S-nitrosylation of proteins at the leading edge of migrating trophoblasts by inducible nitric oxide synthase promotes trophoblast invasion

Nitric oxide regulates many important cellular processes including motility and invasion. Many of its effects are mediated through the modification of specific cysteine residues in target proteins, a process called S-nitrosylation. Here we show that S-nitrosylation of proteins occurs at the leading edge of migrating trophoblasts and can be attributed to the specific enrichment of inducible nitric oxide synthase (iNOS/NOS2) in this region. Localisation of iNOS to the leading edge is co-incidental with a site of extensive actin polymerisation and is only observed in actively migrating cells. In contrast endothelial nitric oxide synthase (eNOS/NOS3) shows distribution that is distinct and non-colocalised with iNOS, suggesting that the protein S-nitrosylation observed at the leading edge is caused only by iNOS and not eNOS. We have identified MMP-9 as a potential target for S-nitrosylation in these cells and demonstrate that it co-localises with iNOS at the leading edge of migrating cells. We further demonstrate that iNOS plays an important role in promoting trophoblast invasion, which is an essential process in the establishment of a successful pregnancy.

Epidermal growth factor-stimulated extravillous cytotrophoblast motility is mediated by the activation of PI3-K, Akt and both p38 and p42/44 mitogen-activated protein kinases

BACKGROUND: Trophoblast invasion is a temporally and spatially regulated scheme of events that can dictate pregnancy outcome. Evidence suggests that the potent mitogen epidermal growth factor (EGF) regulates cytotrophoblast (CTB) differentiation and invasion during early pregnancy. METHODS AND RESULTS: In the present study, the first trimester extravillous CTB cell line SGHPL-4 was used to investigate the signalling pathways involved in the motile component of EGF-mediated CTB migration/invasion. EGF induced the phosphorylation of the phosphatidylinositol 3-kinase (PI3-K)-dependent proteins, Akt and GSK-3β as well as both p42/44 MAPK and p38 mitogen-activated protein kinases (MAPK). EGF-stimulated motility was significantly reduced following the inhibition of PI3-K (P < 0.001), Akt (P < 0.01) and both p42/44 MAPK (P < 0.001) and p38 MAPKs (P < 0.001) but not the inhibition of GSK-3β. Further analysis indicated that the p38 MAPK inhibitor SB 203580 inhibited EGF-stimulated phosphorylation of Akt on serine 473, which may be responsible for the effect SB 203580 has on CTB motility. Although Akt activation leads to GSK-3β phosphorylation and the subsequent expression of β-catenin, activation of this pathway by 1-azakenpaullone was insufficient to stimulate the motile phenotype. CONCLUSION: We demonstrate a role for PI3-K, p42/44 MAPK and p38 MAPK in the stimulation of CTB cell motility by EGF, however activation of β-catenin alone was insufficient to stimulate cell motility.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Diversification of a large genus in a continental biodiversity hotspot: temporal and spatial origin of Muraltia (Polygalaceae) in the Cape of South Africa

Phylogenetic relationships in the largely South African genus Muraltia (Polygalaceae) are assessed based on DNA sequence data (nuclear ribosomal ITS, plastid atpB-rbcL spacer, trnL intron, and trnL-F spacer) for 73 of the 117 currently recognized species in the genus. The previously recognised subgenus Muraltia is monophyletic, but the South African endemic genus Nylandtia is embedded in Muraltia subgenus Psiloclada. Subgenus Muraltia is found to be sister to subgenus Psiloclada. Estimates show the beginning of diversification of the two subgenera in the early Miocene (Psiloclada, 19.3+/-3.4 Ma; Muraltia, 21.0+/-3.5 Ma) pre-dating the establishment of the Benguela current (intermittent in the middle to late Oligocene and markedly intensifying in the late Miocene), and summer-dry climate in the Cape region. However, the later increase in species numbers is contemporaneous with these climatic phenomena. Results of dispersal-vicariance analyses indicate that major clades in Muraltia diversified from the southwestern and northwestern Cape, where most of the species are found today.

The role of biotic and abiotic factors in evolution of ant dispersal in the milkwort family (Polygalaceae)

A phylogenetic approach was taken to investigate the evolutionary history of seed appendages in the plant family Polygalaceae (Fabales) and determine which factors might be associated with evolution of elaiosomes through comparisons to abiotic (climate) and biotic (ant species number and abundance) timelines. Molecular datasets from three plastid regions representing 160 species were used to reconstruct a phylogenetic tree of the order Fabales, focusing on Polygalaceae. Bayesian dating methods were used to estimate the age of the appearance of ant-dispersed elaiosomes in Polygalaceae, shown by likelihood optimizations to have a single origin in the family. Topology-based tests indicated a diversification rate shift associated with appearance of caruncular elaiosomes. We show that evolution of the caruncular elaiosome type currently associated with ant dispersal occurred 54.0-50.5 million year ago. This is long after an estimated increase in ant lineages in the Late Cretaceous based on molecular studies, but broadly concomitant with increasing global temperatures culminating in the Late Paleocene-Early Eocene thermal maxima. These results suggest that although most major ant clades were present when elaiosomes appeared, the environmental significance of elaiosomes may have been an important factor in success of elaiosome-bearing lineages. Ecological abundance of ants is perhaps more important than lineage numbers in determining significance of ant dispersal. Thus, our observation that elaiosomes predate increased ecological abundance of ants inferred from amber deposits could be indicative of an initial abiotic environmental function.

