Lower Chamber Research Articles

The influence of tumor heterogeneity on sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs

Background: Studies have shown that extensive genetic or spatial heterogeneity is present within tumors. The present study explored the influence of heterogeneous cells in the tumor microenvironment (TME) on the sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs and further investigated associated molecular mechanisms. Methods: Tumor heterogeneity was simulated using transwell co-culture technique with H1975, A549 or MRC-5 cells grown in the upper chambers and PC-9 cells cultured in the bottom chamber. Each co-culture system was grouped based on different proportions of cells in the upper and lower chambers (from 1% to 300%), and each group was subdivided into erlotinib treatment group (erlotinib+) and erlotinib non-treatment group (erlotinib−). The viability of PC-9 cells was analyzed by CCK-8; HGF, IGF-1 and TGF-α were determined by ELISA; MET amplification was detected by qRT-PCR, and the relevant proteins were detected by Western blot. Results: The viability of PC-9 cells increased with increased proportion of A549/PC-9 or MRC-5/PC-9 cells from 1% to 300%. HGF increased with increased proportion of H1975 cells from 1% to 300%. In all three co-culture systems, TGF-α production in the erlotinib+ subgroup was significantly lower than that in erlotinib- subgroup, and phosphorylated AKT protein showed an ascending tendency with the increased proportion of upper chamber cells relative to PC-9 cells from 1% to 300%. H1975 cells could induce MET amplification of PC-9 cells. MRC5 cells inhibited MET amplification. Conclusions: Tumor heterogeneity partially accounts for the resistance of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs. The possible mechanisms may be related to AKT signaling pathways activated by growth factors, which are secreted by heterogeneous cells in the TME under the pressure of EGFR-TKIs.

Effect of up-regulating the expression of microRNA-613 on biological behavior of human renal cell carcinoma cells

Objective To observe the effect of microRNA-613 (miR-613) on the biological behavior of human renal cell carcinoma cells and explore its molecular mechanism. Methods The synthesized miR-613 mimics and miR-NC were respectively transferred into 786-O cells by Lipofectamine 3000 and divided into experimental group and control group. Real time quantitative polymerase chain reaction (qPCR) was used to detect the expression of miR-613 in cells of two groups to verify the transfection success. The proliferation of 786-O cells was detected by methyl thiazolyl tetrazolium (MTT) assay and the migration of 786-O cells was detected by Transwell migration assay. Bioinformatics predicts the target gene of miR-613. Dual luciferase reporter assay validates the targeted relationship between miR-613 and target genes, and their binding sites. qPCR and Western blot were used to detect the expression of target genes. Results qPCR test results showed that the expression of miR-613 in the experimental group (16.22±1.08) was significantly higher than that in the control group (1.06±0.20), with statistically significant difference (P<0.01). The results of MTT showed that the cells in the experimental group showed a decrease in absorbance value at the d-th point after transfection (P<0.05). Transwell cell migration test results showed that the cells in the control group and experimental group migrated from the upper chamber of Transwell to the lower chamber of the cells were respectively (95.55±17.88) and (199.10±22.74), with statistically significant difference (P<0.05). Bioinformatics predicts that the target gene of miR-613 is Cortactin (CTTN). Dual luciferase reporter assay confirmed that miR-613 can target CTTN gene (P<0.05). miR-613 can significantly inhibit the CTTN gene expression in renal cell carcinoma cells (P<0.01). Conclusions Up-regulation of miR-613 expression can inhibit the proliferation and migration of renal cell carcinoma cells, and its possible molecular mechanism is that miR-613 inhibits the expression of CTTN gene. Key words: Carcinoma, renal cell/PP; MicroRNAs; Cell proliferation; Cell migration assays

The immunological regulation effects of human umbilical cord mesenchymal stem cells on RF/6A cultured in high glucose

