Effects of gonadotrophin-induced elevation of serum testosterone upon somatomedin C levels and serum thymidine activity in children

3H-thymidine uptake into lectin-activated human lymphocytes allows to measure a growth-stimulating activity of serum, the thymidine activity (TA), which is GH dependent in vivo and related to somatomedins (Sm). In this work, it is shown: that addition of human chorionic gonadotrophin (HCG) or testosterone in vitro does not increase the 3H-thymidine uptake into lymphocytes; that the gonadotrophin-induced elevation of testosterone in children is accompanied by a significant increase of TA and, at a lesser degree, of Sm C; that these two increases are significantly correlated, and that the age-related variation of TA and Sm C after HCG stimulation test are not parallel.     

1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells.

1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

Temperature comparisons for antibody production in vitro by plaque-forming cells from trout, Salmo gairdneri (Richardson), and mice

Anterior kidney and splenic cells were taken from rainbow trout and splenic cells from BALB/c mice immunized with a T-dependent (sheep red blood cells) or T-independent (DNP-Ficoll) antigen. The cells were incubated at different temperatures in Jerne plaque assays (direct or passive haemolytic plaque assays). The optimum numbers of in vitro plaque-forming cells (PFC) after incubation with homologous complement were directly correlated with normal body temperatures of the respective species. The optimum incubation temperature was 37°C for mouse cells and 10°C for fish cells. Incubation of mouse cells at lower temperatures of 30, 20, 10, 4 or 0°C appeared to yield a direct line reduction in numbers of PFC. Trout cells developed significantly fewer PFC at 4 and 20°C and none at 30°C or above; however, significant numbers still appeared at 0°C. More PFC per million white blood cells were obtained from the anterior kidney; however, related to temperatures, no differences in development of numbers of PFC could be seen between the spleen and anterior kidney cells of trout. When the incubation time was lengthened for both trout and mouse cells held at low temperatures, the numbers of PFC approached those of the cells incubated at the optimum temperatures for 10 h.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Hyperthermia as a teratogen: parameters determining hyperthermia-induced head defects in the rat

This study determined the relationship between the duration and extent of temperature elevation, during a critical period of rat embryonic development, and the induction of congenital malformations. Pregnant Sprague-Dawley rats, at 9 days 12 hours gestation (gastrulation stage), were partially immersed in a water bath until their core temperature, monitored by a rectal thermistor probe, was elevated to a nominated temperature. Seven temperatures were tested from 40.5 degrees C to 43.5 degrees C, elevations of 2.0-5.0 degrees C in core temperature. Various durations at each of these temperatures were tested for potential teratogenicity. A single elevation of 5.0 degrees C or 4.5 degrees C needed only a "spike" in duration to be teratogenic, 4.0 degrees C was teratogenic within 5 minutes, 3.5 degrees C within 10 minutes, 3.0 degrees C within 20 minutes, and 2.5 degrees C within 1 hour. An elevation of 2.0 degrees C for 8 hours was not teratogenic. Microphthalmia was the most common malformation at all teratogenic temperatures and was frequently the only malformation seen at the shortest time exposure for a particular temperature. Encephalocele, facial clefting, and maxillary hypoplasia were the other frequently seen malformations. Five control rats were placed in the water bath for 2 hours at 38 degrees C so that their core temperature was not elevated. All the control fetuses were normal. An elevation of 2.5 degrees C for 1 hour was the threshold combination for teratogenesis. As the temperature increased above a 2.5 degrees C elevation the necessary duration of exposure for teratogenesis decreased.     

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function

Summary The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between argB and purD loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and -rays and incorporates little or no ³H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of ³H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42° C before their transfer to 30° C results in higher survival as compared to their incubation at 30° C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.     

