Copper Is Accumulated as Copper Sulfide Particles, and Not Bound to Glutathione, Phytochelatins or Metallothioneins, in the Marine Alga Ulva compressa (Chlorophyta).

To analyze the mechanism of copper accumulation in the marine alga Ulva compressa, it was cultivated with 10 µM of copper, with 10 µM of copper and increasing concentrations of a sulfide donor (NaHS) for 0 to 7 days, and with 10 µM of copper and a concentration of the sulfide acceptor (hypotaurine) for 5 days. The level of intracellular copper was determined as well as the level of glutathione (GSH) and phytochelatins (PCs) and the expression of metallothioneins (UcMTs). The level of intracellular copper in the algae treated with copper increased at day 1, slightly increased until day 5 and remained unchanged until day 7. The level of copper in the algae cultivated with copper and 100 or 200 µM of NaHS continuously increased until day 7 and the copper level was higher in the algae cultivated with 200 µM of NaHS compared to 100 µM of NaHS. In contrast, the level of intracellular copper decreased in the algae treated with copper and hypotaurine. The level of intracellular copper did not correlate with the level of GSH or with the expression of UcMTs, and PCs were not detected in response to copper, or copper and NaHS. Algae treated with copper and with copper and 200 µM of NaHS for 5 days were visualized by TEM and the elemental composition of electrondense particles was analyzed by EDXS. The algae treated with copper showed electrondense particles containing copper and sulfur, but not nitrogen, and they were mainly located in the chloroplast, but also in the cytoplasm. The algae treated with copper and NaHS showed a higher level of electrondense particles containing copper and sulfur, but not nitrogen, and they were located in the chloroplast, and in the cytoplasm. Thus, copper is accumulated as copper sulfide insoluble particles, and not bound to GSH, PCs or UcMTs, in the marine alga U. compressa.

Titanium dioxide and carbon black nanoparticles disrupt neuronal homeostasis via excessive activation of cellular prion protein signaling.

BACKGROUND: Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer's disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. RESULTS: Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aß40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aß rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. CONCLUSION: Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aß peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.

Identification and Unusual Properties of the Master Regulator FNR in the Extreme Acidophile Acidithiobacillus ferrooxidans.

The ability to conserve energy in the presence or absence of oxygen provides a metabolic versatility that confers an advantage in natural ecosystems. The switch between alternative electron transport systems is controlled by the fumarate nitrate reduction transcription factor (FNR) that senses oxygen via an oxygen-sensitive [4Fe-4S]2+ iron-sulfur cluster. Under O2 limiting conditions, FNR plays a key role in allowing bacteria to transition from aerobic to anaerobic lifestyles. This is thought to occur via transcriptional activation of genes involved in anaerobic respiratory pathways and by repression of genes involved in aerobic energy production. The Proteobacterium Acidithiobacillus ferrooxidans is a model species for extremely acidophilic microorganisms that are capable of aerobic and anaerobic growth on elemental sulfur coupled to oxygen and ferric iron reduction, respectively. In this study, an FNR-like protein (FNRAF) was discovered in At. ferrooxidans that exhibits a primary amino acid sequence and major motifs and domains characteristic of the FNR family of proteins, including an effector binding domain with at least three of the four cysteines known to coordinate an [4Fe-4S]2+ center, a dimerization domain, and a DNA binding domain. Western blotting with antibodies against Escherichia coli FNR (FNREC) recognized FNRAF. FNRAF was able to drive expression from the FNR-responsive E. coli promoter PnarG, suggesting that it is functionally active as an FNR-like protein. Upon air exposure, FNRAF demonstrated an unusual lack of sensitivity to oxygen compared to the archetypal FNREC. Comparison of the primary amino acid sequence of FNRAF with that of other natural and mutated FNRs, including FNREC, coupled with an analysis of the predicted tertiary structure of FNRAF using the crystal structure of the related FNR from Aliivibrio fisheri as a template revealed a number of amino acid changes that could potentially stabilize FNRAF in the presence of oxygen. These include a truncated N terminus and amino acid changes both around the putative Fe-S cluster coordinating cysteines and also in the dimer interface. Increased O2 stability could allow At. ferrooxidans to survive in environments with fluctuating O2 concentrations, providing an evolutionary advantage in natural, and engineered environments where oxygen gradients shape the bacterial community.