Objective To observe the immunological regulation effects of human umbilical cord mesenchymal stem cells (hUCMSC) on glucose-damaged rhesus retinal vascular endothelial cells (RF/6A). Methods hUCMSC and RF/6A were co-culture according to 1: 1 ratio in the co-culture system (Transwell plates), hUCMSC cells were added to upper chamber, while the lower chamber containing 25mmol/L glucose and RF/6A. There were three groups including RF/6A blank control group, high glucose treated RF/6A group, and high glucose treated RF/6A with hUCMSC co-culture group. MTT was used to measure the RF/6A cell viability. Western blot was used to to detect protein level of Foxp3. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of interleukin (IL)-17. Results MTT assay revealed that at the first day, the survival rate of the three groups had no significant difference (F=0.030, P>0.05). On day 3 and day 7, the cell viability of the high glucose group was significantly lower than that of the control group(t=36.072, 27.890; P<0.05), the cell viability of the high glucose treated RF/6A with hUCMSC co-culture group was higher than that of high glucose group (t=36.072, 19.650; P<0.05). Western blot analysis showed that Foxp3 in high glucose RF/6A group was significantly lower than that in the control group at day 7 after culture (t=7.826, P<0.05) and high glucose RF/6A with hUCMSC group (t=19.936, P<0.05). ELISA showed that IL-17 in the high glucose group, high glucose with hUCMSC co-culture group was significantly higher than that of the control group (F=1 267.503, P<0.05), while IL-17 in the hUCMSC co-culture group was significantly lower than that in high glucose group (t=17.386, P<0.05). Conclusion hUCMSC can regulate the expression of Foxp3 and IL-17 to increase the proliferative ability of RF/6A, which was suppressed by high glucose. Key words: Mesenchymal stem cells; Endothelial cells/immunology; Immunomodulation; Animal experimentation

Mechanisms of astrocyte elevated gene-1 promoting cancer metastasis through regulation of the micro-RNA-885-5p/ matrix metalloproteinase 9 signaling pathway in liver cancer

Objective To investigate the relationship between astrocyte elevated gene-1 (AEG-1) and microRNA-885-5p, observe the influence of microRNA-885-5p on MHCC-97H migration and invasion, identify the target gene of microRNA-885-5p and uncover the mechanisms of AEG-1 promoting cancer metastasis. Methods The experimental study was adopted. The AEG-1 over-expressed plasmid constructed and the siRNA of silent AEG-1 synthesized were transiently transfected into MHCC-97H cells, respectively. For plasmid trans-fection, MHCC-97H cells tranfected with empty Vector NC were divided into group A as the control, and MHCC-97H cells tranfected with AEG-1 over-expressed plasmid were divided into group B. For AEG-1 siRNA transfection, MHCC-97H cells tranfected with negative siRNA were divided into group C as the control, and MHCC-97H cells tranfected with AEG-1 siRNA were divided into group D. The relative expressions of microRNA-885-5p in MHCC-97H cells of group A, B, C, D were measured by fluorescent quantitative polymerase chain reaction(PCR). For microRNA-885-5p simulator transfection, MHCC-97H cells tranfected with negative microRNA simulator were divided into group E as the control, and tranfected with microRNA-885-5p simulator were divided into group F. Transwell assay was used to evaluate MHCC-97H migration and invasion. Targetscan software was used to predict the target gene of microRNA-885-5p. MHCC-97H cells co-transfected with reporter plasmid of target gene and negative control siRNA were divided into group G as the control, and MHCC-97H cells co-transfected with reporter plasmid of target gene and microRNA-885-5p simulator were divided into group H. MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and negative control siRNA were divided into group I as the control, MHCC-97H cells co-transfected with reporter plasmid of target gene of binding region mutation and microRNA-885-5p simulator were divided into group J. The luciferase activities of MHCC-97H cells in group G, H, I, J were tested. MHCC-97H cells transfected with negative control siRNA were divided into group K, and tranfected with microRNA-885-5p simulator were divided into group L. Western blot test was used to detect the relative expression of target gene of microRNA-885-5p. MHCC-97H cells transfected with siRNA of target gene of microRNA-885-5p were divided into group M, and transfected with negative control siRNA were divided into group N. Transwell assay was used to evaluate MHCC-97H migration and invasion in group M and group N. Measurement data with normal distribution were presented as ±s and comparison between groups was done using the t test. Results The relative expressions of microRNA-885-5p in MHCC-97H cells of group A and group B were (10.68±1.32)×10-4 and (5.02± 0.20)×10-4, respectively, showing significant difference between the 2 groups (t=7.357, P 0.05) . The relative expressions of MMP9 in MHCC-97H cells of group K and group L were 0.75±0.03 and 0.25±0.03, respectively, showing significant difference between the 2 groups (t=19.086, P<0.05). The results of MHCC-97H migration and invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate were 1 210±163 and 1 192±170 in group M, 537±16 and 374±55 in group N, showing significant differences between the 2 groups (t=7.111, 7.916, P<0.05). Conclusions AEG-1 downregulates the expression of microRNA-885-5p, the target gene of which is MMP9. MicroRNA-885-5p inhabits the migration and invasion of hepatoma cells by negative regulation of MMP9. Key words: Liver neoplasms; Metastases; Astrocyte elevated gene-1; MicroRNA-885-5p; Matrix metalloproteinase 9

