Epitope mapping of antibodies to alpha-synuclein in LRRK2 mutation carriers, idiopathic Parkinson disease patients, and healthy controls

Alpha-synuclein (Snca) plays a major role in Parkinson disease (PD). Circulating anti-Snca antibodies has been described in PD patients and healthy controls, but they have been poorly characterized. This study was designed to assess the prevalence of anti-Snca reactivity in human subjects carrying the LRRK2 mutation, idiopathic PD (iPD) patients, and healthy controls and to map the epitopes of the anti-Snca antibodies. Antibodies to Snca were detected by ELISA and immunoblotting using purified recombinant Snca in plasma from individuals carrying LRRK2 mutations (104), iPD patients (59), and healthy controls (83). Epitopes of antibodies were mapped using recombinant protein constructs comprising different regions of Snca. Clear positive anti-Snca reactivity showed no correlation with age, sex, years of evolution, or the disability scores for PD patients and anti-Snca reactivity was not prevalent in human patients with other neurological or autoimmune diseases. Thirteen of the positive individuals were carriers of LRRK2 mutations either non-manifesting (8 out 49 screened) or manifesting (5 positive out 55), three positive (out of 59) were iPD patients, and five positive (out of 83) were healthy controls. Epitope mapping showed that antibodies against the N-terminal (a.a. 1-60) or C-terminal (a.a. 109-140) regions of Snca predominate in LRRK2 mutation carriers and iPD patients, being N122 a critical amino acid for recognition by the anti-C-terminal directed antibodies. Anti-Snca circulating antibodies seem to cluster within families carrying the LRRK2 mutation indicating possible genetic or common environmental factors in the generation of anti-Snca antibodies. These results suggest that case-controls' studies are insufficient and further studies in family cohorts of patients and healthy controls should be undertaken, to progress in the understanding of the possible relationship of anti-Snca antibodies and PD patholog

Polimorfismo genético de citocinas na população do Rio de Janeiro

Citocinas são moléculas que controlam e modulam a atividade de numerosas células por se ligarem a seus receptores específicos. As diferenças observadas na produção de citocinas entre indivíduos podem ser, pelo menos em parte, explicadas pelos polimorfismos genéticos como o polimorfismo de um único nucleotídeo (SNP). Em 181 indivíduos saudáveis não-aparentados da cidade do Rio de Janeiro (região Sudeste - Brasil), nós analisamos os polimorfismos de citocinas em genes que codificam para Fator de Necrose Tumoral-alfa (TNF-a), Fator de Crescimento Transformante-beta (TGF-b), Interleucina-10, Interleucina-6 e Interferon-gama (IFN-g). Reação em cadeia da polimerase utilizando-se iniciadores sequencia-específicos foi realizada com auxílio do kit comercial CytGen (One Lambda Inc. Canoga Park, CA, USA). Ao todo, 8 polimorfismos foram analisados: TNF-a (-308G/A); TGF-b (códon 10C/T, códon 25C/G); IL-10 (-1082A/G, -819T/C, -592A/C); IL-6 (-174C/G) e IFN-g (+874T/A). Os dados observados foram comparados a três grupos de população de diferentes regiões do Brasil (São Paulo, Paraná e Bahia) e a três populações de outros continentes (Itália, Eslováquia e Negros Norte-Americanos). O teste qui-quadrado foi utilizado para as comparações. Nossa análise da população do Rio de Janeiro mostrou que os as freqüências alélicas em IL-10, IL-6 e IFN-g são desigualmente distribuídos entre Brancos, Mulatos e Negros (p<0,05). A comparação com populações de outras regiões do Brasil revelou que Rio de Janeiro e Bahia possuem freqüências alélicas e genotípicas de TGF-b (códon 25) estatisticamente diferentes (p=0,004 e p=0,002, respectivamente). Ainda, a freqüência alélica na população do Rio de Janeiro é significativamente diferente quando comparada à população da Itália [IL-6 (-174), p=0,0092; e IFN-g (+874) p=0,0418)]; Eslováquia [IL-10(-1082), p=0,006; IL-6(-174), p=0,0002; e IFN-g(+874), p=0,0335]; e Afro-Americanos [IL-10(-819), p=0,0446; IL-6(-174), p<0,0001; e IFN-g(+874), p<0,0001]. Adicionalmente, observamos que a diferença na distribuição dos haplótipos em IL-10 (-1082/-819/-592) na população do Rio de Janeiro em comparação com a da Itália (p=0,0293) e Afro-Americanos (p=0,0025) é significativa. Portanto, concluímos que os polimorfismos em IL-10, IL-6 e IFN-g estão distribuídos de acordo com a etnia na população do Rio de Janeiro. A população do Rio de Janeiro possui freqüências de polimorfismos diferentes das populações de Bahia, Itália, Eslováquia e Afro-Americanos, mas semelhantes à população de São Paulo/Paraná. Nossas observações poderão ser úteis para futuros estudos e associação entre polimorfismos genéticos de citocinas e doenças na população do Rio de Janeiro.

