We propose instead a cultural explanation for this late deforestation: the expansion of the Ottoman Empire in Bulgaria (1396), Romanian Principalities (1417 for the Wallachia; 1498 for Moldavia; 1526 for Transylvania) and Serbia (1455). The Ottoman-ruled Bulgaria and Serbia and especially the vassal Romanian

principalities provided a significant part of the empire’s resource provisioning including “wheat, honey, timber, and above all, sheep” ( White, 2011). GS-7340 We propose that deforestation of highly erodible alpine settings that led to the five-fold increase of sediment load on the Danube ( Giosan et al., 2012) reflects this increased demand for timber and especially for sheep by the Ottoman Porte. Indeed, zooarchaeological evaluations

for medieval Moldavian towns ( Stanc and Bejenaru, 2013) shows that before the Ottoman expansion in the region, cattle and pig dominated the local diet. In a short time, by the end of the 16th century, Moldavia alone may have provided 300,000 sheep to Constantinople (Istanbul), out of an estimated 400–500,000 sent by the entire northern Balkans and Romanian principalities ( White, 2011). Such radical changes in animal husbandry suggest that the region adapted to meet the religious dietary requirements and the huge demand of the suzerain Islamic empire by deforesting alpine lands for pasture. Currently, despite selleck compound a 70% sediment deficit accrued after extensive damming in the watershed during the Communist industrialization of Romania in the late 20th century (McCarney-Castle et al., 2012), Danube delta is better positioned compared to other deltas to withstand in the short run the ongoing rise in sea level (e.g., Cazenave et al., 2002). This is due to a combination of reduced subsidence and anthropogenically-augmented sediment trapping on the delta plain (Giosan et al., 2013). That holds true in large part for the internal lobes of Chilia I and II; furthermore, ongoing and planned restoration measures such as dike removal (e.g., Schneider et al., 2008) may re-establish sediment

retention and ecological functions even for their sectors that were drained for agriculture or diked for fisheries. On the other hand, the open coast Chilia III lobe coming under increased BCKDHA wave dominance due to the sediment deficit has become the most dynamic coast of the entire Danube delta (Fig. 4c). Besides the Old Stambul mouth that advances into a shallow lagoon, the only other stable stretch of the coast is linked to the construction of a protecting jetty at the Bastroe mouth, built as a part of a large navigation project. This led to updrift beach ridge progradation as the southward longshore drift is trapped by the jetty and downdrift spit extension under a reversed drift in the lee of the jetty (Fig. 4c).

The increasing use of next-generation

sequencing in a screening role across Europe will revolutionize understanding of both endemic and exotic circulation of low-pathogenicity arboviruses, driven initially by investigations of unexplained clinical cases in affected hosts ( Delwart, 2007 and Radford et al., 2012). The broad-scale sensitivity of next generation sequencing techniques will allow increasing use of sentinel surveillance worldwide by reducing cost/benefit ratios that currently make such schemes unworkable. These data are likely to be complemented in due course by analyses CDK inhibitor of the entire viral populations of Culicoides themselves, an area that has yet to be explored with next-generation sequencing methodologies, but which has already shown promise in identifying novel virus species and strains in mosquitoes ( Bishop-Lilly et al., 2010). In addition, detailed serological surveys of arboviruses currently being conducted for SBV on humans in Europe may prove useful in predicting points of contact between vectors and hosts if a zoonotic Culicoides-borne arbovirus emerges in Europe. Screening for potential clinical disease or seroconversion in human populations should be targeted towards geographic areas that include overlap between C. impunctatus and C. obsoletus populations, as preliminarily characterized for Scotland ( Purse et al., 2012).

A greater understanding of the degree of exposure of humans to Culicoides biting in Europe during both work and recreation would complement these studies. There is also an additional requirement to understand how the host preferences and abundance of livestock- Selleckchem NLG919 and human-biting Culicoides species vary seasonally across heterogeneous suburban and recreational landscapes. Direct examination of potential introduction routes of Culicoides-borne arboviruses into northern Europe

would be helpful in providing a framework for risk assessment ( Napp et al., 2013). If specific cargoes could be defined as presenting a particular risk of containing Culicoides, this would allow analysis of import patterns and habitat of origin and destination ports facilitating inference regarding species of arbovirus that could potentially be introduced ( Tatem and Hay, 2007 and Tatem et al., 2006). Similarly, tracing of human movements into Europe from areas Clomifene of endemicity have already demonstrated utility in mosquito-borne arbovirus research and could be usefully extended to monitor the risk of emerging Culicoides-borne infections ( Tatem and Hay, 2007). Two major areas of uncertainty exist in the degree of vector competence of Culicoides species present in Europe for human-pathogenic arboviruses and the ability of available livestock and wildlife hosts to replicate these arboviruses to transmissible levels. To date, no studies of infection of abundant human and livestock biting species of Culicoides in Europe have been carried out with arboviruses transmitted from animals to humans or among humans.

