TdR Incorporation Research Articles

Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation: comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA

The conventional tritiated thymidine ( 3 H -TdR) incorporation assay is considered as the `gold standard' for the assessment of cell growth. However, the 3 H -TdR incorporation assay has several disadvantages which have prompted the development of nonradiolabelling proliferation assays such as 5-bromo-2-deoxyuridine (BrdU) ELISA, tetrazolium microplate assay and acid phosphatase assay. In studies, these three proliferation assays have shown equivalent sensitivity and reproducibility to the 3 H -TdR incorporation assay. However the results may be affected by the cell type studied. In the present study, we have used these three proliferation assays for the assessment of rat lymph node CD4+ T lymphocyte growth in response to polyclonal antigen stimulation. The proliferation assays were compared on the basis of four criteria: sensitivity, reproducibility, stimulation index and insensitivity of the assay to the cell number. Our results indicated that the BrdU ELISA demonstrated the highest sensitivity, reproducibility and stimulation index but had a limited linear range for cellular growth. The tetrazolium microplate assay also had a relatively good sensitivity, reproducibility, stimulation index and a wider linear response range for cell growth in comparison to the BrdU ELISA. The acid phosphatase assay showed the lowest reproducibility and stimulation index. Because BrdU incorporation in DNA of proliferating cells has been reported to block cell division, we have investigated this possibility in sequential assays. Our results indicated that in our experimental conditions no evidence of an anti-mitogenic action of BrdU was observed. We also compared the performance of the MTS assay and BrdU ELISA in measuring substance P-induced CD4+ T cell proliferation. The results indicated that the MTS assay may reflect change in cell activation leading to an overestimate of cell growth. In conclusion, our results indicate that the BrdU ELISA is the most sensitive of the three proliferation assays used for the assessment of CD4+ T lymphocyte growth and is the preferred assay when small changes in cell growth are expected.

Activity of marine bacteria under incubated and in situ conditions

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 20:213-223 (1999) - doi:10.3354/ame020213 Activity of marine bacteria under incubated and in situ conditions Evelyn B. Sherr*, Barry F. Sherr, Crystal T. Sigmon College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97331-5503, USA *E-mail: sherrb@oce.orst.edu ABSTRACT: We evaluated the extent to which the average metabolic activity of open ocean bacterioplankton changed during 2 to 3 d incubations of 1 μm filtered seawater at in situ temperature. Indices of bacterial activity during incubation experiments were compared to those of in situ bacterioplankton, which were repeatedly sampled at each oceanic site. Indices included: total and cell-specific incorporation rates of tritiated leucine ([3H]-Leu) and thymidine ([3H]-TdR), ratio of Leu to TdR incorporation, and percentage of highly active cells as determined by detectable reduction of the fluorogenic dye, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Patterns of change in metabolic activity were similar for experiments with water collected in 3 regions: midshelf, slope, and offshore, off the Oregon coast, USA, and incubated at in situ temperatures of 9.5 to 18°C. At the beginning of the experiments, cell-specific incorporation rates of [3H]-Leu and [3H]-TdR were lower compared to in situ rates, but after 19 to 28 h, incorporation rates of [3H]-Leu and [3H]-TdR increased dramatically, by more than an order of magnitude, compared to in situ activity. When scaled to the abundance of CTC positive (CTC+) cells, rates of Leu and TdR incorporation were 2.8- to 8.1-fold higher during the incubations compared to activity of in situ CTC+ cells. In this study, marine bacteria in 1 μm, incubated filtrate exhibited a much larger variation in metabolic activity than did in situ bacterioplankton. In addition, the proportion of CTC+ cells was closely related to cell-specific rates of Leu and TdR incorporation. KEY WORDS: Marine bacteria · Incubation · Metabolism · Cell-specific activity · CTC Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 20, No. 3. Publication date: December 30, 1999 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1999 Inter-Research.

