Cell-specific metabolism and pathogenesis of transmembrane prion protein

The C-transmembrane form of prion protein ((Ctm)PrP) has been implicated in prion disease pathogenesis, but the factors underlying its biogenesis and cyotoxic potential remain unclear. Here we show that (Ctm)PrP interferes with cytokinesis in cell lines where it is transported to the plasma membrane. These cells fail to separate following cell division, assume a variety of shapes and sizes, and contain multiple nuclei, some of which are pyknotic. Furthermore, the synthesis and transport of (Ctm)PrP to the plasma membrane are modulated through a complex interaction between cis- and trans-acting factors and the endoplasmic reticulum translocation machinery. Thus, insertion of eight amino acids before or within the N region of the N signal peptide (N-SP) of PrP results in the exclusive synthesis of (Ctm)PrP regardless of the charge conferred to the N region. Subsequent processing and transport of (Ctm)PrP are modulated by specific amino acids in the N region of the N-SP and by the cell line of expression. Although the trigger for (Ctm)PrP upregulation in naturally occurring prion disorders remains elusive, these data highlight the underlying mechanisms of (Ctm)PrP biogenesis and neurotoxicity and reinforce the idea that (Ctm)PrP may serve as the proximate cause of neuronal death in certain prion disorders.     

Octapeptide Analogs of Somatostatin Containing α,α-Dialkylated Amino Acids with Potent Anticancer Activity

We describe the synthesis and anticancer activities of octapeptide analogs of somatostatin incorporating α,α-dialkylated amino acids. The designed analogs of somatostatin are: d-Phe¹-Cys²-Tyr³-d-Trp⁴-Orn⁵-Xxx⁶-Pen⁷-Thr⁸-NH₂ where Xxx=α-Aminoisobutyric acid (Aib), Diethyl glycine (Deg), 1-Aminocyclopentane carboxylic acid (Ac5c), and, d-Phe¹-Cys²-Tyr³-d-Trp⁴-Lys⁵-Ac5c⁶-Pen⁷-Thr⁸-NH₂ (disulphide bond between Cys² and Pen⁷ in all analogs). The conformational studies two of the designed analogs were carried out by NMR techniques and the experimental results suggest a β-turn structure for one of the designed analog. In vivo tumor regression study of two designed analogs on human primary colon tumor xenografts in nude mice demonstrates the anticancer potential of the synthesized analogs.     

Mechanisms in decorin regulation of vascular endothelial growth factor-induced human trophoblast migration and acquisition of endothelial phenotype

Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and utero-placental arteries to establish an efficient exchange of key molecules between maternal and fetal blood. Trophoblast invasion is stringently regulated in situ both positively and negatively by a variety of factors at the fetal-maternal interface to maintain a healthy utero-placental homeostasis. One such factor, decorin, a transforming growth factor (TGF)-beta binding, leucine-rich proteoglycan produced by the decidua, negatively regulates EVT proliferation, migration, and invasiveness independent of TGF-beta. We reported that these decorin actions were mediated by its binding to multiple tyrosine kinase receptors, including vascular endothelial growth factor receptor (VEGFR)-2. The present study explores the mechanisms underlying decorin antagonism of VEGF (VEGF-A) stimulation of endovascular differentiation of EVT using our EVT cell line, HTR-8/SVneo. We observe that decorin inhibits VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel. VEGF activates MAPKs (p38 MAPK, MEK3/6, and ERK1/2) in EVT cells, and the activation is blocked in both cases by decorin. Employing selective MAPK inhibitors, we show that both p38 and ERK pathways contribute independently to VEGF-induced EVT migration and capillary-like tube formation. VEGF upregulates the vascular endothelial (VE) markers VE-cadherin and beta-catenin in EVT and endothelial cells, and this upregulation is blocked by decorin and MAPK inhibitors. These results suggest that decorin inhibits VEGF-A stimulation of trophoblast migration and endovascular differentiation by interfering with p38 MAPK and ERK1/2 activation. Thus decorin-mediated dual impediment of endovascular differentiation of the EVT and angiogenesis may have implications for pathogenesis of preeclampsia, a hypoinvasive trophoblast disorder in pregnancy.     

MAP kinase phosphorylation is dispensable for cell division,but required for cell growth in Drosophila

Proper activation of the Ras/MAPK pathway is broadly required during development, and in many cases, signal transduction downstream of the receptor is linear. Thus, different mechanisms exist to properly regulate the large number of specific developmental outputs that are required by the activation of this pathway. Previously, we have reported a regulated cytoplasmic sequestration of phosphorylated MAPK (pMAPK) in developing Drosophila compound eyes and wings “called MAPK Cytoplasmic Hold”. In the developing wing, we have shown that cytoplasmic hold promotes the differentiation of wing vein tissue, while pMAPK nuclear translocation regulates growth and division. We had also suggested that the Ras pathway signals for inducing cell growth and cell division split upstream of the nuclear translocation of MAPK itself. Here, we further refine the role of MAPK in Drosophila. We report evidence that suggests, for the first time, that the phosphorylation of MAPK is itself another step in the regulation of cell growth and division in both Drosophila wing and eye cells. We show that inhibition of MAPK phosphorylation, or pMAPK nuclear translocation, is sufficient to block cell growth, but not cell division. These data suggest that non-phosphorylated MAPK is sufficient to induce cell division, but not cell growth, once inside the nucleus of the cell.Key words: Drosophila, MAPK, growth, division, proliferation, phosphorylation     

