Objective-Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy for severe ischemic diseases. Recent reports indicated that adipose tissues could supply adipose-derived regenerative cells (ADRCs). Accordingly, we examined whether implantation of ADRCs would augment ischemia-induced angiogenesis. Method and Results-Adipose tissue was obtained from C57BL/6J mice, and ADRCs were isolated using standard methods. ADRCs expressed stromal cell-derived factor 1 (SDF-1) mRNA and proteins. Hind limb ischemia was induced and culture-expanded ADRCs, PBS, or mature adipocytes (MAs) as control cells were injected into the ischemic muscles. At 3 weeks, the ADRC group had a greater laser Doppler blood perfusion index and a higher capillary density compared to the controls. Implantation of ADRCs increased circulating endothelial progenitor cells (EPCs). SDF-1 mRNA abundance at ischemic tissues and serum SDF-1 levels were greater in the ADRC group than in the control group. Finally, intraperitoneal injection of an anti-SDF-1 neutralizing antibody reduced the number of circulating EPCs and therapeutic efficacies of ADRCs. hen tissue is exposed to severe ischemia, new blood vessels develop into the ischemic foci to prevent tissue necrosis. Because circulating endothelial progenitor cells (EPCs) have been shown to participate in postnatal neovascularization after mobilization from the bone marrow (BM), 1,2 we have performed basic and clinical studies related to therapeutic angiogenesis using EPCs or BM cells. [3][4][5] We have performed therapeutic angiogenesis using autologous BM mononuclear cell (BM-MNCs) implantation into the ischemic muscles in patients with critical limb ischemia (TACT). 6 -8 Although the safety and efficiency of the TACT protocol have been established, we recently reported that patients with very severe peripheral artery occlusive disease had poor responses to the TACT procedure. 7 Moreover, recent data indicated that patients with severe obstructive vascular disease or multiple coronary risk factors had diminished functions of EPCs and poor responses to angiogenic cell therapy. 9 -12 Thus, alternative source of stem/progenitor cells for therapeutic angiogenesis has been searched extensively. Conclusions-AdiposeRecently, several investigators have reported that adipose tissues contain multipotent mesenchymal stem cells termed adipose-derived regenerative cells (ADRCs), which have an ability to regenerate damaged tissues. [13][14][15] However, little is known as to how implantation of ADRCs would induce angiogenesis in ischemic tissues. It has been known that ADRCs secrete multiple angiogenic growth factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). 13,14 Such growth factors would mobilize EPCs from the BM into peripheral blood (PB) and finally to ischemic tissues. However, there is limited evidence regarding the effects of in vivo implantation of ADRCs on EPC kinetics during ischemia-induced angiogenesis.Accordingly, we examined ...

We have cloned and characterized the genomic structure of the human gene for Myc-associated zinc finger protein (MAZ), which is located on chromosome 16p11.2. This gene is transcribed as an mRNA of 2.7 kilobases (kb) that encodes a 60-kDa MAZ protein. A 40-kb cosmid clone was isolated that includes the promoter, five exons, four introns, and one 3-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. The promoter region has features typical of a housekeeping gene: a high G ؉ C content (88.4%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. An S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 174 nucleotides (nt) upstream of the ATG codon and such expression was reflected by the promoter activity of a MAZ promoter/CAT (chloramphenicol acetyltransferase) reporter gene. Cis-acting positive and negative elements controlling basal transcription of the human MAZ gene were found from nucleotides (nt) ؊383 to ؊248 and nt ؊2500 to ؊948. Moreover, positive and negative autoregulatory elements were also identified in the regions from nt ؊248 to ؊189 and from nt ؊383 to ؊248 after co-transfection of HeLa cells with plasmids that carried the MAZ promoter/CAT construct and the MAZ-expression vector. Our results indicate that the 5-end flanking sequences are responsible for the promoter activities of the MAZ gene.The c-myc protooncogene is a member of a family of genes that encode DNA sequence-specific transcription factors with basic, helix-loop-helix, and leucine zipper domains. The Myc protein binds to DNA as heterodimers with a related polypeptide, Max (1-4). Appropriate regulation of expression of the human c-myc gene is necessary for the proliferation and differentiation of cells and for progression of the cell cycle, and deregulation of the expression of c-myc is associated with tumorigenesis and apoptosis (1, 4). Regulation of the expression of the human c-myc gene occurs at multiple levels, which include the initiation, the termination, and the attenuation of transcription (2, 4). In proliferating cells, the initiation of transcription of the c-myc gene is controlled by two major promoters, P1 and P2, and the RNA initiated from the P2 promoter accounts for 80 -90% of the total RNA initiated from the P0, P1, and P2 promoters (4, 5). Initiation of transcription from the P2 promoter requires at least three cis-elements: ME1a2, E2F, and ME1a1 (6, 7). Several transcription factors, including Sp1 (5, 8), the Myc-associated zinc finger protein (MAZ) 1 (9, 10), Pur-1 (11), and E2F (12) bind to these elements in vitro and in vivo.The MAZ protein was identified as a transcription factor that binds to a GA box (GGGAGGG) at the ME1a1 site, to the attenuator region of P2 within the first exon of the c-myc gene, and to a related sequence that is involved in the termination of transcription of the gene for ...

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Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism.