Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC.

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

Can we withdraw anticoagulation in patients with antiphospholipid syndrome after seroconvertion?

The current mainstay of treatment in patients with thrombotic antiphospholipid syndrome (APS) is long-term anticoagulation, mainly with Vitamin K antagonist agents. Some recently available studies have created new ground for discussion about the possible discontinuation of anticoagulation therapy in patients with a history of thrombotic APS in whom antiphospholipid antibodies (aPL) are not detected any longer (i.e. aPL seroconversion). We report the main points discussed at the last CORA Meeting regarding the issue whether or not anticoagulation can be stopped after aPL seroconversion. In particular, we systematically reviewed the available evidence investigating the clinical outcome of APS patients with aPL seroconversion in whom anticoagulation was stopped when compared to those in whom therapy was continued regardless the aPL profile. Furthermore, the molecular basis for the aPL pathogenicity, the available evidence of non-criteria aPL and their association with thrombosis are addressed. To date, available evidence is still limited to support the indication to stop oral anticoagulation therapy in patients with a previous diagnosis of thrombotic APS who subsequently developed a negative aPL profile. The identification of the whole risk profile for cardiovascular manifestations and possibly of a second level aPL testing in selected patients with aPL might support the eventual clinical decision but further investigation is warranted.

Oxacillin-resistant and multidrug-resistant Staphylococcus aureus in Lima, Peru.

In a hospital in Lima, Peru, a review of 103 Staphylococcus aureus infections was conducted during 2002. The prevalence of oxacillin-resistant S. aureus strains was 68%; 25% of strains were resistant to multiple drugs. Previous use of antibiotics and undergoing a surgical procedure during the current hospital stay were associated with the presence of an oxacillin-resistant S. aureus strain.

Effects of 3-OM-dopa on monoamine metabolism in rat brain.

We studied the effects of 3-methoxy, 4-hydroxy-phenylalanine (3-OM-dopa) on monoamine turnover, L-3-4-dihydroxy-phenylalanine (L-dopa) reversal of catecholamine depletion induced by alpha-methyl-paratyrosine (alpha-MT), and the passage of L-dopa across the blood-brain barrier of the rat brain. 3-OM-dopa did not affect monoamine turnover but interfered with restoration of dopamine levels by L-dopa. In addition, systemic 3-OM-dopa decreased brain uptake of 14C-L-dopa. High 3-OM-dopa blood levels may interfere with the therapeutic response to L-dopa in parkinsonism.

Nitric oxide induces differentiation in the NB69 human catecholamine-rich cell line.

The nitric oxide (NO) donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), induced differentiation of human neuroblastoma NB69 cells to a dopamine phenotype, as shown by phase-contrast microscopy and tyrosine hydroxylase (TH) immunocytochemistry. NB69 cells were treated with 50 to 750 microM SNAP in serum-free-defined medium for 24 h. SNAP treatment did not increase the number of necrotic or apoptotic cells. However, a decrease in the number of viable cells was observed at 750 microM SNAP. In addition, a decrease in (3)H-thymidine uptake was detected at the highest dose of SNAP. An increase in the antiapoptotic Bcl-2 and Bcl-xL protein levels and a decrease in the proapoptotic Bax and Bcl-xS protein levels were also detected by Western blot analysis after SNAP treatment. At low doses (50-125 microM), SNAP induced an increase in catecholamine levels, (3)H-dopamine uptake, TH activity and monoamine metabolism, while a decrease in all these parameters was observed at high doses (250-750 microM). The TH protein content, analyzed by Western blot, remained unchanged in SNAP-treated cells throughout the range of doses studied, when compared with the control group. SNAP produced a dose-dependent decrease in the glutathione (GSH) content of the culture medium, without altering intracellular GSH. In addition, cGMP levels and nitrite concentration, measured in the supernatant of SNAP-treated cells, increased in a dose-dependent manner, as compared to control levels. The guanylate cyclase inhibitor lH-[1,2, 4]oxadiazolo[4,3a]quinoxaline-l-one (ODQ) did not revert the SNAP-induced effect on (3)H-dopamine uptake to control values. These results suggest that NO, released from SNAP, induces differentiation of NB69 cells and regulates TH protein at the post-transcriptional level through a cGMP-independent mechanism.

