Effect of pepsin pretreatment on pulse-cytophotometric DNA histograms.

Samples of mitotic L-cells were investigated after different preparation and staining procedures using the technique of pulse-cytophotometry. It is shown that most mitotic cells which should appear in the second peak of the DNA histogram are disintegrated or separated into halves by pepsin pretreatment. Hence, the designation 'G2 + M' for the second peak is not correct for this preparative method. This should be taken into account in cell kinetic investigations performed after pepsin pretreatment.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

Epidermal polyamine levels related to cell cycle events during the metamorphosis of Tenebrio molitor L. (insecta,coleoptera): Effect of juvenoid application

• 1.1. Three polyamines were separated by reverse-phase HPLC and detected by fluorescence in Tenebrio epidermis: putrescine, spermidine and spermine.
• 2.2. The levels of these compounds varied during metamorphosis: one peak was observed during the G₂-arrest preceding the pupal-adult mitotic crisis and a second occurred when cells were again G₂-arrested after the mitotic period.
• 3.3. A juvenile hormone analogue, which inhibits the G₂-M transition preparing cells to the adult mitoses, was unable to prevent the first polyamine increase suggesting that juvenile hormone does not act on further development via inhibition of polyamine synthesis.

     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

Regulation of cell division in the subapical shoot meristem of dwarf watermelon seedlings by gibberellic acid and polyethylene glycol 4000

The stimulatory effects of gibberellic acid (GA₃) and the inhibitory effects of polyethylene glycol 4000 (PEG) on hypocotyl elongation and cell cycle kinetics in subapical pith cells of dwarf watermelon seedlings (Citrullus lanatus [Thunb.] Matsu and Nakai) were investigated. Mitotic indices (MI) were determined from direct counts of pith cells stained by a modified Feulgen technique. Labeling indices (LI) were determined from direct counts of labeled pith cells sampled 1.5 h after apical applications of³H-thymidine. Root application of 0.32 mM GA₃ at 96, 120, or 144 h after sowing resulted in significant increases in both mitotic and labeling indices within 4.5 to 7.5 h following treatment. A single mitotic peak at 13.5 h occurred in all three treatment periods. Labeling peaks were often less defined than mitotic peaks; however, a relatively high proportion of labeled nuclei were usually observed between 7.5 and 9 h after GA₃ treatment and at 16.5 h, the latter period coinciding with progression of cells into S phase from the peak period of mitosis. The results suggest that GA₃ increases the proportion of rapidly dividing cells in the subapical meristem by increasing the probability that slowly cycling or nonproliferative cells in both 2C and 4C DNA states will enter the proliferative pool. The addition of PEG (200 g/l, = 1.5 mPA) to the rooting medium of dwarf watermelon seedlings inhibited hypocotyl elongation and reduced both mitotic and labeling indices simultaneously within 4.5 h after treatment. Within 24–28 h after PEG treatment, mitotic and labeling indices approached 0. Seedlings transferred from PEG to either water or GA₃ exhibited rapid recovery of cell division and hypocotyl elongation. Mitotic and labeling indices increased within 4.5–7.5 h into the recovery period in either water or GA₃ and reached control values within 10.5 h. GA₃ hastened the recovery from PEG-induced stress. It is concluded that water stress imposed by PEG 4000 causes arrest of cell division in meristematic cells of watermelon seedlings in both G₁ and G₂ periods. PEG and GA treatments resulted in only a partial and transitory synchronization of the cell cycle.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment.

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.     

Process integration for recovery of recombinant collagen type I α1 from corn seed

Because of safety concerns and product consistency issues with the use of animal‐derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple‐helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia‐produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre‐ and post‐treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13‐fold purification with a 2.7‐fold volume reduction which would reduce the pepsin requirement for post‐recovery foldon cleavage. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:98–107, 2016     

Loading of the centromeric histone H3 variant during meiosis–how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.     

Differential effects of probenecid on the levels of endogenous PGF2α and TXB2 in brain cortex

Probenecid in single or repeated doses does not modify levels of PGF_2α and TXB₂ in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazone (PMT) PGF_2α increases sharply and rapidly declines subsequently, whereas the elevation of TXB₂ is smaller but of longer duration. After probenecid pretreatment PGF_2α levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB₂ tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.     

Influence of gonadotropin (FSH + LH) and thyrotropin (TSH) on the multiplication of Chinese hamster ovary (CHO) cells. Impact of the age of the culture.

CHO cells repeatedly treated with gonadotropin showed peak division rates after their third exposure and a decrease in the mitotic rate after their fourth exposure. Thyrotropin induced a considerable decrease in the mitotic rate following the first exposure, a significant increase after the second and a further decrease following the third and fourth exposures. The pattern did not differ between the two hormones when the cells were exposed further. The age (density of the cell cultures) had an appreciable influence on hormone-provoked changes in the mitotic rate, this differing only in intensity and never in the response following the initial re-exposure.     