Ultrastructure of colorectal adenocarcinoma and peritumoral tissue in untreated patients.

In this study, we describe, compare, and discuss several subcellular alterations found in Colorectal Adenocarcinoma and peritumoral tissue using transmission electron microscopy, morphometry, and statistical analysis. Tissue samples from anterior resections were collected from patients diagnosed with Colorectal Adenocarcinoma in the University Hospital of Caracas. Samples were processed according to the typical protocol for their observation through transmission electron microscopy. The resulting images were analyzed using specialized software for the collection of morphometric data. Several anomalies were common for both tissues, including but not limited to, rough endoplasmic reticulum and mitochondrial swelling, nuclear invagination, nuclear enlargement, and cellular swelling. In general, alterations within the tumor were more frequent and intense. Extensive organellar degradation and other evidences of cellular damage seemed to extend past the edge of the tumor into the peritumoral tissue. There seems to be a clear process of lateral cancerization present in the peritumoral area. The tissue layers composed of smooth muscle cells, probably due to their structural features, may allow greater diffusion of harmful substances produced by the tumor. A more in-depth analysis of peritumoral tissue considering organellar damage and morphometric data may provide relevant insight about the changing microenvironment promoted by the close proximity of a tumor.

Mechanical technology effect in the treatment of anorectocele using transanal repair of rectocele and full rectal mucosectomy with one circular stapler procedure / Efeito mecânico da tecnologia no tratamento da anorretocele com o uso do procedimento TRREMS

ABSTRACT Objective: Transanal repair of rectocele and full rectal mucosectomy with one circular stapler is a procedure designed for the treatment of Obstructive Defecation Syndrome by doctor Fco. Sergio Regadas in 2005. We compare the use of multiple instruments and their mechanical technology effect in the treatment of anorectocele. Patients and methods: Female patients complaining about sensation of incomplete evacuation, ages between 40 and 55. The evaluation was made with the function of evacuation protocol: colonic transit time, colon radiology, ecodefecography, anorectal manometry and psychological test. The technique used was transanal repair of rectocele and full rectal mucosectomy with one circular stapler, using staplers CPH-34, CPH-34HV and EEA-3135-HEM, with measurement of the rectal wall resected: vertical length in centimetres, horizontal length in centimetres, weight in grams and volume in cubic centimetres; afterwards histological study of the tissue thickness, and applied the ANOVA and SPSS 12 tests for the statistical analysis. Results: The results obtained by comparing the resections made with the CPH-34, the CPH-34HV and the EEA-3135-HEM in respect of vertical length, horizontal length, weight and volume, were found to have no significant differences; neither in the histological study of the tissue thickness in respect of characteristics and structure. Conclusion: The effect of mechanical technology in the treatment of anorectocele with transanal repair of rectocele and full rectal mucosectomy with one circular stapler procedure using the CPH-34, the CPH-34HV and the EEA-3135-HEM, does not show any difference. Leaving the application of each to the operator competencies.