Expression of miR-145 in breast cancer and its role in invasion and migration of breast cancer cells

Objective To investigate the expression of microRNA–21 (miR–21) in breast cancer cell lines and serum of patients with breast cancer and the impact on the invasion and migration of breast cancer cells. Methods From Jan 2013 to Feb 2014, miR–21 expression were determined by fluorescent quantity polymerase chain reaction (FQ–PCR) in 4 breast cell lines (HBL–100, MCF–7, MDA–MB–231 and MDA–MB–468) and in serum from breast cancer patients (n=56), breast benign disease patients (n=39) andhealth controls (n=45). The characteristics of cell invasion and migration were examined by transwellinvasion and migration assay afterbreast cancer cell line MDA–MB–231 were transfectedwith miR–21 inhibitor or negative control by lipofectamin. The t test was used to analysis the normal distribution data. Results FQ–PCR results showed that the relative expression of miR–21 in the normal breast epithelial cell line HBL–100 was 1.01 ± 0.04, in the breast cancer cell line MCF–7, MDA–MB–231 and MDA–MB–468 were 1.99 ± 0.11, 4.02 ± 0.38 and 3.73 ± 0.79 respectively. Compared with the normal controls, miR–21 were highly expressed in the three breast cancer cell lines, the difference was statistically significant (t=9.01, 9.20 and 4.55, respectively, P<0.01); and the miR–21 was highly expressed in invasive and metastatic breast cancer cell lines (MDA–MB–231 and MDA–MB–468), compared with weakly invasive breast cancer cell line MCF–7, the difference was statistically significant (t values were 6.14 and 2.91, P<0.05), suggesting that miR–21 is highly expressed in breast cancer cells, and is closely related to the invasion and metastasis. The relative expression of miR–21 in serum of breast cancer was 2.63 (1.57–4.59), in benign breast disease group was 1.34 (1.01–1.78), in healthy control group was 0.81 (0.52–1.59), the miR–21 expression in the serum of breast cancer patients was significantly higher than in patients with benign lesions and normal control group (U values were 208 and 279, P<0.01), whereas no significant difference in serum in patients with benign lesions and normal control group, the miR–21 expression in the serum of breast cancer patients with lymph node metastasis (U=95, P=0.19) was 3.55 (2.44–5.26), significantly higher than those without lymph node metastasis [2.11(1.59–3.25), U=216, P=0.021]. The results of invasion and migration assay showed that cells treated with miR–21 inhibitor invasion was: 44 ± 18, the number of cell migration was: 98 ± 22, while the negative control treated cells after invasion was: 133 ± 44, migration cell number: 255 ± 35; miR–21 inhibitor treatment compared with the negative control, cell invasion and migration was also significantly decreased (t values were 5.46 and 9.08, P<0.01). The cell invasion and migration assay indicated the numbers of MDA–MB–231 cells, which invaded or migrated to lower chamber, were 44±18 and 98±22 respectively after miR–21 inhibitor was applied, The numbers of invaded or migrated cells were 133±44 and 255±35 when the negative control was applied. The ability of cell invasion and migration was decreased significantly in the inhibitor group compared with the negative group (tvalue separately was 5.46, 9.08, P<0.01). The capacity of breast cancer cell invasion and migrationwas significantly decreased after transfection ofmiR–21 inhibitor. Conclusions MiR–21 is highly expressed in breast cancer cell lines and breast cancer patients′ serum. Altered expression of miR–21 maybeplays an important role in breast cancer invasion and migration. MiR–21 may serve as new biomarker to early detectionand prognosis estimation of breast cancer. (Chin J Lab Med, 2015, 38: 186–190) Key words: Breast neoplasms; microRNAs; Neoplasm metastasis; Tumor markers, biological

