Incorporation of 3H-leucine, 3H-lysine, and 3H-tryptophan during the cell cycle of Chinese hamster ovary cells: a flow cytometric analysis

Exponentially growing Chinese hamster ovary cells were pulse labeled with 3H-leucine, 3H-lysine, or 3H-tryptophan, fixed, and stained by either the acriflavine-Feulgen procedure or with fluorescein isothiocyanate (FITC). Protein content as determined by FITC fluorescence was representative of protein content determined biochemically by the method of Lowry. Utilizing a fluorescence-activated cell sorter, 3H-labeled cells were sorted according to their DNA or protein content and the incorporation of 3H-leucine, 3H-lysine, or 3H-tryptophan determined by liquid scintillation counting. The rates of 3H-leucine, 3H-lysine, and 3H-tryptophan incorporation increased with respect to increasing DNA content (G1, mid-S, G2+M). The rate of 3H-lysine incorporation increased continuously with increasing protein content, whereas the rates of 3H-leucine and 3H-tryptophan incorporation were constant initially with an increase in incorporation near mid-cycle followed by a slight decrease. Matrix algebra modeling of the increase in protein content suggests that 3H-lysine incorporation is consistent with a sigmoidal increase in protein content, however, 3H-leucine and 3H-tryptophan incorporation do not follow either the exponential, linear, or sigmoidal models. Matrix algebra simulation of the FITC protein distribution indicates that while the rate of protein accumulation is not linear, the exponential and sigmoidal models fit the experimental data equally well.     

Disappearance of 3H-leucine incorporation circadian rhythm induced by symplasmic isolation of Chara vulgaris antheridium from thallus.

Autoradiographic research with the use of 3H-leucine demonstrates that circadian rhythm of protein synthesis characteristic of manubria at the proliferative phase of spermatogenesis in Chara vulgaris disappears after symplasmic isolation of antheridium from thallus during the time preceding block of DNA endoreplication in manubria following initiation of spermiogenesis insensitive to light.     

Adaptation of the 3H-Leucine Incorporation Technique to Measure Heterotrophic Activity Associated with Biofilm on the Blades of the Seaweed Sargassum spp.

The ecological interaction between microorganisms and seaweeds depends on the production of secondary compounds that can influence microbial diversity in the water column and the composition of reef environments. We adapted the ³H-leucine incorporation technique to measure bacterial activity in biofilms associated with the blades of the macroalgae Sargassum spp. We evaluated (1) if the epiphytic bacteria on the blades were more active in detritus or in the biofilm, (2) substrate saturation and linearity of ³H-leucine incorporation, (3) the influence of specific metabolic inhibitors during ³H-leucine incorporation under the presence or absence of natural and artificial light, and (4) the efficiency of radiolabeled protein extraction. Scanning electron microscopy showed heterogeneous distribution of bacteria, diatoms, and polymeric extracellular secretions. Active bacteria were present in both biofilm and detritus on the blades. The highest ³H-leucine incorporation was obtained when incubating blades not colonized by macroepibionts. Incubations done under field conditions reported higher ³H-leucine incorporation than in the laboratory. Light quality and sampling manipulation seemed to be the main factors behind this difference. The use of specific metabolic inhibitors confirmed that bacteria are the main group incorporating ³H-leucine but their association with primary production suggested a symbiotic relationship between bacteria, diatoms, and the seaweed.     

Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20–30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 m³) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 m³) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.     

Effects of glycyl-histidyl-lysine on Morris hepatoma 7777 cells

Glycyl-histidyl-lysine (GHL) has been shown to have growth stimulatory effects on a number of different cell types including hepatocytes and hepatoma cells. In this study, the effects of GHL on Morris hepatoma 7777 cells were investigated. The greatest stimulatory effects on 3H-thymidine and 3H-leucine incorporation were observed at a GHL concentration of 2 ng/ml. In randomly proliferating cells, the incorporation of 3H-thymidine into DNA increased by 50% and that of 3H-leucine into protein by 29%. In addition, synergistic effects were observed when insulin and glucagon were included with GHL in the incubation mixture. Experiments with cells rendered quiescent by serum starvation indicated that cells in the G1 phase of the cell cycle are more sensitive to GHL stimulation. In these experiments, 3H-thymidine incorporation increased earlier and peaked at a higher value than in the control cells. This finding suggests that GHL may play a role in stimulating quiescent cells to re-enter the cell cycle.     

