Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Local treatment with deep percutaneous electrochemotherapy of different tumor lesions: pain relief and objective response results from an observational study.

OBJECTIVE: The aim of this investigation focuses on the evaluation of the efficacy of deep-seated Electrochemotherapy (ECT) in terms of pain relief and local objective response, in pre-treated patients with neither further available pharmacological treatments nor eligible for surgery. PATIENTS AND METHODS: Deep percutaneous ECT has been performed in 20 patients subjected to systemic anaesthesia. Bleomycin was administrated intravenously before the application of the electrical pulses on the target area, employing multiple single needles depending on the size and location of the target tumor. RESULTS: Pain assessment based on Visual Analogue Scale showed significant pain relief one month after treatment in all patients, reducing from 7.5 to 3 as a median value (p-value at Wilcoxon test <0.001). Local symptom-free survival median value was 5.5 months. At the first follow-up (1-2 months), a local disease control rate (LDCR) was observed in 19/20 (95%) patients: complete responses in 2 (10%), partial responses in 8 (40%) and stable disease in 9 (45%). Local progression-free survival median value was 5.7 months. Overall, no major adverse effects were observed. CONCLUSIONS: Our study indicates that deep percutaneous ECT can produce a significant pain reduction and a high LDCR in different tumor lesions, for anatomical site or histotype. In particular, ECT has demonstrated to be effective in various histotypes and deep-seated tumor lesions never treated before by this approach giving a new chance to physicians for reducing oncological pain in patients not eligible to other therapeutic routes. The innovative peculiarity of our study was the successful application of deep percutaneous ECT on adrenal metastasis, malignant pleural mesothelioma, uterine leiomyosarcoma and the uncommon case of a male müllerian tumor.

Mastitis in the transition period: identification of potential blood markers / Mastite no período de transição: identificação de potenciais marcadores no sangue

This study identified potential blood markers associated with mastitis in dairy cows under different dry therapies during the transition period, using a logistic regression model. Thirty-four Holstein dairy cows were divided into three groups: untreated controls (13 cows, 42 quarters); animals that received an antimicrobial at drying-off (10 cows, 40 quarters); and animals that were administered an internal teat sealant at drying-off (11 cow, 44 quarters). Blood and quarter milk samples were collected 60 days before the expected day of calving, on the day of calving, and three, seven, 15, 21, and 30 days after calving. Milk samples were submitted for bacteriological analysis and somatic cell count. Blood samples were collected for analyses of the following: the erythrogram and leucogram; plasm fibrinogen concentration; hepatic and renal functions; metabolic profile; serum calcium and phosphorous levels; total serum protein and albumin concentrations. The concentration of total serum proteins was associated with a high somatic cell count. Similarly, the concentrations of total serum proteins and triglycerides were associated to milk bacteriological positive samples during the transition period. Thus, the occurrence of mastitis in dairy cows during the transition period was greater in animals that showed higher concentrations of serum total proteins and triglycerides, in contrast to the use of dry cow therapy.(AU)

O presente estudo identificou potenciais marcadores sanguíneos associados tanto à mastite, durante o período de transição em vacas leiteiras submetidas à antimicrobianoterapia, quanto à utilização do selante interno de teto na secagem por modelo de regressão logística. Trinta e quatro vacas da raça Holandesa foram divididas em três grupos experimentais, a saber: 13 animais (42 quartos mamários) que não receberam tratamento na secagem; 10 animais (40 quartos mamários) tratados por via intramamária com uma bisnaga do antimicrobiano para vaca seca à base de cefalônio anidro após a última ordenha em cada quarto mamário; e 11 animais (44 quartos mamários) que receberam, por via intramamária, uma bisnaga de selante à base de subnitrato de bismuto após a última ordenha em cada quarto mamário. As amostras de leite e sangue foram coletadas 60 dias antes da data prevista do parto, na secagem, e no dia do parto e após três, sete, 15 e 21 dias após o parto. As amostras de leite foram utilizadas para o exame microbiológico e a determinação da contagem de células somáticas. As amostras de sangue foram utilizadas para determinação do eritrograma, leucograma, fibrinogênio plasmático, funções hepáticas e renais, perfil metabólico, proteína total e albuminas séricas e concentração sérica de cálcio e fósforo. A concentração total de proteínas séricas foi associada à alta contagem de células somáticas. Similarmente, a concentração total de proteínas séricas e triglicérides foi associada ao isolamento de patógenos causadores de mastite nas amostras de leite durante o período de transição. Desse modo, conclui-se que vacas leiteiras com concentrações séricas maiores de proteína total e triglicérides têm maior chance de apresentar mastite durante o período de transição; em contraste, o uso da antimicrobianoterapia de vaca seca reduz esse risco.(AU)

