Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 andD-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: Role of Mg2+ ions

Agonists modulation of Mg²⁺-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg²⁺ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with higher and lower apparent affinity states, are extrapolated. In the presence ofD-Ala²-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E₂ increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg²⁺-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.     

Entericidin Is Required for a Probiotic Treatment (Enterobacter sp. Strain C6-6) To Protect Trout from Cold-Water Disease Challenge

Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB<pET101::ecnAB>) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens.     

In Vitro and In Vivo Antitumor Activity of [Pt(O,O′-acac)(γ-acac)(DMS)] in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive malignancy highly resistant to chemotherapy. There is an urgent need for effective therapy inasmuch as resistance, intrinsic and acquired, to conventional therapies is common. Among Pt(II) antitumor drugs, [Pt(O,O′-acac)(γ-acac)(DMS)] (Ptac2S) has recently attracted considerable attention due to its strong in vitro and in vivo antiproliferative activity and reduced toxicity. The purpose of this study was to examine the efficacy of Ptac2S treatment in MPM. We employed the ZL55 human mesothelioma cell line in vitro and in a murine xenograft model in vivo, to test the antitumor activity of Ptac2S. Cytotoxicity assays and Western blottings of different apoptosis and survival proteins were thus performed. Ptac2S increases MPM cell death in vitro and in vivo compared with cisplatin. Ptac2S was more efficacious than cisplatin also in inducing apoptosis characterized by: (a) mitochondria depolarization, (b) increase of bax expression and its cytosol-to-mitochondria translocation and decrease of Bcl-2 expression, (c) activation of caspase-7 and -9. Ptac2S activated full-length PKC-δ and generated a PKC-δ fragment. Full-length PKC-δ translocated to the nucleus and membrane, whilst PKC-δ fragment concentrated to mitochondria. Ptac2S was also responsible for the PKC-ε activation that provoked phosphorylation of p38. Both PKC-δ and PKC-ε inhibition (by PKC–siRNA) reduced the apoptotic death of ZL55 cells. Altogether, our results confirm that Ptac2S is a promising therapeutic agent for malignant mesothelioma, providing a solid starting point for its validation as a suitable candidate for further pharmacological testing.     

p8/nupr1 regulates DNA‐repair activity after double‐strand gamma irradiation‐induced DNA damage

The stress protein p8 is a small, highly basic, unfolded, and multifunctional protein. We have previously shown that most of its functions are exerted through interactions with other proteins, whose activities are thereby enhanced or repressed. In this work we describe another example of such mechanism, by which p8 binds and negatively regulates MSL1, a histone acetyl transferase (HAT)‐associated protein, which in turn binds the DNA‐damage‐associated 53BP1 protein to facilitate DNA repair following DNA γ‐irradiation. Contrary to the HAT‐associated activity, MSL1‐dependent DNA‐repair activity is almost completely dependent on 53BP1 expression. The picture that has emerged from our findings is that 53BP1 could be a scaffold that gets the HAT MSL1‐dependent DNA‐repair activity to the sites of DNA damage. Finally, we also found that, although p8 expression is transiently activated after γ‐irradiation, it is eventually submitted to sustained down‐regulation, presumably to allow development of MSL1‐associated DNA‐repair activity. We conclude that interaction of MSL1 with 53BP1 brings MSL1‐dependent HAT activity to the vicinity of damaged DNA. MSL1‐dependent HAT activity, which is negatively regulated by the stress protein p8, induces chromatin remodeling and relaxation allowing access to DNA of the repair machinery. J. Cell. Physiol. 221: 594–602, 2009. © 2009 Wiley‐Liss, Inc.     

SHP-2 regulates myogenesis by coupling to FAK signaling pathway

Transient dephosphorylation of FAK at Tyr-397 is required for cell cycle withdrawal early on during myogenesis. Here, we show that upon serum starvation of C2C12 myoblasts, FAK is transiently dephosphorylated in parallel with SHP-2 activation and association with FAK. SHP-2 knockdown by RNA interference suppressed the transient upregulation of SHP-2 and dephosphorylation of FAK during myogenesis. Furthermore, depletion of SHP-2 retarded the cell cycle withdrawal and the differentiation of serum-starved myoblasts into myotubes. These data provide a mechanistic basis for the reduction in FAK activity in differentiating myoblasts, indicating that myogenesis is critically triggered by FAK/SHP-2 complex.

Structured summary

MINT-7258938: Fak1 (uniprotkb:P34152) physically interacts (MI:0915) with shp2 (uniprotkb:P35235) by anti bait coimmunoprecipitation (MI:0006)     

A Novel Virus Causes Scale Drop Disease in Lates calcarifer

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.     

Drought‐induced changes in flow regimes lead to long‐term losses in mussel‐provided ecosystem services

Extreme hydro‐meteorological events such as droughts are becoming more frequent, intense, and persistent. This is particularly true in the south central USA, where rapidly growing urban areas are running out of water and human‐engineered water storage and management are leading to broad‐scale changes in flow regimes. The Kiamichi River in southeastern Oklahoma, USA, has high fish and freshwater mussel biodiversity. However, water from this rural river is desired by multiple urban areas and other entities. Freshwater mussels are large, long‐lived filter feeders that provide important ecosystem services. We ask how observed changes in mussel biomass and community composition resulting from drought‐induced changes in flow regimes might lead to changes in river ecosystem services. We sampled mussel communities in this river over a 20‐year period that included two severe droughts. We then used laboratory‐derived physiological rates and river‐wide estimates of species‐specific mussel biomass to estimate three aggregate ecosystem services provided by mussels over this time period: biofiltration, nutrient recycling (nitrogen and phosphorus), and nutrient storage (nitrogen, phosphorus, and carbon). Mussel populations declined over 60%, and declines were directly linked to drought‐induced changes in flow regimes. All ecosystem services declined over time and mirrored biomass losses. Mussel declines were exacerbated by human water management, which has increased the magnitude and frequency of hydrologic drought in downstream reaches of the river. Freshwater mussels are globally imperiled and declining around the world. Summed across multiple streams and rivers, mussel losses similar to those we document here could have considerable consequences for downstream water quality although lost biofiltration and nutrient retention. While we cannot control the frequency and severity of climatological droughts, water releases from reservoirs could be used to augment stream flows and prevent compounded anthropogenic stressors.     

Caenorhabditis elegans PTR/PTCHD PTR-18 promotes the clearance of extracellular hedgehog-related protein via endocytosis

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function.     

The process of thermodialysis in bioremediation of waters polluted by endocrine disruptors

Endocrine disruptors are chemicals able to induce adverse effects into wildlife and humans owing to their ability of interfering with the endocrine system. Bisphenol A (BPA) has been chosen as model of endocrine disruptors. To reduce the BPA pollution in waters we proposed the employment of the process of thermodialysis. Two different catalytic membranes have been prepared by covalently immobilizing laccase (from Trametes versicolor) by means of a diazotation process or tyrosinase (from mushroom) by condensation. The support was a nylon membrane. The bioremediation power of both catalytic membranes has been analysed under isothermal and non-isothermal conditions.The advantages in using non-isothermal bioreactors were discussed in terms of reduction of the bioremediation times.     

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.