Analysis of the genetic basis of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome.

PFAPA syndrome is the most common autoinflammatory syndrome in children from Western countries. In spite of its strong familial clustering, its genetic basis and inheritance pattern are still unknown. We performed a comprehensive genetic study on 68 individuals from 14 families. Linkage analysis suggested a susceptibility locus on chromosome 8, but direct molecular sequencing did not support this initial statistical finding. Exome sequencing revealed the absence of any gene that was mutated in all patients. Exhaustive screening of genes involved in other autoinflammatory syndromes or encoding components of the human inflammasome showed no DNA variants that could be linked to PFAPA molecular pathology. Among these, the previously-reported missense mutation V198M in the NLRP3 gene was clearly shown not to co-segregate with PFAPA. Our results on this relatively large cohort indicate that PFAPA syndrome is unlikely to be a monogenic condition. Moreover, none of the several genes known to be involved in inflammation or in autoinflammatory disorders seem to be relevant, alone, to its etiology, suggesting that PFAPA results from oligogenic or complex inheritance of variants in multiple disease genes and/or non-genetic factors.

Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network.

Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal disease.

ClC-5 chloride channel and kidney stones: what is the link?

Nephrolithiasis is one of the most common diseases in the Western world. The disease manifests itself with intensive pain, sporadic infections, and, sometimes, renal failure. The symptoms are due to the appearance of urinary stones (calculi) which are formed mainly by calcium salts. These calcium salts precipitate in the renal papillae and/or within the collecting ducts. Inherited forms of nephrolithiasis related to chromosome X (X-linked hypercalciuric nephrolithiasis or XLN) have been recently described. Hypercalciuria, nephrocalcinosis, and male predominance are the major characteristics of these diseases. The gene responsible for the XLN forms of kidney stones was cloned and characterized as a chloride channel called ClC-5. The ClC-5 chloride channel belongs to a superfamily of voltage-gated chloride channels, whose physiological roles are not completely understood. The objective of the present review is to identify recent advances in the molecular pathology of nephrolithiasis, with emphasis on XLN. We also try to establish a link between a chloride channel like ClC-5, hypercalciuria, failure in urine acidification and protein endocytosis, which could explain the symptoms exhibited by XLN patients.

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

The role of REST in transcriptional and epigenetic dysregulation in Huntington's disease

Huntington's disease (HD) is a devastating disorder that affects approximately 1 in 10,000 people and is accompanied by neuronal dysfunction and neurodegeneration. HD manifests as a progressive chorea, a decline in mental abilities accompanied by behavioural, emotional and psychiatric problems followed by, dementia, and ultimately, death. The molecular pathology of HD is complex but includes widespread transcriptional dysregulation. Although many transcriptional regulatory molecules have been implicated in the pathogenesis of HD, a growing body of evidence points to the pivotal role of RE1 Silencing Transcription Factor (REST). In HD, REST, translocates from the cytoplasm to the nucleus in neurons resulting in repression of key target genes such as BDNF. Since these original observations, several thousand direct target genes of REST have been identified, including numerous non-coding RNAs including both microRNAs and long non-coding RNAs, several of which are dysregulated in HD. More recently, evidence is emerging that hints at epigenetic abnormalities in HD brain. This in turn, promotes the notion that targeting the epigenetic machinery may be a useful strategy for treatment of some aspects of HD. REST also recruits a host of histone and chromatin modifying activities that can regulate the local epigenetic signature at REST target genes. Collectively, these observations present REST as a hub that coordinates transcriptional, posttranscriptional and epigenetic programmes, many of which are disrupted in HD. We identify several spokes emanating from this REST hub that may represent useful sites to redress REST dysfunction in HD.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 aminoacids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressorgene, with possible relevance for glioblastoma therapy. (C) 2009 Elsevier Ltd. All rights reserved.