Aluminum ions stimulate mitosis in murine cells in tissue culture

Addition of aluminum to the culture medium of Nakano mouse lens epithelial (NMLE) cells and Swiss 3T3K cells induced both 3H-thymidine incorporation and mitosis. This is in contrast to other metal ions such as vanadium, which, at concentrations high enough to increase 3H-thymidine incorporation, actually inhibits mitosis (Jones and Reid, J Cell Physiol 121:199, 1984). Aluminum concentrations between 20 microM and 50 microM were most effective. The 3T3 cells respond to aluminum with a 7.6-fold increase, and NMLE cells respond with a 21-fold increase in 3H-thymidine incorporation. DNA synthesis in NMLE cells was also found to be synergistically stimulated by aluminum and low concentrations of insulin (4.5 X 10(-8) M). A 3.25-hr incubation with 50 microM aluminum was sufficient to induce 50% of maximum 3H-thymidine incorporation during the 40-hr assay. Aluminum-stimulated 3H-thymidine incorporation is inhibited by hydroxyurea, and aluminum causes an increase in cell number. Also, by sedimentation equilibrium analysis of the product of aluminum-stimulated DNA synthesis it was found that a single copy of DNA was synthesized following addition of aluminum to quiescent cells. These facts indicate that aluminum induces both S-phase DNA synthesis and mitosis. However, only 48% of the NMLE cells found to be labeled with DNA went on to divide. In contrast, although only a small percentage of 3T3 cells were found to be labeled after aluminum treatment, all of these cells appeared to go through mitosis.     

Families of replicating units in cultured hamster fibroblasts

An examination of the patterns of DNA replication in pseudodiploid Don C and diploid Don cell lines in culture has been made. Pulse-chase labelling experiments with ³H-thymidine in both synchronized and log-phase cells indicate that the newly replicated DNA can be divided into two and three large temporally distinct fractions in Don C and Don cells, respectively. This is shown radiochemically by fluctuations in the incorporation of ³H-thymidine into the DNA of synchronized cells and autoradiographically by fluctuations in counts of labelled metaphases and grain over mitotic figures. Pulse-chase experiments and fluorometric determinations indicate that the periodic incorporation of ³H-thymidine can be accounted for by discontinuous synthesis and turnover of DNA during the cell cycle.A survey of the literature reveals that fluctuations in DNA synthetic activity during the S phase are to be found in a large number of published graphs of cell population kinetics. The phenomenon is observable in both diploid and non-diploid cells. A change in the timing of DNA replicon synthesis during the S phase according to the developmental stage and age of the cell is proposed.     

Time relationship between ambient temperature change and antigen stimulation on immune responses of mice

We investigated the time relationship between ambient temperature change and antigen stimulation on immune responses to sheep red blood cells (SRBC) and polyvinylpyrrolidone (PVP) in mice. In the case of a shift from comfortable (25°C) to cold (8°C) temperatures, suppression in the number of splenic plaque-forming cells (PFC) took place mainly when the shift was done between 1 day before and 2 to 4 days after immunization. The suppression of the PVP response lasted for up to a maximum of 6 days when mice were transferred 1 day before immunization. In the case of a temperature shift from 25° to 36.5°C, the suppressive effect was found when the temperature shift was done between 4 days before and 2 days after immunization. The effect lasted longer than that of the temperature shift to cold, i.e., at least 9 days after the temperature shift. Blood corticosterone levels after the temperature shifts corresponded to changes in the immune responses: elevation of the blood corticosterone levels was observed for only the first 3 days after a temperature shift to 8°C but for 10 days after a temperature shift to 36.5°C during the period time of the experiment. These result suggested that blood corticosterone level contributes to the duration of the effects of temperature shifts on immune responses of mice. Furthermore, it appeared that the early stage of the immune response is more susceptible to temperature shifts than the later stage. To explain these results, the terms effective period in the course of physiological adaptation to changed ambient temperature and susceptible period in the course of the immune response, were proposed.     

Correlation between low-temperature immunosuppression and the absence of unsaturated fatty acid synthesis in murine T cells.

