C-Jun N-terminal kinases are required for oncolytic adenovirus-mediated autophagy.

Oncolytic adenoviruses, such as Delta-24-RGD (Δ24RGD), are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, Δ24RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with Δ24RGDundergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/2 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with Δ24RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered Δ24RGD-treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with Δ24RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between Δ24RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates Δ24RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.

Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells.

BACKGROUND: Current evidence indicates that a stem cell-like sub-population within malignant glioblastomas, that overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters, is responsible for multidrug resistance and tumour relapse. Eradication of the brain tumour stem cell (BTSC) compartment is therefore essential to achieve a stable and long-lasting remission. METHODS: Melatonin actions were analysed by viability cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein expression and quantitative and qualitative promoter methylation methods. RESULTS: Combinations of melatonin and chemotherapeutic drugs (including temozolomide, current treatment for malignant gliomas) have a synergistic toxic effect on BTSCs and A172 malignant glioma cells. This effect is correlated with a downregulation of the expression and function of the ABC transporter ABCG2/BCRP. Melatonin increased the methylation levels of the ABCG2/BCRP promoter and the effects on ABCG2/BCRP expression and function were prevented by preincubation with a DNA methyltransferase inhibitor. CONCLUSION: Our results point out a possible relationship between the downregulation of ABCG2/BCRP function and the synergistic toxic effect of melatonin and chemotherapeutic drugs. Melatonin could be a promising candidate to overcome multidrug resistance in the treatment of glioblastomas, and thus improve the efficiency of current therapies.

Entorno urbano y actividad física en niños escolares de la ciudad de Córdoba / Urban environment and physical activity in scholar children of Córdoba city

NTRODUCCIÓN La prevalencia de exceso de peso infantil se ha incrementado significativamente en todo el mundo. La región con la prevalencia más elevada es América, donde cerca del 32% de los niños tiene sobrepeso y uno de cada cuatro es obeso. La exploración de las causas ha buscado identificar factores ambientales que influyen en los estilos de vida físicamente activos. OBJETIVOS Analizar la relación entre las características sociales y físicas del entorno urbano y los niveles de actividad física (AF) en niños de distintas zonas geográficas de la ciudad de Córdoba. MÉTODOS Se realizó un estudio transversal en áreas periféricas de la ciudad de Córdoba, con un muestreo estratificado de escuelas primarias municipales. A través de cuestionarios, se recogieron datos sobre la AF de los niños y su entorno físico y social. Se estimaron niveles de AF y asociaciones con el entorno urbano. Se utilizó prueba de chi cuadrado y regresión logística. RESULTADOS Se observó que el 79,1% de los escolares de la ciudad no cumplen las recomendaciones de AF establecidas por la Organización Mundial de la Salud. No se encontraron diferencias según zonas geográficas de residencia. Los lugares de recreación más próximos a los hogares de los niños mostraron el uso más frecuente (plaza, parque, baldío, cancha de fútbol, etc.), con diferencias de género. La mayoría de los padres dijo que la inseguridad es una de las principales barreras para que sus hijos realicen AF en el barrio. Esta percepción redujo la frecuencia de uso de las canchas de fútbol y obstaculizó el cumplimiento de las recomendaciones de AF por parte de los varones. DISCUSIÓN Es importante promover iniciativas de salud pública y diseño urbano que faciliten el acceso a lugares de recreación para realizar AF en los barrios de la ciudad, considerando el enfoque de género.

Reduction of nontarget infection and systemic toxicity by targeted delivery of conditionally replicating viruses transported in mesenchymal stem cells.