Survivin and inducible nitric oxide synthase production during 4NQO-induced rat tongue carcinogenesis: A possible relationship

This study was undertaken to investigate, by immunohistochermistry, the expression of survivin and inducible nitric oxide synthase during 4NQO-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, survivin and iNOS were expresssed (P < 0.05) in some cells of the 'normal' oral epithelium. In pre-neoplastic lesions at 12 weeks following carcinogen exposure, the levels of survivin and iNOS were increased (p < 0.05) when compared to negative control, being the strongest effect observed to iNOS. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, survivin and iNOS were expressed in some tumor cells. Lack of immunoreactivity for both markers was observed in the negative control group. Taken together, our results support the belief that expression of survivin and iNOS are early events during malignant transformation and conversion of the oral mucosa. (c) 2007 Elsevier B.V. All rights reserved.

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: Histopathological diagnosis and potential invasiveness mediated by extracellular matrix components

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diabetes evolution in rats after neonatal treatment with alloxan

Physical exercises have been recommended in the prevention of non-insulin dependent diabetes mellitus (NIDDM), but the mechanisms involved in this intervention are not yet fully understood. Experimental models offer the opportunity for the study of this matter. The present study was designed to analyze the diabetes evolution in rats submitted to neonatal treatment with alloxan with the objective of verifying the suitability of the model to future studies with exercises. For this, newly born rats (6 days old) received intraperitoneal alloxan (A = 200 mg/kg of body weight). Rats injected with vehicle (citrate buffer) were used as controls (C). The fasting blood glucose level (mg/dL) was higher in the alloxan group at the day 28 (C=47.25 +/- 5.08; A=54.51 +/- 7.03) but not at the 60 day of age (C=69.18 +/- 8.31; A=66.81 +/- 6.08). The alloxan group presented higher blood glucose level during glucose tolerance test (GTT) (mg/dL. 120 min) in relation to the control group both at day 28 (C=16908.9 +/- 1078.8; A=21737,7 +/- 1106.4) and at day 60 (C=11463.45 +/- 655.30; A=15282.21 +/- 1221.84). Insulinaemia during GTT (ng/mL.120 min) was lower at day 28 (C=158.67 +/- 33.34; A=123.90 +/- 19.80), but presented no difference at day 60 (C=118.83 +/- 26.02; A=97.8 +/- 10.88). At day 60, the glycogen concentration in the soleus muscle (mg/100mg) was lower in the alloxan group (0.3 +/- 0.13) in relation to the control group (0.5 +/- 0.07). No difference was observed between groups in relation to (mu mol/g.h): Glucose Uptake (C = 5.8 +/- 0.63; A = 5.2 +/- 0.73); Glucose Oxidation (C= 4.3 +/- 1.13; A= 3.9 +/- 0.44); Glycogen Synthesis (C= 0.8 +/- 0.18; A= 0.7 +/- 0.18) and Lactate Production (C= 3.8 +/- 0.8; A= 3.8 0.7) by the isolated soleus muscle. The glucose-stimulated insulin secretion (16.7mM) by the isolated islets (ng/5 islets. h) of the alloxan group was lower (14.3 +/- 4.7) than the control group (32.0 +/- 7.9). Thus, we may conclude that this neonatal diabetes induction model gathers interesting characteristics and may be useful for further studies on the role of the exercise in the diabetes mellitus appearance.

Hepatic gluconeogenesis in rats trained to eat a single meal daily. Role of eating periodicity and the amount of food ingested in the last meal

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The monosodium glutamate (MSG) obese rat as a model for the study of exercise in obesity

Obesity is an increasing problem in several countries, leading to health problems. Physical exercise, in turn, can be used effectively by itself or in combination with dietary restriction to trigger weight loss. The present study was designed to evaluate the effects of aerobic exercise training on lipid profile of obese male Wistar rats in order to verify if this model may be of value for the study of exercise in obesity. Obesity was induced by MSG administration (4mg/g, each other day, from birth to 14 days old) After 14 from drug administration, the rats were separated into two groups: MSG-S (sedentary) and MSG-T (exercise trained). Exercise training consisted in 1h/day, 5 days/week, with an overload of 5% bw, for 10 weeks. Rats of the same age and strain, receiving saline at birth, were used as control (C), and subdivided into two groups: C-S and C-T. At the end of the experimental period, MSG-T and C-T rats showed similar blood lactate and muscle glycogen responses to exercise training and acute exercise. MSG-S rats showed significantly higher carcass fat, serum triacylglycerol, serum insulin and liver total fat than C-S rats. On the other hand, MSG-T rats had lower carcass fat, serum triacylglycerol and liver total fat than MSG-S rats. There were no statistical differences in food intake and serum free fatty acids among the groups studied. These data indicate that this model may be of value for the study of exercise effects on tissue and circulating lipid profile in obesity.