Establishment of model of serum-caused damage to pulmonary microvascular endothelial cells of mice with renal ischemia-reperfusion injury

Objective To establish the model of serum-caused damage to pulmonary microvascular endothelial cells (PMVECs) of mice with renal ischemia-reperfusion (I/R) injury. Methods Mice PMVECs were cultured to measure the standard trans-endothelial electrical resistance (TER) in the monolayer of PMVECs. When PMVECs were cultured and arranged in compact monolayer and TER was achieved, they were divided into 4 groups (n = 3 each) using a random number table: serum of normal mice group (NS group) and different concentrations (5%, 10% and 20%) of serum of mice with renal I/R injury groups (IRS5 group, IRS10group and IRS20 group). The PMVECs were cultured for 1 h in the serum-free endothelial culture medium. The 0.8 and 0.2 ml culture medium containing 20% serum of normal mice were then added to the upper and lower chambers, respectively, in group NS. The 0.8 and 0.2 ml culture medium containing 5%, 10% and 20% serum of mice with renal I/R injury were then added to the upper and lower chambers in IRS5, IRS10 and IRS20 groups, respectively.100 μg/ml FITC-BSA 100 μl was added to the upper chamber in the four groups. At 3, 6, 9, 12, 15, 18, 21 and 24 h of incubation, the PMVEC monolayer permeability (apparent permeability coefficient, Pa) was detected. Results Compared with NS group, the Pa was significantly increased at 12 and 15 h of incubation in IRS5 group, and the Pa was increased at 6-24 h of incubation in IRS10 and IRS20 groups. Compared with IRS5 group, the Pa at 21 and 24 h in IRS10 group and at 9-24 h in IRS20 group were significantly increased. Conclusion Both 10% and 20% serum of mice with renal I/R injury can successfully establish the model of damage to PMVECs, and 20% serum causes a more severe damage. Key words: Reperfusion injury; Kidney; Serum; Endothelial cells

What a world assembly could look like

I identify some basic institutional features that can make a world assembly viable in terms of size and degree of complexity. The most potentially satisfactory model is a bicameral assembly formed by a lower chamber with about 2000 seats (of which about one-fourth would be elected in single-member districts and about three-fourths in multimember districts of proportional representation with moderate magnitude) and an upper chamber of territorial representation based on about 700 territorial units all across the world (basically corresponding to the nearly 200 currently independent states plus about 500 regional governments).

Effects of adenovirus-mediated phosphatase and tensin homolog deleted on chromosome ten gene on the prostate cancer PC-3 cells' metastasis and expression of matrix metalloproteinase-2 and matrix metalloproteinase-9

