The Cell Surface Receptor Tartan Is a Potential In Vivo Substrate for the Receptor Tyrosine Phosphatase Ptp52F

Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as “bait.” Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST “pulldown” experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons.Receptor-linked protein-tyrosine phosphatases (RPTPs) are enzymes with extracellular (XC) domains, a single transmembrane domain, and one or two cytoplasmic protein tyrosine phosphatase (PTP) homology domains. Many RPTPs have XC sequences that resemble those of cell adhesion molecules (for a review, see reference 33). This sequence organization suggests that RPTPs can couple cell-cell recognition events to dephosphorylation of cytoplasmic substrates. Interestingly, while phosphotyrosine (PY) pathways involved in cell growth and differentiation typically involve receptor tyrosine kinases that bind to growth factors and are regulated by nontransmembrane PTPs, those that control axon guidance often use RPTPs and nontransmembrane TKs. This implies that the cues that affect PY signaling in axonal growth cones may interact with RPTPs rather than with receptor tyrosine kinases (reviewed in reference 14).There are 17 active RPTPs encoded in the human genome, while Drosophila has six. Most of the mammalian RPTPs are expressed in nonneural tissues, but four of the six fly RPTPs are expressed only by central nervous system (CNS) neurons in late embryos. All published zygotic phenotypes produced by Rptp mutations are alterations in axon guidance or synaptogenesis. These results suggest that the major functions of the Drosophila RPTPs are in neural development (for a review, see reference 16). Analysis of axon guidance phenotypes in embryos bearing single or multiple Rptp mutations is consistent with the idea that RPTP interactions with ligands at growth cone choice points convey “information,” in the form of changes in substrate phosphorylation within growth cones, that is used to determine pathway decisions.In the Drosophila neuromuscular system, 36 motor axons grow out within six nerve bundles in each abdominal hemisegment, and each axonal growth cone makes a series of genetically determined guidance decisions that direct it to the appropriate muscle fiber (for a review, see reference 27). Our work on Rptp mutant combinations suggests that each pathway decision uses a specific subset of the six RPTPs. RPTPs can exhibit functional redundancy, so that the loss of one does not produce a defect unless another RPTP is also absent, or competition, in which loss of one RPTP suppresses the phenotype produced by loss of another (5, 6, 31). Examination of RPTP expression patterns suggests that the RPTPs are expressed by most (or possibly all) CNS neurons, including motor neurons. If so, the requirements for individual RPTPs for execution of particular guidance decisions cannot be due to selective expression of these RPTPs on specific motor axons. These requirements might instead be determined by the expression patterns of RPTP ligands, so that only RPTPs whose ligands were localized to the vicinity of a growth cone choice point would participate in that pathway decision. Alternatively (or in addition), the necessity of a particular RPTP for a pathway decision might arise from selective expression of RPTP substrates, so that an RPTP would be important for guidance decisions made by a growth cone of a specific motor neuron only if that neuron expressed the relevant substrate(s).Evaluation of such models requires identification of specific XC ligands and intracellular substrates for the Drosophila RPTPs. Only one set of ligands has been identified thus far. These are the heparan sulfate proteoglycans Syndecan (Sdc) and Dallylike (Dlp), which bind to the Lar RPTP with nanomolar affinity and contribute to its functions in axon guidance and synapse growth (9, 15). Similarly, little is known about substrate specificity in vivo. Lar can dephosphorylate the Enabled (Ena) protein, which regulates the growth cone cytoskeleton, and genetic interaction studies suggest that Ena may be an in vivo substrate for Lar (35). The transmembrane protein gp150 can be dephosphorylated by Ptp10D in cell culture and intact fly larvae, but genetics has not provided evidence that Ptp10D and gp150 are in the same signaling pathway in vivo (7).The identification of in vivo substrates for RPTPs has been hampered by the fact that purified RPTP cytoplasmic domains often do not exhibit high selectivity in vitro when tested for dephosphorylation activity on peptides or proteins. The most fruitful method for finding substrates for both RPTPs and cytoplasmic PTPs has been the use of “substrate-trapping” mutants. The most effective substrate traps were devised by Tonks and coworkers, and are created by changing an invariant Asp (D) residue within the PTP active site to Ala (A) (8). The D residue has an abnormal pK and is thus able to donate a proton to the phosphorus-oxygen bond, facilitating displacement of the tyrosine (Y) OH by the invariant Cys (C) nucleophile of the enzyme. This creates a phosphoenzyme intermediate. The dephosphorylated substrate then dissociates, and water attacks the Cys-phosphate bond, releasing the phosphate and reconstituting the enzyme. In D→A mutants, the polarization of the phosphorus-oxygen bond by protonation cannot take place, and the PY substrate remains bound to the enzyme. Substrate-trapping mutants expressed in cells often bind to only a few phosphoproteins, suggesting that PTPs exhibit high specificity in vivo (see, for example, reference 11).We conducted a modified yeast two-hybrid screen to find Drosophila phosphoproteins that bind selectively to RPTP substrate-trapping mutants. We identified the cell surface receptor Tartan (Trn) in this screen and showed that it is a substrate for the Ptp52F RPTP in Drosophila Schneider 2 (S2) cells. Axon guidance phenotypes in trn mutants are identical to those seen in Ptp52F mutants, and trn and Ptp52F exhibit dosage-dependent genetic interactions. These results suggest that Ptp52F is a regulator of Trn signaling in motor neurons in vivo.     