MITOTIC ACTIVITY OF VAGINAL EPITHELIAL CELLS FOLLOWING NEONATAL INJECTIONS OF DIFFERENT DOSES OF ESTROGEN IN MICE

In C57Black/Tw mice given injections of 1 μg estradiol-17β (E) for 5 days beginning on the day of birth, and killed a few days after the treatment, the vaginal epithelium showed estrogen-dependent proliferation and parakeratosis. In contrast, in the mice treated neonatally with 30 μg E for 5 days, the vaginal epithelium exhibited estrogen-independent proliferation and cornification or parakeratosis. Two peaks occurred in the mitotic rate in vaginal epithelial cells in the proximal and middle vaginae of the 1 μgE-treated mice, at 1 and 5 days of age, respectively, while the first peak was lacking in the distal vagina. The mitotic activity in 1 μgE-treated mice declined to the control level at 60 days. In the 30 μgE-treated animals also, 2 peaks were found in the mitotic rate at 1 and 7 days in both the proximal and middle vaginae. In contrast to the 1 μgE-treated mice, although the rate dropped once at 10 days, it increased again at 20 days and remained high thereafter. The second peak at 7 days of age coincided with the active proliferation of nodules appearing in the 30 μgE-treated mice. In the distal vagina, a peak occurred in the mitotic rate at 7 days without a preceding peak like that observed in the other parts of the vagina following the first injection of E on the day of birth.     

In vitro excystment of the black spot trematode Uvulifer ambloplitis (Trematoda:Diplostomatidae)

Spellman S. J. and Johnson A. D. 1987. In vitro excystment of the black spot trematode Uvulifer ambloplitis (Trematoda:Diplostomatidae). International Journal for Parasitology17: 897–902. Metacercariae of U. ambloplitis became activated and excysted in both acid pepsin and acidified Locke's balanced salt solution (BSS). The maximum percentage of excystment was 40% in acid pepsin and 4% in Locke's BSS. Acid pretreatment was required for the additional excystment that occurred in the pretreatment reductant sodium dithionite, or in an incubation medium, or in a sodium dithionite-incubation medium sequence. Since larvae excysted during both pretreatments and in incubation media, maximum overall excystment percentages were obtained with three treatments; (1) 0.5% pepsin at pH 2.0 for 30 min, (2) 0.2% sodium dithionite at pH 7.4 for 10 min and (3) an incubation medium of 0.2 or 0.5% ox bile salts a pH 7.4 for 120 min, 66 and 79%, respectively. Excystment occurred in trypsin alone or trypsin containing incubation media following pepsin pretreatment or a pepsin-sodium dithionite sequence, but the larvae were sluggish and died within a few min after excysting. Thus, a synergistic effect between bile salts and trypsin was not found in this study. Excystment was primarily an active process with the larva emerging from the narrow end of the parasite cyst, although a breakdown of the host cyst and some softening of the parasite cyst was observed.     

Evidence against prostaglandin-mediation of somatostatin-inhibition of gastric secretions

The inhibitory activities of somatostatin and PGE2 against pentagastrin-stimulated gastric acid and pepsin secretions were investigated, with and without pretreatment with the cyclooxygenase inhibitor indomethacin, in conscious cats prepared with gastric fistulae. Somatostatin was a potent inhibitor of acid secretion in both vagus intact and vagotomized animals, and its effect was not diminished by indomethacin pretreatment. Somatostatin inhibition of pepsin secretion was diminished after indomethacin, but a similar effect was noted with exogenous PGE2, suggesting a mechanism unrelated to inhibition of prostaglandin synthesis. It is concluded that there is no evidence to implicate endogenous prostaglandins in somatostatin inhibition of feline gastric exocrine secretions.     

A Stochastic Model For Cell Populations With Circadian Rhythms

A mathematical model for cell kinetics, based on a random walk, is developed. the model allows variations with time of the rates of passage of proliferating cells through the four phases of the mitotic cycle. Circadian variations in the mitotic and labelling indices of the Syrian hamster cheek pouch epithelium have previously been observed, and the random walk model has been used to simulate this phenomenon. Assuming that all basal cells are proliferative and that these cells leave the basal layer randomly throughout the mitotic cycle to become differentiated cells, it was found that the experimentally observed circadian rhythms of the mitotic and labelling indices could be reproduced in the model by postulating a circadian rhythm in the rate of passage of cells through the G₁ and S phases only. Moreover, the growth activity of cells in both the G₁ and S phases appears to reach a peak during the dark hours of the light-dark cycle, and to fall off rapidly in the early hours of daylight. the postulate of Møller, Larsen & Faber (1974) that injection of the animals with tritiated thymidine causes a shortening of the G₂ phase duration has been qualitatively confirmed by using the random walk model to simulate the FLM and MI curves after injection with tritiated thymidine.     

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.