Modulation of trophoblast stem cell and giant cell phenotypes: analyses using the Rcho-1 cell model

Trophoblast giant cells are located at the maternal-embryonic interface and have fundamental roles in the invasive and endocrine phenotypes of the rodent placenta. In this report, we describe the experimental modulation of trophoblast stem cell and trophoblast giant cell phenotypes using the Rcho-1 trophoblast cell model. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate into trophoblast giant cells. Differentiated Rcho-1 trophoblast cells are invasive and possess an endocrine phenotype, including the production of members of the prolactin (PRL) family. Dimethyl sulfoxide (DMSO), a known differentiation-inducing agent, was found to possess profound effects on the in vitro development of trophoblast cells. Exposure to DMSO, at non-toxic concentrations, inhibited trophoblast giant cell differentiation in a dose-dependent manner. These concentrations of DMSO did not significantly affect trophoblast cell proliferation or survival. Trophoblast cells exposed to DMSO exhibited an altered morphology; they were clustered in tightly packed colonies. Trophoblast giant cell formation was disrupted, as was the expression of members of the PRL gene family. The effects of DMSO were reversible. Removal of DMSO resulted in the formation of trophoblast giant cells and expression of the PRL gene family. The phenotype of the DMSO-treated cells was further determined by examining the expression of a battery of genes characteristic of trophoblast stem cells and differentiated trophoblast cell lineages. DMSO treatment had a striking stimulatory effect on eomesodermin expression and a reciprocal inhibitory effect on Hand1 expression. In summary, DMSO reversibly inhibits trophoblast differentiation and induces a quiescent state, which mimics some but not all aspects of the trophoblast stem cell phenotype.     

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

Purification and characterization of an alpha-glucosidase from germinating millet seeds

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.     

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells through lysophosphatidylcholine generation and activation of G2A receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.     

Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps

The activity and composition of leafhopper saliva are important in interactions with the host rice plant, and it may play a physiological role in detoxifying toxic plant substances or ingesting sap. We have characterized diphenoloxidase in the salivary glands of Nephotettix cincticeps, its activity as a laccase, and its presence in the watery saliva with the objective of understanding its function in feeding on rice plants. Nonreducing SDS-PAGE of salivary gland homogenates with staining by the typical laccase substrate 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), hydroquinone or syringaldazine revealed a band at a molecular mass of approximately 85 kDa at pH 5. A band also appeared at a molecular mass of approximately 200 kDa when the gels were treated with dopamine, L-3,4-dihydroxyphenylalanine (DOPA) or catechol at pH 7. The ABTS-oxidizing activity of the homogenates was drastically inhibited by N-hydroxyglycine, a specific inhibitor of laccase. However, the dopamine-oxidizing activity was not inhibited by N-hydroxyglycine, while it was inhibited by phenylthiourea (PTU). Thus, the salivary glands of N. cincticeps contain two types of phenoloxidases: a laccase (85 kDa) and a phenoloxidase (200 kDa). Laccase activity was detected in a holidic sucrose diet that was fed on for 16 h by two females, but only a trace of catechol oxidase activity was observed, suggesting that the laccase-type phenoloxidase was the predominant phenoloxidase secreted in watery saliva. The laccase exhibited an optimum pH of 4.75-5 in McIlvaine buffer and had a PI of 4.8. Enzyme activity was histochemically localized in V cells of the posterior lobe of the salivary glands. It remained at the same level throughout the adult stage from 2 days after eclosion. A possible function of N. cincticeps salivary laccase may be rapid oxidization of potentially toxic monolignols to nontoxic polymers during feeding on the rice plant. This is the first report proving that laccase occurs in the salivary glands of Hemiptera species and is secreted in the watery saliva.     

Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster

Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor.