Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon

Pseudoexons occur frequently in the human genome. This paper characterizes a pseudoexon in the GH receptor gene. Inappropriate activation of this pseudoexon causes Laron syndrome. Using in vitro splicing assays, pseudoexon silencing was shown to require a combination of a weak 5' pseudosplice-site and splicing silencing elements within the pseudoexon. Immunoprecipitation experiments showed that specific binding of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1) and U1 small nuclear ribonucleoprotein (snRNP) in the pre-spliceosomal complex was associated with silencing of pseudoexon splicing. The possible role of hnRNP E1 was further supported by RNA interference experiments in cultured cells. Immunoprecipitation experiments with three other pseudoexons suggested that pre-spliceosomal binding of U1 snRNP is a potential general mechanism of suppression of pseudoexons.     

Human urine is an excellent liquid waste for the culture of fish food organism, Moina micrura

With a view to converting human urine into bio-wealth in the form of zooplankton, the nutrient potentials of liquid wastes (0.11 mL L⁻¹)—(i) human urine (♂), (ii) cow urine, (iii) human–cow mixed urine or some solid wastes (0.11 g L⁻¹): (iv) vermi-compost, (v) cow dung, (vi) poultry droppings and (vii) mixed wastes (vermi-cow-poultry)—were evaluated for the mass culture of zooplankton Moina micrura in 24 outdoor tanks (4500 L) in triplicate treatments using life table as indicator during the period of October–December, 2005. Neonates of Moina micrura held in the treatment with human urine started reproduction at least 4 days earlier than other solid wastes tested. Total number of Moina micrura enumerated in the culture tank, related with offspring production per life span, was maximum in case of human urine treatment, followed by human–cow mixed urine, cow urine, vermin-compost, poultry droppings, mixed wastes (vermin–cow–poultry), cow dung and control treatments. The relationship between the total offspring production per female per life span and the nitrogen content of water in different treatments implied that human urine was an excellent liquid waste that can be used for the mass production of zooplankton Moina micrura required for larval and post larval rearing of commercial fishes.     

An experimental model of Actinobacillus suis infection in mice

Actinobacillus suis is an opportunistic pathogen of high health status swine and is associated with fatal septicemia, especially in neonatal pigs. A practical model of A. suis is unavailable currently. However, some evidence suggests that A. suis can infect nonporcine species. We therefore hypothesized that a mouse model of A. suis infection might be possible. To test this idea, we challenged CD1 mice with 3 strains of A. suis (2 porcine [SO4 and H91-0380] and 1 feline [96-2247]) by intranasal and intraperitoneal routes. We also evaluated the effects of coadministration of hemoglobin and immunosuppression by dexamethasone on the susceptibility of mice to A. suis infection. The feline and H91-0380 porcine strains induced clinical signs of acute disease and necrotizing pneumonia in mice similar to those seen in pigs. Although few bacteria were recovered, dissemination of A. suis was widespread. Generally, mice infected with the feline A. suis isolate had more severe clinical signs and higher bacterial titers than did mice infected with either of the porcine strains. Pretreatment of the mice with dexamethasone or addition of 2% porcine hemoglobin to the challenge inoculum appeared to hasten the onset of clinical signs by the porcine strains but had no significant effect on moribundity. These experiments demonstrate that mice can be infected with A. suis and subsequently develop pneumonia and bacteremia comparable to that seen in pigs, suggesting that mice may be used as a model for studying infection in swine.     

