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.     

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.     

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.     

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.     

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.     

Partial purification and characterization of mitotic factors from HeLa cells

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.     

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.     

Role of the hypophysis in thyroid regeneration after partial thyroidectomy.

The role of the hypophysis in thyroid regeneration was investigated by measuring the mitotic activity of the thyroid remnant in hemithyroidectomized rats as well as the blood levels of thyroid hormone at various time-intervals after hemithyroidectomy. Mitotic activity underwent a significant increase to reach a peak (a 5- to 8- fold increase) 2 days after hemithyroidectomy. The thyroid hormone level in blood was lower than in controls. Histologically, the thyroid gland showed signs of an elevated rate of functional activity, as indicated by losses of colloid and cell hypertrophy. In a second approach, the mitotic activity of the thyroid remnant was estimated in hypophysectomized and in thyroxine treated rats. Both hypophysectomy and thyroxine injection prevented occurrence of the mitotic peak at 2 days. The regeneration of the thyroid after hemithyroidectomy, as it occurred in the present work, may be explained by a release of thyroid stimulating hormone from the pituitary, brought about by the low level of circulating thyroid hormone, itself resulting from a loss of thyroid tissue.     

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.     

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.     

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.     

12-O-tetradecanoylphorbol-13-acetate induces an immediate, but transient increase in mitotic activity uncoupled from DNA synthesis in the monolayer cultures of rat keratinocyte

A single wave of mitotic activity was observed in a monolayer culture of rat keratinocytes immediately after exposure to 12-O-tetradecanoylphorbol-13-acetate. A peak for cells in prophase, observed at 10 min after the exposure, was followed by a peak for metaphase at 20 min, for anaphase at 25 min and telophase at 30 min after the exposure. Thereafter, the mitotic activity began to subside. This transient stimulation of mitotic activity resulted in an increase of population density in the monolayer culture. There was neither a stimulation of DNA synthesis during this period nor a change of the DNA content after the mitotic activity was completed. This single burst of synchronous mitotic activity which did not require a substantial stimulation of DNA synthesis suggests that the effect was on the initiation process of mitosis among a subpopulation of cells, presumably cells delayed in the G2 phase of the cell cycle.     

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.     

Mentha piperita (Linn) leaf extract provides protection against radiation induced alterations in intestinal mucosa of Swiss albino mice

Intestinal protection in mice against radiation injury by M. piperita (1 g/kg body weight/day) was studied from day 1 to day 20 after whole body gamma irradiation (8 Gy). Villus height, goblet cells/villus section, total cells, mitotic cells and dead cells/crypt section in the jejunum are good parameters for the assessment of radiation damage. There was significant decrease in the villus height, number of total cells and mitotic cells/crypt section, whereas goblet cells and dead cells showed significant increase after irradiation. Mentha pretreatment resulted in a significant increase in villus height, total cells and mitotic cells, whereas goblet cells and dead cells showed a significant decrease from respective irradiated controls at each autopsy day. The results suggest that Mentha pretreatment provides protection against radiation induced alterations in intestinal mucosa of Swiss albino mice.     

Pathogenic importance of pepsin in ischemia/reperfusion-induced gastric injury

We investigated the role of pepsin in the development of ischemia/reperfusion (I/R)-induced gastric lesions in rats. Under urethane anesthesia, the pylorus was ligated, the celiac artery was clamped, and 1 ml of HCl (50-150 mM) was instilled in the stomach. Then, reperfusion was established 15 min later by removing the clamp, and 2 h later the stomach was assessed for gross mucosal damage. Pepstatin (a specific pepsin inhibitor) or pepsin was given i.g. after the pylorus was ligated while cimetidine, omeprazole, or atropine was given s.c. 30 min before the ligation. I/R produced hemorrhagic gastric injury, with a concomitant increase in the amount of pepsin secreted, and the degree of both these responses was dependent on the concentration of HCl. The formation of lesions by IR in the presence of 100 mM HCl was significantly prevented by atropine or bilateral vagotomy, but neither omeprazole nor cimetidine had any effect. Intragastric administration of pepstatin dose-dependently reduced the severity of the I/R-induced gastric lesions, the effect being significant even at 0.1 mg/kg, while that of pepsin markedly aggravated these lesions. The increased pepsin output during I/R was associated with luminal acid loss and significantly inhibited by bilateral vagotomy or pretreatment with atropine but not cimetidine or omeprazole, while pepstatin significantly inhibited the pepsin activity. In conclusion, we suggest that pepsin plays a pivotal role in the pathogenesis of I/R-induced gastric lesions, and pepsin secretion is increased during I/R, the process being associated with acid back-diffusion and mediated through a vagal-cholinergic pathway.     

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.     

Correspondence of two nuclear networks observed in situ with the nuclear matrix

In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter-chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten-fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.     

Development of Angiostrongylus cantonensis in the lung of Achatina fulica (author's transl)

Twenty-four Achatina fulica from Pai-sha of the Pescardores Islands, where no Angiostrogylus cantonensis has been found in A. fulica(4), were inoculated with the first stage larvae of A cantonensis. One snail was killed every day after 24 days following inoculation. The lung of each snail was separated and examined for larvae under microscope. The same lung was then digested with pepsin. The larvae recovered after pepsin digestion were examined for more detailed morphology. This study confirmed that the first stage larvae of A. cantonensis passed through the respiratory pore to the lung of A. fulica and developed into infectious form (3rd stage larvae) in the lung. The second and third stage larvae were found in the lung on the 7th and 17th day respectively after inoculation. Worm capsules were found in the lung on the 12th day after inoculation and the size of capsule was in proportion to the duration of the infection.     

Quantification of gastric secretions in the wild whiting fed on natural prey in captivity

Quantification of gastric acid and enzyme pepsin in the gastric phase of digestion in wild whiting (Merlangius merlangus L.) were elucidated in the present study. The results showed that after ingestion of a meal, first pepsin and, more slowly, gastric acid (hydrochloric acid) was secreted into the gastric lumen to peak at 2–4 h (49.4–53.8 mEq wt acid × 10³; 652–813 μg pepsin ml Eq min^?1). Fish size had a significant influence on the amount of gastric acid and pepsin secreted; digestive power increased α W^0.67, as predicted by the ‘physiological’ model. However, the predicted effects of meal size [stimulus, distension volume (DV)] were not clear. Instead, the stomach distension model for flatfish from a previous worker showed that muscular contractions (mixing mechanism) were related to DV^0.33 and found to partially support the physiological model in the present study.