Neurons, astrocytes and oligodendrocytes arise from common progenitor cells that reside in the neuroepithelium of the developing brain. We have focused on cytokines as astrocyte-inducing cell external cues, and found that leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2) synergistically induce glial fibrillary acidic protein (GFAP)-positive astrocytes. The synergistic action of these two cytokines is achieved by the complex formation between respective downstream transcription factors, STAT3 and Smad1, bridged by a transcriptional coactivator p300. Simultaneously, BMP2 also represses the differentiation of neurons by inducing anti-neurogenic basic helix-loop-helix (bHLH) transcription factors, Id1, Id3 and Hes-5. Astrocyte-inducing cytokines not only promote astrocytogenesis but also inhibit neurogenesis, suggesting the presence of negative interaction between neuronal and astrocytic differentiation pathways. We also found that oligodendrocytic bHLH factor OLIG2 inhibits the LIF-induced transcription of GFAP gene by abolishing the complex formation between STAT3 and p300. This suggests the presence of negative interaction between astrocytic and oligodendrocytic cell lineages, same as has been observed for neuronal and astrocytic cell lineages. Interestingly, differentiation of neuroepithelial cells depends on cell intrinsic programs, in addition to cell external cues. In contrast to embryonic day 14.5 (E14.5) neuroepithelial cells, LIF is not sufficient to induce GFAP expression in E11.5 neuroepithelial cells. Analysis of the GFAP gene promoter revealed that CpG dinucleotide sequences within the STAT3 recognition site is methylated in E11.5 neuroepithelial cells, and this methylation leads to the inaccessibility of activated STAT3 to its binding elements. The methylation frequency in the STAT3 binding site in the GFAP promoter declines as the developmental process proceeds, allowing the expression of GFAP in astrocytic cell lineage. The results suggest that lineage specification is regulated by cell-external cues and cell-intrinsic programs, where the former involves extracellular cytokines and the latter includes DNA methylation of cell lineage specific gene promoters. Our work also suggests the presence of negative regulatory interactions among the signals that promote differentiation of neurons, astrocytes and oligodendrocytes.

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Epidemiological studies in Western countries indicate that up to 5% of men and 2.5% of women 60 years of age or older have symptoms of intermittent claudication. The symptoms of chronic arterial insufficiency of the lower extremities progress rather slowly over time. Thus, after 5 to 10 years, more than 70% of patients report either no change or improvement in their symptoms, while 20% to 30% have progressive symptoms and require intervention, and less than 10% need amputation.
With respect to affected limbs, the goal is to eliminate ischemic symptoms and prevent progression to vascular occlusion. Accepted treatments include nonsurgical measures such as exercise, risk factor modification, and pharmacological therapy, as well as surgical treatment, which includes interventional radiological procedures such as angioplasty or stent insertion and surgical treatment such as endarterectomy, bypass grafting, and amputation.
Therapeutic angiogenesis by cell transplantation is another promising therapy. Recently, we have reported the effectiveness and safety of therapeutic angiogenesis by transplantation of autologous bone marrow mononuclear cells (BM-MNCs) to ischemic limbs, because of the natural ability of bone marrow cells to supply endothelial progenitor cells and secrete various angiogenic factors or cytokines. Autologous transplantation of BM-MNCs represents a new and promising strategy for clinical application designed to revascularize ischemic tissues.

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The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of hematopoietic stem cells (HSCs) with their particular microenvironments, known as the stem cell niches, is critical for the regulation of the HSCs. HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and anti-apoptotic, and comprise a side-population (SP) of HSCs, which contact closely to osteoblasts. We also found that angiopoietin-1, a ligand for Tie2, was produced by osteoblasts (OBs) in the bone marrow niche. It suggests that Tie2 and Ang-1 are part of a key signaling interaction between HSCs and niche cells. This signaling pathway regulates the feature of HSCs in the BM niche. The interaction of Tie2 with Ang-1 in vitro induced tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhanced the ability of HSCs to become quiescent and also induced their adhesion to bone surface in vivo, resulting protection of HSC compartment from stresses, which suppress hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.

