Regulation of mitotic activity and the cell cycle in primary chick muscle cells by neurotransferrin

We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein-stained mitotic figures and analysis of cell cycle kinetics with a fluorescence-activated cell sorter. In low-density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 μg/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from G_t to S and G₂ + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high-density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9–13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13–14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Mitotic Figures in the Median Eminence of the Hypothalamus

The median eminence of the hypothalamus is part of the avenue by which neurosecreted hormones from the hypothalamic nuclei reach the pars nervosa (neural lobe) of the pituitary and eventually the bloodstream. Lithium treatment and osmotic stress increases the transport of neurosecretory hormones to the pituitary in the adult rat. Specialized astrocytes termed pituicytes in the pars nervosa of the pituitary participate in the secretory process and also develop considerable mitotic activity. The present work reveals similar mitotic figures in cells within the median eminence following 3 days of lithium treatment. The location and appearance of these mitoses add to the evidence that pituicytes are present in the median eminence. Moreover, mitoses occur within the ependymal (tanycyte) layer of the median eminence. Thus, the present results suggest that the tanycyte layer may contain pituicytes, indicating that the hypothalamus possesses specialized cells for modulating neurosecretion in response to osmotic challenges.     

EVOLUTION DE L''INCORPORATION IN VIVO DE L''ACIDE [14C]OROTIQUE DANS L''ARN ET L''ADN DU CERVELET DE JEUNES RATS HYPO- ET HYPERTHYROIDIENS: EFFETS COMPARATIFS DE L''HYPO- ET L''HYPERTHYROIDIE SUR LES MULTIPLICATIONS CELLULAIRES

The change in the body and cerebellar weights, together with the quantity of RNA, DNA and proteins in the cerebellum were studied for the first 10 days and on the 12th, 14th, and 17th days of postnatal life in normal, hypo- and hyperthyroid rats. In the normal animals: (1) The average cellular protein content decreases from the first to the second day, increases to a maximum at 4 days, then decreases. (2) The specific radioactivity of the RNA, 14 h after an intravenous injection of [6-¹⁴C]orotic acid, varies distinctly from birth to 9 days and reaches two maxima at 4 and 6 days. After 9 days it decreases markedly. (3) Mitotic activity (number of replicating cells) increases, reaches a maximum at 9 days, then decreases. (4) The specific radioactivity of the DNA (used as a measure of the percentage of the cellular population in division) reaches a maximum at 6 days. (5) Mitotic efficiency (number of replicating cells in mitotic activity) decreases from 2 to 7 days, and subsequently increases. In the hypothyroid animals: (1) The average cellular protein content increases from the first to the second day and then decreases. (2) The specific radioactivity of the RNA, always significantly higher than that of normal animals, varies from birth to 9 days, reaches two maxima at 4 and 6 days, then decreases after 9 days. (3) Mitotic activity, always significantly lower than that of normal animals, increases from birth, reaches a maximum at 9 days, then decreases. (4) The specific radioactivity of the DNA reaches a maximum at 6 days and the mitotic efficiency a minimum at 7 days. Neither are significantly different from that of normal animals. In the hyperthyroid animals: (1) The average cellular protein content, is maximal at 2 days, then decreases. (2) The specific radioactivity of the RNA, always significantly lower from that of normal animals, decreases from birth. (3) Mitotic activity is similar to that of normal animals, increases from birth up to 6 days, then decreases. (4) The specific radioactivity of the DNA increases from birth up to 5 days, then decreases. It is significantly lower than that of normal animals. (5) Mitotic efficiency is significantly higher than that of normal animals. In the different groups, the maximum of the average cell size, always precedes the maximum of the cellular division. In the hypothyroid animals, the rate of cell death is higher than that of normal animals, and the average cell size is higher during the first fourteen days. In the hyperthyroid animals, the rate of cell death is lower than that of normal animals, and the average cell size is higher at 14 and 17 days.     

