Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

Role of human organic anion transporter 4 in the transport of ochratoxin A

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S(2) hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S(2) hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S(2) hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K(m) value of 22.9+/-2.44 microM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner (K(i)=44.4-336.4 microM), with the following order of potency: probenecid > piroxicam > octanoate >citrinin. The efflux of OTA by S(2) hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S(2) hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.     

Functional integration of serial dilution and capillary electrophoresis on a PDMS microchip

For the quantitative analysis of an unknown sample a calibration curve should be obtained, as analytical instruments give relative, rather than absolute measurements. Therefore, researchers should make standard samples with various known concentrations, measure each standard and the unknown sample, and then determine the concentration of the unknown by comparing the measured value to those of the standards. These procedures are tedious and time-consuming. Therefore, we developed a polymer based microfluidic device from polydimethylsiloxane, which integrates serial dilution and capillary electrophoresis functions in a single device. The integrated microchip can provide a one-step analytical tool, and thus replace the complex experimental procedures. Two plastic syringes, one containing a buffer solution and the other a standard solution, were connected to two inlet holes on a microchip, and pushed by a hydrodynamic force. The standard sample is serially diluted to various concentrations through the microfluidic networks. The diluted samples are sequentially introduced through microchannels by electro-osmotic force, and their laser-induced fluorescence signals measured by capillary electrophoresis. We demonstrate the integrated microchip performance by measuring the fluorescence signals of fluorescein at various concentrations. The calibration curve obtained from the electropherograms showed the expected linearity.     

Expression of vascular endothelial growth factor in the progression of cervical neoplasia and its relation to angiogenesis and p53 status

OBJECTIVE: To evaluate vascular endothelial growth factor (VEGF) expression in the successive steps of cervical neoplasia and to determine its correlation with angiogenesis and p53 status. STUDY DESIGN: Immunohistochemical staining with a VEGF monoclonal antibody was performed on a total of 161 cervical specimens representing 12 normal epithelium, 33 cervical intraepithelial neoplasia (CIN) 1, 30 CIN 3 and 86 squamous cell carcinomas. Microvessels were immunohistochemically labeled with an antibody to CD34. Computerized image analysis was used to evaluate microvessel density (MVD). p53 Status was determined by immunohistochemistry and direct sequencing of exons 5-8 of the p53 gene. RESULTS: VEGF expression progressively increased along the continuum from normal epithelium to squamous cell carcinoma (P < .05). MVD increased significantly with cervical neoplasia progression, from normal epithelium, through CIN, to squamous cell carcinoma (P < .001). A strong correlation was observed between VEGF expression and MVD (P < .001). p53 Protein expression was not detected in the normal epithelium or in CIN 1, while 3 (10%) of 30 CIN 3 and 28 (33%) of 86 squamous cell carcinomas were positive for p53. VEGF expression correlated statistically with p53 protein expression (P < .001). In double VEGF- and p53-stained sections, the 2 markers were generally expressed in the same tumor cells. Of the 4 p53 gene mutations, 3 exhibited strong VEGF expression, and 1 exhibited moderate VEGF expression. VEGF expression did not correlate significantly with outcome variables in patients with squamous cell carcinoma. CONCLUSION: Our results suggest that VEGF expression is involved in the promotion of angiogenesis in cervical neoplasia and that p53 is likely to be involved in the regulation of VEGF expression.     

Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p<0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Site-specific fluorescent labeling of DNA using Staudinger ligation

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.     

Evidence for heme oxygenase-1 association with caveolin-1 and -2 in mouse mesangial cells

The interaction of heme oxygenase-1 (HO-1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO-1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO-1 and caveolin-1 or caveolin-2. The experiments of immuno-precipitation showed complex formation between the HO-1 and caveolin-1 or caveolin-2 in the Cd treated MMCs. Confocal microscopic results also support co-localization of HO-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with HO-1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO-1.