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Embryonic stem (ES) cells can differentiate into a wide range of well-defined cell types. Cell transplantation to restore tissue function after disease or injury is in theory applicable to a huge variety of human diseases. Thus, the use of lineage-restricted differentiation techniques developed for ES cells will promote future cell therapy. Recently, we and several groups have reported that ES cells can be induced to differentiate into insulin-producing cells. However, the efficiency of differentiation is not enough to produce insulin-secreting cells for future therapeutic use. Another potential source of beta cell regeneration is adult pancreatic stem cells. Recently, several reports showed that the prolonged culture of isolated ductal tissues from mice and humans resulted in the production of functional endocrine cells, suggesting that adult pancreatic stem cells locate at or near the ductal tissues. However, these cells tended to spontaneously differentiate and have not been fully characterized. We recently established our original methods to isolate duct epithelial cells from normal adult mouse pancreas and grow in serum-free culture. We investigated the differentiation capacity of these duct-derived cells. We showed that the duct-derived cells retained the capacity to differentiate into both pancreatic endocrine cells and hepatocytes, and were considered to be endodermal stem cells. Further studies to efficiently induce the differentiation of these cells into insulin-producing cells should afford promise of future therapeutic use of human pancreatic stem cells for diabetes patients.

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Although atherosclerosis progresses with age and causes various disorders, the mechanism for the formation of atherosclerosis is complicated and not fully understood. At present, several biochemical markers are known as risk factors for atherosclerosis. A new guideline for the reference value of serum lipid (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride) was proposed by the Japan Atherosclerosis Society in 2002. These values are utilized for the diagnosis and treatment of hyperlipidemia to protect patients from coronary heart disease (CHD). Degenerated lipoprotein has also been involved in the progression of atherosclerosis. Actually, oxidized LDL is increased in patients with CHD. Preβ1-HDL, not electrophoresed on α but on preβ position, is a unique HDL and increased in patients with CHD. Other lipoproteins such as Lp(a)and small dense LDL are also considered to be risk factors of atherosclerosis. Additionally, clinical research papers showed that gene variant of homocystein showed high homocystein level in serum and is strongly associated with the incidence of CHD. On the other hand, it is now clear that atherosclerosis is a chronic inflammatory disease and oxidized LDL is a key factor in the process of plaque inflammation. High-sensitive CRP (hs-CRP) may be thus used for the predictor of CHD, but it can not be available for patients accompanied with other inflammatory diseases. More recently, it is reported that serum LOX-1 is a possible predictor for CHD. These predictors are clinically useful and more sensitive biomarkers for atherosclerosis may be available in the future.

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It has been shown that the matrix metalloproteinase (MMP)-1 promoter polymorphism 1G/2G is associated with an increased risk of developing various cancers including renal cell carcinoma (RCC), and is in linkage disequilibrium (LD) with the MMP-3 promoter polymorphism 5A/6A. However, the relationship between the MMP-3 5A/6A polymorphism and susceptibility to cancer remains ambiguous. In this study, we genotyped eight polymorphisms over the region of the MMP-1 and MMP-3 genes in 177 healthy subjects, and explored the relationships between RCC and these polymorphisms or haplotypes in 156 cases and 230 age- and gender-matched controls. There were three polymorphisms that showed stronger LD with the MMP-1 1G/2G than with the MMP-3 promoter 5A/6A variant. One of these three polymorphisms was found to be present in the second exon of the MMP-3 gene and to cause an amino acid change, Glu45Lys (G/A). When the genotype distribution of Glu45Lys was compared between RCC patients and controls, the frequency of the G/G genotype was significantly higher in the patients (age- and gender-adjusted odds ratio [OR]=1.81). A significant increase in the frequency of the 2G/2G genotype of MMP-1 1G/2G polymorphism was also observed in the patients (age- and gender-adjusted OR=1.86). The frequency of the 2G-G haplotype of MMP-1 1G/2G and MMP-3 Glu45Lys polymorphisms was significantly higher in the patients compared to controls (crude OR=1.95, CI=1.31-2.91). These findings suggest that a haplotype of MMP-1 and MMP-3 variants may be associated with the risk of developing RCC.

