Home About us Contact
|
Home About us Contact | ||
|
|
||
Samples Isolated (sample + isolated)
Selected AbstractsFrequency of and variables associated with the EGFR mutation and its subtypesINTERNATIONAL JOURNAL OF CANCER, Issue 3 2010Tomoaki Tanaka Mutation in the epidermal growth factor receptor (EGFR) is frequently seen in non-small cell lung cancers (NSCLCs), especially in Asian females with adenocarcinoma. The frequency of mutation and the factors associated requires to be elucidated by analyzing a large number of consecutive clinical samples. We summarized the result of the EGFR mutation analysis for 1,176 patients performed at the time of diagnosis or relapse. The PNA-LNA PCR clamp, a highly sensitive detection method for the EGFR mutation, was employed. For fresh cases a portion of samples isolated to establish the diagnosis of lung cancer was used. For cases with a relapsed disease archival tissue were tested. The variables associated with the EGFR mutation after removing the confound factors were investigated by the logistic analysis using the samples collected in our university (n = 308) where detailed information on patients were available. The frequency of the EGFR mutation and its subtypes were investigated using all samples (n = 1,176). The EGFR mutation was significantly associated with adenocarcinoma (p = 0.006) and light-smoking (p < 0.0001), but not gender. The deletions in exon 19 were more frequently associated with male gender while exon 21 deletions were with female gender (p = 0.0011). The overall frequency of the EGFR mutation was 31%. Our result suggests that the female predominance in the EGFR mutation rate is a reflection of a higher frequency of adenocarcinoma in females. The gender difference in the mutation subtypes may provide a clue for the mechanism of the occurrence of the EGFR mutation. [source] The ABC-transporter hutCD genes of Photobacterium damselae subsp. piscicida are essential for haem utilization as iron source and are expressed during infection in fishJOURNAL OF FISH DISEASES, Issue 8 2010C R Osorio Abstract The marine fish pathogen Photobacterium damselae subsp. piscicida utilizes haem compounds as the sole iron source. In a previous work, we characterized a gene cluster with ten potential haem uptake and utilization genes. Two of these genes, hutC and hutD, which are iron-regulated, conform a putative inner membrane haem ABC transporter. In this study, we constructed an insertional mutant, leading to the inactivation of hutCD genes. Reverse transcriptase-PCR analyses demonstrated that an insertion between the hutB and hutC genes abolished transcription of the downstream hutC and hutD genes. The hutCD mutant was unable to utilize haem as the sole iron source, demonstrating that the putative ABC-transporter proteins HutC and HutD are essential for haem utilization as an iron source in P. damselae subsp. piscicida. In addition, reverse transcriptase-PCR assays conducted with RNA samples isolated from experimentally infected fish revealed the presence of hutCD transcripts. The results demonstrate for the first time that haem uptake genes of a fish pathogen are expressed during the infective process in fish. [source] Quantitative assessment of chondrocyte viability after laser mediated reshaping: A novel application of flow cytometryLASERS IN SURGERY AND MEDICINE, Issue 1 2003Alexandre Rasouli BS Abstract Background and Objectives Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. Study Design/Materials and Methods Porcine septal cartilages were irradiated with an Nd:YAG laser (,,= 1.32 ,m, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO®/DEAD RedÔ (Molecular Probes, Eugene, OR). A flow cytometer was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post-irradiation debris in each treatment population. Results Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28,88%, with the least amount of deterioration in untreated and singly irradiated samples. Conclusions Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. Lasers Surg. Med. 32:3,9,2003. © 2003 Wiley-Liss, Inc. [source] Absence of 2,-deoxyguanosine-carbon 8-bound ochratoxin A adduct in rat kidney DNA monitored by isotope dilution LC-MS/MSMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 4 2008Thierry Delatour Abstract The contribution of DNA adduct formation in the carcinogenic action of the mycotoxin ochratoxin A (OTA) has been subject to much debate. Recently, a carbon-bonded ochratoxin A-2,-deoxyguanosine adduct (dGuoOTA) formed by photochemical reaction in vitro has been shown by 32P-postlabeling/TLC to comigrate with a spot detected in DNA isolated from rat and pig kidney following exposure to OTA. Considering the large body of evidence arguing against covalent DNA binding of OTA and the poor resolution and specificity of postlabeling analysis, we developed a stable isotope dilution LC-MS/MS method to analyze dGuoOTA in kidney DNA isolated from rats treated with OTA. dGuoOTA and nitrogen-15-labeled dGuoOTA (15N5 -dGuoOTA) were prepared by photoirradiation of OTA in the presence of dGuo or nitrogen-15-labeled dGuo. Conditions for DNA hydrolysis were optimized using a synthetic oligonucleotide containing dGuoOTA to ensure complete release of dGuoOTA. The LOD of the method (S/N > 3) was 10 fmol dGuoOTA on-column. However, dGuoOTA was not detected in DNA samples isolated from male F344 rats treated with OTA for up to 90 days at doses known to cause renal tumor formation. Detection limits, calculated for each individual sample based on the absolute LOD and the amount of