Diel cycles of heterotrophic bacterioplankton abundance and production in the ocean surface waters

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 17:239-246 (1999) - doi:10.3354/ame017239 Diel cycles of heterotrophic bacterioplankton abundance and production in the ocean surface waters Fuh-Kwo Shiah* PO Box 23-13, Institute of Oceanography, National Taiwan University, Taipei, Taiwan, ROC *E-mail: fkshiah@odb03.gcc.ntu.edu.tw ABSTRACT: To examine the magnitude of phytoplankton-bacterial coupling in low productivity areas, diel changes of bacterial abundance and production as well as primary production (PP) in the oligotrophic Kuroshio surface waters were investigated by on deck carboy incubation and consecutive hydrocast sampling. Both methods yielded similar results. Cell counts varied <13% in 1 diel cycle, while thymidine (TdR) incorporation rates and TdR incorporation per cell (TdR cell-1) varied 2- to 5-fold with higher values appearing at night. Such opposing trends between the bacterial rate parameters and PP were consistent over different locations and months. When incubated under an artificial light source, the TdR cell-1 in whole water samples were negatively correlated with light intensity and PP. In nutrient enrichment experiments, the addition of labile organic carbon (glucose) had no effect on bacterial growth in noon and midnight samples. Values of the TdR cell-1 in midnight samples increased about 70% when the inorganic nutrient mixture (NH4+ and PO4-3) was added; however, such addition did not enhance the TdR cell-1 in noon samples. This study demonstrates that the diel pattern of bacterial growth in the ocean surface water is opposite to that of phytoplankton. UV radiation is not required in driving such a variation since UV was excluded in the experimental set-up. There seems to be no simple explanation for the phenomenon reported by this study. Other potential factors, such as inorganic nutrient, organic substrate supply rates and bacterivory, all might play roles in affecting the bacterial diel cycle. KEY WORDS: Bacteria · Oligotrophic · Kuroshio · Growth rate · Inorganic nutrients · Dissolved organic carbon Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 17, No. 3. Publication date: June 18, 1999 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1999 Inter-Research.

Dynamics in bacterioplankton production in a shallow, temperate lake (Lake Neusiedl, Austria): evidence for dependence on macrophyte production rather than on phytoplankton

The seasonal dynamics in bacterioplankton abundance and production were studied at 3 characteristic stations (open water, large pond within the reed belt and within the reed Phragmites australis) in the shallow Lake Neusiedl, Austria, and related to phytoplankton primary production and dissolved organic carbon (DOC). DOC concentrations ranged from about 1 to 2.5 mm01 1-' with humic DOC contributing between 4 0 % during the winter and 55% during the summer. Phytoplankton production was highest in the pond withln the reed belt, where the attenuation was lowest, reaching 110 mg C m-' d-' during a distinct phytoplankton bloom in August. Bacterial abundance ranged from 2 X 106 cells ml-' during winter to about 10 X 10' cells ml-l during summer. Bacterial production calculated by thymidine (TdR) and leucine (leu) incorporation, respectively, were in good agreement at the stations in the reed belt, but bacterial production based on leu incorporation was significantly lower than bacterial production based on TdR incorporation at the open water station. Based on a bacterial growth yield of 16% determined in an earlier study, bacterioplankton carbon demand was always at least 1 order of magnitude higher than carbon production of phytoplankton, indcating that bacterioplankton metabohsm in Lake Neusiedl is heavily dependent on non-phytoplankton sources of DOC. The bacterial carbon demand (ranging from 225 to 870 mg C m-2 d-' depending on the sampling site and substrate used) could be matched by the production of the reed P. australis amounting to 750 to 4510 mg C m-* d-'. Since there is no major allochthonous organic matter input from other sources, this macrophyte production is obviously channeled to the pelagic food web via the bacterioplankton.

