PKCγ-Mediated Phosphorylation of CRMP2 Regulates Dendritic Outgrowth in Cerebellar Purkinje Cells.

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.

Exchange of extracellular domains of CCR1 and CCR5 reveals confined functions in CCL5-mediated cell recruitment.

The chemokine CCL5 recruits monocytes into inflamed tissues by triggering primarily CCR1-mediated arrest on endothelial cells, whereas subsequent spreading is dominated by CCR5. The CCL5-induced arrest can be enhanced by heteromer formation with CXCL4. To identify mechanisms for receptor-specific functions, we employed CCL5 mutants and transfectants expressing receptor chimeras carrying transposed extracellular regions. Mutation of the basic 50s cluster of CCL5, a coordinative site for CCL5 surface presentation, reduced CCR5- but not CCR1-mediated arrest and transmigration. Impaired arrest was restored by exchanging the CCR5-N-terminus for that of CCR1, which supported arrest even without the 50s cluster, whereas mutation of the basic 40s cluster essential for proteoglycan binding of CCL5 could not be rescued. The enhancement of CCL5-induced arrest by CXCL4 was mediated by CCR1 requiring its third extracellular loop. The domain exchanges did not affect formation and co-localisation of receptor dimers, indicating a sensing role of the third extracellular loop for hetero-oligomers in an arrest microenvironment. Our data identify confined targetable regions of CCR1 specialised to facilitate CCL5-induced arrest and enhanced responsiveness to the CXCL4-CCL5 heteromer.

Familial amyotrophic lateral sclerosis is associated with a mutation in D-amino acid oxidase.

We report a unique mutation in the D-amino acid oxidase gene (R199W DAO) associated with classical adult onset familial amyotrophic lateral sclerosis (FALS) in a three generational FALS kindred, after candidate gene screening in a 14.52 cM region on chromosome 12q22-23 linked to disease. Neuronal cell lines expressing R199W DAO showed decreased viability and increased ubiquitinated aggregates compared with cells expressing the wild-type protein. Similarly, lentiviral-mediated expression of R199W DAO in primary motor neuron cultures caused increased TUNEL labeling. This effect was also observed when motor neurons were cocultured on transduced astrocytes expressing R199W, indicating that the motor neuron cell death induced by this mutation is mediated by both cell autonomous and noncell autonomous processes. DAO controls the level of D-serine, which accumulates in the spinal cord in cases of sporadic ALS and in a mouse model of ALS, indicating that this abnormality may represent a fundamental component of ALS pathogenesis.

Large deletions and point mutations involving the dedicator of cytokinesis 8 (DOCK8) in the autosomal-recessive form of hyper-IgE syndrome.

BACKGROUND: The genetic etiologies of the hyper-IgE syndromes are diverse. Approximately 60% to 70% of patients with hyper-IgE syndrome have dominant mutations in STAT3, and a single patient was reported to have a homozygous TYK2 mutation. In the remaining patients with hyper-IgE syndrome, the genetic etiology has not yet been identified. OBJECTIVES: We aimed to identify a gene that is mutated or deleted in autosomal recessive hyper-IgE syndrome. METHODS: We performed genome-wide single nucleotide polymorphism analysis for 9 patients with autosomal-recessive hyper-IgE syndrome to locate copy number variations and homozygous haplotypes. Homozygosity mapping was performed with 12 patients from 7 additional families. The candidate gene was analyzed by genomic and cDNA sequencing to identify causative alleles in a total of 27 patients with autosomal-recessive hyper-IgE syndrome. RESULTS: Subtelomeric biallelic microdeletions were identified in 5 patients at the terminus of chromosome 9p. In all 5 patients, the deleted interval involved dedicator of cytokinesis 8 (DOCK8), encoding a protein implicated in the regulation of the actin cytoskeleton. Sequencing of patients without large deletions revealed 16 patients from 9 unrelated families with distinct homozygous mutations in DOCK8 causing premature termination, frameshift, splice site disruption, and single exon deletions and microdeletions. DOCK8 deficiency was associated with impaired activation of CD4+ and CD8+T cells. CONCLUSION: Autosomal-recessive mutations in DOCK8 are responsible for many, although not all, cases of autosomal-recessive hyper-IgE syndrome. DOCK8 disruption is associated with a phenotype of severe cellular immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective T-cell activation and T(h)17 cell differentiation, and impaired eosinophil homeostasis and dysregulation of IgE.

