Ministry of Education, Culture, Sports, Science and Technology

The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention.

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro-expanded fetus-derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal-cord-contusion injury model. As the source of graft material, we used a neural stem cell-enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor-derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro-expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.

This treatise is an update to a preceding highlight (CH/π hydrogen bonds in crystals) published in this journal 5 years ago (M. Nishio, CrystEngComm, 2004, 6, 130–156). After the introductory part (sections 1 and 2), we survey recent results (mostly since 2004) relevant to the CH/π hydrogen bond: crystal conformation, packing and host/guest chemistry (section 3). Section 4 summarizes the results obtained by crystallographic database (CSD and PDB) analyses. In section 5, several topics in related fields (selectivity in organic reactions, surface chemistry, structural biology, drug design and high-level ab initio calculations of protein/substrate complexes and natural organic compounds) are introduced, and in the final part we comment on the prospects of this emerging field of chemistry.

A nuclidic mass formula composed of a gross term, an even-odd term and a shell term is presented as a revised version of the mass formula constructed by the present authors and published in 2000. The gross term has almost the same functional form as in the previous formula, but the parameter values in it are somewhat different. The even-odd term is treated more carefully, and a considerable improvement is realized. The shell term is exactly the same as the previous one; it was obtained using spherical single-particle potentials and by treating the deformed nucleus as a superposition of spherical nuclei. The new mass formula is applicable to nuclei with Z ≥ 2 and N ≥ 2. The root-mean-square deviation from experimental masses is 666.7 keV, which is less than that of the previous mass formula, 689.8 keV.

We developed a new endoscopic thyroid surgery by the axillo-bilateral-breast approach (ABBA) method, which is different from the previously described breast approach (BA) in that the port sites are modified to obtain a better view and to prevent the interference of surgical instruments. This modification also improves cosmetic results by eliminating the parasternal incision, which results in hypertrophic scar in a significant number of cases treated with BA. Twelve patients with benign thyroid tumors successfully underwent endoscopic thyroid surgery by ABBA, and their clinical outcomes were compared with those of four patients treated with BA. The mean operation time was significantly shorter in the ABBA group than in the BA group (188 minutes vs. 270 minutes; P < 0.01). Furthermore, the mean blood loss in the ABBA group (53 mL) was half of that in the BA group (108 mL). Neither conversion to open surgery nor significant intraoperative complications were experienced. The operative scars by ABBA became inconspicuous in a few weeks. These results seem to indicate that ABBA is a better method than BA and can be a feasible option, particularly for young patients who opt for the better cosmetic outcome.

Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho. Accumulating evidence has demonstrated that the Rho/Rho-kinase-mediated pathway plays an important role in various cellular functions, not only in vascular smooth muscle contraction but also in actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions, all of which may be involved in the pathogenesis of arteriosclerosis/atherosclerosis. Indeed, animal experiments have demonstrated that Rho-kinase inhibitors effectively suppress coronary artery spasm and that long-term inhibition of Rho-kinase inhibits the development of coronary arteriosclerotic lesions and even causes regression of coronary vascular lesions in vivo. Recent clinical studies also have demonstrated the inhibitory effect of a Rho-kinase inhibitor on coronary artery spasm in patients with vasospastic angina and on exercise-induced myocardial ischemia in patients with stable effort angina with adequate safety. It is possible that Rho-kinase is also involved in the pathogenesis of other forms of cardiovascular diseases. Thus, Rho-kinase could be regarded as a novel therapeutic target in treatment of cardiovascular diseases.