Cellular effects produced by internal contamination of rats with tritiated water

The activity of the erythrocyte glucose-6-phosphate dehydrogenase (G6PDH) and the thymidine (TdR) incorporation in the bone marrow cells have been investigated in rats internally contaminated with tritiated water (HTO). The total doses (4.2 cGy; 19.4 cGy and respectively 5 cGy; 20 cGy) were delivered as chronic (100 days) and acute (5 days) contamination. It was observed a significant increase of Glucose-6-Phosphate- Dehydrogenase (G6PDH) activity for 4.2 cGy (0.4 mGy/day). Both types of contamination produced the reduced incorporation of TdR in the bone marrow cells. These results would allow us to speculate that the reduction of the TdR incorporation could be determined by the changes at the level of membrane transport functions for the lower doses (4.2 cGy; 5 cGy), and by radiation induced phenomena at the intracellular level for the larger doses (19.4 cGy; 20 cGy).

Regulation of alveolar macrophage and type II cell DNA synthesis: effects of ozone inhalation.

A characteristic reaction of the lung to inhaled ozone is an increase in the number of type II epithelial cells and alveolar macrophages (AMs). In the present study, we analyzed mechanisms regulating this response. Acute exposure of rats to ozone (2 parts/million, 3 h) induced expression of proliferating cell nuclear antigen, a marker of cellular proliferation, in both type II cells and AMs. This was maximum 48 h after ozone inhalation. Type II cells and AMs isolated from treated rats at this time also incorporated significantly more [3H]thymidine ([3H]TdR) than cells from control animals. When type II cells and AMs were cocultured, a synergistic increase in [3H]TdR uptake was observed. This appeared to be due to increased DNA synthesis by both cell types. Thus [3H]TdR incorporation by type II cells and AMs cocultured with mitomycin C-treated AMs and type II cells, respectively, was elevated compared with cells cultured alone. Type II cells and AMs plated onto tissue culture inserts, as well as culture supernatants from these cells, were found to stimulate DNA synthesis in AMs and type II cells, respectively. In addition, crude membrane preparations from these cells exhibited growth-promoting activity. Thus the mitogenic effects of both cell types appeared to be mediated by soluble factors and membrane-associated molecules. Ozone inhalation resulted in an increase in the mitogenic activity of AMs treated with mitomycin C and plated on tissue culture inserts toward type II cells and of type II cell culture supernatants toward AMs. These data suggest that type II cell and AM proliferation contributes to the regulation of the number of cells in the lung under normal homeostatic conditions and after ozone-induced injury. Moreover, type II cells and AMs produce paracrine mediators that contribute to cellular proliferative responses.

Evaluation of X-radiation and UV-B radiation for elimination of [formula omitted]-thymidine incorporation into responder cells in the IL-2 producing helper T lymphocyte precursor assay

The helper T lymphocyte precursor (HTLp) assay is of value for predicting graft-vs.-host disease after allogeneic bone marrow transplantation. The assay is based on limiting dilution analysis and requires detection of the amount of interleukin 2 (IL-2) produced by one activated T cell. IL-2 is detected by 3 H -thymidine ( 3 H -TdR) incorporation into the IL-2 dependent cell line, CTLL-2. Detection of IL-2 in the whole culture volume of the wells in the microtiter plates increases sensitivity, but requires elimination of 3 H -TdR incorporation into the responder cells in the HTLp assay. We have compared the ability of X-radiation and ultraviolet B (UV-B) radiation to eliminate 3 H -TdR incorporation. X-irradiation of the cells reduced 3 H -TdR incorporation by 90% with doses up to 400 Gy, but the incorporation was still 10 times higher than in wells with stimulator cells only. UV-B irradiation (with Philips TL 12 tubes) of the cells with ≥2 J/cm 2 decreased 3 H -TdR incorporation to the level of wells with stimulator cells only. Neither X-irradiation nor UV-B irradiation of the cultures affected IL-2 produced in the assay or human recombinant IL-2 measured by 3 H -TdR incorporation into the IL-2 dependent cell line, CTLL-2. HTLp frequencies determined after 25 Gy X-irradiation were higher (mean 1.5, range 1.02–2.2 times higher) than HTLp frequencies determined after UV-B irradiation with 2 J/cm 2.

Growth Rates of Bacterial Communities in Soils at Varying pH: A Comparison of the Thymidine and Leucine Incorporation Techniques.

Abstract The growth rate of bacteria in 19 soils with pH values ranging from 4 to 8 was determined using the thymidine (TdR) and leucine (Leu) incorporation techniques. The variation in isotope dilution and unspecific incorporation was also studied. The mean Leu incorporation into protein was 45% of the incorporation into total macromolecules, and was not affected by soil pH. TdR incorporation into DNA varied between 5 and 20% of that into total macromolecules, with the lowest values in the low-pH soils. Isotope dilution plots for Leu incorporation were linear. This was not the case for TdR incorporation, indicating non-Michaelis-Menten kinetics. The degree of participation (DP) of the added labeled compound in Leu incorporation varied between 0.4 (in low-pH soils) and 0.7 and was directly affected by pH. DP for TdR incorporation varied more (from 0.1 to 1), with the lowest values in the low-pH soils. The variation in DP in TdR incorporation was, however, not directly affected by pH. Calculated bacterial turnover times at 20 degreesC varied between 2.3 and 33 days (mean 9.3 days) using TdR incorporation data, and between 2.1 and 13.1 days (mean 5.9 days) using Leu incorporation data. Turnover times were longer for bacteria in low-pH soils, calculated using the Leu incroporation data, while no effect from pH was found using the TdR incorporation data. Comparing data from aquatic habitats indicated that bacterial growth rates in soil were lower.

Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension.

Hyperinsulinemia (HI) and insulin resistance (IR) are frequently associated with hypertension and atherosclerosis. However, the exact roles of HI and IR in the development of hypertension are unclear. Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, we examined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolated from aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). VSMCs were grown to confluence in culture, serum starved, and examined for DNA synthesis (using [3H]thymidine (TDR), immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1) induction). Basal rate of TDR incorporation into DNA was twofold higher in SHR compared with WKY (P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did not abolish mitogenesis mediated by 10-100 nM insulin, suggesting that insulin effect is mediated via its own receptors. Insulin had a small mitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects on MAPK activity in WKY. In contrast, serum-stimulated MAPK activation was comparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059, completely blocked insulin's effect on MAPK activation and mitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin's effects on MAPK activation and mitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapid induction of MKP-1, the dual-specificity MAPK phosphatase. In contrast, VSMCs from SHR were resistant to insulin with respect to MPK-1 expression. We conclude that insulin is mitogenic in SHR, and the effect appears to be mediated by sustained MAPK activation due to impaired insulin-mediated MKP-1 mRNA expression, which may act as an inhibitory feedback loop in attenuating MAPK signaling.

Differential Involvement of a Fas-CPP32-Like Protease Pathway in Apoptosis of TCR/CD9-Costimulated, Naive T Cells and TCR-Restimulated, Activated T Cells

Abstract Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells. AICD was observed at 12 h after the restimulation of activated T cells with anti-CD3 and reached a peak level at 24 h after this restimulation. CPP32-like protease activity was detected during AICD. Although TCR/CD9 stimulation-associated apoptosis was observed at 24 h after the stimulation of naive T cells and reached a peak level at 36 h after this stimulation, CPP32-like protease activity in these T cells was only marginal at all time points. Nevertheless, both forms of apoptosis were prevented similarly by two different peptide-based caspase inhibitors. These results indicate that the apoptosis that follows the T cell activation which is induced as a result of CD9 costimulation does not involve a Fas-CPP32-like protease pathway, but suggest that different caspase members are likely to be critical in this form of apoptosis.

Fibroblast growth factor-2–toxin induced cytotoxicity: differential sensitivity of co-cultured vascular smooth muscle cells and endothelial cells

Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived ribosome-inactivating toxin saporin (SAP) fused to basic fibroblast growth factor (FGF-2). FGF-2-SAP targets and kills cells bearing upregulated FGF receptors. In vivo, FGF-2-SAP inhibits smooth muscle cell hyperplasia in models of restenosis. The present study examined the potential for a differential effect of FGF-2-SAP on canine vascular endothelial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model. Canine vascular SMC and EC cultures were established separately and made quiescent once cells reached 80% confluence. Following the release from growth arrest, both cell types were treated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h. [ 3H]TdR incorporation was used to determine the growth response of SMC and EC. The co-culture system was created by plating canine vascular SMC and EC on either side of a microporous 13 μm thick polyester membrane insert. Both cell types were grown to 80% confluence and independently made quiescent. Following the release from growth arrest, cells were treated with FGF-2-SAP, or FGF-2, or SAP alone. Negative and positive control groups were untreated wells containing phosphate buffered saline and complete growth media, respectively. After 48 h, both [ 3H]TdR incorporation and total DNA content, by fluorometric measurement, were quantitated in SMC and EC independently. FGF-2-SAP showed a concentration-dependent cytotoxicity in both canine SMC and EC but cytotoxicity for EC required substantially higher concentrations. In co-cultured SMC, FGF-2-SAP significantly decreased both [ 3H]TdR uptake and total DNA content at 0.5, 5, 50, and 500 ng/ml (0.01–10 nM) compared to positive controls. In co-cultured EC, FGF-2-SAP decreased [ 3H]TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive controls. Neither SAP alone nor FGF-2 alone showed a significant effect on [ 3H]TdR uptake or DNA content of either SMC or EC. In this unique co-culture model, which better replicates the relationship between SMC and EC in vivo, we demonstrated a dose–response range of FGF-2-SAP at which both the proliferation and total cell number of SMC, but not EC, is significantly reduced. These data suggest that FGF-2-SAP may have therapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascular reconstructions.

Targeting of human and mouse T-lymphocytes by monoclonal antibody-HPMA copolymer–doxorubicin conjugates directed against different T-cell surface antigens

Binding of HPMA copolymer-conjugated doxorubicin targeted with monoclonal antibodies directed against various T-cell surface receptors, i.e. Thy1.2 (CDw90), I-A (MHC class II. glycoprotein), L3T4 (CD4), IL-2R (CD25) and CD3, is considerably increased in Con A stimulated T-lymphocytes. FACS analysis showed that the binding is most intensive with anti-Thy1.2 and anti-L3T4 targeted derivatives and it is proportional to the antiproliferative effect of the antibody-targeted drug. No binding and no antiproliferative capacity was observed after in vitro incubation of mouse T-cells with a nonspecific mouse IgG-HPMA-DOX conjugate. [ 3H]-TdR incorporation was inhibited considerably more in Con A stimulated T-cell culture and in EL4 mouse T-cell lymphoma as compared with the culture of nonactivated T-lymphocytes. This proves that intensively proliferating cells are more susceptible to the inhibitory action of an antibody-targeted drug. The cytotoxic efficacy of HPMA copolymer with GlyPheLeuGly or GlyLeuPheGly side-chains to which the drug is conjugated was superior to HPMA copolymer with GlyPheGly or GlyLeuGly side-chains. However, there is no direct correlation between the rate of in vitro drug release and the in vitro cytotoxicity of the respective conjugates. This suggests that the rate of drug release from the conjugate is only one factor responsible for the pharmacological efficacy of the preparation. Furthermore, we detected substantial and prolonged inhibition of proliferation of Con A activated T-cells only if doxorubicin was injected in vivo in the form of an anti-Thy1.2-targeted conjugate.

Endothelins: effect on matrix biosynthesis and proliferation in normal and scleroderma fibroblasts.

We investigated the effect of endothelin-1 (ET-1) in normal and systemic sclerosis (SSc) dermal fibroblasts. Collagen type I, collagen type III, and MMP-1 levels in culture supernatants were measured by competition ELISA and cellular mRNA expression was examined by Northern blotting. Mitogenic responses to ET-1 were assessed by [3H]TdR incorporation. ET receptor mRNA expression was examined by RT-PCR analysis of fibroblast RNA and with surface binding studies using radiolabeled ET receptor ligands and specific receptor antagonists. ET-1 enhanced release of collagen types I and III by control and SSc fibroblast strains, but the effects were significantly greater for control cells (p < 0.05). This effect appeared to involve both ETA and ETB receptor subtypes. SSc fibroblasts demonstrated lower constitutive MMP-1 production than control fibroblasts (p < 0.01), but ET-1 treatment decreased MMP-1 in normal fibroblasts to levels observed in SSc. Mitogenic response (percent control [3H]TdR incorporation) to ET-1 for SSc fibroblasts was 130 +/- 34, significantly less (p < 0.01) than that for normal fibroblasts strains (290 +/- 25). This response appeared to be predominantly mediated via the ETA receptor subtype. Surface binding studies suggested a significantly lower level of ETA binding sites in SSc compared with normal fibroblasts (p < 0.05). These data suggest that ET-1 induces a fibrogenic phenotype in normal dermal fibroblasts that resembles that seen in fibroblasts grown from lesional SSc skin. Moreover, SSc cells appear to be refractory to these effects, and this reduced responsiveness is associated with an altered ratio of ETA:ETB receptor expression, supporting a role for ET-1 in the fibrotic pathology of SSc.

Serum deprivation enhances DNA synthesis of human hepatoma SMMC7721 cells.

AIM:To determine the relationship between serum deprivation or serum levels and cell proliferation of human hepatoma SMMC-7721 cells.METHODS:Human hepatoma SMMC-7721 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS)in 5% CO(2) incubator at 37&mgr; for 24h, and culture media were replaced to serum-free or different serum FCS levels (2.5%, 5%, 10%, 20% and 25%). Six h,12h,18h and 24h after the culture,the cells were incorporated &mgr;(3)H&mgr;-TdR for 4h. At last &mgr;(3)H&mgr;-TdR incorpor-ation was detected with liquid scintillation counting.RESULTS:DNA synthesis of SMMC-7721 cells could be sharply stimulated by short-time (6h) serum deprivation (the cpm value of (3)H-TdR incorporation of cells in serum-free was 39.32-fold higher than cells in 25% serum),and the incorporation of (3)H-TdR was negatively related to the serum levels.Longer-time serum starvation (12h, 18h and 24h) also greatly stimulated DNA synthesis, although the cpm value of (3)H-TdR incroporation was less than that in 6h serum deprivation. Morphology of cells cultured in different serum levels also showed significant difference.CONCLUSION:Compared with other cell lines such as BEL7404 and Swiss 3T3,human hepatoma SMMC-7721 cells had different response to the serum deprivation.Short-time serum deprivation could greatly stimulate DNA synthesis of human hepatoma SMMC-7721 cells.Precautions must be given to the changes of serum levels for the detection of growth factors and drugs using SMMC-7721 cells as a model.

Temperature control of bacterioplankton growth in a temperate large lake

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 16:119-130 (1998) - doi:10.3354/ame016119 Temperature control of bacterioplankton growth in a temperate large lake Meinhard Simon*, Christine Wünsch Limnological Institute, University of Constance, PO Box 5560, D-78457 Konstanz, Germany *Present address: Institute for Chemistry and Biology of the Marine Environment (ICBM), University of Oldenburg, PO Box 2503, D-26111 Oldenburg, Germany. E-mail: m.simon@icbm.uni-oldenburg.de ABSTRACT: In order to examine the significance of temperature in controlling growth of bacterioplankton in a large lake with a pronounced pelagic zone we performed 12 temperature shift-up experiments year-round in mesotrophic Lake Constance, Germany, with samples from 1, 6, 10 or 20 and 50 m depth. We measured incorporation of 14C-leucine (Leu) and 3H-thymidine (TdR) at 5, 10, 15, 20 and 25°C including the in situ temperature ±2°C. The results show that during most of the year bacteria at 1 and 6 m were well adapted to the ambient temperature, ranging between 4 and 23°C, because in situ and optimum growth temperatures agreed within 1 to 5°C. At 20 and 50 m, in situ and optimum growth temperatures usually diverged by more than 5°C, and often by more than 10°C, indicating that bacterial growth at these depths was clearly limited by temperature. Hence, in the upper water column, temperature controlled bacterial growth by selecting for a well-temperature-adapted community whereas in the lower part of the water column temperature directly controlled bacterial growth. In several experiments we observed 2 temperature optima. We interpret this observation as an indication of the existence of 2 different subpopulations with different temperature preferences. Q10 values of Leu incorporation and TdR incorporation, ranging from 1.4 to 42.5, exhibited systematic differences. At 1, 6 and 10 m, Q10 of Leu was higher than that of TdR except in 3 cases, whereas at 20 and 50 m Q10 of TdR was higher than that of Leu except in 3 cases. This observation suggests that protein synthesis (Leu) and DNA replication (TdR) are controlled differently by temperature in differently adapted bacterial communities. In order to test the hypothesis that utilization of the most important substrates is optimized at the optimum growth temperature we carried out 5 additional experiments in which we measured uptake and respiration of dissolved free amino acids and monosaccharides. In 3 experiments we found that the fraction of amino acids respired had a distinct minimum at the optimum growth temperature whereas respiration of monosaccharides had such a minimum only in 1 experiment. The experiments gave no indication of enhanced respiration percentages at minimum growth temperatures. KEY WORDS: Temperature · Bacterial production · Amino acid uptake · Monosaccharide uptake · Respiration · Large lake Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 16, No. 2. Publication date: November 27, 1998 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1998 Inter-Research.

Dual action of CD30 antigen: anti-CD30 antibody induced apoptosis and interleukin-8 secretion in Ki-1 lymphoma cells.

CD30 is a member of the tumor necrosis factor superfamily. In this study, we examined the effect of four anti-CD30 (aCD30) antibodies (Abs) on CD30-positive anaplastic large cell lymphoma-derived cell line, Ki-JK. The aCD30 Abs suppressed [3H]thymidine (TdR) incorporation. With a TdT mediated dUTP-biotin nick end labeling method, apoptosis was detected in Ki-JK cells at day 5 after the addition of aCD30 Ab to the culture. Genistein, an inhibitor of protein tyrosine kinase, had no effect on aCD30 Ab-induced apoptosis. The aCD30 Ab simultaneously induced interleukin-8 (IL-8) secretion in the Ki-JK cells. In culture of the Ki-JK cells with aCD30 Ab for 5 days, the IL-8 concentration of the cell free-supernatant increased to 240 +/- 16 pg/ml, though the concentration was < 12.5 pg/ml without aCD30 Ab. In combination with aCD30 Ab, genistein decreased the concentration of IL-8 in day 5 supernatants. Although, doxorubicin and herbimycin-A suppressed [3H]TdR incorporation and induced apoptosis in the Ki-JK cells, they did not induce IL-8 secretion. Only aCD30 Ab-induced apoptosis was accompanied by IL-8 secretion. IL-8 mRNA was not detected in the Ki-JK cells by reverse transcription-polymerase chain reaction assay. IL-8 mRNA was detected 5 days after adding aCD30 Ab to the culture.

A Novel Alternatively Spliced Form of Murine Vascular Endothelial Growth Factor, VEGF 115

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

Negative feedback control of the spermatogenic progression by testicular oestrogen synthesis: insights from the shark testis model.