The fiber-modified adenoviral vector Delta-24-RGD (D24RGD) offers vast therapeutic potential. Direct injection of D24RGD has been used to successfully target ovarian tumors in mice. However, systemic toxicity, especially in the liver, profoundly limits the efficacy of direct viral vector delivery. Mesenchymal stem cells (MSC) have the ability to function as a vector for targeted gene therapy because of their preferential engraftment into solid tumors and participation in tumor stroma formation. We show that MSC-guided delivery of D24RGD is specific and efficient and reduces the overall systemic toxicity in mice to negligible levels compared with D24RGD alone. In our model, we found efficient targeted delivery of MSC-D24RGD to both breast and ovarian cell lines. Furthermore, immunohistochemical staining for adenoviral hexon protein confirmed negligible levels of systemic toxicity in mice that were administered MSC-D24RGD compared with those that were administered D24RGD. These data suggest that delivery of D24RGD through MSC not only increases the targeted delivery efficiency, but also reduces the systemic exposure of the virus, thereby reducing overall systemic toxicity to the host and ultimately enhancing its value as an anti-tumor therapeutic candidate.

Tie2-mediated multidrug resistance in malignant gliomas is associated with upregulation of ABC transporters.

Resistance and relapse are still primary causes that result in poor effectiveness of chemotherapy in malignant gliomas. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating chemoresistance. We previously found that abnormal high expression of the Tie2 receptor in gliomas was associated with tumor malignancy. Here, we studied the role of Tie2 activation in drug resistance by testing the cytotoxicity of several chemotherapeutic drugs in a panel of human glioma cell lines and brain tumor stem cells and found that Tie2 activation was significantly related to chemoresistance. The essential role of Tie2 in this phenotype was illustrated by silencing Tie2 using specific siRNA, and the subsequent abrogation of the angiopoietin 1 (Ang1)-mediated chemoresistance. Using quantitative real-time PCR and functional drug efflux studies, we observed that Tie2 activation resulted in increased expression of ATP-binding cassette (ABC) transporters. Consistent with these results, downmodulation of ABCG2 or ABCC2 resulted in the inability of Tie2 activation to induce a chemoresistant phenotype. Our results indicate that Tie2 activation may be important in modifying the evolution of gliomas during conventional chemotherapy regimens, and open new avenues for the search of more effective therapies to avoid the inevitable brain tumor recurrence.

Oncolytic adenovirus retargeted to Delta-EGFR induces selective antiglioma activity.

The fact that glioblastomas, which are one of the most devastating cancers, frequently express the Delta-EGFR (epithelial growth factor receptor) also called mutant variant III of EGFR (EGFRvIII) suggests that this cancer cell-specific receptor might serve as an ideal target for cancer therapy. To assess its potential as such a target, we constructed an oncolytic adenovirus with Retargeted Infectivity Via EGFR (Delta-24-RIVER) on the backbone of Delta-24. This new oncolytic adenovirus targets, as Delta-24 does, the disrupted Rb pathway in cancer cells; in addition, this adenovirus has also been retargeted through the abrogation of CAR binding (Y477A mutation in adenoviral fiber protein) and insertion of an EGFRvIII-specific binding peptide in the HI loop of the fiber protein. As compared with Delta-24, Delta-24-RIVER induced EGFRvIII-selective cytotoxicity in U-87 MG isogenic cell lines and in tetracycline-inducible EGFRVIII expressing U-251 MG cells. Accordingly, by tittering the viral progeny and examining fiber protein expression in the above cells, we showed that the replication of this new construct also correlated with EGFRvIII expression. Consistently, immunohistochemistry staining of the adenoviral capsid protein hexon in the virus-treated tumors revealed that the virus replicated more efficiently in EGFRvIII-expressing U-87 MG.DeltaEGFR xenografts than in the tumors grown from U-87 MG cells. Importantly, treatment with Delta-24-RIVER prolonged the survival of animals with intracranial xenografts derived from U-87 MG.DeltaEGFR cells. Therefore, our results constitute the first proof of the direct targeting of a cancer-specific receptor using an oncolytic adenovirus.

Invaginación ileocólica en el adulto ¿Es seguro desinvaginar antes de resecar? / Intestinal intussusception in adults. It safe the option of primary reduction before resection?

