RIP1 Protein-dependent Assembly of a Cytosolic Cell Death Complex Is Required for Inhibitor of Apoptosis (IAP) Inhibitor-mediated Sensitization to Lexatumumab-induced Apoptosis

Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.     

Polyelectrolyte complexes of gum kondagogu and chitosan,as diclofenac carriers

Polyelectrolyte complexes (PEC) of gum kondagogu (GKG) and chitosan were prepared by mixing polymeric solutions of different concentrations (0.02–0.18% w/v). The complex formed were loaded with diclofenac sodium, and the release of the drug was measured in vitro and in vivo, along with the measurement of particle size, zeta potential, complex formation, flow properties, and loading efficiency. Maximum yield of PEC was observed at gum kondagogu concentrations above 80%. The PEC showed lower release of diclofenac sodium in 0.1 N HCl as compared to phosphate buffer (pH 6.8). Increasing the concentration of gum kondagogu in PEC led to an increase in drug release. However, PEC 1:3 (gum kondagogu: chitosan) with higher concentration of chitosan showed 98% release with in 4.5 h, owing to the fact that chitosan has a higher degree of swelling in acidic medium. PEC 5:1 and 3:1 showed a 5.3- and 5.8-fold increase in relative bioavailability compared to the free drug when administered orally to the rats.     

A Drosophila systems model of pentylenetetrazole induced locomotor plasticity responsive to antiepileptic drugs

Background  

Rodent kindling induced by PTZ is a widely used model of epileptogenesis and AED testing. Overlapping pathophysiological mechanisms may underlie epileptogenesis and other neuropsychiatric conditions. Besides epilepsy, AEDs are widely used in treating various neuropsychiatric disorders. Mechanisms of AEDs' long term action in these disorders are poorly understood. We describe here a Drosophila systems model of PTZ induced locomotor plasticity that is responsive to AEDs.     

In silico QSAR studies of anilinoquinolines as EGFR inhibitors

Members of the epidermal growth factor receptor (EGFR) family of proteins are frequently overactive in solid tumors. A relatively new therapeutic approach to inhibit the kinase activity is the use of ATP-competitive small molecules. In silico techniques were employed to identify the key interactions between inhibitors and their protein receptors. A series of EGFR inhibitory anilinoquinolines was studied within the framework of hologram quantitative structure activity relationship (HQSAR), density functional theory (DFT)-based QSAR, and three-dimensional (3D) QSAR (CoMFA/CoMSIA). The HQSAR analysis implied that substitutions at certain sites on the inhibitors play an important role in EGFR inhibition. DFT-based QSAR results suggested that steric and electronic interactions contributed significantly to the activity. Ligand-based 3D-QSAR and receptor-guided 3D-QSAR analyses such as CoMFA and CoMSIA techniques were carried out, and the results corroborated the previous two approaches. The 3D QSAR models indicated that steric and hydrophobic interactions are dominant, and that substitution patterns are an important factor in determining activity. Molecular docking was helpful in identifying a bioactive conformer as well as a plausible binding mode. The docked geometry-based CoMFA model with steric and electrostatic fields effect gave q ² = 0.66, r ² = = 0.94 with r ² _predictive = 0.72. Similarly, CoMSIA with hydrophobic field gave q ² = 0.59, r ² = 0.85 with r ² _predictive = 0.63. Bulky groups around site 3 of ring “C”, and hydrophilic and bulky groups at position 6 of ring “A” are desirable, with a hydrophobic and electron-donating group at site 7 of ring “A” being helpful. Accordingly, potential EGFR inhibitors may be designed by modification of known inhibitors.     

Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment

The use of highly discriminatory methods for the identification and characterization of genotypes is essential for plant protection and appropriate use. We utilized the RAPD method for the genetic fingerprinting of 11 plant species of desert origin (seven with known medicinal value). Andrachne telephioides, Zilla spinosa, Caylusea hexagyna, Achillea fragrantissima, Lycium shawii, Moricandia sinaica, Rumex vesicarius, Bassia eriophora, Zygophyllum propinquum subsp migahidii, Withania somnifera, and Sonchus oleraceus were collected from various areas of Saudi Arabia. The five primers used were able to amplify the DNA from all the plant species. The amplified products of the RAPD profiles ranged from 307 to 1772 bp. A total of 164 bands were observed for 11 plant species, using five primers. The number of well-defined and major bands for a single plant species for a single primer ranged from 1 to 10. The highest pair-wise similarities (0.32) were observed between A. fragrantissima and L. shawii, when five primers were combined. The lowest similarities (0) were observed between A. telephioides and Z. spinosa; Z. spinosa and B. eriophora; B. eriophora and Z. propinquum. In conclusion, the RAPD method successfully discriminates among all the plant species, therefore providing an easy and rapid tool for identification, conservation and sustainable use of these plants.     

