGs, but instances of close membrane apposition between pairs of neuronal somata are not uncommon, especially in prenatal rats. Close membrane apposition could permit electrotonic interactions among neighboring DRG neurons. We carried out an ultrastructural examination of DRGs taken from rats 2-5 weeks of age and found that by this age < 2% of cells remain in close apposition with neighbors. The remainder are separated by one or two layers of satellite glial cytoplasm. It is, therefore, unlikely that close apposition between adjacent neurons contributes significantly to functional cross-excitation in the DRG.

Structural basis of neuron-to-neuron cross-excitation in dorsal root ganglia.

Lanthanum was used as a tracer substance to determine whether small molecules in the bulk extracellular space in dorsal root ganglia have access to the narrow cleft that separates sensory neurons from their surrounding satellite cell sheath. Results showed that lanthanum is able to diffuse into this cleft, especially when the tissue is incubated with the tracer before fixation. Lanthanum gained access to the cleft at the seam where adjacent satellite cell processes meet. There appears to be preferential access in the axon hillock-initial segment region. Large diameter light neurons, which generally support fast conducting myelinated axons and carry information about non-nociceptive sensory events, proved more likely to admit lanthanum than small diameter dark neurons, which tend to have thin myelinated and unmyelinated axons and typically carry nociceptive information. Peripheral axotomy triggered a reduction in the access of lanthanum to the neuron-satellite cell cleft. These data bear on the mechanism of non-synaptic cell-to-cell cross-excitation within dorsal root ganglia, and in particular, lend support to the hypothesis that this interaction is mediated chemically rather than electrically.