Etoposide (1 μg/mL) was used as a positive control. The number of cells in both the control and treated cell samples were estimated based on their total nucleic acid content, as described by Cingi et al. (1991). Cells were seeded at 5 × 104 cells/well in 96-well tissue culture plates and exposed to different concentrations of ConA or ConBr lectins (1–200 μg/ml) dissolved Talazoparib purchase in the RPMI medium (with 1% FBS).

After 72 h of incubation, cells were fixed (5% trichloroacetic acid), washed twice with ice-cold PBS, and a soluble nucleotide pool was extracted with cold ethanol. The cell pellet was dissolved in 0.5 M NaOH at 37 °C overnight. Following this, the absorbance at 260 nm of the NaOH fraction was used as an index of the cell number (Bianchi and Fortunati, 1990). The results are expressed as mean percentages of absorbance at 260 nm in treated cells compared to the controls. Etoposide (1 μg/ml) was used as a positive control. In MTT and NAC assays the concentration GSK J4 order that inhibits 50% of cell proliferation (IC50) was determined from plots of cell viability. Proliferating cells can be identified using DNA labeling with nucleotide analogs such as bromodeoxyuridine (BrdU). Leukemic cells were plated in 24-well tissue culture plates (0.3 × 106 cells/mL) and treated with lectins at different concentrations dissolved in RPMI medium (with 1% FBS). After 21 h of exposure, 20 μl of BrdU (10 mM) was added

to each well and incubated for 3 h at 37 °C. To determine the amount of BrdU incorporated into DNA (Pera et al., 1977), cells were harvested and then transferred to cytospin slides and allowed to dry for 2 h at room temperature. Cells that had incorporated BrdU were Erlotinib cell line labeled by direct peroxidase immunocytochemistry using the chromogen diaminobenzidine (DAB). Slides were counterstained with hematoxylin, mounted, and coverslipped. Determination of BrdU positivity was performed by light microscopy (Olympus, Tokyo, Japan). Two hundred cells were counted per sample to determine the percentage of BrdU-positive

cells. Etoposide (1 μg/ml) was used as a positive control. The comet assay, which is used to detect DNA strand breaks, was conducted under alkaline conditions as described by Singh et al. (1988) with minor modifications (Klaude et al., 1996) following the recommendations of the International Workshop on Genotoxicity Test Procedures (Tice et al., 2000). HL-60 and MOLT-4 (0.3 × 106 cells/ml) cells were incubated for 24 h with lectins at 5, 25, and 50 μg/ml. After this, the cells were centrifugated and resuspended in the medium. Subsequently, 20 μl of the cells in suspension (∼106 cells/ml) were dissolved in 0.75% low melting point agarose and immediately spread onto a glass microscope slide precoated with a layer of 1% normal melting point agarose. The agarose was allowed to set at 4 °C for 5 min. The slides were incubated in an ice-cold lysis solution (2.

The calculations also indicate that the maximum monthly mean values for Qin,sur,Gib and Qout,deep,Gib occur in February and March, while the maximum monthly mean values for Qin,sur,Sci and Qout,deep,Sci occur

in August. The net precipitation reaches its minimum monthly mean value in August for the WMB (−0.021 × 106 m3 s−1) and the EMB (−0.065 × 106 m3 s−1). However, the net precipitation reaches its maximum monthly mean value in November for the WMB (−0.003 × 106 m3 s−1) and in December for the EMB (−0.002 × 106 m3 s−1). Generally, Qf for the WMB ranged from 0.002 × 106 m3 s−1 in August to 0.004 × 106 m3 s−1 in February; however, Qf for the EMB Lapatinib purchase ranged from 0.006 × 106 m3 s−1 in August to 0.018 × 106 m3 s−1 in April. The annual mean net flow through the selleck chemicals WMB is larger than through the EMB (Fig. 6); moreover, the net flows through the WMB and EMB display positive trends of 5.2 × 10 m3 s−1 yr−1 and 3.3 × 10 m3 s−1 yr−1, respectively. The net precipitation is negative for the Mediterranean Sea, especially over the EMB, indicating that evaporation is larger than precipitation and without any trends. Annual river discharge into the EMB is larger than river discharge into the

WMB because we have treated the Black Sea outflow as river input into the EMB. The river discharge displays no trend for the WMB. For the EMB, river discharge decreases by a significant 2.1 × 10 m3 s−1 yr−1. This reduction is explained by an approximately 50% decrease in River Nile discharge after the building of the Aswan High Dam in 1964 together with decreased freshwater inflow from the Black Sea. The Black Sea water discharge displays a negative trend over the 1958–2009 period (Shaltout and Omstedt, 2012). Generally, we find no trend in net precipitation over the EMB and WMB, together with no trend in river discharge into

the WMB but a significant decrease in river discharge into the EMB. Accordingly, we would expect to find increased salinity in the EMB but not in the WMB. This agrees well also with the earlier findings of Skliris et al. (2007) Acetophenone and Shaltout and Omstedt (2012). The climatic monthly mean surface temperatures, salinities, and evaporation rates, 1958–2010, calculated from the PROBE-MED version 2.0 model are presented in Fig. 7. The climatic monthly mean surface temperatures for the WMB (EMB) ranged from 13.8 ± 0.4°C in February (15.5 ± 0.4°C in March) to 25.1 ± 0.7°C in August (26.1 ± 0.6°C in August); the climatic monthly mean surface salinities for the WMB (EMB) ranged from 37.4 ± 0.11 (38.2 ± 0.05) in April to 37.6 ± 0.09 (38.6 ± 0.09) in August; and the climatic monthly mean evaporation rates over the WMB (EMB) ranged from 1.78 ± 0.87 mm day−1 in May (2 ± 0.77 mm day−1 in April) to 3.03 ± 1.4 mm day−1 in November (3.69 ± 1.37 mm day−1 in December). In the summer, the surface temperature reaches its maximum values for both studied sub-basins, as do surface salinity and evaporation rate values.