Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

SHANK3 genetic polymorphism and susceptibility to ASD: evidence from molecular,in silico,and meta-analysis approaches

Molecular Biology Reports - The SHANK3 gene encodes a master synaptic scaffolding protein at the excitatory synapse’s postsynaptic density, which is predominantly responsible for constructing...     

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.     

Three- and four-repeat Tau coassemble into heterogeneous filaments: an implication for Alzheimer disease

Tau filaments are the pathological hallmark of numerous neurodegenerative diseases including Alzheimer disease, Pick disease, and progressive supranuclear palsy. In the adult human brain, six isoforms are expressed that differ by the presence or absence of the second of four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half of the isoforms have four repeats (4R tau). Tauopathies can be characterized based on the isoform composition of their filaments. Alzheimer disease filamentous inclusions contain all isoforms. Pick disease filaments contain 3R tau. Progressive supranuclear palsy filaments contain 4R tau. Here, we used site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy to obtain structural insights into these filaments. We find that filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of β-strands. This structure is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassemble into heterogeneous filaments. These filaments share the highly ordered core in the third repeat; however, they differ in their overall composition. Our findings indicate that at least three distinct types of filaments exist: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. These results suggest that individual filaments found in Alzheimer disease are structurally distinct from those in the 3R and 4R tauopathies.     

N-(2-hydroxy phenyl) acetamide produces profound inhibition of c-Fos protein and mRNA expression in the brain of adjuvant-induced arthritic rats

Chronic pain and cognitive decline are characteristic symptoms of rheumatoid arthritis. One of the immediate early gene c-fos is overexpressed during peripheral and central noxious conditions and can be used as a marker for neuronal activity/excitability. In the adjuvant-induced arthritis Sprague–Dawley rat model, we examined the dynamics of c-Fos protein and mRNA expression in the amygdala, cortex, hippocampus, and thalamus and evaluated the effects of N-(2-hydroxy phenyl) acetamide (NA-2), a derivative of salicylic acid. The paw volume was assessed as an indicator of peripheral edema and the hyperalgesia associated with arthritis was monitored by gait analysis. The region of interests of the brain from arthritic and non-arthritic animals were used to isolate the RNA and were then reverse transcribed into cDNA. The PCR products were electrophoresed on 1 % agarose gel and the gels were visualized in gel-doc system. The frozen brain sections were stained for c-Fos using immunohistochemistry. Negative control experiments were performed without the primary and secondary antibodies to rule out the nonspecific tissue binding of antibodies. We report a significant increase in the c-Fos expression in the arthritic control animals. In comparison to the control group, the treatment of NA-2 treatment was found to block the development of the arthritis-induced c-Fos protein and mRNA expression and peripheral edema. It also significantly reduces the gait deficits which were otherwise observed in the arthritic control group. Both the immunohistochemistry and PCR analysis revealed NA-2 to be more potent in comparison to member of non-steroidal anti-inflammatory drug.     

Erythropoietin effects on iron metabolism in rat bone marrow cells

This paper describes a study of the incorporation of ^{5 9}Fe from ^{5 9}Fe-labelled rat transferrin into rat bone marrow cells in culture. ^{5 9}Fe was found in both stroma and cytoplasm of marrow cells, and the cytoplasmic ^{5 9}Fe separated by polyacrylamide gel electrophoresis, into ferritin, haemoglobin and a low molecular weight fraction.The incorporation of ^{5 9}Fe into all three cytoplasmic fractions, but not into the stroma, increased progressively with time. Erythropoietin stimulated the increase of ^{5 9}Fe in ferritin within 1 h, the earliest time examined, and more than 3 h later in the stroma and haemoglobin.A proportion of the ⁵⁹Fe incorporated into the stroma and low molecular weight iron fractions during a 1 h incubation with ⁵⁹Fe-labelled transferrin was mobilised into ferritin and haemoglobin during a subsequent 4-h “cold-chase”. Erythropoietin, when present during the “cold-chase”, did not influence these ⁵⁹Fe fluxes. The erythropoietin stimulation of ⁵⁹Fe incorporation into ferritin, one of the earliest erythropoietin effects to be recorded, was therefore considered to be due to an increase of ⁵⁹Fe uptake by the hormone-responsive cells rather than a direct effect on ferritin synthesis.20-h cultures containing erythropoietin when incubated with ⁵⁹Fe-labelled transferrin for 4 h, showed dose-related erythropoietin stimulation of ⁵⁹Fe incorporation into haemoglobin only.In the presence of 10 mM isonicotinic acid hydrazide, ⁵⁹Fe incorporation into haemoglobin was inhibited, as in reticulocytes (Ponka, P. and Neuwirt, J. (1969) Blood 33, 690–707), while that into the stroma, ferritin and low molecular weight iron fractions, was stimulated; there were no reproducible effects of erythropoietin.     

Conditioned medium enhances the fusion capability of rat bone marrow mesenchymal stem cells and cardiomyocytes

Mesenchymal stem cells (MSCs) show accelerated regeneration potential when these cells experience hypoxic stress. This “preconditioning” has shown promising results with respect to cardio-protection as it stimulates endogenous mechanisms resulting in multiple cellular responses. The current study was carried out to analyze the effect of hypoxia on the expression of certain growth factors in rat MSCs and cardiomyocytes (CMs). Both cell types were cultured and assessed separately for their responsiveness to hypoxia by an optimized dose of 2,4,-dinitrophenol (DNP). These cells were allowed to propagate under normal condition for either 2 or 24 h and then analyzed for the expression of growth factors by RT-PCR. Variable patterns of expression were observed which indicate that their expression depends on the time of re-oxygenation and extent of hypoxia. To see whether the growth factors released during hypoxia affect the fusion of MSCs with CMs, we performed co-culture studies in normal and conditioned medium. The conditioned medium is defined as the medium in which CMs were grown for re-oxygenation till the specified time period of either 2 or 24 h after hypoxia induction. The results showed that the fusion efficiency of cells was increased when the conditioned medium was used as compared to that in the normal medium. This may be due to the presence of certain growth factors released by the cells under hypoxic condition that promote cell survival and enhance their fusion or regenerating ability. This study would serve as another attempt in designing a therapeutic strategy in which conditioned MSCs can be used for ischemic diseases and provide more specific therapy for cardiac regeneration.