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1.
Clin Exp Med ; 23(4): 1251-1263, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36459278

RESUMO

Microbial secondary infections can contribute to an increase in the risk of mortality in COVID-19 patients, particularly in case of severe diseases. In this study, we collected and evaluated the clinical, laboratory and microbiological data of COVID-19 critical ill patients requiring intensive care (ICU) to evaluate the significance and the prognostic value of these parameters. One hundred seventy-eight ICU patients with severe COVID-19, hospitalized at the S. Francesco Hospital of Nuoro (Italy) in the period from March 2020 to May 2021, were enrolled in this study. Clinical data and microbiological results were collected. Blood chemistry parameters, relative to three different time points, were analyzed through multivariate and univariate statistical approaches. Seventy-four percent of the ICU COVID-19 patients had a negative outcome, while 26% had a favorable prognosis. A correlation between the laboratory parameters and days of hospitalization of the patients was observed with significant differences between the two groups. Moreover, Staphylococcus aureus, Enterococcus faecalis, Candida spp, Pseudomonas aeruginosa and Klebsiella pneumoniae were the most frequently isolated microorganisms from all clinical specimens. Secondary infections play an important role in the clinical outcome. The analysis of the blood chemistry tests was found useful in monitoring the progression of COVID-19.


Assuntos
COVID-19 , Coinfecção , Humanos , SARS-CoV-2 , Pandemias , Unidades de Terapia Intensiva
3.
Future Microbiol ; 16: 741-751, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34082567

RESUMO

The value of blood cultures for confirming the clinical diagnosis of sepsis is suboptimal. There is growing interest in the potential of real-time PCR technology by detection of minute amounts of pathogen DNA in patient blood samples with results available within 4-6 h. Adopting a two-step approach, we evaluated the compliance of two versions of the MicrobScan assay on a total of 748 patients with suspected bloodstream infections. The results obtained with a second version of the MicrobScan assay are characterized by increased specificity (from 95.1 to 98.2%) and sensitivity (from 76.7 to 85.1), increased throughput and the possibility of simultaneously testing different kinds of samples collected from the potential sites of infection and utilizing different syndromic panels.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real , Sepse , Humanos , Sepse/diagnóstico
4.
Cells ; 10(2)2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498474

RESUMO

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking, likely by Rab phosphorylation, that in turn may regulate different aspects of neuronal physiology. Here we show that LRRK2 interacts with Sec8, one of eight subunits of the exocyst complex. The exocyst complex is an evolutionarily conserved multisubunit protein complex mainly involved in tethering secretory vesicles to the plasma membrane and implicated in the regulation of multiple biological processes modulated by vesicle trafficking. Interestingly, Rabs and exocyst complex belong to the same protein network. Our experimental evidence indicates that LRRK2 kinase activity or the presence of the LRRK2 kinase domain regulate the assembly of exocyst subunits and that the over-expression of Sec8 significantly rescues the LRRK2 G2019S mutant pathological effect. Our findings strongly suggest an interesting molecular mechanism by which LRRK2 could modulate vesicle trafficking and may have important implications to decode the complex role that LRRK2 plays in neuronal physiology.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos Knockout , Células PC12 , Ligação Proteica , Ratos
5.
Front Microbiol ; 12: 827799, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35095827

RESUMO

BACKGROUND: The SARS-CoV-2 pandemic stimulated an outstanding global sequencing effort, which allowed to monitor viral circulation and evolution. Nuoro province (Sardinia, Italy), characterized by a relatively isolated geographical location and a low population density, was severely hit and displayed a high incidence of infection. METHODS: Amplicon approach Next Generation Sequencing and subsequent variant calling in 92 respiratory samples from SARS-CoV-2 infected patients involved in infection clusters from March 2020 to May 2021. RESULTS: Phylogenetic analysis displayed a coherent distribution of sequences in terms of lineage and temporal evolution of pandemic. Circulating lineage/clade characterization highlighted a growing diversity over time, with an increasingly growing number of mutations and variability of spike and nucleocapsid proteins, while viral RdRp appeared to be more conserved. A total of 384 different mutations were detected, of which 196 were missense and 147 synonymous ones. Mapping mutations along the viral genome showed an irregular distribution in key genes. S gene was the most mutated gene with missense and synonymous variants frequencies of 58.8 and 23.5%, respectively. Mutation rates were similar for the S and N genes with one mutation every ∼788 nucleotides and every ∼712 nucleotides, respectively. Nsp12 gene appeared to be more conserved, with one mutation every ∼1,270 nucleotides. The frequency of variant Y144F in the spike protein deviated from global values with higher prevalence of this mutation in the island. CONCLUSION: The analysis of the 92 viral genome highlighted evolution over time and identified which mutations are more widespread than others. The high number of sequences also permits the identification of subclusters that are characterized by subtle differences, not only in terms of lineage, which may be used to reconstruct transmission clusters. The disclosure of viral genetic diversity and timely identification of new variants is a useful tool to guide public health intervention measures.

