Retinoblastoma-binding protein 2 (RBP2) is frequently expressed in neuroendocrine tumors and promotes the neoplastic phenotype.

Neuroendocrine tumors (NETs), which can have survival rates as low as 4%, currently have limited therapeutic interventions available highlighting the dire need for the identification of novel biological targets for use as new potential drug targets. One such potential target is retinoblastoma-binding protein 2 (RBP2), an H3K4 demethylase whose overexpression has been linked to cancer formation and metastasis in non-endocrine tumor types. We measured RBP2 mRNA and protein levels in enteropancreatic NETs by measuring RBP2 in matched human normal and NET tissue samples. Further, proliferation, migration, invasion and colony formation assays were performed in the physiologically relevant NET cell lines ßlox5, H727 and QGP-1 to understand the role of RBP2 and its demethylase activity on end points of tumorigenesis. Our data indicate a strong correlation between RBP2 mRNA and protein expression in NET specimens. RBP2 was overexpressed relative to tissue-matched normal controls in 80% of the human tumors measured. In vitro studies showed RBP2 overexpression significantly increased proliferation, migration, invasion and colony formation, whereas knockdown significantly decreases the same parameters in a demethylase-independent manner. The cell cycle inhibitors p21 and p57 decreased with RBP2 overexpression and increased upon its depletion, suggesting a regulatory role for RBP2 in cellular proliferation. Taken together, our results support the hypothesis that the aberrant overexpression of RBP2 is a frequent contributing factor to tumor formation and metastasis in enteropancreatic NETs.

Comparative transcriptomic analysis reveals the oncogenic fusion protein PAX3-FOXO1 globally alters mRNA and miRNA to enhance myoblast invasion.

Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression.

Investigation on removal of malachite green using EM based compost as adsorbent.

The discarded materials from different sources can be utilized as effective materials in wastewater remediation. This proposed study was aimed mainly to investigate the possibility of Effective Microorganisms based compost (EMKC), which is derived from the kitchen solid waste, as a non-conventional low cost adsorbent for the removal of malachite green from aqueous solution. Batch experiments were carried out to evaluate the optimum operating parameters like pH (2-9), initial dye concentration (50-1000mg/L), adsorbent particle size (0.6-2.36mm) and adsorbent dosage (2-12g/L). EMKC recorded maximum uptake of 136.6mg/g of MG at pH 8, initial dye concentration 1000mg/L, adsorbent particle size 1.18mm and adsorbent dosage 4g/L. Two and three parameter adsorption models were employed to describe experimental biosorption isotherm data. The results revealed that the Sips model resulted in better fit than other models. The pseudo-first and -second order models were applied to describe kinetic data, of which the pseudo-second order described experimental data better with high correlation coefficient. This investigation suggested that EMKC could be an effective and low cost material for the removal of malachite green dye from aqueous solution.

Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.

OBJECTIVES: Women with a sonographic short cervix in the mid-trimester are at increased risk for preterm delivery. This study was undertaken to determine the efficacy and safety of using micronized vaginal progesterone gel to reduce the risk of preterm birth and associated neonatal complications in women with a sonographic short cervix. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial that enrolled asymptomatic women with a singleton pregnancy and a sonographic short cervix (10-20 mm) at 19 + 0 to 23 + 6 weeks of gestation. Women were allocated randomly to receive vaginal progesterone gel or placebo daily starting from 20 to 23 + 6 weeks until 36 + 6 weeks, rupture of membranes or delivery, whichever occurred first. Randomization sequence was stratified by center and history of a previous preterm birth. The primary endpoint was preterm birth before 33 weeks of gestation. Analysis was by intention to treat. RESULTS: Of 465 women randomized, seven were lost to follow-up and 458 (vaginal progesterone gel, n=235; placebo, n=223) were included in the analysis. Women allocated to receive vaginal progesterone had a lower rate of preterm birth before 33 weeks than did those allocated to placebo (8.9% (n=21) vs 16.1% (n=36); relative risk (RR), 0.55; 95% CI, 0.33-0.92; P=0.02). The effect remained significant after adjustment for covariables (adjusted RR, 0.52; 95% CI, 0.31-0.91; P=0.02). Vaginal progesterone was also associated with a significant reduction in the rate of preterm birth before 28 weeks (5.1% vs 10.3%; RR, 0.50; 95% CI, 0.25-0.97; P=0.04) and 35 weeks (14.5% vs 23.3%; RR, 0.62; 95% CI, 0.42-0.92; P=0.02), respiratory distress syndrome (3.0% vs 7.6%; RR, 0.39; 95% CI, 0.17-0.92; P=0.03), any neonatal morbidity or mortality event (7.7% vs 13.5%; RR, 0.57; 95% CI, 0.33-0.99; P=0.04) and birth weight < 1500 g (6.4% (15/234) vs 13.6% (30/220); RR, 0.47; 95% CI, 0.26-0.85; P=0.01). There were no differences in the incidence of treatment-related adverse events between the groups. CONCLUSIONS: The administration of vaginal progesterone gel to women with a sonographic short cervix in the mid-trimester is associated with a 45% reduction in the rate of preterm birth before 33 weeks of gestation and with improved neonatal outcome.

