The Differential Response of Protein Kinase A to Cyclic AMP in Discrete Brain Areas Correlates with the Abundance of Regulatory Subunit II

Abstract: We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIα, RIIα, and RIIβ) and of 150-kDa RIIβ-anchor proteins for cyclic AMP (cAMP)-dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIβ paralleled the presence of specific RII-anchoring proteins, high levels of membrane-bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C-PKA). Conversely, in brain areas showing low RIIβ levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII-anchoring proteins, low levels of membrane-bound holoenzyme PKA, and high levels of cytosolic dissociated C-PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP-induced dissociation, without, however, a consistent translocation of C-PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C-PKA and phosphorylation of cAMP-responsive element binding protein. Taken together, these data suggest that RIIβ (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP-induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.     

Inflammatory process of CD8+ CD28- T cells in induced sputum from asthmatic patients

Previously unreported CD8(+) CD28(-) and CD8(+) CD28(+) T-cell subsets occur in healthy individuals and expand in patients suffering from autoimmune disease. Here we studied, for the first time, the expression of CD8(+) CD28(+) , CD8(+) CD28(-) , and CD8(+) CD56(+) subpopulations in induced sputum from asthmatics. Using sputum samples, purified CD8(+) T cells were stained for surface antigen CD28, CD56, FITC-conjugated anti-perforin, and anti-IFN-gamma. Cytotoxic activity was evaluated in a chromium releasing test. Induced sputum CD8(+) CD28(-) T cells were found to be more expanded and expressed low levels of IFN-gamma in severe asthmatics than mild asthma and age-matched healthy controls. The predominance of CD8(+) CD28(-) T cells can be in part explained by the expansion of CD8(+) CD56(+). CD8(+) CD28(-) T cells from severe asthmatics produced high intracytoplasmic perforin and exerted a potent cytotoxic activity. Considering their phenotyping and functional properties, the CD8(+) CD28(-) T-cell subset may constitute an intermediate phenotype in the process of CD8(+) T-cell differentiation of effector-type cells in severe asthmatics. Functional studies showed that CD8(+) CD28(-) T cells had cytotoxic function.     

Challenging popular tools for the annotation of genetic variations with a real case,pathogenic mutations of lysosomal alpha-galactosidase

Background

Severity gradation of missense mutations is a big challenge for exome annotation. Predictors of deleteriousness that are most frequently used to filter variants found by next generation sequencing, produce qualitative predictions, but also numerical scores. It has never been tested if these scores correlate with disease severity.

Results

wANNOVAR, a popular tool that can generate several different types of deleteriousness-prediction scores, was tested on Fabry disease. This pathology, which is caused by a deficit of lysosomal alpha-galactosidase, has a very large genotypic and phenotypic spectrum and offers the possibility of associating a quantitative measure of the damage caused by mutations to the functioning of the enzyme in the cells. Some predictors, and in particular VEST3 and PolyPhen2 provide scores that correlate with the severity of lysosomal alpha-galactosidase mutations in a statistically significant way.

Conclusions

Sorting disease mutations by severity is possible and offers advantages over binary classification. Dataset for testing and training in silico predictors can be obtained by transient transfection and evaluation of residual activity of mutants in cell extracts. This approach consents to quantitative data for severe, mild and non pathological variants.

     

THE rhGM-CSF–EPO HYBRID PROTEIN MEN 11300 INDUCES ANTI-EPO ANTIBODIES AND SEVERE ANAEMIA IN RHESUS MONKEYS

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3–4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.     

Deletion polymorphism in the gene for angiotensin converting enzyme is associated with elevated fasting blood glucose levels

The frequency and distribution of angiotensin converting enzyme insertion/deletion (ACE I/D) polymorphism, and its association with other known risk factors for coronary atherosclerosis, has been studied, in a normal south Italian population. Subjects homozygous for deletion showed elevated fasting blood glucose levels when compared with subjects homozygous for insertion. The difference was consistent with an increased number of type 2 diabetics among the former group of subjects.Recipient of a post-doctoral fellowship from the Associazione Italiana Ricerca sul Cancro (A.I.R.C.)     

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile α-motif (SAM) domain (residues 505–579), a region crucial to explaining the human genetic disease ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of free-dom of individual residues and discussion of the possible causes for the pathology.