Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

R-factor-mediated suppression of the galactose-sensitive phenotype of Escherichia coli K-12 galE mutants

Plasmids S-a and Rts1 suppress the galactose-sensitive phenotype of galE mutants of Escherichia coli K-12, giving rise to both galactose-fermenting and nonfermenting strains. Fermenting strains produce normal inducible UDP-galactose epimerase. Plasmids extracted from either a fermenting or a nonfermenting strain are indistinguishable when examined by either measurements of length of relaxed circular molecules by electron microscopy or electrophoretic pattern of restriction endonuclease digestion products. The phenomenon could be explained by reversible recombination between a plasmid-borne epimerase gene and homologous chromosomal sequences.     

Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Fusicoccin stimulates the production of H2O2 in sycamore cell cultures and induces alternative respiration and cytochrome c leakage from mitochondria

Fusicoccin (FC) is a well known toxin acting as a 14-3-3 protein-mediated activator of the plasma membrane H⁺-ATPase and it has been widely used to study the regulatory mechanism and the physiological role of this enzyme's activity. Recently, FC has been shown to induce other responses similar to those occurring under a stress condition, perhaps not strictly dependent on the activation of proton extrusion. In this paper we report that in cultured sycamore ( Acer pseudoplatanus L.) cells FC induces H₂O₂ overproduction as well as other novel, presumably related responses, such as the activation of the alternative oxidase and the leakage of cytochrome c from the mitochondria, accompanied by a decrease of the cytochrome pathway capacity. The relationship between H₂O₂ production and other phenomena has also been studied by means of exogenously added H₂O₂.     

Heregulin targets gamma-catenin to the nucleolus by a mechanism dependent on the DF3/MUC1 oncoprotein

The DF3/MUC1 transmembrane oncoprotein is aberrantly overexpressed in most human breast carcinomas and interacts with the Wnt effector gamma-catenin. Here, we demonstrate that MUC1 associates constitutively with ErbB2 in human breast cancer cells and that treatment with heregulin/neuregulin-1 (HRG) increases the formation of MUC1-ErbB2 complexes. The importance of the MUC1-ErbB2 interaction is supported by the demonstration that HRG induces binding of MUC1 and gamma-catenin and targeting of the MUC1-gamma-catenin complex to the nucleolus. Significantly, nucleolar localization of gamma-catenin in response to HRG is dependent on MUC1 expression. Moreover, mutation of a RRK motif in the MUC1 cytoplasmic domain abrogates HRG-induced nucleolar localization of MUC1 and gamma-catenin. In concert with these results, we show nucleolar localization of MUC1 and gamma-catenin in human breast carcinomas but not in normal mammary ductal epithelium. These findings demonstrate that MUC1 functions in cross talk between ErbB2 and Wnt pathways by acting as a shuttle for HRG-induced nucleolar targeting of gamma-catenin.