New europium-doped carbon nanoparticles showing long-lifetime photoluminescence: Synthesis, characterization and application to the determination of tetracycline in waters.

The growing appearance of antibiotic-resistant strains of microorganisms originated from the widespread use and ubiquitous presence of such drugs is a major concern in the world. The development of methodologies able to detect such substances at low concentration in real water samples is mandatory to overcome this problem. Europium(III) is known to form complexes with tetracycline (TC) with photoluminescent characteristics useful for TC determination. In the present work, we synthesized for the first time carbon nanoparticles (CN) showing delayed photoluminescence using a Europium(III) doping synthesis. The new material (PCNEu) was characterized both morphologically and spectroscopically, showing an analytical photoluminescent signal in presence of TC, arising from the 5D0→7F2 transition of europium, one hundred times higher than that of the europium salt alone in presence of the antibiotic. This enhancement is a consequence of the amplifying effect exerted by nanoparticle structure itself, leading to an efficient synergistic "antenna effect" in the system PCNEu - TC. The analytical signal is affected both by pH and the nature of the buffer used, and it allows the detection of tetracycline in waters with a limit of detection of 2.18 nM and recoveries between 90 and 110%. The analytical performance of the developed methodology enables having lower limits of detection than other luminescent and chemiluminescent reported methodologies.

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Effects of protein malnutrition on muscle fibers of the brachial biceps and medial pterygoid of Wistar rats

Protein malnutrition is a public health problem, and in childhood, it can lead to muscle deficits. Here, our objective was to evaluate the effects of malnutrition upon the muscle fibers in the medial pterygoid and braquial biceps. Ten just weaned rat pups that had been born to parents fed a nourished or malnourished diet (N = 5 per group) were studied. The medial pterygoid and braquial biceps muscles were removed and crosssectioned, and histological staining with picrosirius and histochemistry reaction with nicotinamide adenine dinucleotide - tetrazolium reductase (NADH-tr) were performed. The samples stained with picrosirius were observed under polarized light, and from the qualitative analysis, we observed that type I collagen fibers were only present in the braquial biceps muscles of the nourished animals. The NADH-tr reaction indicated that the pterygoid muscle specimens from the malnourished pups lacked intermediate muscle fibers. The crosssectional area of the muscle was lower in the malnourished group than in the nourished group. The density of muscle fibers was higher in the malnourished group than in the nourished group. The consequences of malnutrition were visible when comparing the muscles. We concluded that the differences in daily muscle action along with the differences in embryological origin are instrumental in establishing the results.

Cell volume and ionic transport systems after cold preservation of coronary endothelial cells.

BACKGROUND: Hypothermia-induced changes in cell volume and ionic transport systems of coronary endothelial cells may play a role in the development of coronary artery disease in cardiac transplant recipients. METHODS: Coronary endothelial cells were incubated in University of Wisconsin solution or culture control medium for up to 48 hours at 4 degrees C. Parallel control cultures were incubated at 37 degrees C. Na/K-ATPase and Na/K/Cl cotransport activities were determined as ouabain- and furosemide-sensitive 86Rb+ uptake, respectively. Cell volume changes and cell death were analyzed by a FACScan flow cytometer and the release of lactate dehydrogenase, respectively. RESULTS: Coronary endothelial cells stored in University of Wisconsin solution up to 6 hours showed an increased Na/K-ATPase activity compared to control cells, whereas no changes were observed in Na/K/Cl cotransport activity or cell volume. Long-term preservation (24 and 48 hours) was associated with a partial loss of cell viability, as demonstrated by lactate dehydrogenase release, and dramatic alterations in ionic transport system activities. CONCLUSIONS: University of Wisconsin solution seems to prevent coronary endothelial cells Na/K/Cl cotransport activity changes during cold preservation, which could alter cell volume regulation and cause cell injury.

Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats.

OBJECTIVE: To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting. RESULTS: Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine. CONCLUSIONS: Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression.

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.

Expresión como proteínas de fusión de las subunidades PB1 y PB2 de la polimerasa del virus de la influenza / Expression how proteins of fusion of the subunits PB1 and PB2 of the influenza viral of polymerase

Los cADNs de los genes que codifican las proteínas PB1 y PB2 de la ARN polimerasa del virus de la influenza, se clonaron en fase en un sitio distal del ATG que codifica la proteína 10 del bacteriógafo T7, en el vector de expresión pAR3040, bajo el control de un promotor de la ARN polimerasa de T7. Los plásmidos recombinantes se introdujeron en la cepa bacteriana. E coli BL21 (DE3)plysS, que contiene en su genoma el gen de la ARN polimerasa del fago T7, bajo el control de un promotor (lac uv5) inducible por IPTG. La inducción de la ARN polimerasa del fago T7 en las células hospederas, resultó en la expresión de las proteínas PB1 y PB2 en forma de proteínas de fusión. Las proteínas expresadas se acumularon en grandes cantidades en el interior de las células bacterianas y fueron recuperadas de los lisados en forma de precipitados insolubles.

Venezuelan haemorrhagic fever.

An outbreak of severe haemorrhagic illness began in the municipality of Guanarito, Portuguesa State, Venezuela, in September, 1989. Subsequent detailed study of 15 cases confirmed the presence of a new viral disease, designated Venezuelan haemorrhagic fever. Characteristic features are fever, toxicity, headache, arthralgia, diarrhoea, conjunctivitis, pharyngitis, leucopenia, thrombocytopenia, and haemorrhagic manifestations. Other features include facial oedema, cervical lymphadenopathy, nausea/vomiting, cough, chest or abdominal pain, and convulsions. The patients ranged in age from 6 to 54 years; all were residents of rural areas in central Venezuela, and 9 died. Infection with Guanarito virus, a newly recognised arenavirus, was shown by direct culture or by serological confirmation in all cases. Epidemiological studies suggest that the disease is endemic in some rural areas of central Venezuela and that it is rodent-borne. Venezuelan haemorrhagic fever has many similarities to Lassa fever and to the arenavirus haemorrhagic fevers that occur in Argentina and Bolivia.