White spot syndrome virus (WSSV) transmission risk through infected cooked shrimp products assessed by polymerase chain reaction (PCR) and bio-inoculation studies

The aim of the study was to evaluate the resistance of white spot syndrome virus (WSSV) in shrimps (Penaeus monodon) to the process of cooking. The cooking was carried out at 1000C six different durations 5, 10, 15, 20, 25 and 30 min. The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). In the single step PCR, the primers 1s5 & 1a16 and IK1 & IK2 were used. While in the nested PCR, primers IK1 &IK2 – IK3 & IK4 were used for the detection of WSSV. WSSV was detected in the single step PCR with the primers 1s5 and 1a16 and the nested PCR with the primers IK1 and IK2 – IK3 & IK4 from the cooked shrimp samples. The cooked shrimps, which gave positive results for WSSV by PCR, were further confirmed for the viability of WSSV by conducting the bio-inoculation studies. Mortality (100%) was observed within 123 h of intra-muscular post injection (P.I) into the live healthy WSSV-free shrimps (P. monodon). These results show that the WSSV survive the cooking process and even infected cooked shrimp products may pose a transmission risk for WSSV to the native shrimp farming systems.

Growth of beta barium borate (beta-BaB2O4) thin films on alpha barium borate (alpha-BaB2O4) substrates

Crystalline beta-BBO thin films have been successfully prepared on (001)-oriented Sr2+-doped alpha-BBO substrates using liquid phase epitaxy and pulsed laser deposition techniques. The films were characterized by X-ray diffraction and X-ray rocking curve (XRC). The present results manifest that the beta-BBO thin films grown on Sr2+-doped alpha-BBO substrates have larger degree of orientation f-value and smaller XRC FWHM than the ones grown on other reported substrates. Compared with other substrates, alpha-BBO has the same UV cutoff and the similar structure to beta-BBO. These results reveal that alpha-BBO single crystal may be a promising substrate proper to the growth of beta-BBO films. (c) 2005 Elsevier B.V. All rights reserved.

Preparation of crystalline beta barium borate thin films on Sr2+-doped alpha barium borate substrates by liquid phase epitaxy

Thin films of beta barium borate have been prepared by liquid phase epitaxy on Si2+-doped alpha-BaB2O4 (alpha-BBO, the high temperature phase of barium berate) (001) and (110) substrates. The results of X-ray diffraction indicate that the films show highly (001) preferred orientation on (001)-oriented substrates while the films grown on (110) substrates are textured with (140) orientation. The crystallinity of these films was found to depend on growth temperature, rotation rate, dip time and orientation of substrate. Growth conditions were optimized to grow films with (001) orientation on (001) substrates reproducibly. The films show second harmonic generation of 400 nm light upon irradiation with 800 nm Ti: Sapphire femtosecond laser light. (c) 2005 Elsevier B.V. All rights reserved.

Fabrication of beta-BaB2O4 layers on (001) Sr2+-doped alpha-BaB2O4 by vapor transport equilibration (VTE)

Crystalline beta-BBO layers have been successfully prepared on (0 0 1)-oriented Sr2+-doped alpha-BBO substrates using vapor transport equilibration technique. The layers were characterized by X-ray diffraction, X-ray rocking curve and transmission spectra. The present results manifest that the VTE treatment time and powder ratio are important factors on the preparation of beta-BBO layers. beta-BBO layers with a highly (0 0 l) preferred orientation were obtained according to XRD profiles. The full width at half-maximum of the rocking curve for the layer is as low as about 1000 in., which shows the high crystallinity of the layer. These results reveal the possibility of fabricating beta-BBO (0 0 1) layers on (0 0 1)-oriented Sr2+-doped alpha-BBO substrates by VTE. (C) 2006 Elsevier Ltd. All rights reserved.

Edge-defined, film-fed growth (EFG) technique for the preparation of alpha-Al2O3 : C crystal with highly sensitive thermoluminescence response

In this work, alpha-Al2O3:C, a highly sensitive thermoluminescence dosimetry crystal, was grown by the EFG method in which a graphite heating unit and shield acted as the carbon source during the growth process. The optical, luminescent properties and dosimetric characteristics of the crystal were investigated. The as-grown crystal shows a single glow peak at 536 K, which is associated with Cr3+ ions. After annealing in H-2 at 1673 K for 80 h, the crystal shows a single glow peak at 460 K and a blue emission band at 415 nm. The thermoluminescent response of the annealed crystal shows linear-sublinear-saturation characteristics in the dose range from 5 x 10(-6) to 100 Gy.

Growth of highly sensitive thermoluminescent crystal alpha-Al2O3 : C by the temperature gradient technique

In this work, an alpha-Al2O3:C crystal with highly sensitive thermoluminescence was directly grown by the temperature gradient technique (TGT) using Al2O3 and graphite powders as raw materials. The optical and luminescent properties and the dosimetric characteristics of the crystal were investigated. An as-grown alpha-Al2O3:C crystal shows a single glow peak at 462 K and a blue emission peak at 415 nm. The thermoluminescence (TL) response of the crystal shows a linear-sublinear-saturation characteristic. In the dose range from 5 x 10(-6) to 10Gy, the alpha-Al2O3:C crystal shows excellent linearity, and saturation was observed at about 30Gy. The sensitivity of the crystal decreases as the heating rate increases. (c) 2008 Elsevier B.V. All rights reserved.