Objective To investigate the effects of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression on the prostate cancer PC-3 cells' metastasis and the expression of matrix metalloproteinase (MMP)-2 and MMP-9.Methods Recombinant adenovirus vectors were infected into prostate cancer cell line PC-3.The protein expression levels of PTEN,MMP-2 and MMP-9 in the three groups were detected by Western blotting and indirect immunofluorescence assay respectively.The enzymatic activities of MMP-2 and MMP-9 in the three groups were determined by gelatin zymography.Wound scratch assay was used to determine the effect of PTEN on migratory ability of PC-3 cells.Invasion ability of PC-3 cells was measured by Transwell chamber assay.Results After infection by adenovirus,the protein expression of PTEN was significantly up-regulated in PC-3 cells,and the expression and enzymatic activities of MMP-2 and MMP-9 were significantly down-regulated.Wound scratch assay showed that migration distance in Ad-PTEN group at 12,24 and 48 h was (112.36 ± 18.38),(214.75 ± 35.62) and (436.61 ±52.69) μm respectively,which was significantly shorter than in Ad-LacZ group [(166.52 ± 19.28) μm,t=4.55,P＜0.01 at 12 h; (361.87 ±46.15) μm,t=5.64,P＜0.01 at 24 h,and (732.56 ±78.30) μm,t =7.01,P ＜0.01 at 48 h correspondingly].Transwell showed that the number of PC-3 cells that invaded the lower chamber in the Ad-PTEN group (22.40 ± 2.41) was significantly decreased as compared with the control group (38.20 ± 4.44,t =6.99,P ＜ 0.01) and the Ad-LacZ group (37.20 4.97,t =5.98,P ＜ 0.01).Conclusion PTEN gene can significantly inhibit metastasis of the PC-3 cells and expression of MMP-2 and MMP-9,suggesting it may paly an important role in metastasis of prostate cancer. Key words: Prostate cancer; Phosphatase and tensin homolog deleted on chromosome ten; Matrix metalloproteinases-2 ; Matrix metalloproteinases-9 ; Metastasis

Differential efficacy of endodontic obturation procedures: an ex vivo study

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers' broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan-Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p < 0.01). Histological evaluation, performed to investigate microbial localization inside specimens, confirmed that this parameter varied according to the obturation procedures and materials employed. This ex vivo study indicates that gutta-percha/PCS, if used with the MCWCT, is as effective as Resilon when coupled to Real Seal with the SCT or, interestingly, to RelyX Unicem with the TCWCT. These data suggest that further improvement of the currently employed root canal filling procedures is achievable, depending on both the filling materials and the technique employed, thus encouraging clinical studies in this direction.

Risananeiza crassaparies n. sp. from the upper Chattian of Porto Badisco (southern Apulia, Italy).

A new species of Rotaliidae, Risananeiza crassaparies n. sp., is described from the upper Chattian of the Porto Badisco Calcarenites (Salento Peninsula, Southern Italy). The studied specimens are assigned to the foraminiferal genus Risananeiza based on the presence of vertical canals in both the ventral and dorsal side of the test, and an intraseptal canal system that evolves into marginal sutural canals. The new species differs from the type species of the genus, R. pustulosa, in having a lower chamber lumen, and smaller dimension.

Pinch-1 was up-regulated in leukemia BMSC and its possible effect

Pinch-1, a widely expressed focal adhesion protein, has been demonstrated to be up-regulated in multiple solid tumor-associated stromal cells, particularly at invasive edges. It was supposed that Pinch-1 was intimately associated with development and progression of tumors. The expression of Pinch-1 in hematopoietic microenvironment in patients with leukemia remains unclear. This study focused on the expression of Pinch-1 in bone marrow stromal cells (BMSCs) from leukemia patients and its possible effect. BMSC was isolated and cultured from bone marrow in leukemia patients and normal healthy donors. RT-PCR and Western blot analysis were performed to determine Pinch-1 mRNA and protein level in BMSC, respectively. Lentiviral vector containing Pinch-1 siRNA was constructed, and the recombinant lentivirus particle was packaged in 293 cells. Effectiveness of Pinch-1 siRNA was determined by Western blot. The proliferation, apoptosis and motility of leukemia BMSC subjected to Pinch-1 knockdown using siRNA were tested by flow cytometry, TUNEL assay and Transwell system, respectively. Pinch-1 mRNA and protein were significantly up-regulated in ALL and AML BMSC compared to normal BMSC (p<0.01). Although there was no difference in Pinch-1 mRNA between ALL and AML BMSC, cellular levels of Pinch-1 protein in ALL BMSC were significantly higher than that in AML BMSC (p<0.01). Overexpressed Pinch-1 was significantly reduced in leukemia BMSC transfected with Pinch-1 siRNA evidenced by Western blot. Flow cytometry analysis showed that the percentage of cells in S+G2 phases in leukemia BMSC transfected with Pinch-1 siRNA was significantly lower than control (p<0.01). The percentage of apoptotic cells in leukemia BMSC transfected with Pinch-1 siRNA was 19.8±1.0%, significantly higher than controls (p<0.01). The number of leukemia BMSC transfected with Pinch-1 siRNA that migrated to the lower chamber after culturing for 24 h was 8.4±1.1 per field, significantly lower than controls (p<0.01). Pinch-1 mRNA and protein in leukemia BMSC were up-regulated drastically compared with BMSC from healthy donors. Leukemia BMSC displayed hypoproliferation, decreased migration and increased apoptosis after transfecting Pinch-1 siRNA.