DNA injected, were as low as 3.5 dGuoOTA/109 nucleotides. These data are consistent with previous results showing lack of DNA adduct formation by OTA and demonstrate that dGuoOTA is not formed in biologically relevant amounts under physiological conditions in vivo. [source] Application of the crypt isolation technique to the assessment of genetic alterations of colorectal carcinomasPATHOLOGY INTERNATIONAL, Issue 10 2002Hiroshi Takahashi The crypt isolation technique (CIT) allows for the isolation of pure tumor crypts from colon tumor tissue. In a previous study we reported on the genetic alterations found in colorectal tumor crypts using the CIT; however, a direct comparison of the genetic alterations found in colorectal carcinomas using either conventional methods (CM) or the CIT has not previously been performed. Here, we analyzed the impact of this method on the genetic analysis of colon tumor cells by comparing the observed frequency of genetic alterations in colon tumors isolated using CM or the CIT. We used a combination of the CIT and the fluorescent polymerase chain reaction assay to accurately assess the incidence of allelic imbalances (AI) at a number of chromosomal loci (17p, 5q, 18q, 1p, 8p, 22q), microsatellite instability (MSI), and mutations of cancer-related genes (p53 and APC genes) in 48 sporadic colorectal carcinomas. In addition, genetic alterations seen in multiploid tumors (defined as tumors with both diploid and aneuploid cell populations) identified by the CIT were also examined. The incidence of AI at the chromosomal loci tested was more frequently detected in samples isolated from tumors using the CIT than in those isolated from the same tumors using CM. In contrast, we observed no differences in the frequency of MSI or cancer-related gene mutation between the two groups. Although there was no difference in the frequency of genetic alterations between tumors with evidence of multiploidy, sorting of diploid and aneuploid populations allowed detection of distinct genetic changes. The crypt isolation method thus appears to be useful in that it allows purification of tumor cells and the accurate assessment of their genetic alterations. In addition, it may also be of benefit in clarifying the genetic profile of multiploid tumor cell populations. [source] Flowering and dwarfism induced by DNA demethylation in Pharbitis nilPHYSIOLOGIA PLANTARUM, Issue 1 2010Yuiko Iwase Flowering and dwarfism induced by 5-azacytidine and zebularine, which both cause DNA demethylation, were studied in a short-day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non-inductive long-day (LD) conditions. The flower-inducing effect of 5-azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non-inductive photoperiodic conditions. These results suggest that the flowering-related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A3 but not by t -zeatin or kinetin. The change in the genome-wide methylation state was examined by methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD-specific fragments were extracted from the gel of the MS-AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins. [source] Effects of policosanol treatment on the susceptibility of low density lipoprotein (LDL) isolated from healthy volunteers to oxidative modification in vitroBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 3 2000Roberto Menéndez Aims The aim of this study was to investigate the effect of policosanol on the susceptibility of LDL-C to in vitro lipid peroxidation in human healthy volunteers. Methods The effect of policosanol (5 and 10 mg day,1) on LDL-C oxidation was studied in a double-blind, randomized, placebo-controlled trial conducted in 69 subjects. LDL-C samples isolated at baseline and after 8 weeks were subjected to in vitro tests of LDL-C oxidation. We tested the susceptibility of LDL-C to lipid peroxidation in a cell-free system by the addition of copper ions as well as in a more physiological system, macrophage-mediated oxidation. Results At baseline all groups were well matched regarding all variables. After 8 weeks of therapy policosanol administered at 5 and 10 mg, significantly and in a dose-dependent manner increased the lag phase of conjugated diene generation (mean ± s.d.) from 83.79 ± 29.16 min to 94.90 ± 25.50 min (5 mg day,1) and from 82.74 ± 17.16 min to 129.89 ± 35.71 min (10 mg day,1), while in the placebo group LDL-C oxidation did not change significantly. Policosanol (10 mg day,1), but not placebo, significantly decreased the rate of conjugated diene generation. Comparison with placebo after therapy also showed significant differences. Macrophage mediated-oxidation was also inhibited by policosanol as evident by measuring thiobarbituric acid reactive substances (TBARS). Policosanol (10 mg day,1) significantly lowered malondialdehyde (MDA) generation from 8.50 ± 0.91 to 5.76 ± 1.01 nmol mg,1 protein. Comparison with placebo after 5 and 10 mg day,1 showed significant differences. Policosanol significantly lowered total cholesterol by 10.5% (5 mg day,1) and 12.4% (10 mg day,1) and LDL-C by 16.7% and 20.2%, respectively. Also, policosanol (10 mg day,1) increased HDL-C by 15.2%. Five subjects withdrew from the study, none because of adverse experiences. No clinical or blood biochemical drug-related disturbances were found. Conclusions The present study demonstrated that policosanol administered within its therapeutic dosage for lowering cholesterol (5 and 10 mg day,1), decreased the susceptibility of LDL-C to lipid peroxidation in vitro. [source] | |||||||||||||