Autoradiographic studies of the effect of desmethylimipramine (DMI, Pertofran) on the incorporation of 3H-leucine into the rat brain]

The influence of Desmethylimipramin (PertofranR) on the regional uptake of 3H-leucine in different areas of rat brains has been investigated with autoradiographic methods. Male rats were injected 10 m/kg Desmethylimipramin (DMI, PertofranR) i.m. and 1 hr later 8,33 mCi 3H-leucine i.p.. 1 and 7 hrs after application 3H-leucine the animals were sacrificed. Concentrations of silvergrains of 3H-leucine activity were countered under surface illumination in varions brain areas by means of strippingfilmautoradiograms. DMI markedly depressed the concentrations of 3H-leucine-activity in all layers of the parietal cortex after 8 hrs and the depression was greater with the increase of nerv- and gliacellvolumendensity of the layers. Within 2 hrs such an influence of DMI on 3H-leucine uptake could not be found. There was a smaller decrease of 3H-leucine incorporation after DMI applications in some layers of ammon's horn, dentate gyrus and cerebellum. Some further effects of DMI and IP (Imipramine) on components concerned with protein metabolism are discussed.     

大鼠骨髓间充质干细胞条件培养液促进心肌细胞肥大

体外模拟心肌缺血微环境,研究骨髓间充质干细胞(MSCs)的旁分泌作用对心肌细胞的影响。以大鼠MSCs各时间点的条件培养液刺激心肌细胞,观察心肌细胞蛋白含量、[3H]-Leu掺入、ANF-荧光素酶(luciferase)表达和心肌细胞面积的变化。MSCs条件培养液处理心肌细胞后,与对照组相比较6h及9h时间点的条件培养液可明显增加心肌细胞蛋白含量、[3H]-Leu掺入、ANF-荧光素酶表达以及心肌细胞面积,其中以6h时间点条件培养液的作用最为显著(P<0.01)。MSCs条件培养液能够通过旁分泌作用刺激心肌细胞肥大,此现象提示移植入心肌缺血区MSCs可能通过旁分泌作用影响心肌细胞,从而参与细胞移植后心功能的改善。     

Effects of dexamethasone and indomethacin on estrogen-induced uterine growth

Certain aspects of estrogen-induced uterine growth are reminiscent of an inflammatory response. Dexamethasone (DEX) and indomethacin (IND), two anti-inflammatory agents that interfere with arachidonic acid metabolism, were examined with respect to their effects on several growth-associated responses of the uterus to estrogen. Ovariectomized rats were given a s.c. injection of either DEX (2 mg) or IND (8 mg) immediately prior to receiving a s.c. injection of estradiol (10 ωg). At 4 hr, DEX inhibited estrogen-stimulated uterine wet weight and ornithine decarboxylase (ODC) activity by 100% and 48%, respectively. At 24 hr, ³H-leucine incorporation into protein was inhibited 44% and ³H-thymidine incorporation into DNA was depressed 83%. Estrogen-stimulated increases in uterine protein/DNA ratio and epithelial microvilli density at 24 hr were not inhibited by DEX. IND inhibited estrogen-stimulated wet weight by 64% and ³H-thymidine incorporation into DNA by 42%, yet did not inhibit the increases in ODC activity, ³H-leucine incorporation into protein or protein/DNA ratio. These results suggest that the inflammation-like component of estrogen-induced uterine growth is mediated, at least in part, by arachidonic acid metabolites and is directed primarily toward stimulating cell division, and not cell growth.     