OBJECTIVES: Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cell (EPC). We investigated the effect of angiotensin II (Ang II) on EPC senescence, leading to the impairment of proliferative activity. METHODS AND RESULTS: EPCs were isolated from peripheral blood and characterized. Both reverse transcription (RT)-polymerase chain reaction (PCR) and Western blotting were used to assess gp91phox expression. Immunofluorescence of nitrotyrosine provided evidence of peroxynitrite formation. Our data indicate that Ang II increased the expression of gp91phox mRNA in a dose-dependent manner, which was attenuated by Ang II type 1 (AT1) receptor antagonist valsartan. Similarly, Western blotting revealed that Ang II stimulated an increase in gp91phox, whereas pre-treatment with Valsartan reduced the Ang II-induced expression of gp91phox protein. Valsartan as well as superoxide dismutase (SOD) also inhibited Ang II-induced peroxynitrite formation. The exposure of cultured EPC to Ang II (100 nmol/l) significantly accelerated the rate of senescence compared to a control during 14 days in culture as determined by acidic beta-galactosidase staining. Ang II-induced EPC senescence was significantly inhibited by pre-treatment of either valsartan or SOD (P < 0.01). Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity by using PCR-enzyme-linked immunosorbent-based assay. Ang II significantly diminished telomerase activity, although the effect was significantly reduced by pre-treatment with either valsartan or SOD (P < 0.01). We examined whether Ang II-induced EPC senescence translates into an impairment of EPC proliferation. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] assay disclosed an inhibitory effect of Ang II on EPC proliferation. CONCLUSIONS: Ang II increases gp91phox expression in EPC, which may contribute to oxidative stress, as evidenced by peroxynitrite formation. Ang II accelerates the onset of EPC senescence via increased oxidative stress, which may be related to telomerase inactivation. In addition, Ang II-induced EPC senescence leads to the impairment of proliferative activity.

BACKGROUND AND PURPOSE: The necessity for structural MRI is greater than ever to both diagnose AD in its early stage and objectively evaluate its progression. We propose a new VBM-based software program for automatic detection of early specific atrophy in AD. MATERIALS AND METHODS: A target VOI was determined by group comparison of 30 patients with very mild AD and 40 age-matched healthy controls by using SPM. Then this target VOI was incorporated into a newly developed automated software program independently running on a Windows PC for VBM by using SPM8 plus DARTEL. ROC analysis was performed for discrimination of 116 other patients with AD with very mild stage (n = 45), mild stage (n = 30) and moderate-to-advanced stages (n = 41) from 40 other age-matched healthy controls by using a z score map in the target VOI. RESULTS: Medial temporal structures involving the entire region of the entorhinal cortex, hippocampus, and amygdala showed significant atrophy in the patients with very mild AD and were determined as a target VOI. When we used the severity score of atrophy in this target VOI, 91.6%, 95.8%, and 98.2% accuracies were obtained in the very mild AD, mild AD, and moderate-to-severe AD groups, respectively. In the very mild AD group, a high specificity of 97.5% with a sensitivity of 86.4% was obtained, and age at onset of AD did not influence this accuracy. CONCLUSIONS: This software program with application of SPM8 plus DARTEL to VBM provides a high performance for AD diagnosis by using MRI.

Adenocarcinomas with a micropapillary pattern (MPP), featuring small papillary tufts lacking a central fibrovascular core, are thought to have a poor prognosis. To examine whether the MPP is a predictor of prognosis, clinicopathologic characteristics of adenocarcinomas were analyzed with particular reference to survival of early-stage patients. The subjects were 344 consecutive patients (female/male ratio 163:181) for whom complete surgical resection was undertaken at the Cancer Institute Hospital, Japan, during 1986-1995. Histologically, they were divided into two groups: MPP-positive (n = 139; 40%) and MPP-negative (n = 205; 60%). The following items were significantly more frequent in the MPP-positive group: metastasis to lymph nodes (p <0.001), pleural invasion (p = 0.02), intrapulmonary metastasis (p <0.001), and nonsmoking status (p = 0.002). In stage I patients (i.e., without lymph node metastasis, n = 154), 5-year survival of the MPP-positive group (n = 45) was 79%, significantly lower than the MPP-negative group (n = 109) of 93% (p = 0.004). In many cases of the c-stage I MPP-positive group, upstaging was necessary on the basis of pathologic findings for metastases, and the survival was between stage I and stage II. Our study clearly indicated that the MPP is a distinct prognostic marker for lung adenocarcinoma, particularly regarding apparent stage I diseases.