RESUMO Objetivo: TRREMS (Transanal Repair of Rectocele and full rectal Mucosectomy with one circular Stapler, Reparo transanal de retocele e mucosectomia retal total com um grampo circular) é um procedimento que visa o tratamento da Síndrome da Defecação Obstrutiva pelo Dr. Francisco Sergio Regadas em 2005. Comparamos o uso de diversos instrumentos e o efeito mecânico da tecnologia no tratamento da anorretocele. Pacientes e métodos: Pacientes do gênero feminino com queixa de sensação de evacuação incompleta (SEI), com idades entre 40 e 55 anos. A avaliação foi efetuada com o protocolo de função de evacuação: tempo de trânsito colônico, radiologia do cólon, ecodefecografia, manometria anorretal e teste psicológico. A técnica empregada foi TRREMS, com o uso de grampeadores CPH-34, CPH-34HV e EEA-3135-HEM, com medição da parede retal ressecada: comprimento vertical em centímetros, comprimento horizontal em centímetros, peso em gramas e volume em centímetros cúbicos; subsequentemente, foi realizado estudo histológico da espessura do tecido, com aplicação de ANOVA e do programa SPSS 12 para a análise estatística. Resultados: Observamos que os resultados obtidos com a comparação das ressecções realizadas com CPH-34, CPH-34HV e EEA-3135-HEM com relação ao comprimento vertical, comprimento horizontal, peso e volume, bem como os resultados do estudo histológico da espessura do tecido com relação às características e estrutura, não apresentavam diferenças significativas.

Changes in the proportions of types I and III collagen in hemorrhoids: the sliding anal lining theory / Mudanças nos percentuais do colágeno dos tipos I e III em hemorroidas: teoria do revestimento anal deslizante

Abstract Objective This study aims to determine changes in the proportions of types I and III collagen in hemorrhoids and to verify the sliding anal canal lining theory. Patients and method The study is focused on a sample of 17 patients, 9 females and 8 males (age range: 30-70 years), with grade III and grade IV hemorrhoids. Tissue from 4 fetuses (age: 16 weeks of gestation) was used as control sample. All the participants gave their informed consent. Samples were gathered in 2014. All patients underwent open hemorrhoidectomy by using the technique described by Milligan and Morgan, published in Lancet journal in 1937. The hemorrhoid samples were stained with hematoxylin-eosin for the histologic study to confirm the hemorrhoidal tissue diagnosis. The picrosirius red staining protocol was used after the histologic analysis. The method used for image processing is described in the text. Images were imported to the Image Tool for Windows software. The same process was used on the embryonic tissue. Data resulting from the analysis of images were processed using STATISTICA, a software for statistical analysis. Results When compared, it was found that the two tissues presented very different values, with hemorrhoids containing the highest type III collagen values. Conclusion Our results seem to imply that hemorrhoids have a larger proportion of type III collagen than fetal tissue. They also suggest a possible age-related deterioration of the tissue.

Resumo Objetivo Esse estudo tem por objetivo determinar mudanças nos percentuais do colágeno dos tipos I e III em hemorroidas e verificar a teoria do revestimento de canal anal deslizante. Pacientes e método O estudo está focado em uma amostra de 17 pacientes (9 mulheres e 8 homes; faixa etária: 30-70 anos), com hemorroidas de graus III e IV. Utilizamos tecido de quatro fetos (idade: 16 semanas de gestação) como amostra de controle. Todos os participantes deram consentimento informado. As amostras foram reunidas em 2014. Todos os pacientes passaram por uma hemorroidectomia aberta; para tanto, foi empregada a técnica descrita por Milligan e Morgan, publicada no periódico Lancet em 1937. As amostras de hemorroida foram coradas com hematoxilina-eosina com vistas ao estudo histológico para confirmação do diagnóstico de tecido hemorroidal. Após a análise histológica, o material foi corado com o protocolo de picrosirius red. O método empregado para o processamento das imagens está descrito no texto. As imagens foram importadas pelo software Image Tool for Windows. O mesmo processo foi empregado no tecido embrionário. Os dados resultantes da análise das imagens fora processados com o programa STATISTICA, um software para análise estatística. Resultados Por comparação, constatamos que os dois tecidos apresentavam valores muito diferentes, e as hemorroidas continham os mais altos valores de colágeno do tipo III. Conclusão Nossos resultados parecem implicar que hemorroidas possuem um percentual mais elevado de colágeno do tipo III versus tecido fetal. Os resultados também sugerem uma possível deterioração do tecido, relacionada à idade.

Peripheral Arterial Disease: Surgical Treatment.