6.
Artigo em Inglês | MEDLINE | ID: mdl-31799215

RESUMO

The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including blaKPC-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.


Assuntos
Bacteriemia/diagnóstico , Bacteriemia/microbiologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Candida/efeitos dos fármacos , Candida/genética , Candidemia/diagnóstico , Candidemia/microbiologia , Reação em Cadeia da Polimerase Multiplex , Bacteriemia/tratamento farmacológico , Candidemia/tratamento farmacológico , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Multiplex/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
Free Radic Biol Med ; 75: 210-21, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25017964

RESUMO

Natural hemozoin, nHZ, is avidly phagocytosed in vivo and in vitro by human monocytes. The persistence of the undigested ß-hematin core of nHZ in the phagocyte lysosome for long periods of time modifies several cellular immune functions. Here we show that nHZ phagocytosis by human primary monocytes severely impaired their chemotactic motility toward MCP-1, TNF, and FMLP, by approximately 80% each, and their diapedesis across a confluent human umbilical vein endothelial cell layer toward MCP-1 by 45±5%. No inhibition was observed with latex-fed or unfed monocytes. Microscopic inspection revealed polarization defects in nHZ-fed monocytes due to irregular actin polymerization. Phagocytosed nHZ catalyzes the peroxidation of polyunsaturated fatty acids and generation of the highly reactive derivative 4-hydroxynonenal (4-HNE). Similar to nHZ phagocytosis, the exposure of monocytes to in vivo-compatible 4-HNE concentrations inhibited cell motility in both the presence and the absence of chemotactic stimuli, suggesting severe impairment of cytoskeleton dynamics. Consequently, 4-HNE conjugates with the cytoskeleton proteins ß-actin and coronin-1A were immunochemically identified in nHZ-fed monocytes and mass spectrometrically localized in domains of protein-protein interactions involved in cytoskeleton reorganization and cell motility. The molecular and functional modifications of actin and coronin by nHZ/4-HNE may also explain impaired phagocytosis, another motility-dependent process previously described in nHZ-fed monocytes. Further studies will show whether impaired monocyte motility may contribute to the immunodepression and the frequent occurrence of secondary infections observed in malaria patients.


Assuntos
Aldeídos/metabolismo , Inibição de Migração Celular/efeitos dos fármacos , Hemeproteínas/farmacologia , Leucócitos Mononucleares/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Actinas/metabolismo , Células Cultivadas , Quimiocina CCL2/farmacologia , Quimiotaxia/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Proteínas dos Microfilamentos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fagocitose/fisiologia , Pigmentos Biológicos/farmacologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/imunologia , Fator de Necrose Tumoral alfa/farmacologia
8.
Haematologica ; 99(3): 570-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24038029

RESUMO

High counts of circulating microparticles, originated from the membrane of abnormal erythrocytes, have been associated with increased thrombotic risk in hemolytic disorders. Our studies indicate that in thalassemia intermedia patients the number of circulating microparticles correlates with the capability of the thalassemic erythrocytes to release microparticles. The microparticles are characteristically loaded with hemichromes formed by denatured α-chains. This finding was substantiated by the positive correlation observed in thalassemia intermedia patients between the amount of hemichromes measured in erythrocytes, their capability to release microparticles and the levels of plasma hemichromes. We observed that hemichromes, following their binding to the cytoplasmic domain of band 3, induce the formation of disulfide band 3 dimers that are subsequently phosphorylated by p72Syk kinase. Phosphorylation of oxidized band 3 appears to be relevant for the formation of large hemichromes/band 3 clusters that, in turn, induce local membrane instability and the release of microparticles. Proteomic analysis of microparticles released from thalassemia intermedia erythrocytes indicated that, besides hemichromes and clustered band 3, the microparticles contain a characteristic set of proteins that includes catalase, heat shock protein 70, peroxiredoxin 2 and carbonic anhydrase. High amounts of immunoglobulins and C3 have also been found to be associated with microparticles, accounting for their intense phagocytosis. The effect of p72Syk kinase inhibitors on the release of microparticles from thalassemia intermedia erythrocytes may indicate new perspectives for controlling the release of circulating microparticles in hemolytic anemias.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Eritrócitos/metabolismo , Hemeproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Talassemia/metabolismo , Ativação Enzimática , Eritrócitos/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxirredução , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinase Syk , Talassemia/sangue
9.
Oxid Med Cell Longev ; 2013: 385214, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24454983

RESUMO

Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.