Production of rotavirus-like particles in Spodoptera frugiperda larvae.

A strategy for the production of virus-like particles (VLPs) from simian rotavirus in larvae of the lepidopteran Spodoptera frugiperda is described. VP2 and VP6 coding sequences were co-expressed in larvae co-infected with recombinant baculovirus and these structural proteins self-assembled into VLPs that were secreted and accumulated in the haemolymph. Under electron microscopy, VLPs produced in larvae were indistinguishable from those produced in Sf9 insect cell cultures. The results showed that it is possible to obtain rotavirus VLPs in larvae reducing significantly the costs of production, making this approach an alternative for the manufacture of live rotavirus vaccines.

Chiral capillary electrophoresis and nuclear magnetic resonance investigation on the structure-enantioselectivity relationship in synthetic cyclopeptides as chiral selectors.

In the present work, synthetic cyclohexa- and cycloheptapeptides previously singled out by a combinatorial chemistry approach have been evaluated as chiral selectors in capillary electrophoresis. By applying the countercurrent migration technique and employing a new adsorbed coating, a series of dinitrophenyl amino acids as well as some chiral compounds of pharmaceutical interest have been evaluated for enantiorecognition. The results thus obtained led to a deeper investigation of the chiral discrimination process, by carrying out nuclear magnetic resonance (NMR) studies on selected cyclopeptide-analyte complexes. These studies shed light on the chemical groups involved in the analyte-selector interaction and provided useful information for a wider application of these cyclopeptides in the separation of other drug enantiomers.

[Recombinant human erythropoietin in anemia associated with pediatric cancer: study of the identification of predictors of response]. / Eritropoyetina recombinante humana en la anemia asociada al cáncer infantil: estudio de la identificación de factores predictivos de la respuesta.

OBJECTIVE: Cancer is frequently associated with anemia and may be related to inadequate erythropoietin production. The objective of this study was to assess the efficacy and safety of recombinant human erythropoietin (r-HuEPO) in increasing hemoglobin levels and reducing the need of blood transfusions in children with cancer, as well as to identify predictors of the response to r-HuEPO. PATIENTS AND METHODS: A pilot trial was performed including 25 patients with solid malignant tumors receiving cyclic chemotherapy, r-HuEPO was administered subcutaneously, 150 U/kg/day 5 times a week for 12 consecutive weeks. Response was defined as the achievement of a Hb increase of at least 2 g/dl without blood transfusions. Patients were compared to history matched controls. RESULTS: Baseline parameters were similar (p > 0.05) in both groups. The mean Hb increase was greater in the r-HuEPO group compared to controls (2.6 vs 0.1 g/dl; p < 0.001) and the mean units of blood transfused were lower in treated patients (0.32 vs 2.36; p < 0.05). Response to r-HuEPO was achieved in 19 (76%) patients. Low baseline erythropoietin levels and an increase in Hb of at least 0.5 g/dl after 3-4 weeks of treatment defined a greater probability of response. No serious adverse effects were observed. CONCLUSIONS: r-HuEPO treatment represents a safe and effective means of increasing Hb levels and reducing blood requirements in pediatric cancer patients receiving chemotherapy and the prediction of response may be based on baseline serum erythropoietin levels and on the variation of Hb levels after 3-4 weeks of treatment.