La invaginación intestinal es una patología frecuente en el niño pero muy infrecuente en el adulto. El mecanismo exacto que precipita la invaginación es todavía desconocido, aunque la mayoría de los casos en los adultos se deben a una tumoración que ejerce como cabeza de la misma. El tratamiento en éstos es siempre quirúrgico y la controversia radica en si se puede desinvaginar antes de la resección. Presentamos el caso de una invaginación ileocólica cuya causa desencadenante fue un tumor miofibroblástico inflamatorio (AU)

Intestinal intussusception is a frequent pathology in children but very rare in adults. The exact mechanism that precipitates this pathology is yet unknown althought in most cases in adults it is due to a neoplasm that acts as the lead point of the intussusception. Treatment in these cases is always surgical and controversy lies in the possibility of initial reduction followed by a more limited resection. We present the case of an ileocolic intussusception that was caused by a inflammatory myofibroblastic tumor (AU)

Tie2: a journey from normal angiogenesis to cancer and beyond.

The tyrosine kinase receptor Tie2 was initially identified as a specific vascular growth factor that governed several properties of endothelial cells under both physiological and pathological conditions. It was subsequently found that angiopoietins, the natural ligands of Tie2, modulate Tie2-dependent signaling, which in turn regulates the survival and apoptosis of endothelial cells, controls vascular permeability, and regulates the capillary sprouting that occurs during normal angiogenesis such as through development and ovarian remodeling. Tie2 also seems to play a crucial role in several vascular abnormalities, such as familial venous malformations. Beyond its critical role in angiogenesis, Tie2 also appears to maintain the long-term population and quiescent status of hematopoietic stem cells in the bone marrow stem cell niche. In cancer, Tie2 was originally found to be overexpressed in tumoral vessels. More recently, our laboratory and others have found that Tie2 is also expressed outside the vascular compartment in several types of cancer, including leukemia and solid neoplasms such as gastric tumors, breast tumors, and gliomas. The role of Tie2 in these tumoral cells is currently being explored. In this regard, our group reported the importance of Tie2-expressing glioma cells in their adhesion to the tumoral microenvironment. Because cancer may be considered as a complex organ with several cellular lineages coexisting in the same tumor, the expression of Tie2 by different tumoral compartments makes this cellular receptor an attractive target for cancer therapy.

Angiopoietin-2 decreases vascular endothelial growth factor expression by modulating HIF-1 alpha levels in gliomas.

Angiogenesis is thought to depend on a perfectly coordinated balance between endogenous-positive and negative regulatory factors. Of these factors, the vascular endothelial growth factor (VEGF) and angiopoietins (Angs) seem to play an essential role. Recently, we reported the expression of the Ang-natural receptor, Tie2, in neoplastic astrocytic cells within gliomas. Because of the VEGF/Ang2 functional partnership together with the presence of Tie2 in gliomas, we hypothesized a role of Ang2 on the modulation of VEGF levels in these tumors. We examined the effect of Ang2 on VEGF expression in a panel of glioma cells, which showed that Ang2 inhibited VEGF expression at both mRNA and protein levels in Tie2-expressing cells, but not in Tie2-negative cells. VEGF promoter analysis showed that Ang2 regulated VEGF expression at the transcriptional level in relation to a decrease in HIF-1alpha expression and HIF-DNA-binding activity. Tie2 silencing by siRNA rescued the Ang2-mediated downmodulation of VEGF, suggesting an essential role for Tie2 in this regulatory loop. To our knowledge, this is the first report on the role of Ang2/Tie2 in the regulation of HIF-1alpha/VEGF expression, providing additional evidence of the intrinsic coordination that occurs among these factors during angiogenesis.

Combination of the oncolytic adenovirus ICOVIR-5 with chemotherapy provides enhanced anti-glioma effect in vivo.