1. Studies were performed to determine if the inability of murine T cells to provide primary helper function at low temperature (27 degrees C) could be correlated with their inability to synthesize unsaturated fatty acids (UFAs). 2. In the absence of exogenous oleic acid (18:1), splenocytes responded to a T-dependent (TD) Ag (trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH) at 37 degrees C but not at 27 degrees C. The addition of 150 microM 18:1 almost completely restored plaque-forming cell (PFC) responses to TNP-KLH at 27 degrees C but markedly suppressed PFC responses to the TD Ag at 37 degrees C. 3. During incubation at 27 degrees C, B cells converted 3- to 5-fold more stearic acid (18:0) to 18:1 and showed a greater accumulation of monounsaturated phospholipid molecular species than did T cells. 4. Following incubation in the presence of a rescuing dose of 18:1 (150 microM), both B and T cells accumulated large amounts of dioleoyl PC. 5. It is proposed that the absence of 18:1 synthesis in T cells is responsible for the unique low temperature susceptibility of this lymphocyte population.     

Sodium orthovanadate stimulation of DNA synthesis in Nakano mouse lens epithelial cells in serum-free medium

Quiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H-thymidine at an accelerated rate; the greatest response occurred at 20 microM vanadate, whereas by 2 microM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 microM had lysed by the end of the 40-hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H-thymidine at a vanadate concentration of 200 microM to 500 microM. Half-maximum incorporation of 3H-thymidine (after a 40-hour incubation) was induced by a 2-hour incubation with 20 microM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H-thymidine incorporation, 20 ng/ml insulin in combination with 20 microM vanadate resulted in a significant increase in 3H-thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S-phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S-phase DNA synthesis demonstrated that vanadate was not causing increased 3H-thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H-thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H-thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.     

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.     

A new mouse cell-surface antigen (Ly-m20) controlled by a gene linked to Mls locus and defined by monoclonal antibodies

Five monoclonal antibodies were established by the fusion of mouse myeloma cells (NS.1) with spleen cells from A and (A x C3H/An)F₁ mice hyperimmunized with 70Z/3 tumor cells. These antibodies recognized a new antigenic specificity provisionally called Ly-m20.2. In direct cytotoxicity assays, 60 percent of cells in spleen, 40 percent in lymph node, 50 percent in bone marrow and less than 5 percent in thymus were found to react with three of the five antibodies, whereas the two others yielded somewhat lower cytotoxicity indices. The Ly-m20.2 antigen was also expressed on cells derived from liver and kidney but not on cells derived from brain. As judged from cytotoxicity assays with separated T and B cells, Ly-m20.2 antigen is carried preferentially on B lymphocytes. Direct plaque-forming cells (PFC) were completely eliminated by Ly-m20.2-specific antibody and complement. Linkage tests by analysis in 20 (CBA/J x C3H/An) x C3H/An backcross mice and by segregation analysis of BXH and SWXL recombinant inbred strains indicate close association of the loci controlling Ly-m20.2 and Mls antigens on chromosome 1.Abbreviations used in this paper MLR mixed lymphocyte reaction - MHC major histocompatibility complex - Mls minor lymphocyte-stimulating antigen - Ia I-region associated - PFC plaque-forming cell - SRBC sheep red blood cell - Con A concanavalin A - LPS lipopolysaccharide - GVH graft-versus-host - HVG host-versus-graft - CML cell-mediated lympholysis - LD lymph ocyte-defined - SD serologically defined The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

Effects of Perfluorocarbons on surfactant exocytosis and membrane properties in isolated alveolar type II cells

Background

Perfluorocarbons (PFC) are used to improve gas exchange in diseased lungs. PFC have been shown to affect various cell types. Thus, effects on alveolar type II (ATII) cells and surfactant metabolism can be expected, data, however, are controversial.

Objective

The study was performed to test two hypotheses: (I) the effects of PFC on surfactant exocytosis depend on their respective vapor pressures; (II) different pathways of surfactant exocytosis are affected differently by PFC.

Methods

Isolated ATII cells were exposed to two PFC with different vapor pressures and spontaneous surfactant exocytosis was measured. Furthermore, surfactant exocytosis was stimulated by either ATP, PMA or Ionomycin. The effects of PFC on cell morphology, cellular viability, endocytosis, membrane permeability and fluidity were determined.

Results

The spontaneous exocytosis was reduced by PFC, however, the ATP and PMA stimulated exocytosis was slightly increased by PFC with high vapor pressure. In contrast, Ionomycin-induced exocytosis was decreased by PFC with low vapor pressure. Cellular uptake of FM 1-43 - a marker of membrane integrity - was increased. However, membrane fluidity, endocytosis and viability were not affected by PFC incubation.

