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.     

The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [³H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [³H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [³H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [³H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G¹-fractions was only 2 and 7%, respectively.     

STUDIES ON LIVER REGENERATION: II. EFFECTS of PHENOBARBITAL ON the ONSET and PATTERN of RAT LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY

Abstract. Partially hepatectomized rats were given a single intraperitoneal injection of a phenobarbital solution or of water immediately after surgery. At various time intervals following the operation, the animals were injected with ¹³¹ iododeoxyuridine (¹¹³IDU), sacrificed 2 hr later, and radioactivity retained in formalin-fixed liver tissue was determined as a measure of DNA synthesis at the time of administration of the labeled precursor. In control animals without phenobarbital treatment, ¹³¹IDU incorporation into liver began to increase between 14 and 16 hr after partial hepatectomy. Phenobarbital treatment (0.1 mg per g of body weight) resulted in a delay of the increase in ¹³¹IDU incorporation by several hours. This delay was observed in animals subjected to partial hepatectomy in the morning as well as in those operated on in the evening. After phenobarbital treatment, the increase of mitotic activity was either delayed or occurred more slowly. the results are compared with the reported effects of partial hepatectomy on the time course of microsomal enzyme induction by phenobarbital.     

Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

Abstract The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like ³H-thymidine ¹⁴C-uracil, ¹⁴C-leucine and ¹⁴C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.     

THE ALTERATION OF THE REGENERATIVE ACTIVITY AND THE CELL CYCLE OF PARTIALLY HEPATECTOMIZED RAT LIVER FOLLOWING ADMINISTRATION OF d-GALACTOSAMINE

A single injection of d-galactosamine given to rats at different times after partial hepatectomy (PH) changes the pattern of regenerative proliferation. When administered during the pre-replicative phase of regeneration, the onset of DNA synthesis and the increase in labelling index after injection of ³H-thymidine are delayed by about 12 hr. The injection of d-galactosamine at 24 hr after PH inhibits the drop in DNA synthesis occurring normally during the following 12 hr period. This was detected by a high labelling index and by an increased specific activity of DNA. The findings indicate a lengthening of the S phase, while G₂ and M remain normal. Two modes of action of d-galactosamine on the cell cycle are discussed.     

Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-β1 in glomerular mesangial cells induced by sublytic C5b-9 complexes

Objectives: To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracelluar matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs.
Material and methods: The expression of cyclin D₂, ³H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-β₁ in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and ³H-thymidine incorporation (³H-TdR), respectively.
Results: The expression of cyclin D₂, ³H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively.
Conclusions: These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-β₁. The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.     

Effect of photoperiod on the incorporation of 3H-thymidine into the gonads of Carassius auratus

Gldfish, Carassius auratus of varying sizes were conditioned in continuous light or darkness f or 10, 20 and 30 days and the incorporation of ³H-thymidine into the gonads was investigated to attempt to develop a bioassay for fish gonadotropins.
The 24-hour gonadal ³H-thymidine of 10-day conditioned fish was significantly less at the dose level of 0.5 μCi ³H-thymidine/fish compared to 1.0, 2.0 and 3.0 μCi/fish which gave gonadal activities not significantly different from each other. Thus, for all subsequent work the dose of 1.0 μCi/fish was used.
Photoperiod of continuous light or darkness had little effect on fish weighing less than 11 g but in fish 11-15 g conditioned for 20 days in darkness and fish greater than 16 g conditioned for 10 days in darkness, depression in gonadal ³H-thymidine incorporation occurred. In fish 11-15 g, prolonged conditioning for 30 days in darkness induced more gonadal activity than was observed at 20 days.
The effect of injection of Channa striatus pituitary extract at doses of 1 mg/10 g body weight and 5 mg/10 g body weight induced a significant increase in ³H-thymidine incorporation in the gonads over saline injected controls. The results suggest the potential of using photoperiod as a means of inducing regression of gonads of suitable fish in a bioassay of gonadotropins.     

