We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations.We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing.We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA.The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.
ABSTRACT We evaluated a novel three-dimensional microarray (PamChip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mecA gene. The results were compared to those obtained for 27 Staphylococcus aureus isolates by conventional DNA sequencing or PCR methods. Hybridization and fluorescence detection were performed using an FD10 system designed for PamChip microarray under conditions optimized for each target/probe on the array. In dilution series analysis using multiplex PCR samples, the sensitivity of the microarray was about 10 times greater than that of conventional PCR methods. A high level of data reproducibility was also confirmed in those analyses. Various point mutations in quinolone resistance-determining regions detected by our system corresponded perfectly to the results obtained by conventional DNA sequencing. The results of the mecA gene detection using our system also corresponded to the PCR method; that is, signal/band was detected in all isolates of methicillin-resistant S. aureus , and no signal/band was detected in any isolate of methicillin-susceptible S. aureus . In conclusion, our novel three-dimensional microarray system provided rapid, specific, easy, and reproducible results for the simultaneous detection of levofloxacin resistance and the mecA gene in S. aureus .
Advancements in Tumor Immunotherapy and Cancer Vaccines 108 types, such as soft tissue sarcoma and lymphoma, the response is marked even by today's standards (Tsung & Norton, 2006).Following the pioneering works by Dr. Coley and his daughter, Dr. Helen Coley Nauts, efforts to treat cancer based on the function of the immune system, i.e., immunotherapy, have continued.As with Coley's toxins, the innate immune response against microbes could provoke anti-tumor effects as a secondary response.Although the exact molecular mechanisms of innate immune cells to recognize the components of microorganisms have not been fully understood, the substances such as bacteria-derived materials which evoke tumor immunity have been categorized as 'biological response modifier (BRM)'.In Japan, several original BRMs have been developed since 1950's.Dr Chisato Maruyama noticed that there were few cancer patients in sanatoriums for tuberculosis or Hansen's disease, and he started research to apply extracts from Mycobacterium tuberculosis to cancer treatment.His preparation, named Specific Substance MARUYAMA (SSM, also called the "MARUYAMA vaccine") Suzuki et al., 1986a; Suzuki et al., 1986b;Sasaki et al., 1990 was composed of deproteinized extracts, and contained lipoarabinomannan, a kind of polysaccharide, as the main component.SSM received much attention from the public as a miracle drug for cancer treatment before being approved by the Ministry of Health and Welfare of Japan at that time.SSM has not been approved to date as a anti-neoplastic drug, but its related preparation, referred to as Z-100 or Ancer, has been approved since 1991 as a drug for radiotherapy associated leucopenia.Ttwo other BRM drugs, krestin (PS-K, polysaccharide-protein complexes extracted from basidiomycetes, Trametes versicolor) (Akiyama et al., 1977;Mizushima et al., 1982) and picibanil (OK-432, a lyophilized preparation of attenuated group A Streptococcus haemolyticus) (Kai et al., 1979;Kataoka et al., 1979) were approved by the Ministry of Health and Welfare of Japan as anti-neoplastic drugs in 1975.Another example of Japanese BRMs is BCG-CWS, a cell wall skeleton preparation of Mycobacterium bovis bacillus Calmette-Guérin, a tuberculosis vaccine strain which is almost nonpathogenic yet retains the immunogenic properties of tuberculosis (Tsuji et al., 2000).BCG-CWS contains a peptidoglycan that is covalently linked to arabinogalactan and mycolic acids (Azuma et al., 1974).Although BCG-CWS has been clinically used for a long time (Hayashi et al., 2009;Kodama et al., 2009), it has not been approved by the Ministry of Health and Welfare of Japan.One of the characteristics of classical BRMs is that they are crude products which are not fully purified.Their multiple components seem to be important for the induction of efficient anti-tumor effects as seen with Coley toxins. Stimulators of innate immunity Toll-like receptors (TLRs)The cells of the innate immune system recognize infectious agents by receptors for characteristic components of pathogenic microorganisms.The structures of these components are highly conserved and are called pathogen-associated molecular patterns (PAMPs).The receptors for PAMPs, referred to as pattern recognition receptors (PRRs), are germline-encoded and highly conserved across species.The innate immune system detects various classes of pathogens and abnormal cells through PRRs, and serves as a first line of defense against microbes.www.intechopen.
We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three‐dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six‐week‐old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N‐diethylnitrosamine (DEN), 0.01% 2‐amino‐3, 8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1′‐acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip® microarray A spotted for 28 genes of drugmetabolizing enzymes in duplicate, and PamChip® microarray B spotted for 131 genes which are known to be up‐ or down‐regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three‐dimensional microarrays.
The frequency and distribution of mitomycin C (MMC)-induced sister chromatid exchanges (SCEs) were investigated in the fibroblast chromosomes of three mammalian species, Microtus montebelli, Apodemus argenteus and Chimarrogale himalayica, by the fluorescence-plus-Giemsa (FPG) and C-band staining methods, paying special attention to the large C-band area (C-block)-carrying and/or nucleolus organizer region (NOR)-carrying chromosomes. The junctions of heterochromatin and euchromatin (HE-junctions) and NORs were found to be "hot spots" of SCEs in all the species examined: their SCE frequencies were 35.3% and 24.2% in the HE-junction of the X chromosomes of M. montebelli and A. argenteus, and 16.7% and 17.8% in the NORs of the No. 1 chromosomes of M. montebelli and C. himalayica, respectively. In M. montebelli and A. argenteus the SCE frequency was apparently lower in the C-block region than in the euchromatic one, when compared with each other based on equal length, while in C. himalayica no such marked difference in the SCE frequency was found between these two regions of the chromosome. These findings may indicate that occurrence of SCEs is significantly suppressed in the C-block region of M. montebelli and A. argenteus, but not in that of C. himalayica. In addition, the C-blocks of the No. 1 homologue of C. himalayica showed a highly varied individual-to-individual heteromorphism in length. The biological implication of SCEs was discussed in connection with the generation of heteromorphism.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)