Evaluation of a protein deficient diet in rats through blood oxidative stress biomarkers

Protein malnutrition leads to functional impairment in several organs, which is not fully restored with nutritional recovery. Little is known about the role of oxidative stress in the genesis of these alterations. This study was designed to assess the sensitivity of blood oxidative stress biomarkers to a dietary protein restriction. Male Wistar rats were divided into two groups, according to the diet fed from weaning (21 days) to 60 day old: normal protein (17% protein) and low protein (6% protein). Serum protein, albumin, free fatty acid and liver glycogen and lipids were evaluated to assess the nutritional status. Blood glutathione reductase (GR) and catalase (CAT) activities, plasma total sulfhydryl groups concentration (TSG) as well as plasma thiobarbituric acid reactive substances (TBARs) and reactive carbonyl derivatives (RCD) were measured as biomarkers of the antioxidant system and oxidative damage, respectively. The glucose metabolism in soleus muscle was also evaluated as an index of stress severity imposed to muscular mass by protein malnutrition. No difference was observed in muscle glucose metabolism or plasma RCD concentration between both groups. However, our results showed that the low protein group had higher plasma TBARs (62%) concentration and lower TSG (44%) concentration than control group, indicating increased reactive oxygen species production in low protein group. The enhancement of erythrocyte GR (29%) and CAT (28%) activities in this group also suggest an adaptation to the stress generated by the protein deficiency. Taken together, the results presented here show that the biomarkers used were able to reflect the oxidative stress level induced by this specific protein deficient diet.

Glucose homeostasis in pregnant rats submitted to dietary protein restriction

In the present work, we examined the effects of feeding a low protein diet during pregnancy on glucose-induced insulin secretion and glucose homeostasis in rats. Young (60 days), pregnant (P) or non-pregnant (NP) rats were fed during pregnancy or for 21 days (the NP) a normal (17%) or a low (6%) protein diet. Serum glucose and insulin levels and pancreas insulin content in the fed state; total area under serum glucose curve (AG) after a glucose load and serum glucose disappearance rate (Kitt) after insulin administration; as well as 86Rb outflow, 45Ca uptake and insulin secretion by isolated pancreatic islets in response to glucose were evaluated. Serum glucose was lower in 17%-P (12%) and 6%-P (27%) than in corresponding NP-rats. Serum insulin was higher in 17%- P (153%) and 6%-P (77%) compared to the corresponding NP-rats. Pancreatic insulin was higher in 6%-rats (55%) than in 17%-rats. No differences were found in AG among the groups whereas Kitt was lower in 6%-NP and higher in 6%-P than in the equivalent 17% rats. Increasing glucose concentration from 2.8 to 16.7 mmol/l, reduced 86Rb outflow from isolated islets from all groups. Increasing glucose concentration from 2.8 to 16.7 mmol/l elevated 45Ca uptake by 17%-NP (47%), 17%-P (40%) and 6%-P (214%) islets but not by 6%-NP ones. The increase in 45Ca uptake was followed by an increase in insulin release by the 17%-NP (2767%), 17%-P (2850%) and 6%-P (1200%) islets. In conclusion, 6%-P rats show impaired glucose induced insulin secretion related to reduced calcium uptake by pancreatic islets. However, the poor insulin secretion did not fully compensate the high peripheral sensitivity to the hormone, resulting in hypoglycemia.

KRAS insertions in colorectal cancer: What do we know about unusual KRAS mutations?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)