Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.     

Marked differences between prone and supine sheep in effect of PEEP on perfusion distribution in zone II lung.

The classic four-zone model of lung blood flow distribution has been questioned. We asked whether the effect of positive end-expiratory pressure (PEEP) is different between the prone and supine position for lung tissue in the same zonal condition. Anesthetized and mechanically ventilated prone (n = 6) and supine (n = 5) sheep were studied at 0, 10, and 20 cm H2O PEEP. Perfusion was measured with intravenous infusion of radiolabeled 15-microm microspheres. The right lung was dried at total lung capacity and diced into pieces (approximately 1.5 cm3), keeping track of the spatial location of each piece. Radioactivity per unit weight was determined and normalized to the mean value for each condition and animal. In the supine posture, perfusion to nondependent lung regions decreased with little relative perfusion in nondependent horizontal lung planes at 10 and 20 cm H2O PEEP. In the prone position, the effect of PEEP was markedly different with substantial perfusion remaining in nondependent lung regions and even increasing in these regions with 20 cm H2O PEEP. Vertical blood flow gradients in zone II lung were large in supine, but surprisingly absent in prone, animals. Isogravitational perfusion heterogeneity was smaller in prone than in supine animals at all PEEP levels. Redistribution of pulmonary perfusion by PEEP ventilation in supine was largely as predicted by the zonal model in marked contrast to the findings in prone. The differences between postures in blood flow distribution within zone II strongly indicate that factors in addition to pulmonary arterial, venous, and alveolar pressure play important roles in determining perfusion distribution in the in situ lung. We suggest that regional variation in lung volume through the effect on vascular resistance is one such factor and that chest wall conformation and thoracic contents determine regional lung volume.     

Tricarboxylic Acid Cycle Activity Measured by <Superscript>13</Superscript>C Magnetic Resonance Spectroscopy in Rats Subjected to the Kaolin Model of Obstructed Hydrocephalus

Evaluating early changes in cerebral metabolism in hydrocephalus can help in the decision making and the timing of surgical intervention. This study was aimed at examining the tricarboxylic acid (TCA) cycle rate and ¹³C label incorporation into neurotransmitter amino acids and other compounds 2 weeks after rats were subjected to kaolin-induced progressive hydrocephalus. In vivo and ex vivo magnetic resonance spectroscopy (MRS), combined with the infusion of [1,6-¹³C]glucose, was used to monitor the time courses of ¹³C label incorporation into the different carbon positions of glutamate in the forebrains of rats with hydrocephalus as well as in those of controls. Metabolic rates were determined by fitting the measured data into a one-compartment metabolic model. The TCA cycle rate was 1.3 ± 0.2 μmoles/gram/minute in the controls and 0.8 ± 0.4 μmoles/gram/minute in the acute hydrocephalus group, the exchange rate between α-ketoglutarate and glutamate was 4.1 ± 2.5 μmoles/gram/minute in the controls and 2.7 ± 2.6 μmoles/gram/minute in the hydrocephalus group calculated from in vivo MRS. There were no statistically significant differences between these rates. Hydrocephalus caused a decrease in the amounts of glutamate, alanine and taurine. In addition, the concentration of the neuronal marker N-acetyl aspartate was decreased. ¹³C Labelling of most amino acids derived from [1,6-¹³C]glucose was unchanged 2 weeks after hydrocephalus induction. The only indication of astrocyte impairment was the decreased ¹³C enrichment in glutamine C-2. This study shows that hydrocephalus causes subtle but significant alterations in neuronal metabolism already early in the course of the disease. These sub-lethal changes, however, if maintained and if ongoing might explain the delayed and programmed neuronal damage as seen in chronic hydrocephalus.     

Access to new highly potent antileukemia,antiviral and antimalarial agents via hybridization of natural products (homo)egonol,thymoquinone and artemisinin

Hybridization of natural products has high potential to further improve their activities and may produce synergistic effects between linked pharmacophores. Here we report synthesis of nine new hybrids of natural products egonol, homoegonol, thymoquinone and artemisinin and evaluation of their activities against P. falciparum 3D7 parasites, human cytomegalovirus, sensitive and multidrug-resistant human leukemia cells. Most of the new hybrids exceed their parent compounds in antimalarial, antiviral and antileukemia activities and in some cases show higher in vitro efficacy than clinically used reference drugs chloroquine, ganciclovir and doxorubicin. Combined, our findings stress the high potency of these hybrids and encourages further use of the hybridization concept in applied pharmacological research.     

Assessment of genetic markers and glioblastoma stem-like cells in activation of dendritic cells

Glioblastoma (GBM) is the most common and aggressive intraparenchymal primary brain tumor in adults. The principal reasons for the poor outcomes of GBM are the high rates of recurrence and resistance to chemotherapy. The aim of this study was to determine the role of tailored cellular therapy for GBM with a poor prognosis and compare the activity of dendritic cells (DCs) that have encountered GBM cells. Detecting the correlations between methylation and expression of MGMT and PTEN genes and GBM cancer stem cells (CSCs) markers after co-cultures with a mononuclear cell cocktail are also aims for this study. Allogenic umbilical cord blood (UCB)-derived DCs were labeled with the CD11a and CD123 for immature DCs, and CD80 and CD11c for mature DCs. CD34, CD45, and CD56 cells were isolated from allogenic UCB for using in DCs maturation. GBM CSCs were detected with CD133/1 and CD111 antibodies after co-culture studies. DC activation was carried out via GBM cells including CD133 and CD111 cells and a mononuclear cells cocktail including CD34, CD45, and CD56 natural killer cells. Real-time PCR was performed to detect the expression and promoter methylation status of PTEN and MGMT genes. The expression of CSCs markers was found in all GBM cases, and a statistically significant correlation was found among them after co-culture studies. The most pronounced affinity of DCs to GBM cells was observed at dilutions between 1/4 and 1/256 in co-cultures. There was a statistically significant correlation between cellularity and granularity ratios for CD123 and CD11c. PTEN and MGMT gene expression and methylation values were evaluated with respect to CSCs expression and no statistical significance was found. Activation of DCs might associate with CSCs and the mononuclear cells cocktail including CD34, CD45, and CD56 cells which were obtained from allogenic UCB.