Community Mobilization in Mumbai Slums to Improve Perinatal Care and Outcomes: A Cluster Randomized Controlled Trial

Introduction

Improving maternal and newborn health in low-income settings requires both health service and community action. Previous community initiatives have been predominantly rural, but India is urbanizing. While working to improve health service quality, we tested an intervention in which urban slum-dweller women''s groups worked to improve local perinatal health.

Methods and Findings

A cluster randomized controlled trial in 24 intervention and 24 control settlements covered a population of 283,000. In each intervention cluster, a facilitator supported women''s groups through an action learning cycle in which they discussed perinatal experiences, improved their knowledge, and took local action. We monitored births, stillbirths, and neonatal deaths, and interviewed mothers at 6 weeks postpartum. The primary outcomes described perinatal care, maternal morbidity, and extended perinatal mortality. The analysis included 18,197 births over 3 years from 2006 to 2009. We found no differences between trial arms in uptake of antenatal care, reported work, rest, and diet in later pregnancy, institutional delivery, early and exclusive breastfeeding, or care-seeking. The stillbirth rate was non-significantly lower in the intervention arm (odds ratio 0.86, 95% CI 0.60–1.22), and the neonatal mortality rate higher (1.48, 1.06–2.08). The extended perinatal mortality rate did not differ between arms (1.19, 0.90–1.57). We have no evidence that these differences could be explained by the intervention.

Conclusions

Facilitating urban community groups was feasible, and there was evidence of behaviour change, but we did not see population-level effects on health care or mortality. In cities with multiple sources of health care, but inequitable access to services, community mobilization should be integrated with attempts to deliver services for the poorest and most vulnerable, and with initiatives to improve quality of care in both public and private sectors.

Trial registration

Current Controlled Trials ISRCTN96256793 Please see later in the article for the Editors'' Summary     

Decorin is a novel VEGFR-2-binding antagonist for the human extravillous trophoblast

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.     

Two putative histidine kinases are required for cyst formation in <Emphasis Type="Italic">Rhodospirillum Centenum</Emphasis>

The photosynthetic bacterium, Rhodospirillum centenum, has a flexible life cycle that permits it to survive starvation as dormant cyst cells. Previous studies have identified some of the key regulators for encystment and demonstrated that the control of development is intricate. This complexity may arise from the need to integrate several environmental signals to mediate a switch from one mode of energy metabolism to another and to ensure that a transition to dormancy is initiated only when necessary. We searched for additional regulators of development by screening for encystment deficient strains after subjecting wild type R. centenum to mini-Tn5 mutagenesis. Analysis of “hypo-cyst” strains led to the identification of two genes that encode putative hybrid histidine kinases (cyd1 and cyd2). Cells with deletions of either gene fail to form cysts under conditions that normally induce development. Furthermore, the deletion strains exhibit altered swarming behavior suggesting that Cyd1 and Cyd2 affect behaviors utilized when the organism is attached to a substrate.     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

Evaluation of antidepressant-like activity of aqueous and ethanolic extracts of Terminalia bellirica Roxb. fruits in mice

The present study was undertaken to investigate the effect of aqueous and ethanolic extracts of T. bellirica on depression in mice using forced swim test (FST) and tail suspension test (TST). The extracts were administered orally for 10 successive days in separate groups of Swiss young male albino mice. Aqueous extract (50, 100 and 200 mg/kg) in a dose-dependent manner and ethanolic extract (100 mg/kg) significantly reduced the immobility time of mice in both FST and TST. The extracts were without any significant effect on locomotor activity of mice. The efficacies of aqueous extract (200 mg/kg) and ethanolic extract (100 mg/kg) were found to be similar to that of imipramine (15 mg/kg, po) and fluoxetine (20 mg/kg, po) administered for 10 successive days. Both extracts reversed reserpine-induced extension of immobility period of mice in FST and TST. Prazosin (62.5 microg/kg, ip; an alpha1-adrenoceptor antagonist), sulpiride (50 mg/kg, ip; a selective D2 receptor antagonist) and p-chlorophenylalanine (100 mg/kg, ip; an inhibitor of serotonin synthesis) significantly attenuated the aqueous and ethanolic extract-induced antidepressant-like effect in TST. Thus, both the aqueous and ethanolic extracts of T. bellirica elicited a significant antidepressant-like effect in mice by interaction with adrenergic, dopaminergic and serotonergic systems.