Novel therapies are clearly needed for gliomas, and the combination of oncolytic vectors with chemotherapy possesses a significant hope for the treatment of this malignancy. In addition, combination with chemotherapy allows for lower virus doses to achieve anticancer effect, thus resulting in lower undesirable toxicities due to viral proteins. In this work, we sought to determine whether combination of an oncolytic adenovirus ICOVIR-5, with RAD001 or temozolomide (TMZ) could result in enhanced anti-glioma effect in vivo. We assessed the in vitro cytotoxic effect and replication properties of ICOVIR-5 in combination with RAD001 or TMZ in U87 MG glioma cell line by MTT and TCID(50), respectively. Our data showed that in vitro treatment with RAD001 or TMZ not only interfered with adenovirus replication but, in addition, enhanced its oncolytic properties. To evaluate the in vivo anticancer effect, athymic mice bearing glioma xenografts (5 x 10(5) U87 MG cells/animal) received a single intratumoral injection of ICOVIR-5 (10(7) PFU/animal). RAD001 was given as a regimen of 5 mg/kg 5 days per week until the end of the experiment and TMZ was administered for 5 days at 7.5 mg/kg/mice. Of significance, combination of ICOVIR-5 with RAD001 or TMZ showed a potent anti-glioma effect in vivo, resulting in a dramatic extension of the median animal survival and in 20-40% animals becoming free of disease beyond 90 days.

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.

Cytotoxin and pyrogenic toxin superantigen gene profiles of Staphylococcus aureus associated with subclinical mastitis in dairy cows and relationships with macrorestriction genomic profiles.

A set of 84 Staphylococcus aureus isolates collected from the milk of cows with subclinical mastitis in Asturias (a cattle region of Spain) and six control strains were tested for sequences of genes encoding hemolysins (hla, hlb, hld, hlg, and hlg-2), leukotoxins (lukPV, lukM, and lukED), toxic shock syndrome toxin (tst), and enterotoxins (sea to see, seg to ser, and seu) by conventional and multiplex PCR. It was found that 84, 83, 11, and 39 isolates carried some type of hl, luk, tst, or se gene, respectively, which were arranged in 14 exotoxin genotypes. All of the isolates were negative for lukPV, hlg, sea, sed, see, sej, sek, sep, seq, and ser. Two gene groupings could be related with pathogenicity islands-[lukED, seg, sei, sem, sen, seo +/- seu] with Sabeta-1 and [tst, sec, sel] with SaPIbov, present in 45 and 13.1% of the isolates, respectively-while 11.9% of them carried both islands. Only one contained seb (together with upsilonSabeta-1), and another contained seh (together with lukED). The isolates were also analyzed by pulsed-field gel electrophoresis performed with SmaI. Thirty-nine SmaI profiles (similarity coefficient [S] = 0.94 to 0.21) were differentiated; 12, 1, and 10 of these, respectively, were generated by isolates presumptively carrying Sabeta-1, SaPIbov, or both. Five SmaI profiles (S > or = 0.8) formed a cluster, which contained 20 and 10 isolates carrying one (upsilonSabeta-1) or both islands. These data show the high frequency of genes encoding cytotoxins and pyrogenic toxin superantigens, their relationship with pathogenicity islands, and their distribution among a diversity of genetic types of S. aureus related to subclinical mastitis.

Mastectom´çia de urgencia por Sarcoma de mama / No disponible

El sarcoma phyllodes es una neoplasia poco común, que suponemenos del 1 % de los tumores malignos de la mama.Se presenta como una neoformación de dimensiones variables,a menudo voluminosa.El tratamiento es la exéresis amplia o la mastectomía simple. Lalinfadenectomía axilar rutinaria es innecesaria.Presentamos un caso de sarcoma phyllodes gigante que precisótratamiento quirúrgico urgente(AU)

Sarcoma phyllodes is an uncommon neoplasm that supposes<1 % of all malignant breast tumors.It presents like a lump of variable dimension, often voluminous.The treatment is the wide local excision or simple mastectomy.Routine axilar linfadenectomy is unnecesary.We present a case of gigantic sarcoma phyllodes that neededurgent surgical treatment(AU)

Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks.