Assuntos
Aterosclerose/sangue , Endarterectomia das Carótidas , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Proteômica/métodos , Proteína Amiloide A Sérica/metabolismo , Apolipoproteínas/sangue , Apolipoproteínas/química , Aterosclerose/cirurgia , Biomarcadores/sangue , Western Blotting , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/metabolismo
10.
J Proteomics ; 76 Spec No.: 251-8, 2012 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-22960126

RESUMO

Malaria parasites interact with the host cell membrane inserting new proteins and inducing oxidative and phosphorylative changes of erythrocyte proteins. In the present report we monitored the time dependent oxidative and phosphorylative modifications induced by parasites in heterozygous beta thalassemia (Het-ßThal). Het-ßThal causes mild anemia and is known to determine a pro-oxidant milieu and a protective effect against severe malaria. In malaria cultures Het-ßThal has been reported to induce accumulation of hemoglobin denaturation products. At early parasite development stages (rings), tyrosine hyper-phosphorylation of band 3 was the most notable modification, and at later development stages (trophozoites), additional membrane proteins displayed significant hyper-phosphorylation of their serine and tyrosine residues (adducins, ankyrin, catalase). Het-ßThal also caused membrane destabilization. Free radical scavengers effectively inhibited the phosphorylative response and membrane destabilization. Kinase inhibitors exerted similar effects suggesting a causal relationship between oxidative stress, membrane protein hyper-phosphorylation and increased membrane damage exacerbated by Het-ßThal. In conclusion, different lines of evidence suggest that Het-ßThal enhances the redox stress caused by malaria parasites inducing its protective effect destabilizing the host cell membrane. This article is part of a Special Issue entitled: Integrated omics.


Assuntos
Membrana Eritrocítica/metabolismo , Heterozigoto , Malária Falciparum/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Plasmodium falciparum/metabolismo , Talassemia beta/metabolismo , Adulto , Membrana Eritrocítica/genética , Membrana Eritrocítica/parasitologia , Feminino , Humanos , Malária Falciparum/genética , Masculino , Oxirredução , Estresse Oxidativo , Fosforilação , Talassemia beta/genética , Talassemia beta/parasitologia
11.
Electrophoresis ; 33(16): 2546-52, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22899262

RESUMO

A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein-protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development of mAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 µg of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available for monoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the high-throughput monoclonal antibody process providing a vast array of high quality antigens at very low cost.


Assuntos
Anticorpos Monoclonais/biossíntese , Antígenos/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas/isolamento & purificação , Animais , Anticorpos Monoclonais/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Extratos Celulares/química , Células HT29 , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/administração & dosagem , Proteínas/imunologia
12.
FEBS J ; 279(12): 2182-91, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22536838

RESUMO

Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and α-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins.


Assuntos
Doxorrubicina/farmacologia , Ubiquitinação/efeitos dos fármacos , Western Blotting , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Imunoprecipitação , L-Lactato Desidrogenase/metabolismo , Fosfopiruvato Hidratase/metabolismo , Pirazinas/farmacologia
13.
Free Radic Biol Med ; 52(2): 527-36, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22142474

RESUMO

Although indolone-N-oxide (INODs) genereting long-lived radicals possess antiplasmodial activity in the low-nanomolar range, little is known about their mechanism of action. To explore the molecular basis of INOD activity, we screened for changes in INOD-treated malaria-infected erythrocytes (Pf-RBCs) using a proteomics approach. At early parasite maturation stages, treatment with INODs at their IC(50) concentrations induced a marked tyrosine phosphorylation of the erythrocyte membrane protein band 3, whereas no effect was observed in control RBCs. After INOD treatment of Pf-RBCs we also observed: (i) accelerated formation of membrane aggregates containing hyperphosphorylated band 3, Syk kinase, and denatured hemoglobin; (ii) dose-dependent release of microvesicles containing the membrane aggregates; (iii) reduction in band 3 phosphorylation, Pf-RBC vesiculation, and antimalarial effect of INODs upon addition of Syk kinase inhibitors; and (iv) correlation between the IC(50) and the INOD concentrations required to induce band 3 phosphorylation and vesiculation. Together with previous data demonstrating that tyrosine phosphorylation of oxidized band 3 promotes its dissociation from the cytoskeleton, these results suggest that INODs cause a profound destabilization of the Pf-RBC membrane through a mechanism apparently triggered by the activation of a redox signaling pathway rather than direct oxidative damage.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Antimaláricos/farmacologia , Membrana Celular/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Radicais Livres/química , Indóis/farmacologia , Malária Falciparum/parasitologia , Fosfotirosina/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Membrana Celular/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Fosforilação , Plasmodium falciparum/efeitos dos fármacos , Multimerização Proteica , Proteoma/metabolismo
14.
Neurobiol Dis ; 43(3): 642-50, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21624464