Thymidine incorporation in Lake Cisó: Problems in estimating bacterial secondary production across oxic-anoxic interfaces

Abstract Heterotrophic bacterial activity was measured by means of the ³H-thymidine (³H-TdR) incorporation technique in Lake Cisó, a small holomictic lake with anoxic hypolimnion. We tested several methodological questions across the vertical profile: TdR concentration at which maximal incorporation is reached, linearity of incorporation and isotope dilution, during holomixis and stratification periods. The TdR concentration at which maximal incorporation is reached changed seasonally and vertically. During holomixis, maximal incorporation was not always reached at concentrations up to 40 nM. Uptake was always linear in short incubation times and decreased from epi- to hypolimnion. The isotope dilution technique indicated a degree of participation in DNA synthesis higher than 50%, although a linear relationship between the inverse of ³H-TdR incorporation and increasing ‘cold’ thymidine concentration was not always observed. Autoradiographic experiments showed a low percentage of bacteria taking up ³H-TdR in both aerobic and anaerobic samples. The percentage of total labeled bacteria seemed to be generally higher in the metalimnion (11% maximal value) than in the hypolimnion. Labeled Amoebobacter and Chromatium cells were detected in field samples. Amoebobacter cells photoassimilated TdR in culture. Therefore, our results show that ³H-TdR incorporation is not an appropriate technique to estimate bacterial secondary production in anaerobic systems and in oxic-anoxic interfaces.     

CELL KINETICS IN MOUSE THYMUS STUDIED BY SIMULTANEOUS USE OF 3H-THYMIDINE AND COLCHICINE

Following an injection of ³H-thymidine to mice there is no initial incorporation in small thymocytes, only in larger ones. In the course of time small thymocytes aquire the label. Whether the delayed uptake in small thymocytes is due to a direct cell to cell transfer of labelled nuclear material from inititally labelled larger cells to small thymocytes, or whether it is due to small thymocytes being formed from larger cells by mitotic division was investigated by the administration of Colcemid® immediately after one injection of ³H-thymidine. In the absence of cell division no labelled small thymocytes appeared with time. This finding does not support the idea of a cell to cell transfer of DNA; it rather lends support to the view that small thymocytes arise by mitotic division of larger cells in the thymus. During the treatment with Colcemid® the migration of cells took place from peripheral to central cortex just like under normal conditions.     

Some Effects of Pentamidine Di-isethionate on Crithidia fasciculata

SYNOPSIS. Growth-inhibitory concentrations of pentamidine inhibit to a similar extent net synthesis of DNA, RNA, protein and phospholipid by washed cell suspensions of Crithidia fasciculata. The incorporation of ³H-thymidine into DNA, ¹⁴C-adenine into DNA and RNA and ¹⁴C-lysine into protein is similarly inhibited. The same concentrations of drug have little or no effect on viability, motility, 1-C metabolism, respiration or K⁺ content of organisms, altho they do cause increased amounts of intracellular ATP. Lysine uptake (in the absence of arginine) is, however, inhibited. At higher concentrations of drug respiration is inhibited and organisms lose K⁺. The mode of action of pentamidine is considered in the light of these observations and a mechanism of uptake of pentamidine into organisms is suggested.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

Cytokinin-induced switch in development in excised cotyledons of radiata pine cultured in vitro

Cotyledons of Pinus radiata D. Don were cultured under shoot-forming (plus cytokinin) and elongating (minus cytokinin) conditions. Using. autoradiographic and precursor incorporation techniques, the sites and rate of macromolecular synthesis were examined during the first five days in culture. Active incorporation of ³H-thymidine, ³ H-uridine and ³H-leucine occurred. In shoot-forming cotyledons the incorporation became preferentially located in the epidermal and sub-epidermal cell layers in contact with the medium. In elongating cotyledons, in contrast, incorporation was randomly distributed, and the amount of incorporation declined with time. Biochemically, differences in DNA, RNA and total protein synthetic patterns were observed. In elongating cotyledons the rates of RNA and protein synthesis were higher during the first 48 h than in shoot-forming tissues, after which the synthetic rates were similar. Two peaks of newly formed DNA were observed in both tissues. These findings indicate that the cytokinin-induced changes in developmental pathways began within 24 h in culture.     

Hormone-Induced Alterations in Nonhistone Protein

Methylation and phosphorylation of chromosomal nonhistone protein (NHP) has been demonstrated in the salivary gland cells of diptera [5, 7] and implicated in the control of gene expression [35, 36]. Furthermore, hormones can stimulate methyl and phosphoryl side chain metabolism and thus enhance template activity. Salivary glands from late fourth instar female larvae of Sciara coprophila (cortisone-supplemented and normal diet) were incubated in ³H-uridine (10 μCi/ml), ³H-thymidine (10 μCi/ml), ³H-methyl-methionine (20 μCi/ml), ³⁵S-methionine (10 μCi/ml) and ³²P-orthophosphate (1 mc/ml), for varying time periods, to measure RNA synthesis, DNA synthesis, methylation, protein synthesis and phosphorylation, respectively. Following selective extraction of lipid, histone and nucleic acids, glands were prepared for light microscope autoradiography. A more specific labelling pattern, as well as increased grain number on particular bands, interbands and bulbs, was noted on chromosomes from cortisone-fed larvae incubated in ³H-methyl-methionine for 1 min when compared with larvae on the standard diet. Cortisone also increased RNA synthesis and nucleoprotein phosphorylation, but not DNA or protein synthesis. In summary, cortisone enhances the specificity and degree of NHP methylation and phosphorylation at discrete chromosomal loci, i.e. alterations in side chain metabolism which may be responsible for increased RNA synthesis.     