Influence of carbon on the thermoluminescence and optically stimulated luminescence of α -Al2O3:C crystals

alpha-Al2O3:C crystal shows excellent thermoluminescence (TL) and optically stimulated luminescence (OSL) properties but the real role carbon plays in this crystal is still not clearly understood so far. In this work, alpha-Al2O3:C crystal doping with different amounts of carbon were grown by the temperature gradient technique, and TL and OSL properties of as-grown crystals were investigated. Additionally, a mechanism was proposed to explain the role of carbon in forming the TL and OSL properties of alpha-Al2O3:C. TL and OSL intensities of as-grown crystals increase with the increasing amount of carbon doping in the crystal, but no shift is found in the glow peak location at 465 K. As the amount of carbon doping in the crystals decreases, OSL decay rate becomes faster. With the increase in heating rate, the integral TL response of as-grown crystals decreases and glow peak shifts to higher temperatures. TL response decrease rate increases with the increasing amount of carbon doping in the crystals. All the TL and OSL response curves of as-grown crystals show linear-sublinear-saturation characteristic, and OSL dose response exhibits higher sensitivity and wider linear dose range than that of TL. The crystal doping with 5000 ppm carbon shows the best dosimetric properties. Carbon plays the role of a dopant in alpha-Al2O3:C crystal and four-valent carbon anions replace the two-valent anions of oxygen during the crystal growth process, and large amounts of oxygen vacancies were formed, which corresponds to the high absorption coefficient of F and F+ centers in the crystals.

The separation of beta-BBO phase in alpha-BBO crystal by the irradiation of femtosecond laser

We observed and described some phenomena, which were that when a alpha-BBO crystal was irradiated by a focused femtosecond laser beam, the temperature effect happened in a minute area of focus, then the induced beta-BBO phase was separated within the minute area in the alpha-BBO crystal. (C) 2007 Elsevier B.V. All rights reserved.

Growth of alpha-Al2O3:C crystal with highly sensitive optically stimulated luminescence

In this work. an alpha-Al2O3:C crystal was directly grown by the temperature gradient technique (TGT) using Al2O3 and graphite powders as the raw materials. The optical, optically stimulated luminescence (OSL) properties and dosimetric characteristics of as-grown crystal were investigated. As-grown alpha-Al2O3:C crystal shows strong absorption band at 205, 230 and 256 nm. Three-dimensional thermoluminescence (TL) emission spectrum of the crystal shows a single emission peak at similar to 415 nm. The OSL decay curve can be fitted to two exponentials, the faster component and the slower component. The OSL response of the crystal shows a linear-sublinear-saturation characteristic. As-grown alpha-Al2O3:C crystal shows excellent linearity in the dose range from 5 x 10(-6) to 50 Gy. For doses higher than the saturation dose (100 Gy). the OSL sensitivity decreases as the dose increases. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved.

Differential activity of GSK-3 isoforms regulates NF-kappa B and TRAIL- or TNF alpha induced apoptosis in pancreatic cancer cells

While TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in neoplastic cells, many tumors, including pancreatic ductal adenocarcinoma (PDA), display intrinsic resistance, highlighting the need for TRAIL-sensitizing agents. Here we report that TRAIL-induced apoptosis in PDA cell lines is enhanced by pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) or by shRNA-mediated depletion of either GSK-3 alpha or GSK-3 beta. In contrast, depletion of GSK-3 beta, but not GSK-3 alpha, sensitized PDA cell lines to TNF alpha-induced cell death. Further experiments demonstrated that TNF alpha-stimulated I kappa B alpha phosphorylation and degradation as well as p65 nuclear translocation were normal in GSK-3 beta-deficient MEFs. Nonetheless, inhibition of GSK-3 beta function in MEFs or PDA cell lines impaired the expression of the NF-kappa B target genes Bcl-xL and cIAP2, but not I kappa B alpha. Significantly, the expression of Bcl-xL and cIAP2 could be reestablished by expression of GSK-3 beta targeted to the nucleus but not GSK-3 beta targeted to the cytoplasm, suggesting that GSK-3 beta regulates NF-kappa B function within the nucleus. Consistent with this notion, chromatin immunoprecipitation demonstrated that GSK-3 inhibition resulted in either decreased p65 binding to the promoter of BIR3, which encodes cIAP2, or increased p50 binding as well as recruitment of SIRT1 and HDAC3 to the promoter of BCL2L1, which encodes Bcl-xL. Importantly, depletion of Bcl-xL but not cIAP2, mimicked the sensitizing effect of GSK-3 inhibition on TRAIL-induced apoptosis, whereas Bcl-xL overexpression ameliorated the sensitization by GSK-3 inhibition. These results not only suggest that GSK-3 beta overexpression and nuclear localization contribute to TNF alpha and TRAIL resistance via anti-apoptotic NF-kappa B genes such as Bcl-xL, but also provide a rationale for further exploration of GSK-3 inhibitors combined with TRAIL for the treatment of PDA.