Effect of bifolar and chlofiden on DNA synthesis of sarcoma 45 cells and on mitotic activity of rat small intestine epithelium

Results of the study on the influence of new antitumor preparations chlofiden and bifolar on sarcoma 45 DNA synthesis and on mitotic activity are given. It was shown that bifolar and chlofiden led to inhibition of label incorporation into DNA (3H-thymidine). Inhibition of mitotic activity after injection of both preparations in toxic doses (DL50) was revealed. During injection of the bifolar functional character of changes was established that is mitotic activity of the preparation during injection was restored.     

Demonstration of tubulin, actin and alpha-actinin by immunofluorescence in the microtubule-microfilament complex of the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells : II. Murine leukemia virus gp70 increases in S phase

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: A comparison and immunological study of purified solubilized preparations,and alteration of enzyme levels by cholestyramine feeding

Some properties of various preparations of solubilized 3-hydroxy-3-methylglutaryl CoA reductase from rat liver are described. One, prepared by solubilization with deoxycholate, has been brought to a level of purity such that only a sińgle component is detected by polyacrylamide gel electrophoresis. A second preparation, solubilized by high salt concentration and heat treatment, has also been purified to a high level of purity so that only minor contaminants are detected. The deoxycholate-solubilized 3-hydroxy-3-methylglutaryl CoA reductase has a molecular weight of 197,000–202,000. Electrophoresis of both preparations treated with mercaptoethanol on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed one band with a molecular weight of 65,000. The data are consistent with the trimeric structure consisting of three polypeptide chains of apparently identical molecular weight. An antiserum to the deoxycholate-solubilized preparation has been prepared. Despite major differences among these preparations in specific activity, in stability to cold, and in the requirement of high salt concentration for preservation, both samples react in the same manner to the antibody and are immunologically indistinguishable. A preparation solubilized by freeze-thawing also reacts with the antiserum. Possible reasons for the variations in specific activity are considered, and it is concluded that specific activity changes cannot be reliably related to protein concentration unless the protein is isolated.Application of the immunological assay to an analysis of the effect of feeding cholestyramine to rats shows that compared to normals the diurnal cycle is unchanged but the rate of enzyme protein synthesis in the cholestyramine-fed rats is greatly accelerated. However, the first-order rate constant for degradatation of enzyme protein remains essentially unchanged throughout the falling phases of the cycle. The specific activity relationships of the enzyme protein of cholestyramine-fed rats appear to be altered when compared to that of normally fed controls.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome: accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of: cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as: rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G0/G1, S and G2/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

Mechanism for differential sensitivity of the chromosome and growth cycles of mammalian cells to the rate of protein synthesis.

It has been documented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosome cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the G1 phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the G1 phase.     

HeLa cell plasma membranes. Changes in membrane protein composition during the cell cycle.

HeLa cells grown in suspension culture were synchronized by amethopterin block and thymidine reversal. In some cases an additional Colcemid block was used to obtain mitotic cells. From the various phases of the cell cycle, cells were harvested and the plasma membranes isolated. The membrane proteins were solubilized in sodium dodecyl sulphate and separated by gel electrophoresis in the presence of sodium dodecyl sarcosinate. About 35 protein bands, five of which were stained with periodic acid-Schiff reagent, appeared. Most of the bands were identical in all membrane preparations, but a few minor bands seemed to be associated with limited periods of the cell cycle. In particular, the cells in mitosis apparently contained plasma membrane proteins which did not occur in other phases. Amino acid analyses of the plasma membranes revealed no significant cell cycle-dependent changes in the amino acid composition.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of

cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G₀/G₁, S and G₂/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

Properties of the Bacillus pumilus Glutamyl endopeptidase at different growth stages of its recombinant strain

The Bacillus pumilus 3–19 Glutamyl peptidase (EC 3.4.21.19) was isolated from the culture medium of the B. subtilis recombinant strain at the following stages of the bacillus growth: a decelerating growth phase and a stationary growth phase. The action of the purified preparations of the enzyme on different phases of its growth was studied on the insulin B-chain and various protein and peptide substrates. Physicochemical properties of the enzyme were compared for different phases of its growth. The glutamyl endopeptidase preparations differed in their catalytic characteristics and their sensitivity to the metal cations.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.     

Hydrogen shuttles in gills of water versus air breathing osteoglossids.