IN VITRO INCORPORATION OF 3H-THYMIDINE, 3H-URIDINE, 3H-LEUCINE AND 3H-MELPHALAN BY PLASMACYTOMA PLASMA CELLS

Bone marrow plasma cells from fifteen cases of multiple myeloma, immunologically typed, were incubated with different tritiated compounds. The labelling index with tritiated thymidine is generally low, while the mean grain count is fairly normal in the active cells. The labelling index of ³H-uridine and ³H-leucine was very high, while the mean grain count per cell lies within the normal range. The results obtained with ³H-phenylalanine-mustard (melphalan), which is a drug used in the treatment of the plasmacytoma, show also incorporation values roughly comparable to those of ³H-leucine. The present data seem to support the clinical use of melphalan as a compound that is actively incorporated into the plasma cells of plasmacytoma although inhibition of protein synthesis due to specific binding to protein was not demonstrated.     

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Abstract Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis , and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.     

Comparative study of a new composite biomaterial fluor-hydroxyapatite on fibroblast cell line NIH-3T3 by direct test

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to use the embryonal mouse fibroblast cell line NIH-3T3 for comparative study of basal cytotoxicity of fluoridated biomaterials FHA and FA discs. Hydroxyapatite (HA) disc, high-density polyethylene as negative control and polyvinyl chloride (PVC) containing organotin stabilizer as positive control were used as standard biomaterials. The appropriateness of the use of NIH-3T3 cells and their sensitivity for tested biomaterials were evaluated on the basis of five cytotoxic end points: cell proliferation, cell morphology, lactate dehydrogenase (LDH) released, protein and DNA cell content. The basal cytotoxicity of FHA, FA and HA discs was measured by direct contact method. FHA composite, FA and HA demonstrated in cell line NIH-3T3 nearly similar basal cytotoxicity increasing with the time of treatment. After 72 h of biomaterials treatment, about 25% inhibition of cell number, unchanged morphology of dividing cells, 6.31–0.16% increase of released LDH, about 10% inhibition of cell protein content and about 20% inhibition of DNA content was found. On the other hand, from the growth rates it resulted that NIH-3T3 cells, affected by tested biomaterials, divided about 20% slowlier than the control (untreated cells). Using the linear regression analysis we found out that deviations in measurements of cytotoxicity by four methods were as follows: less than 10% for cell number, protein and DNA content methods and 12.4% for released LDH method. Based on a good correlation of the cytotoxicity of biomaterials obtained from all end points we could conclude that fibroblast NIH-3T3 cell line was appropriate for measuring the basal cytoxicity of tested biomaterials.     

Functional activity of uremic erythroblast incubated in autologous and homologous plasma.

The uptake of 3H-thymidine, 3H-uridine and 3H-leucine in the erythroid precursors of patients with chronic renal failure (CRF) was examined by radioautography. The pattern of incorporation of the radioactive precursors was similar to that observed in erythroblasts of control subjects, i.e., the uptake decreased with cell maturation. CRF erythroblasts incubated with normal, homologous plasma, showed significant increase in the uptake of the radioactive precursors, compared to the activity of these cells incubated in autologous plasms, the only exception being the incorporation of 3H-leucine in the proerythroblasts, in which the increase was not statistically significant. These results suggest that the impaired function of CRF erythroblasts related to DNA, RNA and protein synthesis is due not to a defective mechanism in the cells themselves, but most probably to the effect of factors present in uremic plasma, the nature of which remains to be detected.     

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.     

Nucleic acid and protein synthesis during lateral root initiation in Marsilea quadrifolia (Marsileaceae)

The pattern of DNA, RNA, and protein synthesis during lateral root initiation in Marsilea quadrifolia L. was monitored by autoradiography of incorporated of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively. DNA synthesis was associated with the enlargement of the lateral root initial prior to its division. Consistent with histological studies, derivatives of the lateral root initial as well as the cells of the adjacent inner cortex and pericycle of the parent root also continued to synthesize DNA. RNA and protein synthetic activities were found to be higher in the lateral root initials than in the endodermal initials of the same longitudinal layer. The data suggest a role for nucleic acid and protein synthesis during cytodifferentiation of a potential endodermal cell into a lateral root initial.     