Since the first report of serum IgG4 elevation in sclerosing pancreatitis in 2001, various systemic disorders have been reported to elevate IgG4, and many names have been proposed from the perspective of the systemic condition. Despite similarities in the organs damaged in IgG4-related Mikulicz's disease and Sjögren's syndrome, there are marked clinical and pathological differences between the 2 entities. The majority of cases diagnosed with autoimmune pancreatitis in Japan are IgG4-related sclerosing pancreatitis, and it should be recognized that this is distinct from the Western type. Diagnosis of IgG4-related disease is defined by both elevated serum IgG4 (> 1.35 g/l) and histopathological features, including lymphocyte and IgG4+ plasma cell infiltration (IgG4+ plasma cells/IgG+ plasma cells > 50% on a highly magnified slide checked at 5 points). Differential diagnosis from other distinct disorders is necessary: these include sarcoidosis, Castleman's disease, Wegener's granulomatosis, lymphoma, cancer, and other existing conditions. The Japanese IgG4 research group has begun multicenter prospective studies to improve diagnostic criteria and treatment strategies.

OBJECTIVES: Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cells (EPCs). Although hypertension is an important coronary risk factor, the influence to the EPCs is not fully understood. We investigated the effect of hypertension on EPC senescence. METHODS: Experimental study We investigated the number and senescence of EPCs in spontaneously hypertensive rats (SHR/Izm) and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. EPCs were isolated from peripheral blood of rats and were characterized. EPC senescence was detected by acidic beta-galactosidase staining. In addition, we measured the telomerase activity using polymerase chain reaction-enzyme-linked immunosorbent assay. CLINICAL STUDY: EPCs were isolated from peripheral blood samples in 37 patients with essential hypertension. After ex-vivo cultivation, we detected senescence and measured the telomerase activity. The total severity index of hypertension-induced organ damage was calculated by the summation of each severity index in the classification of hypertension severity by Tokyo University (1984). RESULTS: Experimental study The EPC senescence in SHR/Izm and DOCA-salt hypertensive rats was significantly increased compared with that of control rats. The telomerase activities in SHR/Izm and DOCA-salt hypertensive sensitive rats were also significantly lowered compared with those of control rats. Clinical study Compared with the control group, EPCs from hypertensive patients showed accelerated senescence and also showed reduced telomerase activity. In hypertensive patients, the degree of hypertension-induced organ damage was negatively correlated with telomerase activity, and was positively correlated with EPC senescence. CONCLUSIONS: EPC senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension, which may be related to telomerase inactivation. The hypertension-induced EPC senescence may affect the process of vascular remodeling.

