Target recognition by calmodulin

Calmodulin (CaM) inhibits mammalian phosphofructokinase (PFK) in a calcium dependent manner; however the putative CaM binding sequence is not homologous to those of other well characterized systems. This work addresses the structural and functional properties of complexes of CaM with selected target peptides from PFK using calcium binding and optical spectroscopic methods. Wild-type CaM, site-directed mutants of CaM, and cleaved domains of CaM were studied in complex with PFK target peptides of different length and amino-acid composition. Comparison of equilibria and structural data from these complexes allowed definition of the sequence involved in the interaction and conformation of the complexes. The effect of binding different peptides on the calcium affinity of CaM has been determined by an indicator method. The presence of the PFK target peptide apparently causes a 200 fold increase in the affinity of the first two stoichiometric binding constants (K1 & K2), however there is little effect on K3 & K4. Interestingly the change in far-UV, near-UV CD. and fluorescence spectroscopic signals generated by the interaction of CaM with the target sequences are essentially complete on addition of two molar equivalents of calcium to 1 molar equivalent of CaM:PFK peptide complex, consistent with a differential function of the two domains. The dissociation rates of calcium from CaM:PFK peptide complexes has been determined in stopped-flow experiments using the calcium chelators EGTA and Quin-2. Calcium dissociation kinetics are biphasic, with 2 sites having higher affinity. Enzyme activity studies show PFK inactivation to be CaM concentration dependent and the addition of PFK target peptide reverses the inhibition. A mechanism for the calcium dependent interaction of CaM with the PFK target sequence is presented. In comparison to the three closely related known CaM:target peptide structures the interaction of CaM with the PFK target sequence apparently results in a complex with a conformation of lower peptide helicity and with significantly less calcium dependence of interactions. Hence the CaM:PFK complex appears to define a new type of CaM interaction, distinctively different in structure from that currently considered for the binding of CaM in apo- or holo-form to sequences from other target enzymes. This novel interaction has also been addressed by crystallographic methods and details of optimal crystallization conditions for the CaM:PFK peptide complex are described

Ruddlesden-Popper phase materials for solid oxide fuel cell cathodes: A short review

In the last couple of decades, researchers have been working on Ruddlesden-Popper phases to realise them as components of solid oxide cells. Ruddlesden-Popper phase materials have been particularly proposed as materials for intermediate temperature solid oxide fuel cells (IT-SOFCs). As such a sizeable literature was produced on Ruddlesden-Popper phases and in this short review we look at these studies with a particular focus on the structural chemistry, oxygen transport and electrical conductivity, electrochemical performance, and stability of these materials under operating conditions. More specifically, the materials have been studied for cathodes and, therefore, we believe a review dedicated to cathode applications of these materials will be beneficial for the community. A brief outlook on the future directions in the field will also be provided

Concomitant Carcinoma in situ in Cystectomy Specimens Is Not Associated with Clinical Outcomes after Surgery

Objective: The aim of this study was to externally validate the prognostic value of concomitant urothelial carcinoma in situ (CIS) in radical cystectomy (RC) specimens using a large international cohort of bladder cancer patients. Methods: The records of 3,973 patients treated with RC and bilateral lymphadenectomy for urothelial carcinoma of the bladder (UCB) at nine centers worldwide were reviewed. Surgical specimens were evaluated by a genitourinary pathologist at each center. Uni- and multivariable Cox regression models addressed time to recurrence and cancer-specific mortality after RC. Results: 1,741 (43.8%) patients had concomitant CIS in their RC specimens. Concomitant CIS was more common in organ-confined UCB and was associated with lymphovascular invasion (p < 0.001). Concomitant CIS was not associated with either disease recurrence or cancer-specific death regardless of pathologic stage. The presence of concomitant CIS did not improve the predictive accuracy of standard predictors for either disease recurrence or cancer-specific death in any of the subgroups. Conclusions: We could not confirm the prognostic value of concomitant CIS in RC specimens. This, together with the discrepancy between pathologists in determining the presence of concomitant CIS at the morphologic level, limits the clinical utility of concomitant CIS in RC specimens for clinical decision-making. Copyright (C) 2011 S. Karger AG, Base

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus' Lateral Body Protein

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection

Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses

Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, coexpressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5-and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9-and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15-vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens