Thymidine Incorporation Method Research Articles

Expression, menstrual cycle-dependent activation, and bimodal mitogenic effect of transforming growth factor-β1 in human myometrium and leiomyoma

Objective: Transforming growth factor-β1 is the prototype of a bimodal regulator of cell growth, which can either inhibit or stimulate the proliferation of smooth muscle cells. Part of transforming growth factor-β1-mediated stimulation of growth is associated with the increased production of platelet-derived growth factor. The conversion of latent-to-active transforming growth factor-β provides a pivotal mechanism for the regulation of the biologic activity of transforming growth factor-β. We investigated the differential expression and production of the active form of transforming growth factor-β1 in the myometrium and leiomyoma throughout the menstrual cycle. We also studied the mitogenic effects of transforming growth factor-β1 and plateletderived growth factor on myometrial and leiomyoma cells in culture. Study Design: Myometrium and leiomyoma tissue pairs were obtained from 28 women who underwent hysterectomy. Total RNA from each tissue was extracted, and Northern blot analysis was performed for the detection of TGF-β1 messenger RNA. Active and total transforming growth factor-β1 protein was quantified with enzyme-linked immunosorbent assay. Cell proliferation of cultured human myometrial and leiomyoma cells that are treated with TGF-β1 (0.01-1 ng/mL), anti–transforming growth factor-β antibody (0.01-10 ng/mL), or platelet-derived growth factor (10 ng/mL) was assessed by the [3H]thymidine incorporation method. Results: Overall, the transforming growth factor-β1 messenger RNA level in myometrial samples was 1.2-fold higher than in the leiomyoma samples (P < .05). Active transforming growth factor-β1 protein levels in follicular and luteal phase myometrial and leiomyoma samples were significantly greater than the levels in samples from women with atrophic endometrium (P < 0.05). Transforming growth factor-β1, at low concentrations (0.01 ng/mL), induced an increase in cell proliferation (2- to 3-fold; P < .05). When cells were treated with anti-transforming growth factor-β antibody, there was a larger magnitude of increase observed (7- to 20-fold; P < .05). Platelet-derived growth factor (10 ng/mL) consistently increased the rate of cell proliferation both in myometrium and leiomyoma cells (5- to 6-fold; P < .05). Conclusion: Levels of active transforming growth factor-β1 that were produced in follicular and luteal phases indicate a stimulatory role for ovarian hormones. The finding that transforming growth factor-β1, only at low concentrations, stimulates cell proliferation mainly in leiomyoma cells is in agreement with the bimodal and dose-dependent effects of transforming growth factor-β1 that is observed in smooth muscle cells of other tissues. The persistent and high rate of cell proliferation with platelet-derived growth factor suggests that the growth stimulatory effect of transforming growth factor-β1 may be mediated through its up-regulatory effect on platelet-derived growth factor. (Am J Obstet Gynecol 2003;188:76-83.)

Effect of Scutellariae radix extract on the high glucose-induced apoptosis in cultured vascular endothelial cells.

Endothelial cell apoptosis has been postulated as the initial trigger of the progression of microvascular disease in patients with diabetes mellitus. To investigate the role of Scutellariae radix extract, we examined its effect on the endothelial cell proliferation using the [3H]-thymidine incorporation method. Scutellariae radix extract significantly stimulated endothelial cell proliferation in a dose-dependent manner. We focused on the protective action of Scutellariae radix extract on the endothelial cell apoptosis induced by high glucose concentrations. Determination of endothelial cell apoptosis was performed using DNA gel electrophoresis, terminal deoxynuclotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and an ELISA kit. Exposure of vascular endothelial cell to high glucose (16.7 mM) for 72 h resulted in a significant increase in apoptosis, compared with the normal glucose concentrations (5.5 mM). Scutellariae radix extract inhibited high glucose-induced endothelial cell apoptosis. This result suggests that Scutellariae radix extract may contribute to antiapoptotic action against vascular endothelial cells, resulting in a beneficial effect in preventing diabetes-associated microvascular complications.

Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line

To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [ 3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50–100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D ( P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis.

Nodularia spp. (Cyanobacteria) incorporate leucine but not thymidine: importance for bacterial-production measurements

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 28:99-104 (2002) - doi:10.3354/ame028099 Nodularia spp. (Cyanobacteria) incorporate leucine but not thymidine: importance for bacterial-production measurements Susanna Hietanen1,*, Jaana Maritta Lehtimäki2, Liisa Tuominen1, Kaarina Sivonen2, Jorma Kuparinen3 1Finnish Institute of Marine Research, PO Box 33, 00931 Helsinki, Finland 2Department of Applied Chemistry and Microbiology, Division of Microbiology, PO Box 56, Biocentre Viikki, 00014 University of Helsinki, Finland 3Department of Ecology and Systematics, Division of Hydrobiology, PO Box 17, 00014 University of Helsinki, Finland *E-mail: susanna.hietanen@fimr.fi ABSTRACT: Six strains of Nodularia spp., both toxic and non-toxic, were tested for their ability to incorporate leucine and thymidine. All axenic cyanobacterial strains studied showed high leucine incorporation (1.4 to 12 pmol μgchl a-1 h-1 at a concentration of 212 nM leucine), whereas thymidine was either taken up at a very low rate or not at all (0 to 0.2 pmol μgchl a-1 h-1 at a concentration of 121 nM thymidine). We therefore recommend using the thymidine incorporation method instead of that of leucine for measuring heterotrophic bacterial production during cyanobacterial blooms. KEY WORDS: Leucine · Thymidine · Bacterial production · Cyanobacteria Full text in pdf format PreviousExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 28, No. 1. Online publication date: May 16, 2002 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2002 Inter-Research.

Copper mobility and toxicity of soil percolation water to bacteria in a metal polluted forest soil

In order to assess the success of in situ remediation of coniferous forest soil polluted by a Cu–Ni smelter, the total Cu concentration in soil percolation water, the fluxes of Cu down through the soil profile, and the toxicity of soil percolation water to soil bacteria were studied. Total Cu in percolation water was also fractionated into free ionic and complexed forms. The toxicity of the percolation water was measured by the [3H]-thymidine incorporation method, which measures bacterial growth rates. Soil percolation water was collected during one growing season by zero tension lysimeters inserted at depths of 0.2 and 0.4 m in the soil. The treatments consisted of a control, mulch application to the forest floor (M) and mulch application after removing the polluted organic soil layer (MR). The mulch consisted of a mixture of compost and woodchips (1/1; vol/vol). Analysis of Cu species and dissolved organic carbon (DOC) indicated that DOC leached from the mulch and complexed Cu into forms that were less toxic to soil bacteria. At 0.2 m depth percolation water toxicity was 19% lower in the M and 42% lower in the MR treatment than in the control. Toxicity correlated with the Cu2+ concentration, which was 61 and 84% lower in the M and MR treatments, respectively, compared to the control. However, there were signs that total Cu had leached down through the soil profile, the leaching being more pronounced in the MR treatment.

Heterotrophic bacterioplankton production in the East China Sea

Heterotrophic bacterial production (BP) in the East China Sea was measured using tritiated thymidine incorporation (TTI) method in winter 1997 and in summer 1998. The results showed BP in summer (1998, 3.50–15.70 μgC/(L·h) was higher than that in winter (1997, 0.46–2.62 μgC/(L·h). The high values of BP occurred around the Changjiang River estuary and around Station 410. The results at two anchor stations showed that vertical variation of BP was bottom > middle > surface in winter and middle > bottom > surface in summer. Compared with primary production (BP:PP), the average ratio of BP:PP was 0.17(0.04–0.30) in winter and 0.32(0.21–0.43) in summer. There were high ratios around the Changjiang River estuary in winter and around Station 111 in summer.

A New Non-radioactive Method for IL-2 Bioassay

An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine ([3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absor-bance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the [3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the [3H]thymidine employed in the conventional proliferation assays.

Effects of Sphingosylphosphorylcholine on Vascular Smooth Muscle Cell Growth and on the Delayed Growth Response Cell Cycle Proteins Cdk2 and Cdc2

P160 Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important pathophysiological role in the development of vascular disease. We examined the effect of Sphingosylphosphorylcholine (SPC) on DNA synthesis ([ 3 H]thymidine-incorporation method), cell number, cell volume, and cell diameter (CASY-1 coulter counter) as well as on the delayed response cell cycle proteins cyclin-dependent kinase2 (Cdk2) and Cdc2 (a serine/threonineprotein kinase) after stimulation of quiescent VSMCs with 5 μg/mL SPC for 2, 4, 6, 8, 12, 14, 16, 18, 20, 24, and 36 h. Cdk2 protein level is reduced when cells are in the G0-phase and its level is increased by the progression from G1- to S-phase upon addition of growth factors. Progression of the cells from the G2-phase (high level of tyrosine15-phosphorylated form of Cdc2) into M-phase is regulated by dephosphorylation of the Tyr15 residue of Cdc2 (34 kDa). Cdc2 phosphorylation status at Tyr15 and Cdk2 protein level were examined by chemiluminescence western blotting analysis using primary antibodies which recognizes Tyr15-phosphorylated Cdc2 and Cdk2 protein, respectively. Stimulation of VSMCs for 12, 14, 16, 18, 20, 24, and 32 h caused a 50±6, 50±11, 143±3, 192±15, 304±32, 221±29 and 245±26% increase in [ 3 H]thymidine incorporation. Stimulation of VSMCs with SPC for 24, and 32 hour resulted in an 8 and 32% increase in cell number. Cell volume and radius was increased by 40 and 11%, respectively. Stimulation of quiescent VSMCs with SPC for 12, 16, 20, 24 and 32 h caused an 7, 51, 50, 76, and 32% increase of Cdk2 level. After stimulation of VSMCs for 20 and 24 h a 91 and 103% increase of Tyr15-phosphorylation, respectively, occurred. An almost complete Tyr15-dephoshorylation occurred after 32 h. We might conclude that SPC is a potent mitogenic and hyperthrophic factor for VSMCs acting through stimulation of Cdk2 and Cdc2.

Measurement of Human and Murine Interleukin 2 and Interleukin 4

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL-2), and interleukin 4 (IL-4), and of human IL-2 and IL-4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The Basic Protocol describes the use of the CTLL-2 line to detect murine IL-2 and IL-4 in supernatants. One alternate protocol describes the detection of IL-2 in samples of human serum or supernatants using CTLL-2 cells, while other alternate procedures describe the detection and quantitation of murine IL-4 using a mutagenized subline of CTLL-2, CT.4S, and the detection of human IL-4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL-2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Support Protocol 1). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL-2, CT.4S, and CT.h4S cell lines.

Some observations on the copper tolerance of bacterial communities determined by the ( 3H)-thymidine incorporation method in heavy metal polluted humus

Changes in pH after filtration of bacterial suspensions are important when applying the radioactive thymidine incorporation method to heavy-metal polluted soils with low microbial activity. In the original method (Bååth 1992; Soil Biology and Biochemistry 24, 1157–1165) the blended and centrifuged suspension was filtered through glass wool to remove humus particles from the suspension. When we filtered the bacterial suspension through glass wool the pH increased by 2 units and the thymidine incorporation rate decreased. This made the community copper tolerance measurement ambiguous. When using soil samples with very low activity, we recommend the use of acid-washed glass wool or polyester net filtration which eliminates changes in pH.

Bacterial production measured by leucine and thymidine incorporation rates in French lakes

1. Production of heterotrophic bacterioplankton was estimated monthly by the tritiated thymidine and leucine incorporation methods during the draining and filling of the mesotrophic Lake Pareloup (over a 2.5‐years sampling program).2. Rates of 3H‐leucine (leu) and 3H‐thymidine (thy DNA) incorporation generally paralleled each other but the ratio of leu/thy DNA incorporation rates was higher for the draining period (34.5 mean) than during and after filling (11.5 mean).3. After draining, the highest ratios were observed during periods of low temperature and low bacterial specific activity, while DNA labeling by 3H‐thymidine was reduced. However, bacterial production estimates obtained by 3H‐leucine (BPL) and 3H‐thymidine (BPT) incorporation methods were generally well correlated and the average BPL/BPT ratio was equal to 0.78.4. In addition, both methods were applied during a diel cycle in three lakes of different trophic status. An increase of leu/thy DNA incorporation rates was noted from the oligotrophic to the eutrophic system. In the absence of Cyanobacteria, BPL and BPT values were quite concordant on average.5. In situations of unbalanced growth, BPL and BPT values can diverge but when considered over a sufficient period of time they were found to be in agreement.

Tissue biocompatibility of a new caprolactone-coated self-reinforced self-expandable poly-L-lactic acid bioabsorbable urethral stent.

The bioabsorption time as well as other properties of bioabsorbable polymers can be affected by the choice of the basic molecule, by the degree of its polymerization, and by the coating material used in the device. The aim of our present study was to evaluate the biocompatibility of a new caprolactone copolymer-coated, self-reinforced poly-L-lactic acid (SR-PLLA) urethral stent by means of a rabbit muscle implantation test. This new material has previously been tested for cytotoxicity using the thymidine incorporation method (DNA synthesis inhibition test), no toxicity being evidenced. Fifteen male rabbits were used as experimental animals. Rods made from pure lactic acid, pure caprolactone copolymer, and caprolactone-coated lactic acid were placed on both sides of the dorsal muscles, eight implants per rabbit. Rods made from latex and silicone were used as positive and negative controls, respectively. The animals were sacrificed after 1 week, 1 month, or 6 months. Tissue reactions around the implants were analyzed and scored semiquantitatively. Acute tissue reactions attributable to operative trauma were seen in all specimens at 1 week. After 6 months, chronic inflammatory changes and foreign-body reactions were seen only in the positive controls. The new caprolactone copolymer material is highly biocompatible.

Cytotoxicity testing of a new caprolactone-coated self-expanding bioabsorbable self-reinforced poly-L-lactic acid urethral stent.

Forty to 75% of strictures recur within 2 years of urethrotomy and the essential problem remains how to prevent the edges of the cut stricture from adhering to each other and the scar from shrinking. To solve this problem different kinds of urethral stents have been used. Recently a new bioabsorbable self-expanding urethral stent was developed by our group. The core consists of self-reinforced (SR) poly-L-lactic acid (PLLA) and the coating is made of an amorphous copolymer of caprolactone and D,L-lactic acid [P(epsilon-CL/ D,L-LA)]. The aim of our present study was to test the cytotoxicity of the new material used to coat the SR-PLLA urethral stent utilizing the thymidine incorporation method (DNA synthesis inhibition test). The new materials showed no acute toxicity and can be regarded as biocompatible in medical devices.

CORRIGENDA

CORRIGENDACORRIGENDAPublished Online:01 Apr 1999https://doi.org/10.1152/ajpgi.1999.276.4.Ga1Original articleMoreSectionsPDF (29 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 275, December 1998 Volume 38, December 1998 Pages G1394–G1401: K. Imai, T. Mine. M. Tagami, K. Hanaoka, and T. Fujita. “Zonal differences in effects of HGF/SF and EGF on DNA synthesis in hepatocytes under fed or starved conditions.” On page G1397, Fig.2 A appeared incorrectly. Corrected Fig. 2appears below:Fig. 2.Stimulation of [3H]thymidine incorporation into hepatocytes under fed conditions. DNA synthesis was assessed by [3H]thymidine incorporation method as described in materials and methods. [3H]thymidine was included from 48 to 72 h. Values are means ± SD for 12 determinations.A: efficacy of HGF/SF on DNA synthesis assumed by [3H]thymidine incorporation in cultured PVH and PPH under fed conditions.B: efficacy of EGF on DNA synthesis assumed by [3H]thymidine incorporation in cultured PVH and PPH under fed conditions. * P < 0.05, ** P < 0.01, PPH vs. PVH at same concentration.Download figureDownload PowerPoint This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesZonal differences in effects of HGF/SF and EGF on DNA synthesis in hepatocytes under fed or starved conditions 01 Dec 1998American Journal of Physiology-Gastrointestinal and Liver Physiology More from this issue > Video Abstract Volume 276Issue 4April 1999Pages Ga1-Ga1 Copyright & PermissionsCopyright © 1999 the American Physiological Societyhttps://doi.org/10.1152/ajpgi.1999.276.4.Ga1History Published online 1 April 1999 Published in print 1 April 1999 Metrics

Rapid Determination of Bacterial Abundance, Biovolume, Morphology, and Growth by Neural Network-Based Image Analysis

Annual bacterial plankton dynamics at several depths and locations in the Baltic Sea were studied by image analysis. Individual bacteria were classified by using an artificial neural network which also effectively identified nonbacterial objects. Cell counts and frequencies of dividing cells were determined, and the data obtained agreed well with visual observations and previously published values. Cell volumes were measured accurately by comparison with bead standards. The survey included 690 images from a total of 138 samples. Each image contained approximately 200 bacteria. The images were analyzed automatically at a rate of 100 images per h. Bacterial abundance exhibited coherent patterns with time and depth, and there were distinct subsurface peaks in the summer months. Four distinct morphological classes were resolved by the image analyzer, and the dynamics of each could be visualized. The bacterial growth rates estimated from frequencies of dividing cells were different from the bacterial growth rates estimated by the thymidine incorporation method. With minor modifications, the image analysis technique described here can be used to analyze other planktonic classes.