In Brief Objective Prognosis of severe coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting remains poor. We have recently demonstrated that shock wave therapy effectively induces neovascularization and improves myocardial ischemia in a porcine model in vivo. Methods With permission from the Ethical Committee of our Institute, we treated nine patients with end-stage coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting (55–82 years old, five men and four women) with our cardiac shock wave therapy (200 shots/spot at 0.09 mJ/mm2 for 20–40 spots, 3 times a week/series). We followed-up the patients at 1, 3, 6, and 12 months after the therapy to examine the amelioration of myocardial ischemia. When needed, shock wave therapy was performed up to three series at 0, and 1, 3 or 6 months. Results The cardiac shock wave therapy improved symptoms (Canadian Cardiovascular Society functional class score, from 2.7±0.2 to 1.8±0.2, P<0.01) and reduced nitroglycerin use (from 5.4±2.5 to 0.3±0.3/week, P<0.05). The treatment also improved myocardial perfusion as assessed by dipyridamole stress thallium scintigraphy (severity score, 25.2±7.2% improvement, P<0.05; extent score, 23.3±9.0% improvement, P=0.10; washout rate, 20±3 to 34±3, P<0.05). Myocardial perfusion was improved only in the ischemic area treated with the therapy. These beneficial effects persisted for 12 months. No procedural complications or adverse effects were noted. Conclusion These results indicate that our extracorporeal cardiac shock wave therapy is an effective and non-invasive treatment for end-stage coronary artery disease, although further careful evaluation is needed. Prognosis of severe coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting remains poor. We have recently demonstrated that shock wave therapy effectively induces neovascularization and improvesmyocardial ischemia in a porcinemodel in vivo. The aimof this study was to test this notion in patients with severe coronary artery disease. We treated nine patients with severe coronary artery disease with our cardiac shock wave therapy. We followed-up the patients at 1, 3, 6, and 12 months after the therapy to examine the amelioration ofmyocardial ischemia. When needed, shock wave therapy was performed up to three series at 0, and 1, 3 or 6 months. The cardiac shock wave therapy improved symptoms, nitroglycerin use, andmyocardial perfusion as assessed by dipyridamole stress thallium scintigraphy. These beneficial effects persisted for 12 months. No procedural complications or adverse effects were noted. These results suggest that our extracorporeal cardiac shock wave therapy is an effective and non-invasive treatment for severe coronary artery disease.

OBJECTIVE: To examine the role of heme oxygenase-1 (HO-1) induction in the recovery of renal function in rats with ischemic acute renal failure. DESIGN: Randomized, masked, controlled animal study. SETTING: University-based animal research facility. SUBJECTS: Sprague-Dawley male rats, weighing 200-250 g. INTERVENTIONS: Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia was performed by left renal microvascular clamping, followed by reflow of the blood. MEASUREMENTS AND MAIN RESULTS: Ischemia of the kidney in the uninephrectomized rat significantly induced HO-1 messenger RNA, protein, and enzyme activity, reaching a maximum at 6 hrs, which was mediated in part through an increase in microsomal heme concentration. Heat shock protein 70 was induced extremely rapidly, reaching a maximum at 1 hr, suggesting that HO-1 and heat shock protein 70 gene expression are regulated separately. Inhibition of HO activity by tin mesoporphyrin, which resulted in an increase in microsomal heme concentration, significantly exacerbated renal function, as judged by the sustained increase in serum creatinine concentration and extensive tubular epithelial cell injuries. In contrast, animals that did not receive tin mesoporphyrin showed normal creatinine concentration and microsomal heme concentration 24 hrs after reperfusion, as well as restoration of abnormal renal histology. CONCLUSION: These findings indicate that the expression of HO-1 in the ischemic kidney may be critical in the recovery of renal cell function in this animal model. These findings also suggest that H0-1 induction may play an important role in conferring protection on renal cells from oxidative damage caused by heme.

Abstract Noise‐based crustal seismic velocity changes are known to be affected by environmental perturbations, such as rainfall, atmospheric pressure loading, and temperature changes. Similar to geodetic observations, these external perturbations can mask the effects of tectonic and volcanic processes. In this study, we benefit from the dense Hi‐net short‐period seismic network that covers the entire Japan to measure continuous changes in seismic velocities over a few years, using noise‐based seismic monitoring. Some strong seasonal seismic velocity changes are observed in both southern Japan (Kyushu Island) and northern Japan (Hokkaido Island). Decreasing of seismic velocities in summer in southern Japan can be clearly explained by a model of increased crustal fluid pore pressure associated with high rainfall. In northern Japan, it is necessary to adopt a more complex model to explain the observed seismic velocity variations, which takes into account precipitation, snow depth, and sea level changes. Moreover, western and eastern Hokkaido Island show very different responses to these different external perturbations. The models developed are used to remove the seasonal components of the seismic velocity changes. The minimum remaining detectable seismic velocity change reduces to 10 −5 , which allows detection of crustal responses to small earthquakes that are previously hidden in the strong seasonal perturbations.