In silico mutational studies of Hsp70 disclose sites with distinct functional attributes

The Mutation‐Minimization Method (MuMi) to study the local response of proteins to point mutations has been introduced here. The heat shock protein Hsp70 as the test system since it displays features that have been studied in great detail has been used here. It has many conserved residues, serves several different functions on each of its domains, and displays interdomain allostery. For the analysis of spatial arrangement of residues within the protein, the network properties of the wild type (WT) protein as well as its all single alanine residue mutants using MuMi has been investigated. The measures to express the amount of change from the WT structure upon mutation and compare these deviations to find potential critical sites have been proposed. The functional significance of the potential sites to the parameter that uncovers them has been mapped. It was found that sites directly involved in binding were sensitive to mutations and were characterized by large displacements. On the other hand, sites that steer large conformational changes typically had increased reachability upon alanine mutations occurring elsewhere in the protein. Finally, residues that control communication within and between domains reside on the largest number of paths connecting pairs of residues in the protein. Proteins 2015; 83:2077–2090. © 2015 Wiley Periodicals, Inc.     

Vibrio cholerae MARTX toxin heterologous translocation of beta‐lactamase and roles of individual effector domains on cytoskeleton dynamics

The Vibrio cholerae MARTX_Vc toxin delivers three effector domains to eukaryotic cells. To study toxin delivery and function of individual domains, the rtxA gene was modified to encode toxin with an in‐frame beta‐lactamase (Bla) fusion. The hybrid RtxA::Bla toxin was Type I secreted from bacteria; and then Bla was translocated into eukaryotic cells and delivered by autoprocessing, demonstrating that the MARTX_Vc toxin is capable of heterologous protein transfer. Strains that produce hybrid RtxA::Bla toxins that carry one effector domain in addition to Bla were found to more efficiently translocate Bla. In cell biological assays, the actin cross‐linking domain (ACD) and Rho‐inactivation domain (RID) are found to cross‐link actin and inactivate RhoA, respectively, when other effector domains are absent, with toxin autoprocessing required for high efficiency. The previously unstudied alpha‐beta hydrolase domain (ABH) is shown here to activate CDC42, although the effect is ameliorated when RID is also present. Despite all effector domains acting on cytoskeleton assembly, the ACD was sufficient to rapidly inhibit macrophage phagocytosis. Both the ACD and RID independently disrupted polarized epithelial tight junction integrity. The sufficiency of ACD but strong selection for retention of RID and ABH suggests these two domains may primarily function by modulating cell signaling.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-Troglitazone-induced Apoptosis in Prostate Cancer Cells Involve AMP-activated Protein Kinase

Prostate cancer (PCa) is one of the most frequently diagnosed cancers in men with limited treatment options for the hormone-resistant forms. Development of novel therapeutic options is critically needed to target advanced forms. Here we demonstrate that combinatorial treatment with the thiazolidinedione troglitazone (TZD) and TNF-related apoptosis-inducing ligand (TRAIL) can induce significant apoptosis in various PCa cells independent of androgen receptor status. Because TZD is known to activate AMP-activated protein kinase (AMPK), we determined whether AMPK is a molecular target mediating this apoptotic cascade by utilizing PCa cell lines stably overexpressing AMPKα1 dominant negative (C4-2-DN) or empty vector (C4-2-EV). Our results indicated a significantly higher degree of apoptosis with TRAIL-TZD combination in C4-2-EV cells compared with C4-2-DN cells. Similarly, results from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a larger reduction of viability of C4-2-EV cells compared with C4-2-DN cells when treated with TRAIL-TZD, thus suggesting that C4-2-DN cells were more apoptosis-resistant. Additionally, siRNA-mediated knockdown of endogenous AMPKα1 expression showed a reduction of TRAIL-TZD-induced apoptosis, further confirming the participation of AMPK in mediating this apoptosis. Apoptosis induction by this combinatorial treatment was also associated with a cleavage of β-catenin that was inhibited in both C4-2-DN cells and those cells in which AMPKα1 was knocked down. In addition, time course studies showed an increase in pACC^S79 (AMPK target) levels coinciding with the time of apoptosis. These studies indicate the involvement of AMPK in TRAIL-TZD-mediated apoptosis and β-catenin cleavage and suggest the possibility of utilizing AMPK as a therapeutic target in apoptosis-resistant prostate cancer.