Cytogenetic evaluations in human lymphocytes exposed to methyl acetimidate, a lysine-specific protein crosslinking agent

The cytogenetic effects of methyl acetimidate (MAI), a lysine-specific protein crosslinking reagent, were investigated using human peripheral lymphocytes in culture. Lymphocytes were treated with the chemical either prior to PHA exposure or 2-3 days following mitogenic stimulation and assessed for perturbations in cellular proliferation and induction of SCEs. Severe reductions in the mitotic index (MI) and pronounced decreases in the proportion of metaphases proceeding beyond M(1) were observed following G0 exposure to MAI concentrations of as low as 2 mM; with complete suppression of mitotic activity in all cultures exposed to levels of 3 mM MAI or greater. Concentrations resulting in severe depression in MI caused only moderate increases in SCEs. Cells exposed to less than 10 mM MAI during the late S-G2 stages of the cell cycle and harvested at the first metaphase following treatment exhibited profound mitotic delay, impaired prophase to metaphase transitions and abnormal mitotic configurations. These findings demonstrate that protein-specific crosslinking agents may induce a wide spectrum of adverse cytogenetic outcomes in both cycling and noncycling lymphocytes.     

Alpha-fetoprotein serum levels and the development of estrogen-sensitive cell multiplication in the hamster uterus

Subcutaneous injections of 5 or 25 micrograms estradiol-17 beta (E2)/kg in ovariectomized adult hamsters produced substantial increases in uterine wet weight, protein content and the mitotic indices of the glandular and luminal epithelia. However, no significant increase was seen in total uterine DNA. Intact hamsters from 2 to 25 days of age received a daily subcutaneous injection of 5 micrograms E2/kg for 2 consecutive days. Significant increases in uterine wet weight and protein content first occurred at 8 and 17 days, respectively. No significant increase was observed in uterine DNA. In a separate experiment, hamsters between 2 and 20 days of age received one subcutaneous injection of 5 micrograms E2/kg. Mitotic indices in the stroma were increased at 6 and 10 days of age. Mitotic indices in the luminal epithelium were significantly increased only at 6 days of age. Rocket immunoelectrophoresis revealed a sharp decline in serum alpha-fetoprotein (AFP) concentrations after 2 days of age. Estradiol concentrations in the sera of immature hamsters gradually decreased from 55 pg/ml at 0 days of age to 17 pg/ml at 20 days of age. These results provide a quantitative analysis of the effects of E2 upon cell proliferation in the hamster uterus. The correlation of declining AFP levels and the incipience of the mitotic response to estrogen suggests that AFP may directly inhibit estrogen-sensitive cell multiplication in the neonate. Other possible causes for the lack of a mitotic response in the uterus of the newborn hamster to the administration of E2 are also discussed.     

The basalis of the primate endometrium: a bifunctional germinal compartment

Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.     

GROWTH AND MITOTIC ACTIVITY OF LUNG COLONIES DERIVED FROM MOUSE TERATOCARCINOMAS

This study dealt with the mitotic activity of lung colonies in the process of differentiation of mouse teratocarcinoma cells injected in vivo . The results showed that three stages were identifiable in respect to the mitotic activity: 1) a lag stage for a few days after injection, 2) a stage during which maximum mitotic activity is observed for several days following the first stage and 3) a third stage displaying a decrease in mitotic activity. There is a correspondence between these three steps in mitotic activity and differentiation in lung colonies previously identified by histological observations.     