PURPOSE: To review the condition of dry eye associated with chronic graft-versus-host disease (GVHD). METHODS: The immunopathogenic processes and therapeutic options for lacrimal gland chronic GVHD are discussed. RESULTS: Dry eye is the most frequent ocular complication after hematopoietic stem cell transplantation. The condition typically occurs around 6 months post-operation and is recognized as a complication of chronic GVHD. Lacrimal gland specimens from patients with dry eye show prominent fibrosis and an increase in CD34+ stromal fibroblasts in the glandular interstitium in addition to infiltration of T cells into the periductal areas. In periductal areas, CD4+ and CD8+ T cells colocalize with stromal fibroblasts that express the full component of surface molecules necessary for antigen presentation. These findings strongly suggest that periductal fibroblasts are involved in fibrogenic and immune processes by interacting with T cells in the lacrimal gland of patients with chronic GVHD, resulting in rapidly progressive dry eye. Current therapies for dry eye related to chronic GVHD include tear supplements and nonspecific immunosuppressants. CONCLUSION: We report a significant role for stromal fibroblasts in the pathogenic processes of dry eye related to chronic GVHD. Although several supportive therapies can reduce the symptoms, specific therapies that suppress fibrotic and immune processes in the lacrimal glands are necessary to control dry eye associated with chronic GVHD.

Abstract Addressing many of the world’s contemporary challenges requires a multifaceted and integrated approach, and interdisciplinary research (IDR) has become increasingly central to both academic interest and government science policies. Although higher interdisciplinarity is then often assumed to be associated with higher research impact, there has been little solid scientific evidence supporting this assumption. Here, we provide verifiable evidence that interdisciplinarity is statistically significantly and positively associated with research impact by focusing on highly cited paper clusters known as the research fronts (RFs). Interdisciplinarity is uniquely operationalised as the effective number of distinct disciplines involved in the RF, computed from the relative abundance of disciplines and the affinity between disciplines, where all natural sciences are classified into eight disciplines. The result of a multiple regression analysis ( n = 2,560) showed that an increase by one in the effective number of disciplines was associated with an approximately 20% increase in the research impact, which was defined as a field-normalised citation-based measure. A new visualisation technique was then applied to identify the research areas in which high-impact IDR is underway and to investigate its evolution over time and across disciplines. Collectively, this work establishes a new framework for understanding the nature and dynamism of IDR in relation to existing disciplines and its relevance to science policymaking.

Mesencephalic precursor cells may one day provide dopaminergic neurons for the treatment of Parkinson's disease. However, the generation of dopaminergic neurons from mesencephalic precursors has been difficult to follow, partly because an appropriate means for recognizing mesencephalic ventricular zone precursors has not been available. To visualize and isolate mesencephalic precursor cells from a mixed population, we used transgenic mice and rats carrying green fluorescent protein ( GFP ) cDNA under the control of the nestin enhancer. nestin -driven GFP was detected in the mesencephalic ventricular zone, and it colocalized with specific markers for neural precursor cells. In addition, data from flow-cytometry indicated that Prominin/CD133, a cell-surface marker for ventricular zone cells, was expressed specifically in these GFP-positive (GFP + ) cells. After sorting by fluorescence-activated cell sorting, the GFP + cells proliferated in vitro and expressed precursor cell markers but not neuronal markers. Using clonogenic sphere formation assays, we showed that this sorted population was enriched in multipotent precursor cells that could differentiate into both neurons and glia. Importantly, many neurons generated from nestin-GFP -sorted mesencephalic precursors developed a dopaminergic phenotype in vitro . Finally, nestin-GFP + cells were transplanted into the striatum of a rat model of Parkinson's disease. Bromodeoxyuridine–tyrosine hydroxylase double-labeling revealed that the transplanted cells generated new dopaminergic neurons within the host striatum. The implanted cells were able to restore dopaminergic function in the host striatum, as assessed by a behavioral measure: recovery from amphetamine-induced rotation. Together, these findings indicate that precursor cells harvested from the embryonic ventral mesencephalon can generate dopaminergic neurons able to restore function to the chemically denervated adult striatum.