收缩活动促进新生大鼠培养心室肌细胞的^3H—亮氨酸...

To determine whether contraction could influence cell growth, the rate of protein synthesis (3H-leucine incorporation) and cell diameter and volume were measured in cultured neonatal rat cardiac myocytes beating spontaneously or arrested by high potassium. In medium supplemented with 10% calf serum, the 3H-leucine incorporation for 24 h in contracting myocytes (CMC) was significantly higher by 14.2% than that in quiescent myocytes (QMC), i.e. 1,229 +/- 29 cpm/10(5) cells vs. 1,076 +/- 60 cpm/10(5) cells (P < 0.01, n = 5 for each group). The cell diameter and cell volume in QMC group were respectively 15.14 +/- 0.42 microns and 1,842 +/- 123 microns3, while in the CMC group the corresponding figures reached to 16.82 +/- 0.64 microns3 and 2,495 +/- 210 microns3, increased by 11.1% and 35.5% respectively (P < 0.01, n = 6 for each group). With prolongation of culture time, the differences in these parameters between CMC and QMC became even more significant. In all these experiments, there was no significant difference in cell number between the two groups (P > 0.05). It is concluded that contraction per se can accelerate protein synthesis and cell growth in neonatal rat ventricular myocardium.     

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.     

A comparative morphometric and cytophotometric study of intraductal hyperplasia and intraductal carcinoma of the breast

The Feulgen DNA content and the nuclear measurements of four groups of intraductal proliferations of the breast (hyperplasia, atypical hyperplasia, well-differentiated carcinoma without cytologic atypia and intraductal carcinoma with cytologic atypia) were compared. Intraductal carcinoma with atypia was the only group distinct from the others on the basis of DNA content, nuclear area and perimeter. Although the other groups were separable from intraductal carcinoma with atypia, they could not be reliably distinguished from each other by any combination of measurements. At best, 69% of well-differentiated intraductal carcinomas could be distinguished from atypical hyperplasias using a combination of DNA content and nuclear perimeter measurements. Thus, the difficult distinction of atypical hyperplasia from well-differentiated intraductal carcinoma by light microscopy was not aided by DNA analysis or by nuclear measurements.     

Effects of thiamine deficiency on the biosynthesis of insulin in rats.

The effect of thiamine deficiency on insulin biosynthesis was studied. In thiamine deficient rats the total pancreatic protein content was not altered when compared to control rats whereas the pancreatic insulin content was decreased. Though the in vivo incorporation of 3H-leucine and the in vivo conversion of U-14C-glucose into proinsulin and insulin were not affected in thiamine deficient rats, the tolbutamide induced increased in vivo incorporation of 3H-leucine and in vivo conversion of U-14C-glucose into proinsulin and insulin was not seen in thiamine deficient rats. These results suggest that the biosynthesis of insulin is impaired in thiamine deficiency. Even tolbutamide could not increase the biosynthesis of insulin in this condition.     

Integral method for measuring the quantity of cellular DNA content by digital microphotography

Quantitative measurements of nuclear DNA content based on Feulgen reaction and the analysis of CCD images has been proposed. The measurements were performed in the monochrome CCD option (650 × 514 pixels) with a wavelength of 551 nm. The linear dependence of photomatrix element signals on the falling light was shown with a multigrade light absorption filter. The optimal microscope and camera settings and an approach for elimination of the optic blur are proposed. It was found that the contribution of background fluorescence of Feulgen-stained nuclei into the measurements was negligible. Densitometric measurements of the DNA content in blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens, Rana arvalis) were consistent with the literature data. The precision of our approach is comparable to other known cytometry methods (). The current improvement of CCD technical parameters and the widespread use of CCD cameras in biological applications give perspectives for the development of the suggested approach for measuring the quantity of cellular DNA.     

Bcl-3 regulates UVB-induced apoptosis

B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.