Metabolism of Acetate to Amino Acids in Brains of Rats After Complete Hepatectomy

Abstract: The concentration of glutamine increases in the brain after hepatectomy. In the present studies the conversion of intravenously given [¹⁴C]acetate to [¹⁴C]glutamate and [¹⁴C]glutamine was studied in control rats and in rats at 6 h after complete hepatectomy. The incorporation of label into glutamate was only slightly inhibited, but the further incorporation into glutamine was greatly inhibited, after hepatectomy. These data, and previous data using [¹⁴C]glucose as precursor, indicate that synthesis of glutamine in brain is inhibited after hepatectomy, and suggest that its concentration must increase because degradation is inhibited to an even greater extent.     

THE CELL SPECIFIC EFFECT OF THE GRANULOCYTE CHALONE DEMONSTRATED WITH THE DIFFUSION CHAMBER TECHNIQUE

The granulocytic chalone is secreted by mature granulocytes and inhibits ³H-thymidine incorporation of proliferating granulocytes in vitro . The effect and the cell line specificity of this chalone was assessed with the in vivo diffusion chamber culture technique. Tests were carried out on cultures from normal mouse bone marrow cells and mouse and rat blood leucocytes. The majority of the DNA synthesizing cells in marrow cultures were proliferating granulocytes. Macrophages and immunoblasts proliferated in rat leucocyte cultures, when the chambers had been carried for 5 days in host mice. Repeated chalone or control injections were given i.p. to the host mice during 6–7 hr prior to ³H-thymidine injection. Isotope uptake of proliferative granulocytes was reduced by the chalone treatment. No such effect was found on the rat immunoblasts and macrophages. The viability of cultured cells was apparently not affected by the chalone treatment.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

The Effect of Radiation Upon Lymphocyte Response to Pha

Abstract. The proliferative response of lymphocytes to phytohaemagglutinin has been studied following incremental doses of ⁶⁰Co gamma radiation delivered at the beginning of culture. A dose-dependent inhibition of ³H-thymidine uptake is apparent in 48 hr and 72 hr cultures throughout the 250–2000 rad range, and the effect appears more marked at the later labelling time. Parallel autoradiographic studies have revealed changes in the cell cycle kinetics of irradiated cultures which underlie the effects observed at the whole-culture level. Irradiation reduces the number of cells reaching the first period of DNA synthesis and causes dose-dependent delays at specific points in the cycle of proliferating cells.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

Morphofunctional Differentiation in the Liver

The influence of endotoxin and cortisone on the function of hepatic cells was studied in relation to the effect of partial hepatectomy on their activity. The overall rate of clearance of carbon (K) suffered a diminution within the first few hours after surgery but carbon uptake per unit weight tissue was elevated as a result of hepatic resection. The K values were increased by endotoxin (100°g i.p.) alone or together with cortisone (1 mg i.p.) but the hormone alone lowered the clearance rates in high doses (5 mg i.p.). Treatment with either substance increased liver weight over and above the control level in early phases of regeneration and lowered it in the later phases. The actions of cortisone and endotoxin were not additive on either process. As regards the influence of cellular cycle on hormonally modified gene activity, refractoriness to the hypertrophic effect of cortisone (5 mg i.p. a dose which lowered the K value at all times) developed soon after hepatic resection and continued for at least 36 h after surgery. These and other results indicate that the RE cells remain susceptible to external influences throughout the regenerative cycle and do not passively follow the behaviour pattern of parenchyma. An active contribution of RE cells is further supported by the observation that regeneration was accompanied by a progressive increase in spleen weight. Furthermore, because uptake and binding of cortisone to 'specific' receptors progressed at least as well as in control mice at all these time periods, the refractoriness to cortisone action must lie at some stage beyond uptake, processing or association of steroid with the appropriate receptor. These data indicate the necessity to consider the behaviour of individual cell types in order to comprehend the integrated responses observed during growth, development and differentiation in the liver.