Peripheral arterial disease is a frequent under-diagnosed and poorly recognized clinical entity that can affect a great number of patients. Recognition of risk factors is crucial and a through evaluation of symptoms and use of diagnostic tools to better decide when an intervention is warranted. Lower extremity bypass surgery is one alternative method for treatment of PAD. It is indicated for type D and C lesions with low cardiac risk according to the TASC classification system. Preoperative assessment is imperative for every vascular procedure since it has been associated with major postoperative cardiovascular events; myocardial infarction being the most common. After excluding active disease, functional capacity and clinical risk predictors must be determined via METs and the Revised Cardiac Risk Index (RCRI), respectively. If a patient is considered to have a high cardiac risk, then noninvasive studies should be performed. Aspirin and a statin should be administrated preoperatively and postoperatively. Clopidogrel can be utilized as an alternative if a contraindication to aspirin exists. Periodic follow up consisting of clinical evaluations assessing and return or progression of symptoms of claudication, presence of pulses, ankle-brachial index (ABI) measurement, and smoking cessation counseling should be performed in every patient after vascular surgery.

Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in the extremophile Acidithiobacillus ferrooxidans.

Gene transcription (microarrays) and protein levels (proteomics) were compared in cultures of the acidophilic chemolithotroph Acidithiobacillus ferrooxidans grown on elemental sulfur as the electron donor under aerobic and anaerobic conditions, using either molecular oxygen or ferric iron as the electron acceptor, respectively. No evidence supporting the role of either tetrathionate hydrolase or arsenic reductase in mediating the transfer of electrons to ferric iron (as suggested by previous studies) was obtained. In addition, no novel ferric iron reductase was identified. However, data suggested that sulfur was disproportionated under anaerobic conditions, forming hydrogen sulfide via sulfur reductase and sulfate via heterodisulfide reductase and ATP sulfurylase. Supporting physiological evidence for H2S production came from the observation that soluble Cu(2+) included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H(2)S reduces ferric iron to ferrous in acidic medium, its production under anaerobic conditions indicates that anaerobic iron reduction is mediated, at least in part, by an indirect mechanism. Evidence was obtained for an alternative model implicating the transfer of electrons from S(0) to Fe(3+) via a respiratory chain that includes a bc(1) complex and a cytochrome c. Central carbon pathways were upregulated under aerobic conditions, correlating with higher growth rates, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth, probably as a result of more limited access to carbon dioxide. These results are important for understanding the role of A. ferrooxidans in environmental biogeochemical metal cycling and in industrial bioleaching operations.

A peripheral artery disease screening study in Puerto Rico.

UNLABELLED: Peripheral artery disease (PAD) of the lower extremities is frequently underdiagnosed and undertreated. The results of screening for PAD in adults attending outpatient clinics at different sites in Puerto Rico from 2007 to 2010 are presented. METHODS: A total of 33 outpatients screening clinics were conducted at different sites throughout the Island. Following the ACC/AHA Guideline recommendations, asymptomatic patients who qualified were screened for PAD using the ankle-brachial index (ABI). An ABI < 0.9 was considered positive for PAD. We estimated the prevalence of PAD in the study population and used logistic regression models to assess factors associated with a positive screening test for PAD. RESULTS: A total of 933 patients were screened for PAD. Out of the 933 patients, the ABI was < 0.9 in 390 (41.8%) of them. Bivariate analysis showed a significant difference in PAD screening results by gender (P = 0.004) and history of arterial hypertension (P = 0.004). Regarding clinical characteristics, leg edema 44.7% (P = 0.001), intermittent claudication 40.3% (P = 0.002), distal extremity coldness 29.0% (P = 0.012), and weak lower extremity pulses 67.5% (P < 0.001) were more prevalent on patients with an ABI < 0.9. In the multivariate analysis, male gender (OR = 1.92, 95% CI: 1.18, 3.11) and arterial hypertension (OR = 2.16, 95% CI: 1.28, 3.65) were significantly associated with PAD after adjusting for specific confounders. CONCLUSIONS: Arterial hypertension, cigarette smoking, diabetes mellitus, and dyslipidemia are known key factors in development of PAD. Practicing physicians must be aware of the importance of an early diagnosis of PAD, particularly in the asymptomatic patient, so as to institute preventive and management measures.