Climate data were gathered from multiple sources (Assel, 2003, Hunter and Croley, 1993, International Great Lakes Datum, 1985 and Quinn and Norton, 1982) as well as from weather stations from the NOAA National Climatic Data Center (see Fig. 1 and S1) and Hunter and Croley (1993), which has been continuously updated online since the original publication date (http://www.glerl.noaa.gov/data/arc/hydro/mnth-hydro.html). Relationships between variables were analyzed with Pearson’s correlation and linear regression, all with alpha = 0.05 level. Land use, population, employment, income and households were used as indicators to represent direct and indirect drivers of change induced by human activities

and to better understand

the economic status of the human population. While we are aware of the selleck screening library differences between the political and watershed boundaries, our analysis of the socioeconomic system is based on the data obtained at the county level. We also obtained historical data from the Detroit metropolitan area on the USA side because it is a significant driver of change and provides a comparison to the other counties within the LSC watershed. Estimates of the area of the watershed and the land use characteristics selleck chemicals llc were obtained from land use classifications produced by Agriculture and Agri-Food Canada (date of access 8 April 2012, ftp://ftp.agr.gc.ca/pub/outgoing/aesb-eos-gg/LCV_CA_AAFC_30M_2000_V12) and the US Multi-Resolution Land Characteristics Consortium (Fry et al., 2011). Because there were little land use data readily available in 1900, we used a USGS image (United States Geological Survey, access date 31 January 2013, http://www.epa.gov/med/grosseile_site/indicators/landuse.html) of Reverse transcriptase the Detroit metropolitan area to display snapshots of developed land use from 1905, 1938, 1968, and 2001. Socioeconomic data (human

population, households, water and waste water infrastructure, employment and income data) were gathered from USA sources: US Census Bureau (census data accessed 2 May 2012, http://www.census.gov/prod/www/abs/decennial/index.html), Southeast Michigan Council of Governments (SEMCOG, 2002), Camp Dresser and McKee (2003), CH2M HILL (2003), City of Detroit (1959), Detroit Water Service (1966), Morrill (1939), SEMCOG, 1971 and SEMCOG, 2001, St. Clair Regional Planning Commission, 1960 and St. Clair Regional Planning Commission, 1969, State of Michigan (1966), Tetra Tech MPS (2003), Michigan Department of Environmental Quality (access data 11 April 2012, http://www.deq.state.mi.us/owis/Page/main/Home.aspx), and Drinking Water Protection Network (access date 11 April 2012, www.rwqims.com) and from Canadian sources: Ontario Department of Economics and Development (1967), Statistics Canada (date of access, 10 July 2012, http://www12.statcan.gc.ca/census-recensement/2011/dp-pd/prof/index.cfm?Lang=E&TABID=1#tab1 and http://www.statcan.gc.ca/start-debut-eng.

Evidence from this study suggests

that we are dealing see more with higher C-values than other studies use for forest cover. Average annual sheet and rill erosion across the US for forested landcover is estimated at ∼0.91 ton/acre/yr ( Gianessi et al., 1986), slightly exceeding model estimates of 0.002 and 0.85 ton/acre/yr, based on the minimum and maximum C-values obtained from literature review ( Table 1); however, this metric incorporates values from pristine forests that show very little erosion to silviculture operations that resemble bare soil conditions and are therefore associated with extremely high C-factor values (approaching 1). The absolute maximum C-factor for any type of land cover is a value of 1 in cases of exposed bare soil. Using a C-factor of 1 in the model would generate an estimate of soil loss that would overlap with the range of sediment weight estimates ( Fig. 11), furthermore suggesting that, although we are looking