Pancreatic cancer remains difficult to treat and has a high mortality rate. It is difficult to diagnose early, mainly due to the lack of screening imaging modalities and specific biomarkers. Consequently, it is important to develop biomarkers that enable the detection of early stage tumors. Emerging evidence is accumulating that tumor cells release substantial amounts of RNA into the bloodstream that strongly resist RNases in the blood and are present at sufficient levels for quantitative analyses. These circulating RNAs are upregulated in the serum and plasma of cancer patients, including those with pancreatic cancer, compared with healthy controls. The majority of RNA biomarker studies have assessed circulating microRNAs (miRs), which are often tissue-specific. There are few reports of the tumor-specific upregulation of other types of small non-coding RNAs (ncRNAs), such as small nucleolar RNAs and Piwi-interacting RNAs. Long ncRNAs (lncRNAs), such as HOTAIR and MALAT1, in the serum/plasma of pancreatic cancer patients have also been reported as diagnostic and prognostic markers. Among tissue-derived RNAs, some miRs show increased expression even in pre-cancerous tissues, and their expression profiles may allow for the discrimination between a chronic inflammatory state and carcinoma. Additionally, some miRs and lncRNAs have been reported with significant alterations in expression according to disease progression, and they may thus represent potential candidate diagnostic or prognostic biomarkers that may be used to evaluate patients once detection methods in peripheral blood are well established. Furthermore, recent innovations in high-throughput sequencing techniques have enabled the discovery of unannotated tumor-associated ncRNAs and tumor-specific alternative splicing as novel and specific biomarkers of cancers. Although much work is required to clarify the release mechanism, origin of tumor-specific circulating RNAs, and selectivity of carrier complexes, and technical advances must also be achieved, such as creating a consensus normalization protocol for quantitative data analysis, circulating RNAs are largely unexplored and might represent novel clinical biomarkers.

BACKGROUND: Recent studies have demonstrated that aging or senescence constitutes a potential limitation to the ability of endothelial progenitor cells (EPCs) to sustain ischemic tissue and repair. Conversely, estrogens have been shown to accelerate recovery of the endothelium after vascular injury. OBJECTIVE: To investigate whether estrogens are able to prevent senescence of EPCs. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood and characterized. After ex-vivo cultivation, the cells became senescent as determined by acidic beta-galactosidase staining. 17beta-estradiol dose-dependently inhibited the onset of EPC senescence in culture. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. 17beta-estradiol significantly increased telomerase activity. Interestingly, reverse transcriptase-PCR analysis demonstrated that 17beta-estradiol dose-dependently increased the catalytic subunit, telomerase reverse transcriptase (TERT) - an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (either wortmannin or LY294002). Because the expression of TERT is regulated by the PI3-K/Akt pathway, we examined the effect of 17beta-estradiol on Akt activity in EPCs. Immunoblotting analysis revealed that 17beta-estradiol dose-dependently led to phosphorylation and, thus, to activation of Akt in EPCs. We also examined whether the protective effect of 17beta-estradiol on EPC senescence translates into the augmentation of mitogenic activity in EPCs. A [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay demonstrated that the mitogenic potential in EPCs treated with 17beta-estradiol exceeded that in untreated (control) EPCs (P < 0.01). In addition, EPCs released vascular endothelial growth factor (VEGF) protein--an effect that was significantly augmented by 17beta-estradiol. Finally, in a Matrigel assay, EPCs treated with both 17beta-estradiol and VEGF were shown to be more likely to integrate into the network formation than those treated with VEGF alone. CONCLUSION: The inhibition of EPC senescence by estrogen in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.

Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.