In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Time course of phorbol ester-induced contraction and protein kinase C activation in rat aorta.

This study investigates the relationship between the rate of phorbol ester-induced contraction of intact rat aorta and protein kinase C activation, as assessed by the translocation of protein kinase C from the cytosolic to the particulate fraction. Aorta was exposed to Ca(2+)-free physiologic salt solution prior to phorbol ester to prevent Ca(2+)-induced protein kinase C translocation during tissue homogenization. Phorbol myristate acetate, as well as phorbol dibutyrate, decreased cytosolic and/or increased particulate protein kinase C activity as early as 5 s following phorbol ester addition, which was prior to, or coincident with, the onset of contraction. These results suggests that phorbol ester-induced contraction of intact vascular smooth muscle is associated in a time-dependent manner with protein kinase C activation.

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats.

This study investigates the effects of phorbol dibutyrate (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity from the cytosolic to the particulate fraction, in aortas and mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The basal distribution of PKC activity between the cytosolic and particulate fractions of SHR and WKY aortas, and mesenteric arteries, was not significantly different. PDB induced a concentration-dependent decrease in cytosolic PKC activity in SHR and WKY aortas. PDB (0.01 microM) decreased cytosolic PKC activity to a greater magnitude in SHR aorta as compared to WKY aorta, while 1.0 microM PDB decreased cytosolic PKC activities to similar magnitudes in SHR and WKY aortas, and mesenteric arteries. These results suggest that the increased sensitivity of SHR vessels to contraction by phorbol esters may be due, at least in part, to the greater sensitivity of PKC in these vessels to phorbol ester activation.

Effects of protein kinase C activation on norepinephrine-induced phosphatidylinositide hydrolysis in intact rat aorta.

The purpose of this study was to investigate the role of protein kinase C in the regulation of alpha 1-adrenoceptor-mediated phosphatidylinositide hydrolysis in intact vascular smooth muscle. Phorbol myristate acetate (0.1 and 1 microM) and staurosporine inhibited and potentiated, respectively, norepinephrine-induced inositol phosphate formation in intact rat aorta. In contrast, 30 microM prostaglandin F2 alpha, which activated protein kinase C to a similar magnitude as 1 microM phorbol myristate acetate, was without effect on norepinephrine-induced inositol phosphate formation. These results suggest that protein kinase C activated in response to physiologic agonists, but not in response to phorbol esters, may be compartmentalized within the smooth muscle cell.

Phosphorylation of initiation factor 2 alpha subunit and apoptosis in Ca2+ ionophore-treated cultured neuronal cells.

The initial step of protein synthesis is regulated by the eukaryotic initiation factor 2 (eIF-2) whose phosphorylation in the alpha subunit by specific kinases, as double-stranded RNA-dependent protein kinase (PKR), produces an inhibition of the translational rates. Besides, intracellular Ca2+ mobilization has been associated with an increase in the PKR activity. Our results show, in primary neuronal cultures, that the treatment with the Ca2+ ionophore A23187 induces an increase in the phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) and inhibition of protein synthesis. Those biochemical changes run parallel to the appearance of specific apoptosis characteristics such as cell shrinkage, segmentation of chromatin into small round bodies, and cleavage of DNA into 180 bp multimers. These results indicate that one of the targets during the process of apoptosis induced by the rise in the intracellular Ca2+, could be eIF-2 factor.

Methodological considerations for the measurement of protein kinase C translocation in intact smooth muscle.