at a broad envelope of values for sediment sequestered within the pond, we are looking at a very high C-value, possibly on the order of those published by Teh (2011) or Özhan et al. (2005) or higher, which would bring the soil-loss click here estimate into the ballpark of sediment-weight calculations. The C-factor is assumed to have remained constant through time as the extent of forest cover was already well established by 1974 when pond sedimentation initiated; no changes in forest cover are recognized from subsequent aerial

photographs ( Fig. 5). Given that the studied watershed has not undergone significant human-imposed changes, it is surprising to see so much erosion is inferred. Studies of silviculture operations Cell press show erosion rates from clear-cut harvests returning to baseline levels within the first few years after harvesting ( Hood et al., 2002). Assuming that forest conditions have remained unchanged over the last 38 years, we conclude that urban forest cover is highly erosive. The forest ecosystem lacks ecologic complexity that would likely characterize a more natural forest condition, resulting in a higher C-value. In this respect, logging of the old-growth forest in the 1800s has left a continuing mark on the region as second growth forests are less ecologically complex and more susceptible to soil erosion. Refining the C-factor estimate could be undertaken to factor in amount of bare soil, canopy cover, organic content of soil, and on-site storage across the watershed ( Dissmeyer and Foster, 1981); however, this would require much additional field work, arguing against use of the simple USLE for useful soil-loss estimates.

In the case of Polynesia, the Caribbean, and the Channel Islands, human transformation of island ecosystems began at initial colonization and often accelerated

through time as populations grew and human activities intensified. The maritime agriculturalists that occupied Polynesia and the Caribbean often had a similar pattern of occupation with early records documenting significant anthropogenic burning and landscape clearance, a new suite of intentionally and accidentally introduced plants and animals that were part of transported landscapes, followed by soil erosion and later highly buy ISRIB managed anthropogenic landscapes. The pattern identified in these two island regions is similar to the records of islands in the North Atlantic occupied by Neolithic and Viking Age peoples (McGovern et al., 2007 and Perdikaris and McGovern, 2008) and Mediterranean islands (Patton, 1996; Zeder, 2009). Island archeology also reveals important differences in the scale and magnitude

of human environmental impacts. On the Channel Islands and some Caribbean islands, initial human occupations were by maritime hunter-gatherers. The environmental impacts of these early peoples Volasertib manufacturer is often not as rapid, easy to discern, or as clear as those of pastoralists or agriculturalists. Without domesticated plants and animals (except dogs) or the need to clear land for horticulture, for example, early records of human occupation from California’s Channel Islands generally lack the initial burning, landscape clearing, and soil erosion typical of many Polynesian sequences. Anthropogenic burning is evident on the Channel Islands in the past, but these events are not easy to differentiate from natural fires (Anderson et al., 2010b). Still hunter-gatherers transformed their island ecosystems in major ways, including the translocation of animals, direct and indirect influences on the extinction of mammals and birds, fire and burning, and significant impacts on marine resources. On the Channel Islands, these include translocation of island deer mice, island foxes, and perhaps other organisms

(Rick, 2013), and strong influences on island marine ecosystems and organisms (Erlandson and Rick, 2010). The early record of some Caribbean islands also documents extinction of island sloths and other vertebrates, and translocation of plant resources by hunter-gatherer Astemizole populations (Newsom and Wing, 2004:128; Steadman et al., 2005). These data suggest that there was no single, overarching human influence or impact on island ecosystems in the past—the patterns and processes on islands were complex and related to the subsistence strategies of people occupying the island (i.e., agriculturalists, hunter-gatherers), the population densities of those people, their sociocultural systems and technologies, differences in island physical characteristics (size, age, nutrients, etc.), and the collective decisions made by individual societies.

The cytotoxic

effect of 20(S)-Rg3 in MCF-7 cells unexpectedly showed no significant difference. These results were consistent when Rg3 was treated in MDA-MB-453 cells (Figs. 4A, 4B). The results from flow cytometric analysis [i.e., fluorescence-activated cell sorting (FACS)] indicated that Rg5 significantly induced cell cycle arrest (Figs. 5A, 5B). This was further confirmed by the cell cycle assay with the data representing suppressed cell proliferation in MCF-7 cells after Rg5 treatment. Rg5 increased the number of cells in the G0/G1 phase and decreased the number of cells in the S phase. Based on these results, Rg5 may induce cell cycle arrest at the G0/G1 phase. Protein expression of cyclin D1, cyclin E2 and CDK4 was decreased, whereas the expression of p15INK4B, SCH772984 mouse p53 and p21WAF1/CIP1 was increased (Figs. 6A, 6B). As Fig. 7A shows, treatment with EPZ-6438 clinical trial Rg5 induced caspase-8 and caspase-9, caspase-7, caspase-6. The full-length Bid consequently disappeared in a dose-dependent manner. Poly (ADP-ribose) polymerase