Seasonal Dynamics of Bacterio‐ and Phytoplankton in Large and Shallow Eutrophic Lake Võrtsjärv, Estonia

AbstractThe seasonal dynamics of bacterioplankton abundance, biomass and production was measured in the large (surface area 270 km2) and shallow (mean depth 2.8 m) Lake Võrtsjärv during one year. Bacterial biomass and heterotrophic activity (production) was measured using thymidine and leucine incorporation methods. The bacterial biomass production was estimated by leucine incorporation, the cell number production by thymidine incorporation. Primary production and photosynthetic extracellular release was measured simultaneously to evaluate their role on heterotrophic bacterial activity. Relationships between released fraction of primary production and heterotrophic bacteria, as well as between biomass of bacteria and biomass/composition of phytoplankton are discussed. In terms of the carbon balance, the total primary production could support carbon need of heterotrophic bacteria in Lake Võrtsjärv. Neither biomass nor activity of bacteria showed tight coupling to primary production over the whole investigation period. In spring and summer the relationship between bacterial production and primary production was weak. The development of cyanobacterial bloom in summer provided more substrates for heterotrophic bacteria than diatoms in spring. In autumn both activity of bacteria and algae decreased rapidly in a coupled manner.

Comparison of leucine uptake methods and a thymidine incorporation method for measuring bacterial activity in sediment

Three [ 14C]leucine uptake methods and a [ 3H]thymidine method for measuring bacterial activity in sediment were compared. For leucine uptake, a combustion method, in which prior to combustion sediment was washed with ethanol, yielded the most consistent results. The TCA extraction methods with a filtration step yielded very variable and much lower results than the combustion method. A high concentration of leucine was needed to reach the saturation level (13.7 μM), but that was not found to enhance bacterial activity. No clear saturation level for thymidine incorporation was achieved for two of the four lake sediments tested. Therefore, and because of other weaknesses of the method, the thymidine incorporation underestimated the bacterial carbon production in sediment.

Comparison of cytotoxicity of a quaternary pyridinium metabolite of haloperidol (HP+) with neurotoxinN-methyl-4-phenylpyridinium (MMP+) towards cultured dopaminergic neuroblastoma cells

Haloperidol has recently been found to be metabolized to its pyridinium ion (HP+). This conversion of haloperidol to HP+ appears to be similar to the activation of the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to N-methyl-4-phenyl pyridinium ion (MPP+). MPP+ is responsible for the damage of striatal dopaminergic neurons induced by MPTP in humans and animals. It seemed sensible to investigate whether or not HP+ might be toxic towards dopaminergic neurons and perhaps associated with some of the residual moto-function side effects of haloperidol. We therefore investigated the neurotoxicity of HP+ toward cultured human dopamine neuroblastoma cells (SH-SY5Y) and compared it with that of MPP+. HP+ reduced the viability as measured by MTT and [3H]thymidine incorporation methods in SH-SY5Y cells. Cell membrane integrity is reduced by the treatment of HP+ as measured by intracellular LDH levels. The toxicity was concentration and time dependent. Interestingly, HP+ appeared to be more toxic than MPP+ towards the SH-SY5Y cells in early phase in cultures. The toxicity of MPP+ appear to be progressive and subsequently become more than HP+ with prolonged cultivation. In contrary to MPP+, the toxic effect of HP+ towards a dopamine transporter transfected SK-N-MC cell line is not different from its wild type. This indicates that dopamine uptake system is probably not involved in the cytotoxicity caused by HP+.

Pretreatment with Oleic Acid Accelerates the Entrance into the Mitotic Cycle of EGF-Stimulated Fibroblasts

We have previously demonstrated that pretreatment of several cell lines with cis -unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acid accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.

Bacterioplankton growth associated with physical fronts during a cyanobacterial Bloom

The main compartments of the nucrobial food web were studied in a hydrodynamically complicated area to determine the response of bacterioplankton to spatio-temporal discontinuities in the water column structure. The samples were divided according to water masses into those repre- senting frontal areas and those representing low-saline areas. In the upper mixed water layer (UML) bacterial production was higher in the frontal water (average 4.5 mg C m3 dl) than in the low-saline water (3.7 mg C irr3 d-I). However, the proportion of bacterial production of the primary production was about the same in the frontal water (15 5%) and in the low-saline (16 %) water. The data implied that the recorded frontal upwelling event did not drastically change the mode of production from regenerated to new production. Furthermore, the data indicated that heterotrophic flagellates did not respond to increased bacterial abundance during the intervals between hydrodynamic events. Below the thermochne, the turnover time of bacterial numbers was less than in the UML, as was thymidine incorporation (TdR) per cell, but leucine incorporation (Leu) per cell was highest in the UML. The average molar ratio of Leu to TdR was 7.7 in the UML and 3 below the thermocline. The molar ratio showed an increase in the growth rate during a storm event. Leu and TdR methods did not give equivalent rates of bacterial production over the daily timescale, although they gave quite similar estimates when averaged over the whole study penod (11 d). Our data indicated that one should be very cautious in using conversion factors, which are derived from surface water, to calculate bactenal production throughout the water column, and that sometimes even higher conversion factors should be used below the thermocline than in the UML.