For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCRbased identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.

We found that the second intron of Stat5a was one of the common integration sites of the endogenous ecotropic murine leukemia virus, i.e., SL͞Kh virus integration-1 (Svi1), in early pre-B lymphomas in SL͞Kh mice. The high expression of STAT5A induced by Svi1 integration and activation accelerated the transcription of its target genes such as c-Myc. Transfection of the constitutively active Stat5a mutant cDNA, but not of the wild-type cDNA, to the bone marrow cells induced colony formation of pre-B cells in a methylcellulose medium and escaped from dependence on IL-7. Such growth depended on a genetic factor in the SL͞Kh strain. Consitutively high expression of Stat5a either by retrovirus integration or transfection of active mutant cDNA can be lymphomagenic to early pre-B cells in collaboration with a certain genetic background factor of mice.provirus ͉ hot spot ͉ lymphomagenesis T he SL͞Kh strain of mice develop spontaneous pre-B lymphomas at Ͼ90% incidence by 6 mo of age (1, 2). There are two morphological distinct types: the major lymphomas, which show systemic lymphatic tissue involvement, and the minor type, which shows restricted growth in bone marrow (BM) (1). Both are pre-B lymphomas expressing BP-1 and B220 (2, 3). Several lines of evidence indicate that the endogenous murine leukemia virus (MuLV) plays an etiologic role in SL͞Kh lymphomas; in particular, somatically acquired proviruses frequently are observed in lymphoma DNAs (3, 4).To elucidate the mechanism of lymphomagenesis, we cloned the virus-host junctional segments from lymphoma DNAs by an inverse PCR technique and performed sequence analysis. In three of 60 SL͞Kh lymphomas, clonal ecotropic provirus integration was found within a 400-bp stretch in the second intron of the Stat5a gene, a member of the STAT family. In this article, we examined the effect of the provirus integration in this hot spot, named SL͞Kh virus integration-1 (Svi1). The effect of the constitutive high expression of Stat5a on early B cells was further verified by colonial growth of early pre-B cells in soft agar medium and loss of IL-7 dependence, when SL͞Kh BM cells were transfected with an active Stat5a mutant cDNA. It was shown that the immortalization of early pre-B cells by Stat5a depended on an unidentified host factor in SL͞Kh mice. There are several reports on the relevance of Stat5a in hemopoietic malignancies (5-12). Stat5b is a human myeloid cell oncogene, but neither Stat family member has been shown to have a direct pathogenetic role in lymphoid disease. Herein, we report that Stat5a is one of the hot spots of MuLV integration and subsequent constitutive up-regulation of Stat5a perturbs signaling in early B lineage leading to lymphoma development in a certain genetic background. Materials and MethodsMice. SL͞Kh is an inbred strain of mice with a high incidence of spontaneous pre-B lymphomas. Previously, we reported their origin (13) and virus expression (1), the pathology of lymphomas (1, 2), and host genetic factors in lymphomagenesis (3,4,14). They were maint...

Age prediction with epigenetic information is now edging closer to practical use in forensic community. Many age-related CpG (AR-CpG) sites have proven useful in predicting age in pyrosequencing or DNA chip analyses. In this study, a wide range methylation status in the ELOVL2 and FHL2 promoter regions were detected with methylation-sensitive high resolution melting (MS-HRM) in a labor-, time-, and cost-effective manner. Non-linear-distributions of methylation status and chronological age were newly fitted to the logistic curve. Notably, these distributions were revealed to be similar in 22 living blood samples and 52 dead blood samples. Therefore, the difference of methylation status between living and dead samples suggested to be ignorable by MS-HRM. Additionally, the information from ELOVL2 and FHL2 were integrated into a logistic curve fitting model to develop a final predictive model through the multivariate linear regression of logit-linked methylation rates and chronological age with adjusted R(2)=0.83. Mean absolute deviation (MAD) was 7.44 for 74 training set and 7.71 for 30 additional independent test set, indicating that the final predicting model is accurate. This suggests that our MS-HRM-based method has great potential in predicting actual forensic age.

No abstract

No abstract