(PARP) cleavage was detected in Rg5-treated MCF-7 cells, which indicated that Rg5 reduced cell viability by inducing apoptosis. Promotion of mitochondria-mediated intrinsic apoptotic pathway by Rg5 was evidenced by Bax/Bcl-2 dysregulation, activation of caspase-9, and release of cytochrome C (Fig. 7A). Apoptosis was evaluated by annexin V/FITC/PI dual staining. After 48 h, Rg5 significantly increased apoptosis at 25μM and 50μM and reduced apoptotic cells at 100μM, whereas necrotic cells were increased (Fig. 7B). The increased expression

of DR4 and DR5 on the cell surface was obvious when cells were treated at the 100μM concentration of Rg5 (Fig. 8A). Activation of p38 mitogen-activated protein kinases (MAPKs) is necessary for apoptosis induced by exposure to ultraviolet radiation, cytokines, chemotherapy, ceramide, and serum deprivation [24]. When Idoxuridine cells were treated with Rg5 (50μM and 100μM), p38 MAPKs were activated with the generation of reactive oxygen species (data not shown) (Fig. 8C). Survivin, an inhibitor of apoptotic proteins, is highly expressed in most types of cancer and is a regulator of mitosis; survivin-targeting cancer treatment is validated with great efficacy and no serious toxicity [25]. The expression of survivin was suppressed at high concentrations of Rg5 (Fig. 8D). Apoptotic cells were visualized with DAPI as fluorescent probes. When cells were incubated for 48 h with Rg5 at indicated concentrations (i.e., 0μM, 50μM, and 100μM), the cells displayed the typical apoptosis morphology such as fragmented and condensed nuclei with cellular shrinkage (Fig. 9B). Cells treated with Rg5 at the 100μM concentration showed a necrosis-like morphology (Fig. 9C). Red ginseng is fresh ginseng that is dry-steamed once using water vapor. Black ginseng refers to ginseng that is steamed nine times. Fine Black ginseng refers to the fine roots (i.e., hairy roots) of BG steamed nine times. As Fig.

Some researchers assume that inter-fluvial forests were not occupied extensively and thus not altered by people (Bush and Silman, 2007, Denevan, 1996, McMichael et al., 2012 and Steege et al., 2013). But many of the documented cultural forests are indeed in interfluves away from the mainstream (Balee, 1989, Balee, 2013, Balick, 1984, Goulding and Smith, 2007, Levis et al., 2012, Politis, 2007 and Smith Epigenetics inhibitor et al., 2007). My surveys along the Curua River in the middle Xingu interfluves also encountered anthropic forests at current

and former villages and at archeological sites (Fig. 13) (Roosevelt et al., 2009:465–466). Many researchers depict oligarchic forests as “uninhabited” (Pitman et al., 2001 and Steege et al.,

2013) and assume they are a natural phenomenon, without conducting research to exclude a human influence, however. Amazonian forests in different regions differ significantly from one another in topography, climate, geology, hydrology, structure, seasonality, and history, but, nonetheless, they often resemble each other in having this pattern of unexpected dominance and density of a small group of plant species. This pattern has been found wherever Amazon Compound C forests have been inventoried and has yet to be explained by natural factors. The diverse regional and local forests of Amazonia are in essence united by these dominants, most of which have an association with humans. The so-called oligarchs (from Greek for “rule by few”) in the Amazon forests are a group of more than 200 predominant species that make up only 1.4% of all the Amazon forest species but almost half of the trees in any given forest

(Steege et al., 2013). Traditionally, Amazonian tropical forests are considered to be taxonomically very diverse floras in which individuals of most species are locally rare and widely separated from one another, limiting the intensity of exploitation possible in any one place (Longman and Jenik, 1987:115–123; Junk et al., 2010, Pires, 1984, Whitmore and Prance, 1987 and Whitmore, 2010:149–152). Therefore, where a small group of species are significantly more common than the others, in contrast to this pattern, and no natural reason has been suggested, these groupings may not be Forskolin in vitro a solely natural product but a partly human one. Researchers recognize that trees and shrubs are much affected by numerous faunal species, so it’s hardly a reach to consider human effects. The dominant tree species tend to be ones valued and actively managed by Amazonian people today, or ones that benefit from the effects of human occupation. People influence them variously: planting them, concentrating or dispersing their propagules, clearing around them, protecting them, attracting or eliminating their animal predators, and/or fertilizing them with their refuse.