Extractive biofilm membrane bioreactor with energy recovery from excess aeration and new membrane fouling control

Hybrid biofilm membrane bioreactor (BF-MBR) system featuring new mechanisms for recovering the excess energy from air bubbling flow in the biofilm reactor and for controlling membrane biofouling was preliminarily investigated in this study. Alternative design of the biofilm reactor was developed to utilize the bubbling flow from the lower aerobic chamber to generate a mechanical mixing in the upper anoxic chamber in the vertical biofilm reactor. Suspended solid (SS) concentration in the system was hydrodynamically controlled to be lower than 70 mg/L. The ultraviolet (UV) inactivation unit was integrated with the membrane filtration tank to limit biological activities for biofoulant productions and to decelerate the unwanted biofilm formation in the permeate tube. Membrane relaxations at various operating conditions were studied for optimum membrane fouling reductions under low SS environment. Combinations of membrane relaxation and the UV inactivation significantly prolonged sustainable operation periods of the membrane filtration in the BF-MBR process.

Regulation of the Low-Dose Radiation Paracrine-Specific Anchorage-Independent Growth Response by Annexin A2

Here we identify the release of annexin A2 into the culture medium in response to low-dose X-radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we demonstrate that annexin A2 is secreted into the medium by irradiated cells (seeded in upper chamber) and is capable of binding to nonirradiated neighboring cells (seeded in lower chamber). The paracrine factor-mediated anchorage-independent growth response to low-dose X irradiation is reduced when irradiated annexin A2-silenced (shRNA) JB6 cells are co-cultured with nonirradiated cells relative to co-culture with irradiated annexin A2-competent vector control cells. Consistent with this observation, purified bovine annexin A2 tetramer induces anchorage-independent growth. These observations suggest that annexin A2 regulates, in part, the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.

Beyond Couples

Two elderly sisters who lived together complained of discrimination on the ground that, when one of them died, the other would face a heavy inheritance tax bill, unlike the survivor of a marriage or civil partnership who enjoys a “spousal exemption” under the Inheritance Tax Act 1984. They lost in both the lower chamber of the European Court of Human Rights and on appeal to the Grand Chamber. At first instance, discrimination was found but held to be proportionate and justifiable; in the Grand Chamber, no discrimination was found, as siblings and spouses/civil partners were held not to be in an analogous situation. As an attempt to avoid a tax borne only by the comparatively wealthy, this case might not naturally engage feminist sympathies. But it demonstrates how unworthy claims can produce positive results by drawing attention to society’s dismissive treatment of old people and calling into question the legal and economic privileges enjoyed by legally-bound couples at the expense of everyone else.

W1991 Upregulation of Epithelial Mesenchymal Transition in Colorectal Cancer After PTEN Knockdown