Comparison of three pyrethroid treatments of top-sheets for malaria control in emergencies: entomological and user acceptance studies in an Afghan refugee camp in Pakistan

Insecticide-treated bedding materials (sheets and blankets) could be protective against vectors of malaria and leishmaniasis--especially in complex emergencies, epidemics and natural disasters where people are more likely to sleep in exposed situations. Comparison of cotton top-sheets impregnated with different pyrethroids (permethrin 500 mg/m2, deltamethrin 25 mg/m2 or alphacypermethrin 25 mg/m2) for effectiveness against mosquitoes (Diptera: Culicidae) was undertaken in a refugee camp in Pakistan. Predominant species encountered were Anopheles stephensi Liston, An. pulcherrimus Theobald, An. nigerrimus Giles, Culex quinquefasciatus Say, Cx. tritaeniorhynchus Giles and other culicine mosquitoes. All three pyrethroid treatments performed significantly better than the untreated sheets in deterrence and killing of mosquitoes. No significant differences were found between the three insecticides tested in terms of entomological effect. Washing of the treated sheets greatly reduced their effectiveness. In a user acceptance study conducted among 88 families (divided into four groups), six families complained of irritation of the skin and mucous membranes. Of these reports, one was from the placebo group (using untreated sheets) and the other five (5/22=23%) from families using deltamethrin-treated sheets. All families allocated to permethrin and alphacypermethrin groups declared an appreciation for the intervention and reported no side-effects. Ten of the placebo group disliked the intervention, citing no prevention of mosquito biting as the reason. Side-effects associated with deltamethrin indicate that alphacypermethrin and permethrin are more appropriate first choice insecticides for treatment of sheets and blankets.     

Isoflavonoids inhibit catabolism of vitamin D in prostate cancer cells

The high ingestion of soybean products in Asian countries has been suggested to be responsible for a reduced incidence of prostate cancer. The mechanism of action, however, is unknown. Our data demonstrate that genistein and some isoflavone metabolites reduce the activity of 25-D3-24-hydroxylase (CYP24) in the human prostate cancer-derived cell line DU-145. CYP24 is also responsible for degradation of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 which is known to be antimitotic and prodifferentiating in prostate cancer cells. High levels of CYP24 frequently found in prostate cancer cells may thus degrade the active metabolite. This could be prevented by ingestion of genistein-containing food such as soybeans.     

Bacterially synthesized folate and supplemental folic acid are absorbed across the large intestine of piglets

A large pool of folate exists in the large intestine of humans. Preliminary evidence, primarily in vitro, suggests that this folate may be bioavailable. The purpose of this study was to test the hypothesis that supplemental folic acid and bacterially synthesized folate are absorbed across the large intestine of piglets. The pig was used as an animal model because it resembles the human in terms of folate absorption, at least in the small intestine. A tracer of [3H]-folic acid or [3H]-para-aminobenzoic acid ([3H]-PABA), a precursor of bacterially synthesized folate, was injected into the cecum of 11-day-old piglets. Feces and urine were collected for 3 days. Thereafter, piglets were killed, and livers and kidneys harvested. [3H]-Folate was isolated from biological samples by affinity chromatography using immobilized milk folate binding proteins and counted using a scintillation counter. In piglets injected with [3H]-folic acid, the feces, liver, urine and kidneys accounted for 82.1%, 12.3%, 3.9% and 1.7% of recovered [3H]-folate, respectively. In piglets injected with [3H]-PABA, the amount of recovered bacterially synthesized folate in the feces, liver and urine was 85.1%, 0.4% and 14.6%, respectively. Twenty-three percent and 13% of tritium were recovered in samples examined (liver, kidney, fecal and urine) from piglets injected with [3H]-folic acid and [3H]-PABA, respectively. Using our estimates of [3H]-folic acid absorption and the total and percent monoglutamyl folate content of piglet feces, we predict that at least 18% of the dietary folate requirement for the piglet could be met by folate absorption across the large intestine.     

Endothelial Cell mTOR Complex-2 Regulates Sprouting Angiogenesis

Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.     

Polysialylation of NCAM Characterizes the Proliferation Period of Contractile Elements during Postnatal Development of the Epididymis

Polysialic acid (polySia) attached to the neural cell adhesion molecule (NCAM) regulates inter alia the proliferation and differentiation via the interactions with neurotrophins. Since in postnatal epididymis neurotrophins and their receptors like the Low-Affinity Nerve Growth Factor Receptor p75 and TrK B receptor are expressed, we wanted to analyze if the polysialylation of NCAM is also involved during the development of the epididymis. To this end, we monitored the developmental changes in the expression of the polysialyltransferases and NCAM polysialylation using murine epididymis at different time points during postnatal development. Our results revealed that during postnatal development of the epididymis both polysialyltransferases, ST8SiaII and ST8SiaIV, were expressed and that the expression levels dropped with increasing age. In agreement with the expression levels of the polysialyltransferases the highest content of polysialylated NCAM was present during the first 10 days after birth. Interestingly, proliferating smooth muscle cell populations prevalently expressed polysialylated NCAM. Furthermore, we observed that inverse to the decrease in polysialylation of smooth muscle cells a strong up-regulation of collagen takes place suggesting a functional relationship since collagen was recently described to induce the turnover of polysialylated NCAM via an induction of endocytosis in cellulo. The same time course of polySia and collagen synthesis was also observed in other regions of the male reproductive system e.g. vas deferens and tunica albuginea (testis). Together, we identified a spatio-temporal expression pattern of polySia-NCAM characterized by high proliferation rate of smooth muscle cells and low collagen content.