The organisation of the testis of the dogfish shark is technically advantageous for stage-by-stage analysis of spermatogenesis in vivo and in vitro. Prior studies using this model show that total oestrogen receptors (ER) are concentrated in regions where spermatocysts ("follicle-like" germ cell-Sertoli cell units) are in stem cell and spermatogonial stages: respectively, germinal zone (GZ) and premeiotic (PrM) regions. By contrast, key enzymes regulating oestrogen (E) concentrations (aromatase, 17 alpha-hydroxylase) are maximal in meiotic (M) and postmeiotic (PoM) regions, respectively, which are upstream in the intratesticular vascular pathway. To investigate the hypothesis that E is part of a signalling mechanism between stages of development, studies were undertaken to test direct effects of oestradiol-17 beta (E2) on processes in ER-rich regions. As measured by [3H]thymidine (-Tdr) incorporation. DNA synthesis in GZ and PrM regions was inhibited by E2 (0-1000 nM) in a dose-response fashion. The maximal response (30-40%) was significant, reproducible and observed within 72 hr of treatment. Insulin differentially affected DNA synthesis and the response to E2 in GZ in GZ and PrM regions. As measured by [3H]Tdr release after prelabelling spermatocysts of GZ regions, apoptosis progressively decreased with increasing concentrations of E2. At the maximal dose of E2 used, there was no effect on total protein synthesis or secretion in combined GZ/PrM cysts, indicating that effects on DNA synthesis and cell death were authentically physiological, not pharmacological, and consistent with a state of developmental arrest. These results support the hypothesis that E synthesised within the testis is part of a negative feedback regulatory mechanism whereby more mature stages regulate the developmental advance of less mature stages. A growth control mechanism of this type could explain the strict temporal, spatial and quantitative order of succeeding stages characteristic of normal spermatogenesis in all vertebrates. Further study is required to determine whether E signalling in this model is restricted to Sertoli cells or has a germ cell component.

Inhibitory effect of solar radiation on thymidine and leucine incorporation by freshwater and marine bacterioplankton

We studied the effect of solar radiation on the incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) and [(sup14)C]leucine ([(sup14)C]Leu) by bacterioplankton in a high mountain lake and the northern Adriatic Sea. After short-term exposure (3 to 4 h) of natural bacterial assemblages to sunlight just beneath the surface, the rates of incorporation of [(sup3)H]TdR and [(sup14)C]Leu were reduced at both sites by up to (symbl)70% compared to those for the dark control. Within the solar UV radiation (290 to 400 nm), the inhibition was caused exclusively by UV-A radiation (320 to 400 nm). However, photosynthetically active radiation (PAR) (400 to 700 nm) contributed almost equally to this effect. Experiments with samples from the high mountain lake showed that at a depth of 2.5 m, the inhibition was caused almost exclusively by UV-A radiation. At a depth of 8.5 m, where chlorophyll a concentrations were higher than those in the upper water column, the rates of incorporation of [(sup3)H]TdR were higher in those samples exposed to full sunlight or to UV-A plus PAR than in the dark control. In laboratory experiments with artificial UV light, the incorporation of [(sup3)H]TdR and [(sup14)C]Leu by mixed bacterial lake cultures was also inhibited mainly by UV-A. In contrast, in the presence of the green alga Chlamydomonas geitleri at a chlorophyll a concentration of 2.5 (mu)g liter(sup-1), inhibition by UV radiation was significantly reduced. These results suggest that there may be complex interactions among UV radiation, heterotrophic bacteria, and phytoplankton and their release of extracellular organic carbon. Our findings indicate that the wavelengths which caused the strongest inhibition of TdR and Leu incorporation by bacterioplankton in the water column were in the UV-A range. However, it may be premature to extrapolate this effect to estimates of bacterial production before more precise information on how solar radiation affects the transport of TdR and Leu into the cell is obtained.

CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis.

The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. In this study, we show that the costimulation of CD9 on naive T cells during TCR stimulation results in transient, albeit potent, activation followed by apoptosis, rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation. [3H]TdR incorporation determined 2 days after CD9 costimulation was as potent as that induced by CD28 costimulation. In contrast to progressive T cell proliferation induced by CD28 costimulation, CD9 costimulation led to the induction of apoptosis of once-activated T cells. Although IL-2R expression was induced significantly earlier and to a greater degree after CD9 costimulation than after CD28 costimulation, CD9 costimulation only transiently produced a small amount of IL-2 and induced apparently low levels of bcl-xL compared with those observed in CD28 costimulation. Addition of rIL-2 to cultures of CD9 costimulation induced strikingly enhanced expression of bcl proteins, especially of bcl-xL, and protected TCR-stimulated T cells from apoptosis. These data indicate that CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis as the result of failure to generate a positive signal for sufficient levels of IL-2 production.