[Clinical application of supersensitive and multiplex assay, MUSTag technology].

Recently, various sets of protein biomarkers have been discovered in important diseases such as cancers, brain stroke, heart attack, diabetes, and so on. Many of these biomarkers are expected to be extremely valuable as targets for clinical diagnosis and drug development; however, the clinical validation is difficult and time-consuming by individual assays or due to very low concentration in an early stage of disease. For the super-sensitive and multiplex detection of target biomarkers, we have developed MUSTag (Multiple Simultaneous Tag) assay technology with innovative modification of the immuno-PCR method. In MUSTag technology, specific antibodies against several important biomarkers were linked to 100-300bp long oligonucleotides as detection tags. Each different oligo-tag simultaneously detects multiplex protein targets with extremely high sensitivity(more than 10 fg (10(-15) g)/ml) in a dose-dependent manner by qRT-PCR-based (maximum 3 plexes) or capillary electrophoretic amplification (over 30 plexes). Here we report our recent results of multiple cytokine assay or disease-specific biomarker assay using MUSTag technology, and further, clinical results from patients with cancers, ischemic brain or heart attack, who need prompt and predictive diagnosis for adequate treatment.

The temporal profile of genomic responses and protein synthesis in ischemic tolerance of the rat brain induced by repeated hyperbaric oxygen.

Repeated hyperbaric oxygen (HBO) exposure prior to ischemia has been reported to provide neuroprotection against ischemic brain injury. The present study examined the time course of neuroprotection of HBO (3.5 atmosphere absolute, 100% oxygen, 1 h for 5 consecutive days) and the changes of gene/protein expression in rats. First, at 6 h, 12 h, 24 h, and 72 h after HBO sessions, rats were subjected to forebrain ischemia (8 min). Histopathological examination of hippocampal CA1 neurons was done 7 days after ischemia. Second, temporal genomic responses and protein expression were examined at the same time points after HBO sessions without subjecting animals to ischemia. HBO significantly reduced loss of hippocampal CA1 neurons that normally follows transient forebrain ischemia when the last HBO session was 6 h, 12 h, or 24 h before ischemia (survived neurons 55%, 75%, and 53%, respectively), whereas if there was a 72-h delay before the ischemic insult, HBO was not protective (survived neurons only 6%). Statistical analysis on microarray data showed significant upregulation in 60 probe sets including 7 annotated genes (p75NTR, C/EBPdelta, CD74, Edg2, Trip10, Nrp1, and Igf2), whose time course expressions corresponded to HBO-induced neuroprotection. The protein levels of p75NTR, C/EBPdelta, and CD74 were significantly increased (maximum fold changes 2.9, 2.0, and 7.9, respectively). The results suggest that HBO-induced neuroprotection against ischemic injury has time window, protective at 6 h, 12 h and 24 h but not protective at 72 h. Although the precise interaction is to be determined, the genes/proteins relevant to neurotrophin and inflammatory-immune system may be involved in HBO-induced neuroprotection.

Capillary morphogenesis gene (CMG)-1 is among the genes differentially expressed in mouse male germ line stem cells and embryonic stem cells.

We recently established a technique to expand male germ line stem (GS) cells in long-term culture without losing their spermatogenic capacity. To gain insight into the genetic program of these cells, we compared the mRNA expression profile of GS cells with that of embryonic stem (ES) cells using DNA microarrays. We found 79 genes that were upregulated in GS cells compared to ES cells, including synaptonemal complex protein-1, deleted in azoospermia-like, ubiquitin-conjugating enzyme E2B, and ubiquitin carboxy-terminal hydrolase L1, all of which are functionally important for spermatogenesis. In addition, we identified a cDNA encoding the mouse ortholog of capillary morphogenesis gene (CMG)-1. CMG-1 transcripts were predominantly produced in spermatogonia and spermatocytes in mouse testis. When CMG-1 expression was attenuated in a mouse spermatocyte-derived cell line, GC-2spd(ts), by a target-specific short interfering RNA, the morphology of the cells was changed and the expression of cyclin D2 was abrogated. A reporter assay using a genomic region upstream of the mouse cyclin D2 gene revealed that this downmodulation occurs at the transcriptional level. We detected FLAG-tagged CMG-1 protein in the nuclei of transfected COS7 cells, suggesting that CMG-1 may play a unique role in the transcriptional regulation of the cyclin D2 gene. The upregulated GS genes identified in this study will provide useful information for the future investigation of spermatogonial stem cells and the early phase of male germ cell differentiation.