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Advances in molecular biotechnologies and elucidation of molecular etiology of diseases in combination have facilitated laboratory uses of the molecular diagnostic tests. Uses of the molecular diagnostic tests have been essential in performing medical practice of infectious, neoplastic as well as genetic diseases. Automated systems have been developed for amplification and detection, and lately for extraction, allowing improvement of not only assay efficiency but also quality control of the tests. The information on the genome sequences as the outcome of human genome project has been studied to elucidate functions of genes and proteins and clinical significance of nucleic acid sequences. There has been further technological innovations for post-genomics such as expression profiling using DNA microarray, proteomics, and single nucleotide polymorphisms analysis, in conjunction with bioinformatics. Such emerging technologies will continue to be investigated for usage of the molecular diagnostic tests for therapeutic and preventive health care.

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One of most important reasons of the advancement of treatment of diabetes mellitus after the 1980s was the clinical application of hemoglobin A_1c (HbA_1c). However, HbA_1c is not the name of a substance, but the name of one fraction of HPLC, so standardization has been difficult. In Japan, by using the primary calibrators, Lot 1 and Lot 2, standardization has been achieved and maintained. The international standardization of the measurement of HbA_1c will be completed in the near future according to the method proposed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). In order to avoid expected confusions, the Japan Diabetes Society (JDS) and the Japan Society of Clinical Chemistry (JSCC) committees propose A1c-ISP (International Standardized Percent) as the new test name.

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Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful tool for the analysis of glycosylation sites and of site-specific glycosylation in a glycoprotein. The glycopeptides in a complex mixture of tryptic digest can be separated and monitored by using oxonium ions produced from a carbohydrate moiety through CID-MS/MS. Based on b and y ions in the product ion mass spectra, peptides can be identified, and the structure of carbohydrates can be deduced from B ions and the molecular weight of precursor glycopeptide. Here we show the site-specific glycosylation analysis of α-fetoprotein and an SDSPAGE gel-separated GPI-anchored protein.

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The principle of free-flow electrophoresis (FFE) was first introduced by Barrolier (1958) and Hannig (1961). FFE provides a liquid-based separation in three different operating modes: zone electrophoresis-separation of particles (cells, organelles) due to their electrophoretic mobility, isotachphoresis-separation of proteins and peptides in pH step gradient and isoelectric focusing-separation of proteins and peptides due to their isoelectric point. The key feature of FFE technology is as follows. (i) The separation is performed continuously and enables us to obtain as much as hundreds of milligrams or even gram amounts pure substances. (ii) the separation is performed in a thin aqueous film without gels and enables us to collection of matrix-free fraction with high reproducibility. Therefore, FFE technology ensures that the separated samples are compatible with all downstream concentration procedures (e.g. ultrafiltration), whose enrichment allows to visualize less abundant proteins for subsequent 2-DE analysis and separate poor soluble proteins (e.g. membrane proteins) for subsequent SDS-PAGE. Moreover, FFE can be coupled with such analytical methods as liquid chromatography/mass spectrometry. In the field of proteomics, FFE is a highly versatile technology to support key applications due to prefractionation of samples.

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A routine assay for the determination of plasma protein that requires a smaller sample size, shorter analysis time, and can be performed in batch process for a number of samples is required. Micro two-dimensional polyacrylamide gel electrophoresis (M2D-PAGE) using microdose sample volumes on miniature gels is achievable by shortening the migration time while maintaining the advantages of two-dimensional electrophoresis. This assay is a modification of M2D-PAGE, with improved heat discharge efficiency and higher loading voltage achievable through the modification of gel size. As a result, that is reduced migration, staining, destaining times and small sample sizes is possible assay for accurate determination. We succeeded in developing a rapid determination method using M2D-PAGE that requires about 30 minutes to complete. In addition, it is possible to detect a protein spot with the same sensitivity as conventional methods with a sample volume of 0.1μL or 1/12 of that required in conventional methods employing plasma protein staining by Coomassie Brilliant Blue. With silver staining, this rapid M2D-PAGE has the potential to detect protein spots in samples as small as 1nL.

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Oxidation of sulfur by microorganisms is a topic of interest for a number of researchers worldwide. Literature reports indicate that several different pathways for sulfur oxidation may be present within a single organism. In order to discriminate between activities within these pathways, methods must be available which allow discrimination between specific sulfur oxidizing activities. We describe herein a general method of increasing contrast of ferricyanide reducing zymograms and simultaneously fixing gels for storage.

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