Avaliação microbiológica de alimentos isentos de registrono Ministério da Saúde / Microbiological analysis of foods exempt of legal registration procedures

O objetivo deste trabalho foi avaliar o padrão higiênico-sanitário de alimentos comercializados na Regiãode Londrina cadastrados no Ministério da Saúde, porém, dispensados de registro, conforme a RDC nº.23 da Agência Nacional de Vigilância Sanitária (ANVISA). Os alimentos foram escolhidos de acordo comas sugestões da Vigilância Sanitária de Londrina, PR, que levou em consideração o risco epidemiológico.Os padrões microbiológicos foram avaliados conforme a RDC nº. 12 da ANVISA. Contagens de E. colie S. aureus acima do permitido foram observadas respectivamente em 11 (18,3%) e 13 (21,6%) dos 60alimentos analisados. Os demais resultados microbiológicos estavam dentro do estabelecido pela RDCnº. 12. Ficou clara a necessidade de maior fiscalização do processamento e armazenagem de váriosalimentos analisados, em especial, sorvetes, massas recheadas, pães de queijo, lasanha, doces comcreme e salgados recheados. Esses resultados serão encaminhados às Vigilâncias Regional e Municipalalertando sobre a real potencialidade desses alimentos veicularem doenças e na intenção de abrir umadiscussão sobre a liberação ou não de registro desses alimentos junto ao Ministério da Saúde. Alémdisso, os resultados obtidos confirmam que a análise microbiológica do produto final é um instrumentoessencial de validação e verificação das Boas Práticas de Fabricação (BPF) e da Análise de Perigos ePontos Críticos de Controle (APPCC).

The aim of this study was to analyze the sanitary quality of foods commercialized in Londrina, Paraná that are exempt of Brazilian Health Ministry registration procedures, according to Resolution n°. 23 from the National Sanitary Administration Agency (ANVISA). The food categories analyzed were chosenaccording to ANVISA, Londrina, PR which took the epidemiological risk in account. Microbiologicalstandards were evaluated according to ANVISA Resolution n°. 12. Escherichia coli and coagulase positive Staphylococcus counts were above standards in 11 (18.3%) and 13 (21.6%) of food samples analyzed, respectively. The others microbiological results were in conformity to ANVISA Resolution n°.12. These results indicate inadequate sanitary conditions during food processing especially for ice-cream, stuffed pasta, "pães de queijo", lasanha, cream felled pastries, and "salgado recheado". The results could help the State and Local Sanitary Administration Agencies to evaluate the potentiality of these foods cause food-borne diseases and the adequacy of their liberation from Brazilian Health Ministry registration procedures. The results also show that microbiological analysis of the final product is a valuable validation and verification procedure for the GMP and HACCP system.

Gadolinium-based contrast media compared with iodinated media for digital subtraction angiography in azotaemic patients.

BACKGROUND: To determine whether gadolinium-based contrast media (CM) could be used safely for angiographies in patients with renal dysfunction we investigated renal function after gadobutrol exposure and compared the results with standard iodinated CM (iohexol) in a randomized clinical study. METHODS: Twenty-one patients (aged 67+/-11 years, nine female and 12 male) with severely impaired renal function [mean serum creatinine 3.2+/-1.3 mg/dl, mean glomerular filtration rate (GFR) 31+/-16 ml/min/1.73 m(2)] who needed to have angiography because of severe peripheral vascular disease, renal artery stenosis or aortic aneurysms were randomized to receive in a blinded manner either gadobutrol (Gadovist 1.0 mmol/ml) or iohexol (Omnipaque 350) as contrast agents. GFR was measured by CM clearance (Renalyzer) at baseline and 48 h after CM administration. The primary end point was the mean change of GFR from baseline at 48 h, the secondary one the incidence of CM-induced acute renal failure, defined as a decrease in GFR of >50% from baseline within 48 h of CM administration. RESULTS: In the gadobutrol group (n = 10) we observed a statistically significant decrease in GFR of 10.6+/-13.8 ml/min/1.73 m(2) within 48 h after CM administration (P<0.05, paired t test). The incidence of CM-induced ARF amounted to 50%. In comparison, the iohexol group (n = 11) also showed a statistically significant GFR reduction of 8.7+/-8.8 ml/min/1.73 m(2) (P<0.05, paired t test), and of ARF by 45%. The percentile of differences of GFR decreases between the two groups was not significant (P = 0.70). No patient demonstrated other adverse effects of gadobutrol or iohexol administration, apart from GFR reduction. Despite the decline in GFR, no patient required haemodialysis in the 10 following days. CONCLUSIONS: In our study, gadolinium-based angiography showed no benefit over iohexol angiography with respect to preventing GFR reduction in patients with severely impaired renal function.