Gold-coated capillary based 2,4-dichlorophenoxyacetic acid chemi-luminescent assays: possibilities towards multianalysis

The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.     

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.     

An information theoretic approach for analyzing temporal patterns of gene expression

MOTIVATION: Arrays allow measurements of the expression levels of thousands of mRNAs to be made simultaneously. The resulting data sets are information rich but require extensive mining to enhance their usefulness. Information theoretic methods are capable of assessing similarities and dissimilarities between data distributions and may be suited to the analysis of gene expression experiments. The purpose of this study was to investigate information theoretic data mining approaches to discover temporal patterns of gene expression from array-derived gene expression data. RESULTS: The Kullback-Leibler divergence, an information-theoretic distance that measures the relative dissimilarity between two data distribution profiles, was used in conjunction with an unsupervised self-organizing map algorithm. Two published, array-derived gene expression data sets were analyzed. The patterns obtained with the KL clustering method were found to be superior to those obtained with the hierarchical clustering algorithm using the Pearson correlation distance measure. The biological significance of the results was also examined. AVAILABILITY: Software code is available by request from the authors. All programs were written in ANSI C and Matlab (Mathworks Inc., Natick, MA).     

Retinoblastoma: from bench to bedside

Retinoblastoma (Rb) is the most common primary ocular malignancy of children and is caused by a mutation in the gene RB1. Approximately 40% of cases are associated with one or more constitutional mutations, and are therefore heritable, whereas the other 60% are sporadic. Rb is exclusively found in young children. In some cases, Rb tumours metastasise to extraocular organs including bone, lung and brain. Although there is no effective treatment for metastatic disease, non-metastatic cases can be cured by removal of the eye(enucleation). Newer treatment strategies emphasise salvaging the affected eye whenever possible. Animal models of Rb have been developed with xenograft and transgenic techniques. Each model has both strengths and weaknesses for exploring the mechanisms of disease development and progression and the efficacy of new treatment strategies.     

Sensitive liquid chromatography-mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma

We have developed a high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 micrometer, 100x2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol-water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 nM and is linear between 0.3 nM and 1 microM for docetaxel. For paclitaxel, the LLOQ was 1 nM, and the assay is linear between 1 nM and 1 microM. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m(2) of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m(2) of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC-MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.     

Effect of chronic treatment with losartan on streptozotocin-induced renal dysfunction

The present investigation was undertaken to study the effect of chronic treatment with angiotensin (AT₁) receptor antagonist losartan (2 mg/kg, p.o., 6 weeks) on streptozotocin (STZ) induced (45 mg/kg, i.v., single dose) renal dysfunctions in diabetic rats. Injection of streptozotocin produced not only the cardinal symptoms of diabetes mellitus like loss of body weight, hyperglycemia, and hypoinsulinemia but also the renal dysfunctions. Losartan treatment significantly prevented all these changes except STZ-induced hypoinsulinemia. There was a significant elevation of blood pressure in diabetic rats and treatment with losartan significantly brought it back to normal. Renal dysfunction in diabetic rats was characterized by a significant decrease in creatinine clearance, elevated levels of electrolytes and renal hypertrophy. Treatment with losartan prevented these changes. A good correlation was found between biochemical parameters and histopathological abnormalities. Our data suggests that, losartan may be considered as the drug of choice when there is a co-existence of diabetes mellitus and hypertension with compromised kidney function.     

Ascorbic acid and alpha-tocopherol as potent modulators on arsenic induced toxicity in mitochondria

Arsenic exists ubiquitously in our environment and various forms of arsenic circulate in air, water, soil and living organisms. Since arsenic compounds have shown to exert their toxicity chiefly by generating reactive oxygen species, we have evaluated the effect of antioxidants ascorbic acid and alpha-tocopherol on lipid peroxidation, antioxidants and mitochondrial enzymes in liver and kidney of arsenic exposed rats. A significant increase in the level of lipid peroxidation and decrease in the levels of antioxidants and in the activities of mitochondrial enzymes were observed in arsenic intoxicated rats. Co-administration of arsenic treated rats with ascorbic acid and alpha-tocopherol showed significant reduction in the level of lipid peroxidation and elevation in the levels of ascorbic acid, alpha-tocopherol, glutathione and total sulfhydryls and in the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, NADH-dehydrogenase and cytochrome c oxidase. From our results, we conclude that ascorbic acid and alpha-tocopherol alleviate arsenic- induced alterations in mitochondria.     

Effect of uracil on rifamycin SV production by Amycolatopsis mediterranei MV35R

The effect of different organic nitrogen compounds on the production of rifamycin SV by Amycolatopsis mediterranei MV35R and their optimum concentrations have been described. Results obtained indicate that rifamycin SV production increased from 4020 mg l-1 to 4575 mg l-1 when organic nitrogen compound uracil was added at 0.2% (w/v) concentration to the fermentation medium by A. mediterranei MV35R. The rifamycin SV yield was enhanced by 505 mg l-1 using uracil (2 g l-1) when compared with barbital.     

What Is the Signal for the Posttranslational Arginylation of Proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [³H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.