Microbial iron management mechanisms in extremely acidic environments: comparative genomics evidence for diversity and versatility.

BACKGROUND: Iron is an essential nutrient but can be toxic at high intracellular concentrations and organisms have evolved tightly regulated mechanisms for iron uptake and homeostasis. Information on iron management mechanisms is available for organisms living at circumneutral pH. However, very little is known about how acidophilic bacteria, especially those used for industrial copper bioleaching, cope with environmental iron loads that can be 1018 times the concentration found in pH neutral environments. This study was motivated by the need to fill this lacuna in knowledge. An understanding of how microorganisms thrive in acidic ecosystems with high iron loads requires a comprehensive investigation of the strategies to acquire iron and to coordinate this acquisition with utilization, storage and oxidation of iron through metal responsive regulation. In silico prediction of iron management genes and Fur regulation was carried out for three Acidithiobacilli: Acidithiobacillus ferrooxidans (iron and sulfur oxidizer) A. thiooxidans and A. caldus (sulfur oxidizers) that can live between pH 1 and pH 5 and for three strict iron oxidizers of the Leptospirillum genus that live at pH 1 or below. RESULTS: Acidithiobacilli have predicted FeoB-like Fe(II) and Nramp-like Fe(II)-Mn(II) transporters. They also have 14 different TonB dependent ferri-siderophore transporters of diverse siderophore affinity, although they do not produce classical siderophores. Instead they have predicted novel mechanisms for dicitrate synthesis and possibly also for phosphate-chelation mediated iron uptake. It is hypothesized that the unexpectedly large number and diversity of Fe(III)-uptake systems confers versatility to this group of acidophiles, especially in higher pH environments (pH 4-5) where soluble iron may not be abundant. In contrast, Leptospirilla have only a FtrI-Fet3P-like permease and three TonB dependent ferri-dicitrate siderophore systems. This paucity of iron uptake systems could reflect their obligatory occupation of extremely low pH environments where high concentrations of soluble iron may always be available and were oxidized sulfur species might not compromise iron speciation dynamics. Presence of bacterioferritin in the Acidithiobacilli, polyphosphate accumulation functions and variants of FieF-like diffusion facilitators in both Acidithiobacilli and Leptospirilla, indicate that they may remove or store iron under conditions of variable availability. In addition, the Fe(II)-oxidizing capacity of both A. ferrooxidans and Leptospirilla could itself be a way to evade iron stress imposed by readily available Fe(II) ions at low pH. Fur regulatory sites have been predicted for a number of gene clusters including iron related and non-iron related functions in both the Acidithiobacilli and Leptospirilla, laying the foundation for the future discovery of iron regulated and iron-phosphate coordinated regulatory control circuits. CONCLUSION: In silico analyses of the genomes of acidophilic bacteria are beginning to tease apart the mechanisms that mediate iron uptake and homeostasis in low pH environments. Initial models pinpoint significant differences in abundance and diversity of iron management mechanisms between Leptospirilla and Acidithiobacilli, and begin to reveal how these two groups respond to iron cycling and iron fluctuations in naturally acidic environments and in industrial operations. Niche partitions and ecological successions between acidophilic microorganisms may be partially explained by these observed differences. Models derived from these analyses pave the way for improved hypothesis testing and well directed experimental investigation. In addition, aspects of these models should challenge investigators to evaluate alternative iron management strategies in non-acidophilic model organisms.

Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity.

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.