Conclusions

We conclude that PFC effects can be explained by modest, unspecific interactions with the plasma membrane rather than by specific interactions with intracellular targets.     

The dynamics of proliferation and differentiation of osteogenic cells under supportive unloading

With the use of radioactive marker of DNA synthesis--3H-thymidine we have studied the dynamics, peculiarities of proliferation and differentiation of osteogenic cells under hind limb unloading of white rats ("tail suspension" method at an angle 35 degrees) during 28 days. The 3H-thymidine was administered at a single dose at the end of the experiment, the biosamples were taken from femoral bones in 1, 48, 96 hr. Light and electron-microscopic radioautography with 3H-thymidine (in 1 hour) have shown, that basic fraction of DNA synthesizing cells in the zones of adaptive remodelling of bone tissue is represented by little-differentiated perivascular cells (that include osteogenic cell precursors). A tendency for a decrease of a labelling index in the 3H-thymidine osteogenic cells on metaphyseal bone trabeculae under hind limb unloading has been established. The dynamics of labelled cells during various time intervals after 3H-thymidine injection testifies to a delay in the differentiation precursors in osteoblasts and their transformation to osteocytes in experiment animals. The obtained data have shown that a long-term supportive unloading leads to lowering the intensity of osteogenetic processes in long bones and reducing bone mass.     

Bovine thecal cells secrete factor(s) that promote granulosa cell proliferation

To determine if soluble factors (other than steroids) secreted by bovine thecal cells may be involved in local regulation of follicular development, we examined the effects of thecal cell secretory products on the growth of granulosa cells obtained from the same follicles. DNA synthesis (assessed by the incorporation of 3H-thymidine) by granulosa cells plated on coverslips and cocultured with, but not directly in contact with, thecal cells in organ culture dishes in a serum-free medium was 5-fold greater than controls. The effect of the thecal cell-secreted products on DNA synthesis by granulosa cells was significantly higher than the maximum response produced by epidermal growth factor (EGF). Thecal cell-conditioned medium stimulated 3H-thymidine incorporation into the DNA of granulosa cells and a normal rat kidney cell line in a dose-dependent manner. The increases in 3H-thymidine incorporation into granulosa cell DNA subsequently lead to an increase in cell number. Preliminary characterization studies using ultrafiltration membranes indicated that the mitogenic factor was retained in the greater than 10,000 molecular weight fraction. The activity was stable to heating at 90 degrees C for 5 min and was not extracted in ether. The thecal cell-generated growth factor may act as a paracrine regulator of granulosa cell growth, thus providing the dominant follicle with autonomy over other follicles in the cohort.     

Evidence for luteal cell hyperplasia during pregnancy

The pronounced increase in luteal size and weight that occurs during rat pregnancy has been largely attributed to luteal cell hypertrophy. It is generally believed that hyperplasia does not play a role in luteal growth, since it is thought that luteal cell division in vivo does not occur. Recent data suggest that this may not be the case. Thus, to determine whether luteal cell hyperplasia occurs during rat pregnancy, osmotic minipumps filled with 3H-thymidine were implanted in timed-pregnant rats on Day 6 or Day 11 of pregnancy. These pumps provided a continuous infusion of 10 microCi 3H-thymidine per hour for up to 7 days. Seven days later (Day 13 and Day 18 respectively), rats were killed, and the ovaries were removed and prepared for autoradiography. Labeled cells, which have the morphological characteristics of luteal cells, were clearly observed in autoradiographs of ovaries exposed to 3H-thymidine. The labeling index of these cells from ovaries exposed to 3H-thymidine on Days 6-13 of pregnancy was 6.0% and from ovaries exposed to 3H-thymidine on Days 11-18 of pregnancy was 1.2%. Whether the presence of labeled cells signifies proliferation of luteal cells or whether these cells are derived from another cell type that develops into cells morphologically similar to luteal cells is not known at present. However, regardless of origin, these data clearly demonstrate that the number of parenchymal cells in the corpus luteum does increase during pregnancy.