Increased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction.

Telomeric Repeat Amplification Protocol (TRAP) is a sensitive procedure to measure telomerase activity in small samples of cell or tissue extracts. Due to the strict correlation between high levels of telomerase activity and neoplastic transformation, TRAP assay could provide an important diagnostic marker of malignancy. Although the original TRAP assay is very sensitive and some improvements have been described, occasional artifacts still persist in the modified procedures. Here we describe how changes in the sequence of the primer used for the amplification step enhance the reproducibility and sensitivity in the TRAP assay.

Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: complementation of AMCV p33 and p92 replication-defective mutants.

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

Dilataciones quisticas de la via biliar. / Cystic dilatations of the biliary tract

Las dilataciones quisticas de la via biliar son alteraciones congenitas que si bien no constituyen una patologia frecuente, conviene que el cirujano las conozca para que al enfermo se le resuelva su problema de la mejor manera y en una sola operacion

Oclusion experimental de la arteria mesenterica superior. Accion del oxigeno por via digestiva. / Experimental occlusion to the superior mesenteric artery. Oxygen action by digestive way

La oclusion experimental de la arteria mesenterica superior fue realizada en 9 perros para obtener una isquemia intestinal aguda. Este lote fue dividido en 3 grupos: I. de control, II. con oxigenacion endoluminal gastrica y III. con oxigenacion endoluminal yeyunal. Para evaluar el efecto terapeutico del oxigeno se controlo la presion arterial media, el funcionamiento cardiaco con monitoreo electrocardiografico, y los valores de la glucemia, enzimas y gases sanguineos en condiciones basales y a las 2 y 5 horas de la oclusion vascular. Para el estudio histopatologico se tomaron biopsias de la mucosa intestinal y otros parenquimas. Los resultados obtenidos indicaron una perfecta adaptacion a la difusion del oxigeno a traves de la mucosa gastroenterica y su accion protectora en la evolucion de la isquemia intestinal. Se comprobo una correlacion entre los cambios histologicos de la mucosa intestinal y los parametros hemodinamicos y humorales estudiados. Con el oxigeno endoluminal las alteraciones histologicas celulares fueron de escasa intensidad y, consiguientemente, la homeostasis o equilibrio organico general se mantuvo sin modificaciones durante el tiempo que duro la experimentacion

Effect of a single oral dose of oxymetholone on the metabolism of human erythrocytes.

Androgenic steroids have been shown to enhance erythrocyte 2,3-DPG production in vivo and in vitro, and to stimulate the pentose shunt oxidative reactions in vitro. Furthermore, a 3 beta- and a 17 beta-hydroxysteroid dehydrogenase have been identified in red cells. The present study was carried out to explore a cumulative effect of androgens on glycolysis and androgen reduction in human erythrocytes in vivo following a single 50 mg oral dose of 17 beta-hydroxy-2 (hydroxymethylene)-17 methyl-5 alpha-androstan-3-one (oxymetholone). The rate of erythrocyte glycolysis was measured by quantitative determination of: fructose-1,6-diphosphate (FDP); dehydroxyacetone phosphate (DAP); 2,3-diphosphoglycerate (2,3-DPG); adenosine triphosphate (ATP); and lactate. Serum and erythrocyte steroids were separated by thin layer chromatography. The reduction of 5 alpha-androstan-17 beta-ol-3-one by red cell hemolysate was measured in the presence of NADPH as an index of 3(17)beta-hydroxysteroid dehydrogenase activity. Our results show that oxymetholone administration is followed by the appearance of an unidentified steroid fraction in chromatograms of serum and erythrocytes, simultaneously with the enhancement of glycolysis and of hydroxysteroid dehydrogenase activity in erythrocytes. A direct effect of androgen on erythrocyte metabolism, which is independent of the hormone erythropoietic effect, is discussed.