Three food poisoning restaurant outbreaks due to Staphylococcus aureus, occurring during June-October 2002 in the Principality of Asturias (PA), Spain, provided the basis for investigating some aspects of the molecular epidemiology of this organism. The methods applied to identify strains and lineages included multiplex-polymerase chain reaction (PCR) to detect nine enterotoxin (se) genes, and three DNA fingerprinting procedures: pulsed-field gel electrophoresis (PFGE) with SmaI, randomly amplified polymorphic DNA (RAPD) with two selected primers, and plasmid restriction analysis with HindIII. Thirty-two isolates were differentiated into three non-se and 12 se strains, which were outbreak-specific, except for one that was represented in two of the outbreaks. In outbreak 1, the 16 food isolates analyzed had sec, seg and sei genes and generated a distinctive DNA fingerprint, being assigned to a single strain. This strain could be categorized as endemic in the PA and associated to manually handled dairy products and nasal carriers. In outbreak 2, the four food isolates analyzed fell into three strains, each one displaying a different se-gene profile (sea, sec and seg-seh-sei) and a distinctive DNA fingerprint. In outbreak 3, the five food isolates tested fell into four seg-sei strains generating identical RAPD but different PFGE and plasmid profiles, and one sea strain also collected from two nasal carriers. This last strain had also been found in manually handled vegetables in outbreak 2, and it belongs to a not very frequently found sea lineage in the PA. Multiplex-PCR to detect se genes together with the three applied DNA fingerprint typing procedures proved therefore to be useful tools in subclassifying S. aureus for epidemiological purposes.

Thiopurine methyltransferase activity in a Spanish population sample: decrease of enzymatic activity in multiple sclerosis patients.

The present study was performed in order to obtain the thiopurine methyltransferase (TPMT) activity frequency distribution histogram in a Spanish population. A total of 3640 Spanish clinical laboratory samples were evaluated, which included 1249 patients with Crohn's disease, 589 with ulcerative colitis, 348 with multiple sclerosis (MS), 487 with several autoimmune diseases different from the above-mentioned diseases and 967 a donor group. We have measured the TPMT activity in red blood cells (RBCs) by a radiochemical method, using S-adenosyl-L-[methyl-3H]methionine as methyl donor. The different groups present in their entirety a normal distribution histogram and a wide range of TPMT activity from 0 to 41 U/ml RBCs. The differences found between the Spanish population TPMT activity frequency distribution histogram and the pattern previously described in a North American population were not due to azathioprine treatment or gender. The effect of autoimmune diseases on TPMT activity was evaluated: the enzymatic activity was similar in the donor group (19.9 +/- 6.3 U/ml RBCs) and in the patients with Crohn's disease (20.0 +/- 5.8 U/ml RBCs) and ulcerative colitis (19.7 +/- 6.1 U/ml RBCs); however, it decreased significantly (p<0.0001) in MS patients (17.1 +/- 6.1 U/ml RBCs) with respect to the donor group. In conclusion, our results show that the Spanish population TPMT distribution is closer to that of the Jewish population of Israel than to North American populations, and that in MS the enzymatic activity of TPMT decreases significantly. This observation may take into account the usage of azathioprine as therapeutic agent in Spanish MS patients.