1. Using subcellular preparations of gills from Arapaima, an obligate air breather, and aruana, a related osteoglossid that is an obligate water breather, a comparison was made of the relative roles of the malate-aspartate cycle and the alpha-glycerophosphate (alpha-GP) cycle in transferring reducing equivalents from the cytosol to the mitochondria. 2. In aruana gill preparations, the alpha-GP cycle could be most clearly demonstrated by reconstructing it with purified isolated mitochondria, using the oxidation rate of exogenous NADH as a measure of the cycling activity. 3. Subcellular preparations of Arapaima gill, in contrast to the aruana gill, were not responsive to exogenous alpha-glycerophosphate, but a glutamate-malate stimulated O2 uptake was sensitive to aminooxyacetate, an aminotransferase inhibitor, a result that would be expected if the respiration were based on malate-aspartate cycling. 4. It was concluded that, compared to the alpha-glycerophosphate cycle, the malate-aspartate cycle was relatively more active in Arapaima gill than in aruana gill, and possible implications were discussed.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Abstract. Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G¹, S and G₂+ M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about twofold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle ( T _c) was 15-3 h for PB-3 cells and 12-4 h for PB-1 cells. Shortening in T _c for the transformed cells was due to a decrease of nearly 30% in mean duration of the G ₁ phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G₂+ M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G¹ phase of the cell cycle.     

Single-cell mass cytometry adapted to measurements of the cell cycle

Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.     

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.     

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.     

The simulation of thymidine kinase enzyme kinetics in cultured cells using the computer language cellsim

Thymidine kinase is an enzyme that occurs in cells actively synthesizing DNA. In studies of synchronized cell populations, it has been shown that the enzyme activity disappears during the G1 phase of the cell cycle and reappears during the S and G2 phases. Its reappearance is consistent with the synthesis of the mRNA for this enzyme during the S and G2 phases and its immediate translation into active enzyme by the protein synthesis machinery within the cell. The disappearance of the enzyme is consistent with the cessation of mRNA synthesis by mitotic cells. We have now tested this concept by computer simulation of a growing cell population in which a specific mRNA is generated while cells are in the S and G2 phases of the cell cycle. The computer simulation was done using the simulation language Cellsim designed for modeling populations of cells. The Cellsim program which we developed allowed each cell to make about 1 mRNA molecule per min during the S and G2 phases. Every 3 min each mRNA molecule generated a protein enzyme molecule. The mRNA had a half-life of about 9 min, and the enzyme had a half-life of about 150 min. When these molecular parameters were coupled to the cell cycle parameters for Chinese hamster fibroblasts, the resulting curve of enzyme production with time closely matched the observed kinetics of enzyme activity seen in synchronized cells. The only part of the curve that did not fit was the rapid drop in enzyme activity which was seen as the population of mitotic cells was permitted to enter G1. This drop in activity was not seen in mitotic cells blocked with Colcemid where mRNA synthesis must be lacking. Earlier studies have shown that the Gl cells do not contain any inhibitor of enzyme activity. It therefore appears that the enzyme molecule is more unstable during the G1 phase than in any of the other phases of the cell cycle.     

Self‐reported activation during the premenstrual and menstrual phases of the menstrual cycle

Abstract

A total of 171 female subjects completed a self‐report questionnaire dealing with activation (AD‐ACL) during the premenstrual and menstrual phases of their cycles. Significant variation between the two phases was found for all four activation factors: activation was highest premenstrually. Contraceptive use interacted significantly with cycle phase for the General Activation factor. Subjects taking oral contraceptive preparations also had lower scores on the Deactivation‐Sleep factor.     

Effect of oestrus cyclicity on the number of brain opioid mu receptors in the rat

The present experiments have been performed in order to analyse whether the binding characteristics of brain opioid receptors of the mu type vary during the different phases of the oestrous cycle in the female rat. To this purpose different groups of females with a regular 4-day oestrous cycle were killed by decapitation in different phases of their oestrous cycle, i.e. at 10.00 and 16.00 h of the first and second day of dioestrus, at 10.00, 12.00, 14.00, 16.00 18.00 and 20.00 of the day of pro-oestrus, and at 10.00, 12.00 14.00, 16.00 and 18.00 of the day of oestrus. The total brains, after discarding the cerebellum, were homogenized and crude membrane preparations were obtained. On these preparations the maximal binding capacity (Bmax, index of the number of receptors) and the constant of affinity (Ka) for dihydromorphine, a typical ligand of mu opioid receptors were evaluated. Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured by specific radioimmunoassays in order to exactly ascertain the different phases of the oestrous cycle. The results obtained show that the number of mu opioid receptors in the whole brain presents significant changes during the different phases of the oestrous cycle. In particular, an increase in the concentration of these receptors was observed at 12.00 h of the day of pro-oestrus and at 18.00 h of the day of oestrus; these fluctuations of the number of mu receptors were not accompanied by any change of their affinity for the ligand.(ABSTRACT TRUNCATED AT 250 WORDS)