Background: Colonic carcinogenesis is driven by complex interacting signals including epidermal growth factor receptor signals between transforming colonocytes and supporting stromal cells. EGFR effectors include cyclin D1 and cyclooxygenase-2 (Cox-2). Cyclin D1 regulates proliferation by controlling G1 to S cell cycle transition and is over-expressed in colonic epithelial cells during colonic carcinogenesis. Cox-2 is the rate-limiting enzyme for prostaglandin biosynthesis and is initially increased in stromal fibroblasts and later in colonocytes during malignant progression. In order to begin to dissect the cross talk between these cell types, we measured cyclin D1 and Cox-2 expression in colonic fibroblasts and colon cancer cells grown under monoculture or co-culture conditions. Methods: Colonic embryonic fibroblasts CCD-18Co and Caco-2 colon cancer cells were plated under identical conditions alone, or together in close contact on opposing sides of the transwell membrane. Cells were untreated (basal conditions), stimulated with EGF, an important growth factor, or IL-1β, an important pro-inflammatory cytokine secreted by these cells. After 4 or 24 hrs cells were harvested and lysates probed for cyclin D1 and Cox 2 expression by Western blotting. Results: Compared to cells in monoculture, co-culture conditions that facilitated close interaction of these cells significantly increased basal Cox-2 (>5-fold in CCD18-Co and >2-fold in Caco-2 cells) and cyclin D1 (>2-fold in Caco-2 cells). When added to upper and lower transwell chambers, EGF and IL 1β further enhanced expression of Cox 2 (Caco2 and CCD-18Co) and cyclin D1 (Caco-2 cells) over basal co-culture conditions by > 2fold that persisted for up to 24 hrs (p 70% (p<0.05). Conclusion: We have demonstrated potentially important tumor-promoting effector responses In Vitro that emanate from cross talk between colonic fibroblasts and malignant colonocytes. EGFR-dependent mechanisms appear to mediate some of these responses. Using bioengineered Caco-2 cells that selectively up or down-regulate EGFR signals, we are further dissecting paracrine and autocrine signals of this important growth factor receptor that controls effector responses in stromal cells and malignant colonocytes.

Regulation of axon guidance cue netrin-1 on angiogenesis

To study the effect of netrin-1, an axon guidance cue, on angiogenesis. Human umbilical cord vein endothelial cells (HUVECs) were isolated and cultured. Reverse transcription and polymerase chain reaction (RT-PCR) was used to detect all the known receptors of netrin-1 in the HUVECs. Netrin-1 and vascular endothelial growth factor (VEGF) of the concentration of 10 ng/ml were added into the culture fluid respectively for 72 h, cholecystokinin-8 (CCK-8) was added, and then enzyme mark instrument was used to calculate the relative absorbance (A value). Other HUVECs were added into the upper chamber of the Transwell co-culture system and netrin-1 of different concentrations and VEGF of the concentration of 10 ng/ml were added into the lower chamber respectively for 6 h, and then invert microscopy were used to observe the migration of HUVECs. Matrigel was added into the 96-well plate, and HUVECs and netrin-1 of different concentrations were added into the wells, then contrast microscopy was used to calculate the tube formation. Corneal micropocket assay was performed on 96 rabbits to determine the corneal neovascularization (CNV) with treatment with different concentrations of netrin-1. Of the 6 ligands only the receptor UNC5B was expressed in the HUVECs. The cell counting kit-8 assay showed that the proliferation of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 50 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the proliferation of the HUVECs was inhibited (P<0.05). The migration of HUVECs was promoted when the concentration of netrin-1 was under 500 ng/ml, especially when the concentration was 100 ng/ml, however, when the netrin-1 concentrations were 1000 to 5000 ng/ml the migration of the HUVECs was inhibited (P<0.05). Normally HUVECs formed branch-like and tube-like structure in the culture plate, netrin-1 did not influence the tube formation when the concentration was under 500 ng/ml, and inhibited the tube formation when the concentration was 1000 approximately 5000 ng/ml (P<0.05). Rabbit corneal micropocket assay showed that netrin-1 of the concentration of 100 ng/ml and VEGF of the concentration of 10 ng/ml promoted the angiogenesis, however, the netrin-1 of the concentration of 5000 ng/ml inhibited the angiogenesis. Netrin-1 shows has a dual function, both promotive or inhibitory effects, on angiogenesis, depending on the concentration and its inhibitive effect is mediated by UNC5B.