Effect of heparin and fractionated heparin on trophoblast invasion

BACKGROUND: Heparin can significantly reduce pregnancy complications in women with certain thrombophilias, such as antiphospholipid syndrome. Recent reports suggest that heparin may act by mechanisms other than anticoagulation. However, the effect of heparin on trophoblast biology in the absence of thrombophilia has not been extensively investigated. Therefore, this study aimed to evaluate trophoblast invasion, using an established cell line and primary extravillous trophoblasts (EVTs), following exposure to heparin and fractionated heparin. METHODS: An EVT cell line (SGHPL-4) was used to study invasion in the presence of hepatocyte growth factor (HGF) and varying concentrations of fractionated and unfractionated heparin. These experiments were repeated using first trimester primary EVTs. RESULTS: Both forms of heparin significantly reduced HGF-induced invasion in the SGHPL-4 cell line. This suppression of invasion appeared to be dose-dependent for fractionated heparin. In primary EVT cells, fractionated heparin also demonstrated significant suppression of invasion. CONCLUSIONS: Heparin has the potential to reduce trophoblast invasion in cell lines and first trimester EVT cells. This article highlights the need for further evaluation of these medications in vitro and in vivo, especially when used in the absence of thrombophilic disorders.

Increased apoptosis in first trimester extravillous trophoblasts from pregnancies at higher risk of developing preeclampsia

Preeclampsia complicates 5 to 10% of pregnancies and is a leading cause of maternal and fetal mortality and morbidity. Although the cause is unknown, inadequate invasion and remodeling of maternal uterine arteries by extravillous trophoblasts (EVTs) in the first trimester is a common feature. Uterine spiral artery resistance as detected by Doppler ultrasound is commonly used in the second trimester to identify pregnancies destined to develop preeclampsia. Correlation between high uterine resistance and the failure of trophoblast invasion has been reported as early as 12 weeks. However, the reason for this failure has not been established. Understanding the processes involved would significantly improve our diagnostic potential. In this study, we correlated increased first trimester uterine artery resistance with a biological abnormality in trophoblast function. EVTs derived from high-resistance pregnancies were more sensitive to apoptotic stimuli than those from normal-resistance pregnancies. Survival of EVTs from high-resistance pregnancies could be increased by nitric oxide, whereas inhibition of nitric oxide in cells from normal-resistance pregnancies increased apoptotic sensitivity. This predates the onset of symptoms by several weeks and provides evidence for a mechanism responsible for the incomplete uterine vessel remodeling and the differences in artery resistance between preeclamptic and normal pregnancies.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

An elevated PSA, which normalizes, does not exclude the presence of prostate cancer

The purpose of this study was to determine the incidence of prostate cancer in patients who have an elevated referral prostate-specific antigen (PSA), which subsequently falls to within their normal age-specific reference range prior to prostate biopsy. The study demonstrated that of the 160 patients recruited, 21 (13%) had a repeat PSA level which had fallen back to within their normal range. Five of these 21 patients (24%) were diagnosed with prostate cancer following biopsy, two of whom had a benign prostate examination. The study, therefore, demonstrates that normalisation of the PSA level prior to biopsy does not exclude the presence of prostate cancer even when the prostate feels benign.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Intra-individual variation of serum prostate specific antigen levels in men with benign prostate biopsies

To determine the intra-individual (physiological) variation of prostate-specific antigen (PSA) measurements in men after a benign prostatic biopsy. Sixty-four men were prospectively assessed, all of whom had a benign prostatic biopsy within the preceding 13 months. The degree of intra-individual variability was established by calculating the coefficient of variation on four PSA levels obtained from each patient weekly over a month. Six patients were subsequently diagnosed with prostate cancer and their data are presented separately. In the remaining 58 patients the median (range) individual mean PSA value was 6.3 (0.5-34.1) ng/mL. The median (range) coefficient of variation within the group was 9.5 (2.4-76.1)%. There was a clear linear relationship between mean PSA level and the standard deviation. In 48 of the 63 patients analysed, the coefficient of variation for serum PSA values in the group as a whole was greater than the variation claimed for the assay technique. The significance of the linear relationship between PSA and the standard deviation is discussed, with particular reference to those men who had a benign prostate biopsy.

The biocontrol fungus pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

A seven-year follow-up of men following a benign prostate biopsy

Objectives: To determine the incidence and clinical relevance of newly diagnosed cases of prostate cancer in a group of men who had an elevated PSA and benign prostate biopsy 7 years previously. Patients and Method: Patients under the age of 80 years with an elevated PSA who had had a benign prostate biopsy in the 12 months between March 1, 1994 and February 28, 1995 were studied. One hundred and sixty four patients with a mean age of 66.8 years (range 47-79 years) were identified. The mean PSA for this group was 10.3 ng/ml (range 4.1-81 ng/ml). One hundred and fifty nine of the 164 (97%) hospital records were available for review and all but 21 (12.8%) of the General Practitioners were contacted. Results: Eighteen (11%) of the original 164 patients were subsequently diagnosed with prostate cancer, 2 died from their disease. Conclusions: In a population where the follow-up of patients with a benign biopsy was arranged on clinical grounds alone, 11% of the study group were diagnosed with prostate cancer during a seven-year follow-up. Although some of these cancers appear to be slow growing, most of those diagnosed in the initial follow-up period were deemed to be clinically significant and a small proportion progressed rapidly to metastases. All patients who have an elevated PSA, but benign biopsy, should undergo a period of PSA monitoring until it is clear that their PSA is not rising. We propose an initial intensive monitoring period to avoid missing those with clinically aggressive disease. (C) 2003 Elsevier Science B.V. All rights reserved.