Early failure of a mobile-bearing unicompartmental knee replacement.

Concerned by a perceived high revision rate, we retrospectively reviewed the survivorship of a series of 43 cemented, medial, mobile-bearing Preservation unicompartmental knee replacements implanted during a 2-year period at a single institution. The initial post-operative AP and lateral radiographs were independently assessed to test the hypothesis that suboptimal implantation of the prosthesis was responsible for early failure. An X-ray scoring system based on the criteria for assessing the Oxford mobile-bearing unicompartmental knee replacement was devised. The components of this score included assessment of prosthesis alignment, sizing and cementation. Nine (21%) LCS Preservation mobile-bearings prostheses had required revision at a mean of 22 months post-implantation. The commonest causes for failure were pain (44%) and tibial component loosening (33%). Analysis of post-operative radiographs showed no difference (n.s.) between the compound error scores for the revised and the surviving prostheses. No particular surgical error was identifiable leading to subsequent need for revision. The high failure rates shown in this study have led us to cease using this implant. The clinical relevance of this study is that the captive running track of the LCS mobile-bearing prosthesis may over constrain the meniscal component leading to early failure.

Vaginal progesterone is associated with a decrease in risk for early preterm birth and improved neonatal outcome in women with a short cervix: a secondary analysis from a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: To investigate the efficacy of vaginal progesterone to prevent early preterm birth in women with sonographic evidence of a short cervical length in the midtrimester. METHODS: This was a planned, but modified, secondary analysis of our multinational, multicenter, randomized, placebo-controlled trial, in which women were randomized between 18 + 0 and 22 + 6 weeks of gestation to receive daily treatment with 90 mg of vaginal progesterone gel or placebo. Cervical length was measured with transvaginal ultrasound at enrollment and at 28 weeks of gestation. Treatment continued until either delivery, 37 weeks of gestation or development of preterm rupture of membranes. Maternal and neonatal outcomes were evaluated for the subset of all randomized women with cervical length < 28 mm at enrollment. The primary outcome was preterm birth at

Progesterone vaginal gel for the reduction of recurrent preterm birth: primary results from a randomized, double-blind, placebo-controlled trial.

OBJECTIVE: Preterm birth is the leading cause of perinatal morbidity and mortality worldwide. Treatment of preterm labor with tocolysis has not been successful in improving infant outcome. The administration of progesterone and related compounds has been proposed as a strategy to prevent preterm birth. The objective of this trial was to determine whether prophylactic administration of vaginal progesterone reduces the risk of preterm birth in women with a history of spontaneous preterm birth. METHODS: This randomized, double-blind, placebo- controlled, multinational trial enrolled and randomized 659 pregnant women with a history of spontaneous preterm birth. Between 18 + 0 and 22 + 6 weeks of gestation, patients were assigned randomly to once-daily treatment with either progesterone vaginal gel or placebo until either delivery, 37 weeks' gestation or development of preterm rupture of membranes. The primary outcome was preterm birth at

Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase).

On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P'1 residues. With a small P'1 residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P'1 residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24-100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P'1 residues showed a 10-40-fold decrease in Vmax with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S'1 binding subsite, of neutral endopeptidase 24.11.

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase).

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.

Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin.

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pKa is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pKa is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases kappa cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pKa of 7 for NEP and a pKa of 6 for thermolysin. In the case of thermolysin this pKa was decreased to 5.3 in the enzyme-inhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of Ki on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pKa value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pKa of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pKa of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.

Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis.

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopeptidase, which has been shown to inactivate a variety of peptide hormones including enkephalin, chemotactic peptide, substance P, neurotensin, oxytocin, bradykinin, and angiotensins I and II, had not been identified in lymphoid cells. To determine whether CALLA cDNA derived from human acute lymphoblastic leukemia cells (Nalm-6 cell line) encodes functional neutral endopeptidase activity, we generated CALLA+ stable transfectants in the CALLA- murine myeloma cell line J558 and analyzed them for enzymatic activity in a fluorometric assay based upon cleavage of the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total lysates as well as whole-cell suspensions of the Nalm-6 line and of the CALLA+ transfectants, but not of the CALLA- J558 cells, possessed neutral endopeptidase activity. This enzymatic activity was associated with the cellular membrane fraction and was abrogated by the specific neutral endopeptidase inhibitor phosphoramidon. The unequivocal identification of CALLA as a functional neutral endopeptidase provides insight into its potential role in both normal and malignant lymphoid function.