RESUMO

Expression of mutant SOD1 typical of familial amyotrophic lateral sclerosis (ALS) induces the expression of Bcl2-A1, a member of the Bcl2 family of proteins, specifically in motor neurons of transgenic mice. In this work, we have used immortalized motor neurons (NSC-34) and transgenic mice expressing mutant SOD1 to unravel the molecular mechanisms and the biological meaning of this up-regulation. We report that up-regulation of Bcl2-A1 by mutant SOD1 is mediated by activation of the redox sensitive transcription factor AP1 and that Bcl2-A1 interacts with pro-caspase-3 via its C-terminal helix α9. Furthermore, Bcl2-A1 inhibits pro-caspase-3 activation in immortalized motor neurons expressing mutant SOD1 and thus induction of Bcl2-A1 in ALS mice represents a pro-survival strategy aimed at counteracting the toxic effects of mutant SOD1. These data provide significant new insights on how molecular signaling, driven by expression of the ALS-causative gene SOD1, affects regulation of apoptosis in motor neurons and thus may have implications for ALS therapy, where prevention of motor neuronal cell death is one of the major aims.


Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Apoptose/genética , Caspase 3/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/enzimologia , Animais , Caspase 3/metabolismo , Inibidores de Caspase , Linhagem Celular Transformada , Sobrevivência Celular/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Neurônios Motores/enzimologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Oxirredução , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/genética
15.
PLoS One ; 6(5): e18254, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21573212

RESUMO

BACKGROUND: Neuroblastic tumors account for 9-10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied. CONCLUSIONS/SIGNIFICANCE: Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Neuroblastoma/metabolismo , Fosfoproteínas/metabolismo , Tretinoína/farmacologia , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Microscopia de Contraste de Fase , Nucleosídeo NM23 Difosfato Quinases/metabolismo
16.
PLoS One ; 6(1): e15847, 2011 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21246053

RESUMO

BACKGROUND: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/patologia , Deficiência de Glucosefosfato Desidrogenase/patologia , Proteínas Tirosina Quinases/metabolismo , Membrana Eritrocítica/ultraestrutura , Hemoglobinas/metabolismo , Hemólise , Humanos , Oxidantes/farmacologia , Fosforilação/efeitos dos fármacos , Tirosina/metabolismo
17.
Proteomics ; 10(19): 3469-79, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20799346

RESUMO

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.


Assuntos
Membrana Eritrocítica/parasitologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Plasmodium falciparum/fisiologia , Serina/metabolismo , Tirosina/metabolismo , Membrana Eritrocítica/metabolismo , Interações Hospedeiro-Parasita , Humanos , Malária Falciparum/parasitologia , Malária Falciparum/fisiopatologia , Fosforilação , Plasmodium falciparum/crescimento & desenvolvimento
19.
PLoS One ; 4(3): e4782, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19277125

RESUMO

BACKGROUND: Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis. METHODOLOGY AND PRINCIPAL FINDINGS: A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association. CONCLUSION AND SIGNIFICANCE: 5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF. TRIAL REGISTRATION: ClinicalTrials.gov NCT00730509.


Assuntos
Fibrose Cística/tratamento farmacológico , Suplementos Nutricionais , Membrana Eritrocítica/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Tetra-Hidrofolatos/uso terapêutico , Vitamina B 12/uso terapêutico , Antiporters/sangue , Cátions/sangue , Criança , Pré-Escolar , Fibrose Cística/sangue , Fibrose Cística/patologia , Eritrócitos/química , Eritrócitos/ultraestrutura , Feminino , Proteínas de Choque Térmico HSP70/sangue , Homocisteína/sangue , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Malondialdeído/sangue , Lipídeos de Membrana/análise , Estresse Oxidativo , Fosfolipídeos/sangue , Tetra-Hidrofolatos/sangue
20.
Biochem J ; 418(2): 359-67, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18945214

RESUMO

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Eritrócitos/patologia , Deficiência de Glucosefosfato Desidrogenase/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Multimerização Proteica , Proteínas Tirosina Quinases/metabolismo , Anticorpos/metabolismo , Anticorpos/fisiologia , Diamida/farmacologia , Eritrócitos/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/sangue , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Glicosilação , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Oxirredução , Fosforilação , Ligação Proteica , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Especificidade por Substrato , Reagentes de Sulfidrila/farmacologia , Quinase Syk , Tirosina/metabolismo
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