Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.     

Effect of Human WEE1 and Stem Cell Factor on Human CD34^＋ Umbilical Cord Blood Cell Damage Induced by Chemotherapeutic Agents

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE l （WEElHu） and proliferation-stimulating stem cell factor （SCF） was generated. In this study, we selected human umbilical cord blood CD34^＋ cells as the in vitro model in an attempt to investigate whether WEEIHu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEElHu and SCF in CD34^＋ cells provided the cells with some protection. These findings suggest that the expression of WEElHu and SCF might rescue CD34^＋ cells from chemotherapyinduced myelosuppression.     

Application of cationic conjugated polymers in microarrays using label-free DNA targets

A fluorescence-based microarray technique that does not require target DNA labeling is detailed. This 'label-free' approach utilizes a cationic, water-soluble conjugated polymer PFBT (poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide]), and neutral PNA (peptide nucleic acid) hybridization probes. DNA hybridization to immobilized PNA spots results in a change in the net charge at that particular surface. Electrostatic interactions between the cationic polymer and negatively charged DNA bind the polymer to the hybrid DNA/PNA complex. By exciting the conjugated polymer at 488 nm on a commercial microarray scanner, the presence of the target is directly indicated by the fluorescence emission of the polymer. This feature eliminates the necessity of target labeling required in traditional microarray protocols. There are five steps involved in the procedure before scanning or imaging the array: (i) slide hydration, (ii) target hybridization, (iii) post-hybridization washing, (iv) polymer application and (v) polymer washing. Each step takes 20 min to 1 h. The overall protocol requires approximately 2-3 h.     

Prefrontal cortex and somatosensory cortex in tactile crossmodal association: an independent component analysis of ERP recordings

Our previous studies on scalp-recorded event-related potentials (ERPs) showed that somatosensory N140 evoked by a tactile vibration in working memory tasks was enhanced when human subjects expected a coming visual stimulus that had been paired with the tactile stimulus. The results suggested that such enhancement represented the cortical activities involved in tactile-visual crossmodal association. In the present study, we further hypothesized that the enhancement represented the neural activities in somatosensory and frontal cortices in the crossmodal association. By applying independent component analysis (ICA) to the ERP data, we found independent components (ICs) located in the medial prefrontal cortex (around the anterior cingulate cortex, ACC) and the primary somatosensory cortex (SI). The activity represented by the IC in SI cortex showed enhancement in expectation of the visual stimulus. Such differential activity thus suggested the participation of SI cortex in the task-related crossmodal association. Further, the coherence analysis and the Granger causality spectral analysis of the ICs showed that SI cortex appeared to cooperate with ACC in attention and perception of the tactile stimulus in crossmodal association. The results of our study support with new evidence an important idea in cortical neurophysiology: higher cognitive operations develop from the modality-specific sensory cortices (in the present study, SI cortex) that are involved in sensation and perception of various stimuli.     

Glutamate regulates neurite outgrowth of cultured descending brain neurons from larval lamprey

In larval lamprey, descending brain neurons, which regenerate their axons following spinal cord injury, were isolated and examined in cell culture to identify some of the factors that regulate neurite outgrowth. Focal application of 5 mM or 25 mM L-glutamate to single growth cones inhibited outgrowth of the treated neurite, but other neurites from the same neuron were not inhibited, an effect that has not been well studied for neurons in other systems. Glutamate-induced inhibition of neurite outgrowth was abolished by 10 mM kynurenic acid. Application of high potassium media to growth cones inhibited neurite outgrowth, an effect that was blocked by 2 mM cobalt or 100 microM cadmium, suggesting that calcium influx via voltage-gated channels contributes to glutamate-induced regulation of neurite outgrowth. Application of glutamate to growth cones in the presence of 2 microM omega-conotoxin MVIIC (CTX) still inhibited neurite outgrowth, while CTX blocked high potassium-induced inhibition of neurite outgrowth. Thus, CTX blocked virtually all of the calcium influx resulting from depolarization. To our knowledge, this is the first direct demonstration that calcium influx via ligand-gated ion channels can contribute to regulation of neurite outgrowth. Finally, focal application of glutamate to the cell bodies of descending brain neurons inhibited outgrowth of multiple neurites from the same neuron, and this is the first demonstration that multiple neurites can be regulated in this fashion. Signaling mechanisms involving intracellular calcium, similar to those shown here, may be important for regulating axonal regeneration following spinal cord injury in the lamprey.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Glucosamine causes overproduction of reactive oxygen species, leading to repression of hypocotyl elongation through a hexokinase-mediated mechanism in Arabidopsis

Glucosamine (GlcN) is a naturally occurring amino-sugar that is synthesized by amidation of fructose-6-phosphate. Although a number of reports have examined the biological effects of GlcN on insulin resistance in mammalian systems, little is known about its effects on plant growth. In this study, we have shown that exogenous GlcN inhibits hypocotyl elongation in Arabidopsis, whereas glucose and its analogs alleviate this inhibitory effect. The hexokinase (HXK)-specific inhibitor mannoheptulose also restored hypocotyl elongation. The gin2-1 mutants with an alteration in AtHXK1 exhibited higher tolerance to GlcN. We also found that GlcN induces a significant increase in the production of reactive oxygen species (ROS). In addition, the GlcN-mediated inhibition of hypocotyl elongation was relieved by reducing agents such as ascorbic acid and glutathione. GlcN treatment resulted in significant induction of expression of GST1, GST2 and GST6, which are marker genes for ROS production. The gin2 mutation also represses the ROS production and the GST2 induction by GlcN treatment. Taken together, these results provide evidence that GlcN induces HXK-mediated induction of oxidative stress, leading to growth repression in Arabidopsis thaliana.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

A small molecule inhibitor of alpha4 integrin-dependent cell migration

A small molecule inhibitor of alpha4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with gamma-parvin. This molecule should serve as a useful tool to study alpha4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune diseases.