No abstract

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV)has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) ␣ proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V max of 14,925 units/ mg⅐min and a K m of 18.88 M. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mM orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/ threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mM sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V max of 28,600 units/mg⅐min and a K m of 76 M. Mutagenesis of residue Cys 95 to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

Abstract-Proteins that bind to the intracellular expanses, particularly cytoplasmic tail regions, of heptahelical integral membrane receptors are of particular interest in that they can mediate or modulate trafficking or intracellular signaling.In an effort to distinguish new proteins that might promote angiotensin II type 1 (AT 1 ) receptor intracellular events, we screened a yeast 2-hybrid mouse brain library with the rat AT 1A receptor (AT 1 R) carboxyl terminus and identified GABARAP, a protein involved in intracellular trafficking of the GABA A receptor, as a binding partner for the AT 1 R. Interaction of GABARAP with the AT 1 R carboxyl terminus was further substantiated using GST pull-down assays, and binding of the full-length tagged AT 1 R to GABARAP was verified using coimmunoprecipitation. Bioluminescence resonance energy transfer assays further confirmed specific interaction of GABARAP with AT 1 R. Moreover, GABARAP clearly increased the steady-state level of plasma membrane-associated AT 1 R in PC-12 cells. Cotransfection of GABARAP with an AT 1 R fluorescent fusion protein increased PC-12 cell surface expression of the AT 1 R more than 6-fold when standardized to the level of intracellular expression. Furthermore, GABARAP overexpression in CHO-K1 cells engineered to express AT 1 R increased angiotensin II binding sites 3.7-fold and angiotensin II-induced phospho-extracellular signal-regulated kinase 1/2 and cellular proliferation significantly over levels obtained with AT 1 R overexpression alone. In addition, small interfering RNA-mediated knockdown of GABARAP reduced the steady-state levels of the AT 1 R fluorescent fusion protein by 43% and its cell surface expression by 84%. Immunoblot analyses confirmed the quantitative image data. We conclude that GABARAP binds to and promotes trafficking of the AT 1 R to the plasma membrane. receptors are 7-transmembrane G protein-coupled receptors (GPCRs) of the largest GPCR subfamily, family 1, or the rhodopsin-like family. The GPCR superfamily has more than 860 members 1 and more than 50 "GPCR-associated" proteins have now been discovered, the majority of which interact with GPCR cytoplasmic carboxyl termini. 2 Most of these are involved in trafficking, subcellular targeting, and intracellular signaling. Our preliminary studies were designed to identify proteins that bind to the cytoplasmic carboxyl terminus of the AT 1 receptor (AT 1 R), the most prevalent and best characterized of the Ang receptors. Such proteins are expected to be involved in trafficking of the AT 1 R through the secretory pathway and to the plasma membrane, as well as in ligand-mediated internalization and recycling. Moreover, our recent published studies suggest that the AT 1 R is cleaved in a ligand-dependent manner to liberate the cytoplasmic domain, a significant quantity of which traffics to the nucleus. 3 Presumably, this nuclear trafficking event also involves sequence-specific binding proteins. Using a yeast 2-hybrid (Y2H) approach to screen a mouse brain library, we have ide...

Background Rapid-response extracorporeal membrane oxygenation (RR-ECMO) has been implemented at select centers to expedite cannulation for patients placed on ECMO during cardiopulmonary resuscitation (ECPR). In 2008, we established such a program and used it for all pediatric veno-arterial ECMO initiations. This study was designed to compare outcomes before and after program implementation. Methods Between 2003 and 2011, 144 pediatric patients were placed on veno-arterial ECMO. Records of patients placed on ECMO before (17 ECPR and 62 non-ECPR) or after (14 ECPR and 51 non-ECPR) RR-ECMO program implementation were retrospectively compared. Results The peak performance of the ECMO team was assessed by measuring ECMO initiation times for the ECPR patient subgroup (n=31). There was a shift towards more ECPR initiations achieved in under 40 minutes (24% pre-RR-ECMO vs. 43% RR-ECMO, P=0.25) and fewer requiring more than 60 minutes (47% pre-RR-ECMO vs. 21% RR-ECMO, P=0.14) after program implementation, although these changes did not reach statistical significance. After multivariable risk-adjustment, RR-ECMO was associated with a 52% reduction in neurologic complications for all patients (adjusted odds ratio, 0.48; confidence interval, 0.23–0.98; P=0.04), but the risk of in-hospital death remained unchanged (adjusted odds ratio, 0.99; confidence interval, 0.50–1.99; P=0.99). Conclusion Implementation of a pediatric RR-ECMO program for veno-arterial ECMO initiation was associated with reduced neurologic complications but not improved survival during the first three years of program implementation. These data suggest that development of a coordinated system for rapid ECMO deployment may benefit both ECPR and non-ECPR patients, but further efforts are required to improve survival.