DEHIDROERGOSTEROL: UN ARTEFACTO GENERADO DURANTE EL PROCESO DE EXTRACCIÓN DE ESTEROLES EN EL HONGO Pleurotus sajor-caju / DEHYDROERGOSTEROL: AN ARTIFACT GENERATED DURING EXTRACTION OF STEROLS FROM Pleurotus sajor-caju

Durante el estudio químico de la fracción esterólica del hongo Pleurotus sajor-caju se estableció que el dehidroergosterol (ergosta-5,7,9(11),22-tetraen-3-ol) presente en el extracto posiblemente no es un producto natural sino un artefacto producido durante el proceso de extracción de los esteroles con cloroformo. Este hecho se atribuyó a la presencia de sustancias fenólicas en el extracto.

During the extraction of sterolic fraction of Pleurotus sajor-caju was established that dehydroergosterol (ergosta-5,7,9 (11), 22-tetraen-3-ol) present in the extract is possibly not a natural product but an artifact produced during the process of extraction of sterols with chloroform. This was attributed to the presence of phenolic substances in the extract.

TRAF interactions with raft-like buoyant complexes, better than TRAF rates of degradation, differentiate signaling by CD40 and EBV latent membrane protein 1.

The CD40 receptor and the Epstein-Barr virus oncoprotein LMP1 are both members of the TNF-receptor family and share several signaling mediators, including TRAF2 and TRAF3. Depending on the cell lineage and stage of maturation, LMP1 and CD40 can have synergistic, antagonist or unrelated effects. Previous publications have suggested that both TRAF2 and TRAF3 move into lipid rafts upon LMP1 expression or CD40 activation, whereas their proteolysis is only enhanced by CD40. However CD40-induced proteolysis of TRAF2 has only been reported in murine cells, and there are conflicting data regarding translocation of TRAF2 into lipid rafts. We therefore investigated TRAF2 and TRAF3 modifications induced by CD40 and LMP1 signaling in a panel of human cell lines of lymphoid and epithelial origins. Upon CD40 stimulation, a marked redistribution of TRAF2 into the buoyant raft fraction was observed in all cell lines and was often associated with a similar redistribution of TRAF3. In contrast, only TRAF3 was redistributed into the raft fraction upon LMP1 expression. Moreover parallel changes in subcellular distribution of TRAF2 and TRAF3 were recorded by electron microscopy. A significant decrease in TRAF2 and TRAF3 concentrations triggered by CD40 ligation was observed in only 1 cell line and there was no evidence that this decrease was required for the negative feed-back on JNK activation. TRAF2 redistribution into raft-like complexes thus appears as the most significant event distinctive of CD40 and LMP1 signaling. On the other hand, the parallel influence of CD40 and LMP1 on TRAF3 redistribution is consistent with functional similarities between the CD40-TRAF3 and LMP1-TRAF3 axes.

Apoptosis and TRAF-1 cleavage in Epstein-Barr virus-positive nasopharyngeal carcinoma cells treated with doxorubicin combined with a farnesyl-transferase inhibitor.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinomas (NPC) are much more sensitive to chemotherapy than other head and neck carcinomas. Spectacular regressions are frequently observed after induction chemotherapy. However, these favorable responses are difficult to predict and often of short duration. So far there have been only few experiments to investigate the mechanisms which underline the cytotoxic effects of anti-neoplastic drugs against NPC cells. In addition, these studies were performed almost entirely on EBV-negative cell lines therefore not truly representative of NPC cells. For the first time, we have used two EBV-positive NPC tumor lines derived from a North African (C15) and a Chinese (C666-1) patient as in vitro targets for a panel of anti-neoplastic agents. Doxorubicin, taxol and in a lesser extent cis-platinum efficiently inhibited NPC cell proliferation at clinically relevant concentrations, but all three agents failed to induce apoptosis. However, massive apoptosis of C15 cells was achieved when doxorubicin (1 microM) was combined with a farnesyl-transferase inhibitor, BIM 2001 (5 microM). Moreover, this apoptotic process was associated with a caspase-dependent early cleavage of the TNF-receptor associated factor 1 (TRAF-1) molecule, a signaling adaptor which is specifically expressed in latently EBV-infected cells. TRAF-1 cleavage might become a useful indicator of chemo-induced apoptosis in EBV-associated NPCs.