A docking model of human ribonucleotide reductase with flavin and phenosafranine

Ribonucleotide Reductase (RNR) is an enzyme responsible for the reduction of ribonucleotides to their corresponding Deoxyribonucleotides (DNA), which is a building block for DNA replication and repair mechanisms. The key role of RNR in DNA synthesis and control in cell growth has made this an important target for anticancer therapy. Increased RNR activity has been associated with malignant transformation and tumor cell growth. In recent years, several RNR inhibitors, including Triapine, Gemcitabine and GTI-2040, have entered the clinical trials. Our current work focuses on an attempted to dock this inhibitors Flavin and Phenosafranine to curtail the action of human RNR2. The docked inhibitor Flavin and Phenosafranine binds at the active site with THR176, which are essential for free radical formation. The inhibitor must be a radical scavenger to destroy the tyrosyl radical or iron metal scavenger. The iron or radical site of R2 protein can react with one-electron reductants, whereby the tyrosyl radical is converted to a normal tyrosine residue. However, compounds such as Flavin and Phenosafranine were used in most of the cases to reduce the radical activity. The docking study was performed for the crystal structure of human RNR with the radical scavengers Flavin and Phenosafranine to inhibit the human RNR2. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2.     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

Choroidal eye metastases from (recurrent) primary peritoneal carcinoma: case report and review of the literature

Background

Choroidal metastases from gynaecological primary are extremely rare. There is no documented case in the literature of choroid metastasis in a patient with primary peritoneal carcinoma (PPC).

Methods & Results

We describe the first case of a 54-year-old woman with a history of borderline mucinous tumour who presented 17 months later with PPC and 21 months after with recurrent disease metastatic to the eye, and review pertinent literature.

Conclusion

High index of suspicion is warranted when patients with history of primary peritoneal carcinoma present with visual complaints in order to treat and/or relieve symptomatology from metastatic eye disease.

     

Adaptive mechanisms to compensate for overnutrition-induced cardiovascular abnormalities

In conditions of overnutrition, cardiac cells must cope with a multitude of extracellular signals generated by changes in nutrient load (glucose, amino acids, and lipids) and the hormonal milieu [increased insulin (INS), ANG II, and adverse cytokine/adipokine profile]. Herein, we review the diverse compensatory/adaptive mechanisms that counter the deleterious effects of excess nutrients and growth factors. We largely focus the discussion on evidence obtained from Zucker obese (ZO) and Zucker diabetic fatty (ZDF) rats, which are useful models to evaluate adaptive and maladaptive metabolic, structural, and functional cardiac remodeling. One adaptive mechanism present in the INS-resistant ZO, but absent in the diabetic ZDF heart, involves an interaction between the nutrient sensor kinase mammalian target of rapamycin complex 1 (mTORC1) and ANG II-type 2 receptor (AT2R). Recent evidence supports a cardioprotective role for the AT2R; for example, suppression of AT2R activation interferes with antihypertrophic/antifibrotic effects of AT1R blockade, and AT2R agonism improves cardiac structure and function. We propose a scenario, whereby mTORC1-signaling-mediated increase in AT2R expression in the INS-resistant ZO heart is a cardioprotective adaptation to overnutrition. In contrast to the ZO rat, heart tissues of ZDF rats do not show activation of mTORC1. We posit that such a lack of activation of the mTOR?AT2R integrative pathway in cardiac tissue under conditions of obesity-induced diabetes may be a metabolic switch associated with INS deficiency and clinical diabetes.     

Acute exposure to ultraviolet‐B radiation modulates sex steroid hormones and receptor expression in the skin and may contribute to the sex bias of melanoma in a fish model

Using the Xiphophorus fish melanoma model, we show a strong male bias for sunlight‐induced malignant melanoma, consistent with that seen in the human population. To examine underlying factors, we exposed adult X. couchianus fish to a single, sublethal dose of UVB and measured circulating sex steroid hormones and expression of associated hormone receptor genes over a 24‐h period. We found that a single exposure had profound effects on circulating levels of steroid hormones with significant decreases for all free sex steroids at 6 and 24 h and increases in conjugated 2‐estradiol and 11‐ketotestosterone at 6 and 24 h, respectively. Whereas ARα expression increased in male and female skin, neither ARβ nor either of the ERs showed significant responses to UVB in either sex. The rapid response of male androgens and their receptors in the skin after UVB irradiation implicates hormones in the male bias of skin cancer and suggests that the photoendocrine response immediately after UV exposure may be relevant to melanomagenesis.     

Down-regulated Peroxisome Proliferator-activated Receptor γ (PPARγ) in Lung Epithelial Cells Promotes a PPARγ Agonist-reversible Proinflammatory Phenotype in Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.     

Molecular phylogeny of mangroves. VI. Intraspecific genetic variation in mangrove species Excoecaria agallocha L. (Euphorbiaceae).

Genomic DNA from 84 individuals of Excoecaria agallocha from seven mangrove populations were analysed for random amplified polymorphic DNAs (RAPDs) using 16 random 10-mer primers. Polymorphism within populations varied from 20% to 31%. At the interpopulation level, 111/149 (74%) of RAPDs were polymorphic. Restriction fragment length polymorphism (RFLP) analysis of 21 individuals (3 individuals randomly selected from the 7 populations) using 30 probe-enzyme combinations revealed a high level of interpopulation polymorphism (62.2%) indicating interpopulation genetic divergence. The polymorphic RAPDs and RFLPs were pooled, and clustering was carried out based on mean similarity for individual populations. The dendrogram showed groupings of populations from the West and East Coasts of India into separate clusters, at 60% similarity level. Further, RAPD and RFLP analysis of male and female plants showed approximately the same level of variation in both sexes, and no sex-linked markers were found. These results demonstrate that considerable intrapopulation and interpopulation genetic variations exist in E. agallocha, and that lack of genetic variation is not the reason for the morphological uniformity observed across the range of the species.     

Evaluation of inhibition of the carbenicillin-hydrolyzing β-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing β-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k_cat/k_inact) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 β-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.