Clima organizacional y estrés en una unidad de alto riesgo / Organizational atmosphere and stress in a high risk hospital department

Objetivo: Conocer y medir el clima organizacional y el estrés percibido por los profesionales que trabajan en el Servicio de Urgencias del Hospital de Cabueñes de Gijón, determinando los factores que los definen. Métodos: En el estudio participaron 80 profesionales (médicos, DUE/ATS, MIR, auxiliares de enfermería, servicio de información de urgencias, celadores, auxiliares administrativos) de un total de 145 que trabajan en el Servicio de Urgencias. El diseño del estudio fue transversal de carácter analítico descriptivo, utilizando un cuestionario confidencial. El análisis estadístico incluye una estadística básica, un Chi cuadrado y un análisis factorial de componentes principales. Resultados: El perfil del profesional estudiado es una mujer de 36 años, médico, casada, con contrato fijo, con experiencia laboral de 11 años y de 5 en el Servicio de Urgencias y que trabaja a tres turnos. No ha experimentado baja laboral en el último año y su trabajo afecta moderadamente a su vida familiar. Valora positivamente el clima organizacional, es extravertida, estable emocionalmente y no parece presentar estrés. Encontramos relaciones significativas entre el clima organizacional, la personalidad y el estrés, así como entre estas variables y las variables sociodemográficas y socio-laborales. Se han obtenido tres factores que definen el clima organizacional y cinco el estrés con una representatividad del 74,3 por ciento y 65,4 por ciento, respectivamente. Conclusiones: Deberían introducirse modificaciones en la dinámica de la organización y en la estrategia empresarial, por parte de los equipos de dirección y de los responsables de recursos humanos (AU)

Adenovirus-mediated wt-p16 reintroduction induces cell cycle arrest or apoptosis in pancreatic cancer.

Pancreatic cancer has long carried poor prognosis. The development of new therapeutic approaches is particularly urgent. Inactivation of the tumor-suppressor gene p16(INK4a/CDKN2), a specific inhibitor of the cyclin-dependent kinases CDK4 and CDK6, is the most common genetic alteration in human pancreatic cancer, making it an ideal target for gene replacement. Here we transfected tumor cells using a recombinant adenovirus containing the wt-p16 cDNA (Ad5RSV-p16). The overexpression of p16 decreased cell proliferation in all four human pancreatic tumor cell lines (NP-9, NP-18, NP-29, and NP-31). However, G1 arrest and senescence were observed in only three. In contrast, the fourth (NP-18) showed a significant increase in apoptosis. This differential behavior may be related to the differences found in the expression level of E2F-1. Experiments on subcutaneous pancreatic xenografts demonstrated the effectiveness of p16 in the inhibition of pancreatic tumor growth in vivo. Taken together, our results indicate that approaches involving p16 replacement are promising in pancreatic cancer treatment.

Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method.

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

Transfer of E2F-1 to human glioma cells results in transcriptional up-regulation of Bcl-2.

Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-x(L) by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.

Enterotoxin production and DNA fingerprinting in Staphylococcus aureus isolated from human and food samples. Relations between genetic types and enterotoxins.

A total of 224 Staphylococcus aureus strains from human carriers (110 strains) and manually handled foods (114 strains) collected in the Principality of Asturias, Spain over 1995-1999 were analysed for the production of enterotoxins (SEs) A, B, C, and D by a reversed passive latex agglutination test and by amplification of ent genes (A, B, C, D, E, and J) using PCR. Sixty-two strains were enterotoxigenic and a good relation between detection of SEs and their ent genes was found. No strain carried entE and all strains producing SED carried entD and entJ genes. Among the enterotoxigenic strains the percentages registered were 29, 8, 35, 18, 2, 2, and 6 for SEA, SEB, SEC, SEDJ, SEAC, SEADJ and SECDJ, respectively. DNA fingerprinting of 77 strains (the SE prototypes, 62 enterotoxigenic and 10 non-enterotoxigenic [NE]) was carried out by randomly amplified polymorphic DNA using two selected primers independently. Combining results from both primers, 10 genetic types were defined, which showed a different degree of relationship (similarity coefficient: 0.9-0.36) and were clustered into three lineages. One lineage clustered five genetic types and a wide diversity of strains, mainly SEA, SEB, SEDJ, and NE. Another lineage clustered only SEC, SECDJ and NE strains. These two lineages showed a low genetic relationship and appeared as endemic in healthy humans living in the Principality of Asturias. The third lineage included only the prototype strains for SEA and SEE.