To establish the conventional BP age of the sedimentary features, 11 organogenic samples were taken for 14C analysis

using fragments of shells of lagoonal mollusks, vegetal and peat remains (Table 1). The CEDAD laboratories at the University of Lecce, Italy, measured radiocarbon ages. The samples were analyzed using the accelerator mass spectrometry (AMS) technique to determine the 14C content. The conventional 14C ages BP include the 13C/12C corrections and were calibrated using the Calib 7.0 program (Stuiver and Reimer, 1993), and the calibration data sets Intcal13 and Marine13 for terrestrial and marine samples, respectively (Reimer this website et al., 2013). The regional correction (delta R) for marine reservoir effect was 316 ± 35 (Siani et al., 2000). This study used the following archive documents and historical cartography:

(a) the map of the central lagoon by Domenico Margutti of 1691, (b) the hydrographical map of the lagoon by Augusto Dénaix of ca 1810 and (c) the map of the Genio Civile di Venezia of 1901. The original historical maps are the property of the Archivio di Stato di Venezia where they can be found, but a recent collection of historical map reproductions is available in Baso et al. (2003) and D’Alpaos (2010). The map of Margutti was digitized within the Image Map Archive Gis Oriented (IMAGO) Project ( Furlanetto et al., 2009), covering an area in the central lagoon of about 160 km2. MK-1775 in vitro The map of Augusto Dénaix of ca 1810 is a military topographical hydrographical map of the Venice Lagoon and its littoral between the Adige and Piave rivers. It comprises 36 tables, out of which only the ones covering the study area were used. The scale is 1:15,000. The map of the Genio Civile di Venezia M.A.V. of 1901 is a topographic and hydrographic map of the Venice Lagoon and its littoral between the Adige and Sile

rivers. It comprises 18 tables, out of which only the ones covering the Acesulfame Potassium study area were used. The scale is 1:15,000. The description of the georeferencing procedure can be found in Furlanetto and Primon (2004). For the study area we extracted information about the hydrography by digitizing the spatial distribution of palaeochannels. The interpretation of the acoustic profiles is based on a classical seismic stratigraphic method (in terms of reflector termination and configuration) (Mitchum and Vail, 1977). Detailed analysis of acoustic profiles produced a 2D map of the sedimentary features. The initial and final coordinates of each acoustic reflector, with its description, were saved in a Geographical Information System (GIS) through the software GeoMedia®, for further mapping and interpretation (Madricardo et al., 2007, Madricardo et al., 2012 and de Souza et al., 2013). In the GIS it was possible to correlate the acoustic reflectors and to draw the areal extent of each sedimentary feature.

1C and D).

Their nucleus is eccentrically located. Tunic compartment cells are characterized by the presence of two or more cytoplasmic large globules giving the cells a compartmentalized appearance and containing electron-dense granular inclusions of different size and shape, often surrounded by a translucent halo (Fig. 1C), and nucleus centrally located. Because only slight morphological differences distinguish compartment and morula cells, we here collectively refer to them as “tunic compartment/morula cells”. Using the anti-Ci-MAM-A antibody the immunostaining was observed inside several small granules, present among the globules or at the periphery learn more of the cytoplasm in both of the latter cell types. These granules appear oval to spindle-shaped, they are surrounded by a membrane and contain granular

or floccular material which is moderately dense (Fig. 1D and F). To investigate whether an inflammation state alters the distribution of AMPs in the tunic cell population, the immunolocalization was performed on samples during an experimentally induced inflammatory-like reaction. It has been shown previously that, following the initiation of an inflammation by the application of an elicitor, the cell number is massively increased in the inflamed area of the tunic as hemocytes infiltrate from the hemocoel or the mesenchymal space to encapsulate foreign material and release substances in order to destroy it [38] and [39]. Thus, considering that most of the IPI-145 clinical trial cells present in the tunic during an inflammatory response are hemocytes (the classification of which is a controversial issue), and in order to facilitate their identification, cells are here termed according to the nomenclature reported by De Leo [40]. He suggested to distinguish four types of granulocytes:

clear, clear vesicular, micro- and vacuolar granulocytes (with unilocular and globular subtypes). The infiltrating granulocytes observed in the inflamed area are identified SSR128129E as (i) “globular granulocytes”, cell types closely resembling compartment/morula cells; (ii) “unilocular granulocytes” both possessing a single electron-dense large granule, and with a large electron-transparent granule occupying entirely the cytoplasm, like “signet ring cells”; (iii) “microgranulocytes”; (iv) “clear granulocytes and clear vesicular granulocytes”. The cells are often in close contact to one another and appear frequently to be in a degranulating active state, releasing vesicles and showing drastic structural changes so that many cellular ghosts are observed in the tunic matrix (Fig. 2A). Unilocular granulocytes in the inflamed area were immunostained with both anti-Ci-MAM-A (Fig. 2C) and anti-Ci-PAP-A (data not shown) antibodies in their large granule. Many more gold particles were observed with the anti-Ci-PAP-A and anti-Ci-MAM-A antibodies in the cytoplasmic small granules (Fig.

This suggests that ATP does not sufficiently activate P2X7R in swine macrophages cultured

in vitro. ATP, BzATP, lipopolysaccharide (LPS), LPC (1-palmitoyl-sn-glycero-3-phosphocholine), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). A438079 was purchased from Tocris (Bristol, UK). Biotinylated anti-mouse IL-1β (BAF401) and anti-swine IL-1β (BAF681) antibodies were purchased CHIR-99021 datasheet from R&D Systems (Minneapolis, MN). Horseradish peroxidase (HRP)–streptavidin conjugate was purchased from Zymed (South San Francisco, CA). Anti-P2X7R rabbit polyclonal (Epitope: KIRKEFPKTQGQYSGFKYPY from the C-terminus of rat P2X7R, mouse-18/20 and swine-15/20 residues identical) and goat polyclonal (Epitope: YETNKVTRIQSMNY from the N-terminus of human P2X7R, mouse-13/14 and swine-14/14 residues identical) antibodies were purchased from Alomone Labs (Jerusalem, Israel) and Covalab (Villeurbanne, France), respectively. Selleckchem Ku0059436 Anti-actin mouse monoclonal antibody was

purchased from Chemicon International (Temecula, CA). HRP-conjugated rabbit anti-goat, goat anti-rabbit, and goat anti-mouse immunoglobulins antibodies were purchased from ICN Pharmaceutical, Inc. (Aurora, OH). Swine neonates (1–14-days-old crossbred pigs) were obtained from the animal facility at the National Institute of Livestock and Grassland Science, according to the institutional guidelines for animal experiments. After anesthesia had been induced and the animals had been euthanized, their kidneys were dissected out. After the removal of the fibrous renal capsule, the renal cortex was cut into small pieces, and the tissue pieces (3–5 g)

were then forced through a nylon mesh (pore size: 500 µm) in phosphate-buffered saline (PBS) using a scraper. The minced tissue was digested by incubation with collagenase–dispase (Roche Diagnostics, Basel, Switzerland)/PBS solution (1 mg/ml) containing DNase I (Roche) (40 µg/ml) for 1 h at 37 °C. Then, the digested tissue fragments were collected and re-suspended in growth medium composed of Dulbecco’s modified Eagle’s medium (Sigma) containing 10% heat-inactivated Vasopressin Receptor fetal bovine serum (Sanko Junyaku Co., Ltd., Tokyo, Japan), supplemented with 100 µM β-mercaptoethanol (Sigma), 10 µg/ml insulin (Sigma), 100 µg/ml streptomycin (Life Technologies, Carlsbad, CA), 100 U/ml penicillin (Life Technologies), and 5 µg/ml Fungin (InvivoGen, San Diego, CA). The cell suspension was split into 10 T-75 tissue culture flasks (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) and cultured at 37 °C in a humidified atmosphere of 95% air/5% CO2. The culture medium was replaced every 3–4 days. After the cells had been cultured for 2–3 weeks, the cultured cells were harvested by treatment with Accumax™ (Innovative Cell Technologies, Inc., San Diego, CA), suspended in Cell Banker 1 (Nippon Zenyaku Kogyo Co., Ltd.