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ABSTRACT:Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K m for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ ml) and hBVR (0.025-0.05 M), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment.

SUMMARYThymocyte responses to functional activation are of relevance to the evaluation of the ef®cacy of in ovo immunotherapies and vaccines in chickens. In this study we have demonstrated differences in chicken thymocyte responses according to developmental age. RNA samples from stimulated and unstimulated chicken thymocytes were assayed for messenger RNA encoding the cytokines interleukin-1b (IL-1b), IL-2, interferon-a (IFN-a), IFN-b, IFN-g and transforming growth factor-b 4 (TGF-b 4 ), and also components of the major histocompatibility complex (MHC), b 2 -microglobulin (b 2 M) and the MHC class I a-chain (MHC IA). At embryonic day 14 thymocytes were least responsive to functional activation and differences existed even between thymocyte populations at embryonic day 18 and day 1 post-hatch. The duration of proliferation in response to stimulation was found to increase with increasing embryonic age. Mitogen stimulation of embryonic day 18 and day 1 post-hatch thymocytes induced up-regulation of IFN-g, IL-1b and TGF-b transcripts, and down-regulation of IFN-a, IFN-b and IL-2 transcripts, with a higher induction of IFN-g, IL-1b and TGF-b transcripts in more immature T-cell-receptor-negative (TCR À ) than TCR (TCR1 , TCR2 , or TCR3 ) subsets. In contrast, in the mouse and human, both mature and immature thymocytes respond to mitogen stimulation with up-regulation of IL-2. Thymocytes from embryonic day 14 chicks responded to mitogen with a short burst of unsustained proliferation, and transcriptional down-regulation of the cytokines IL-2, IL-1b, IFN-a, IFN-b and IFN-g. These results suggest that embryonic day 14 thymocytes are largely unresponsive to mitogen. Transcripts encoding TGF-b and type I interferons (IFN-a and IFN-b) were constitutively expressed at high levels in very early thymocytes at embryonic day 14. Thymocytes at embryonic days 14 and 18 and day 1 post-hatch responded to mitogen stimulation with up-regulation of MHC IA transcript. The pattern of b 2 M transcription following mitogen stimulation was distinct from that of the globally up-regulated MHC IA transcript, with up-regulation of b 2 M transcription observed at embryonic day 18 and day 1 post-hatch but not at embryonic day 14. In thymocyte subsets, up-regulation of b 2 M transcription was found to be speci®c to the CD8 TCR population. The balance of responses in the embryonic thymus suggests that at all stages thymocytes have a reduced capacity for activation in comparison to mature thymocyte populations.

Chicken anemia virus (CAV) is an immunosuppressive pathogen of chickens. To further examine the role of viral protein 2 (VP2), which possesses dual-specificity protein phosphatase (DSP) activity, in viral cytopathogenicity and its influence on viral growth and virulence, an infectious genomic clone of CAV was subjected to site-directed mutagenesis. Substitution mutations C87R, R101G, K102D and H103Y were introduced into the DSP catalytic motif and R129G, Q131P, R/K/K150/151/152G/A/A, D/E161/162G/G, L163P, D169G and E186G into a region predicted to have a high degree of secondary structure. All mutant constructs were infectious, but their growth curves differed. The growth curve for mutant virus R/K/K150/151/152G/A/A was similar to that for wild-type virus, a second cluster of mutant viruses had an extended latent period and a third cluster of mutant viruses had extended latent and eclipse periods. All mutants had a reduced cytopathogenic effect in infected cells and VP3 was restricted to the cytoplasm. Mutation of the second basic residue (K102D) in the atypical DSP signature motif resulted in a marked reduction in virus replication efficiency, whereas mutation of the first basic residue (R101G) attenuated cytopathogenicity, but did not reduce replication efficiency. Expression of major histocompatibility complex (MHC) class I was markedly downregulated in cells infected with wild-type CAV, but not in those infected with mutants. This study further demonstrates the significance of VP2 in CAV replication and shows that specific mutations introduced into the gene encoding this protein can reduce virus replication, cytopathogenicity and downregulation of MHC I in infected cells.