The Role of the Senate in the Legislative Process

In most countries with a bicameral system, the Upper House plays a restrained role with regard to the legislature. In this contribution, the position of the Upper Houses in the Netherlands, France, the United Kingdom and Germany is characterised in terms of rivalry and assistance. ‘Rivalry’ is to be taken to mean here the (im)possibility of the Upper House to wholly or partially force through its own political policy desires. ‘Assistance’ in parliamentary legislative practice implies first and foremost correction as to lawfulness, in particular improvements of a legal-technical nature. Suggestions for ‘enhancing’ the effectiveness and social desirability of legislative texts, as well as their politico-bicameral and non-controversial nature are also placed under the heading ‘assistance’. Only the position of the German Bundesrat can be seen as providing a rival to the Lower Chamber. Neither the French Sénat, the House of Lords nor the Dutch Eerste Kamer provide (political) rivalry. The Sénat and the House of Lords do, however, have a strong assisting position: these Upper Houses play a significant role by using their right of amendment. The Dutch Upper House has a energetic position ‘on paper’, but, compared to the Upper Houses in France, the United Kingdom and Germany, its position is rather weak. Assistance in the area of co-legislation is virtually impossible.

MIGRATORY ABILITY OF ORCONECTES LIMOSUS THROUGH A FISHPASS AND NOTES ON ITS OCCURRENCE IN THE CZECH REPUBLIC

The occurrence of the spiny-cheek crayfish in the Czech Republic was first reported in the 1980´s in the Elbe River and is a result of its upstream migration from Germany. This study confirms that this species occurs in many other localities across the Czech Republic. Its migration ability was experimentally studied at a thirty-chambers fishpass located at a hydroelectric power station in the Elbe River. Group-marked crayfish were placed into 3 selected chambers. Their up- and downstream movement was then registered for 30 min after crayfish stocking. We found that a large portion of the experimental crayfish was passively carried along the stream and was caught in the lowest chamber. The movement of crayfish against the water current towards the higher-positioned chambers was not recorded. However, crayfish showed to have a high ability to hold their position in a strong water flow. Overall, 56.7 ± 9.43%, 6.7 ± 9.43% and 3.3 ± 4.71% of crayfish remained in the chamber of insertion and 23.3 ± 4.71%, 30.0 ± 14.14% and 26.7 ± 17.00% of crayfish migrated to the lower-positioned chambers.

A simple device for concentration of parasite eggs, larvae, and protozoa.

A device for the concentration of parasite eggs, larvae and protozoa from a 1 gram fecal sample is described. It consists of separable upper chamber and lower 15 ml conical centrifuge tube connected by a midpiece which incorporates a filter of stainless steel mesh. A small plastic tube for air venting passes through the filter. Emulsification of the sample is done in the upper chamber by shaking with 10% formalin, glass beads, and Triton X-100. The emulsified sample is then filtered into the lower chamber. Ether is added through the upper chamber to wash remaining eggs and larvae into the lower chamber, which is then capped, shaken, and centrifuged. Following centrifugation, the top plug of debris is removed and the tube is drained and swabbed clean, leaving a small, packed sediment, containing the parasite eggs and larvae. The device yielded as much as 300 times the egg yield of the direct wet mount and was comparable to standard concentration methods. The increased separation of eggs and larvae in this device is probably due to the physical characteristics that are imparted to the fecal solids during shaking with ether, glass beads, and Triton, and passage through the wire gauze.

Dislocation in the Italian Political System: an Analysis of the 1968 Parliamentary Elections

relative stability in the distribution of popular votes among the major Italian political parties. Votes for the Camera (lower chamber) with few exceptions have varied less than 4 percent for any political party from election to election, and for the Senato (upper chamber) less than 5 percent for any party from election to election. With respect to the distribution of votes among the leading parties the 1968 consultazione (election) appears to be no outstanding exception to past patterns. Nevertheless, analysis in greater depth suggests that despite the apparent continuation of previous patterns, certain fundamental changes occurred in the 1968 elections which signify potential dislocation in the Italian political system. The first significant change is the shift to the political left in the overall sympathies of the electorate. Secondly, the 1968 campaign presented a change in the meaning and relevance of the choices put before the Italian voter. Thirdly and significantly, the election results point to a change in the viability of the recently established governmental alliance the center-left formula. In 1968, the political consultazione produced different consequences and held different meanings for the several segments of the Italian party system, particularly for the major parties. Each major party interpreted the 1968 election results as having an important bearing on its future and on that of the Italian party system as a whole.