This study investigated several potential artifacts that may influence agonist-induced distribution of protein kinase C (PKC) activity between cytosolic and membrane fractions of intact smooth muscle. Protein kinase C activity in the membrane fraction prepared from rat aorta exposed to phorbol myristate acetate (PMA) was only partially extracted by 0.2% Triton (T)X-100, while 1% TX-100, or repeated 0.2% TX-100 extractions completely extracted PKC activity. Extraction of PKC activity from the membrane fraction with TX-100 concentrations of 0.2% or higher was problematic, however, since TX-100 concentrations as low as 0.04% nearly abolished Ca(2+)+ phosphatidyserine+diolein-induced phosphorylation of histone substrate in the PKC assay. Substitution of PMA for diolein, however, restored histone phosphorylation to the level observed in the absence of TX-100. Triton X-100 concentrations as low as 0.025% also abolished Ca(2+)-induced histone phosphorylation, while Ca(2+)+ phosphatidylserine-induced phosphorylation was little affected. In contrast to our previous demonstration that exposure of rat aorta to phorbol ester increased PKC activity in the membrane fraction in aorta washed in Ca(2+)-free solution following phorbol ester exposure (Chuprun et al., Am J Physiol 261:C675-C684, 1991; Bazan et al., Eur J Pharmacol-Molec Pharmacol Section 227:343-348, 1992), PMA decreased PKC activity in the initial 0.2% TX-100 extraction of the membrane fraction in the absence of tissue wash in Ca(2+)-free solution following PMA exposure. This study, along with our previous reports, suggest that partial PKC extraction from the membrane, and Ca(2+)-dependent homogenization-induced translocation of PKC from the cytosol to the membrane fraction, may complicate measurements of agonist-induced PKC translocation. The reliability of PKC assays in crude fractions may be increased in the presence of TX-100, due to the ability of TX-100 to inhibit Ca(2+)-induced phosphorylation, and through the substitution of PMA for diolein, which maximally stimulates PKC in the presence of detergent.

Effects of phorbol esters on contraction and protein kinase C activation in rabbit aorta.

This study investigates the relationship between the contractile efficacy of phorbol esters and their ability to activate protein kinase C in intact rabbit aorta. Phorbol dibutyrate (PDB) induced a maximal contraction approximately 3.5-fold greater than that to phorbol myristate acetate (PMA). The magnitude of maximal PDB- and PMA-induced contraction correlated with the magnitude of protein kinase C activation, as assessed by the decrease in cytosolic protein kinase C activity. KCl (60 mM) did not potentiate the PMA-induced decrease in cytosolic protein kinase C activity. These results suggest that the lack of efficacy of PMA is due to its inability to activate protein kinase C in the intact rabbit aorta. It is speculated that the different contractile efficacies of phorbol esters result from selective activation of protein kinase C isoforms, and that the amounts of these isoforms varies amongst vascular tissues.

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment.

Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGF-expanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs.

[Nonthermal levels of electric currents applied in capacitive electric transfer therapy provokes partial cytotoxic effects in human neuroblastoma cultures]. / Niveles atérmicos de corrientes eléctricas usadas en terapia por transferencia eléctrica capacitiva inducen efectos citotóxicos parciales en cultivos de neuroblastoma humano.

The present study investigates the cellular response to weak, sine wave, 0.5-MHz electric currents. The experimental exposure to identical signals at an intensity high enough as to significantly increase the temperature in target tissues, has provided positive responses in clinical treatments of tumors with capacitive electric transfer (CET) thermal therapy. The present results show that the in vitro exposure to CET signals at athermal doses causes cytotoxic effects in human neuroblastoma cells. Such a response seems to be due to signal-induced alterations in the cell cycle. As a whole, the results suggest that the potential therapeutic effects of the CET strategy could be due to the thermal response of the tissues to the currents, added to an athermal response of the cells to the electric current itself.

[Cytomegalovirus pneumonitis in immunocompetent adults: report of a case]. / Neumonitis por citomegalovirus en pacientes adultos inmunocompetentes: a propósito de un caso.

Cytomegalovirus produces congenital and acquired infections most of them asymptomatic. The percentage of clinical cases with visceral affection increases in patients suffering from a deficiency cellular immunity, showing the great importance of this defensive system in the control of the infection. This case presents an infection by Cytomegalovirus with visceral affection in an immunocompetent adult host. This is a mean duration of fever as the only symptom with lymphocytosis, biochemical hepatic changes and an interstitial pulmonary radiologic image. We think this case is a matter of considerable interest because it is a challenge in the study of the pathogenic mechanisms of this infection.