Initiation of roots on hypocotyl cuttings of Pinus contorta in vitro

The origin of roots and wound tissue after treatments for induction of roots on hypocotyl cuttings of three-week-old Pinus contorta Dougl. ex Loud, is discussed. The cuttings were cultured in vitro and treated with 1.2 μ M to 1.5 M IBA (indole-3-butyric acid) for 6 h to 10 days. The control, which was not treated with IBA developed a wound tissue from which roots formed. Cuttings treated with IBA developed roots directly from the hypocotyl. Direct rooting was faster than indirect rooting via a wound tissue. Rooting was considered to be optimal if more than 80% of the cuttings rooted within 19 days and half of the cuttings which possessed roots after one month had acquired them within 14 days. This type of rooting was obtained after treatment with either 80 μ M IBA for 4 to 6 days or 1.25 to 5.0 m M IBA for 6 h. Suboptimal treatments gave lower rooting percentages and superoptimal treatments resulted in delayed rooting. In IBA-treated cuttings, large increases in mitotic activity (number of mitoses per mm hypocotyl) were found in the pericycle and parenchyma inside endodermis. However, the control also had similar mitotic activities as the IBA-treated cuttings but closer to the cut surface. This led us to the conclusion that similar tissues may produce either wound tissue or roots. Almost all roots obtained through direct rooting originated outside resin ducts.     

Aneuploidy in male germ cells induced by alcohol ingestion

The effects of ethyl alcohol ingestion, at levels comparable to human drinking, have been evaluated by cytogenetic analysis of mouse spermatogonia and primary spermatocytes. The alcohol was administered intragastrically at different doses and for various periods of time. An increase in mitotic and first division meiotic aneuploidy was observed in direct proportion to the alcohol dose. Approximately a three-fold increase in mitotic and meiotic aneuploidy over control levels was observed in animals exposed to a daily 3 gm/kg bw dose for 6 to 28 days. The level of aneuploidy was found to return to control levels by 14 days after cessation of alcohol ingestion. Analysis of the kinetics of decrease in aneuploidy following cessation of alcohol suggests that the primal event resulting in altered chromosome numbers occurs at a stage of the cell cycle other than anaphase and therefore is not due to nondisjunction nor to anaphase lagging.     

Changes in the proliferative activity of the corneal epithelium and the regenerating liver in partial splenectomy in mice

The mitotic activity in epithelial cells of the mouse cornea was studied 4 h, 1, 2, 5, 8 and 14 days after a sham operation or partial (2/3) splenectomy. The decrease in the number of dividing cells in the corneal epithelium was observed within two days after a sham operation and within five days after partial splenectomy. On the contrary, partial hepatectomy increased the number of mitoses in the corneal epithelium. Liver regeneration against the background of a sham operation or partial splenectomy was accompanied by a lesser number of mitoses (by a factor of 2.5-4) in hepatocytes than in the animals subjected to partial hepatectomy only.     

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.     

Cellular composition of structural components of the lymph nodes of rats during rehabilitation after dimethyl sulfate poisoning

By means of microanatomical methods the inferior tracheobronchial lymph nodes have been investigated in 48 Wistar rats in 2 weeks and 3 months after discontinuance of inhalation of dimethylsulfate (DMS) vapours for 2 and 14 days by the animals in concentration 2.0 mg/m3, that is to say during rehabilitation period. Comparison of relative parameters of the structural components areas and cell composition of the lymph nodes has been carried out. During rehabilitation period after DMS inhalation for 2 days the cortical and medullary areas in histological preparations do not essentially differ from corresponding parameters of an acute experiment (2 days, 2.0 mg/m3, without rehabilitation). Amount (%) of cells with mitotic figures in the lymphoid nodules++ increases in 2 weeks and in 3 months. Contents of poorly differentiated cells during rehabilitation periods increase in the cortical plateau, but keeps nearly at the same low level as during the acute experiment in the lymphoid nodules++. In 2 weeks after DMS influence for 14 days, the cortical and medullary area in the histological preparations reach the control levels. In the lymphoid nodules++ a relative amount of reticular, poorly differentiated, mitotically dividing cells increases, and in the medullary cords contents of middle and small lymphocytes become greater in comparison with the acute experiment (14 days, 2.0 mg/m3, without rehabilitation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell Cycle Responses to Red or Far-red Light, or Darkness, in the Shoot Apex of Silene coeli-rosa L. During Floral Induction

Twenty-eight-day-old plants of Silene coeli-rosa L. were maintainedin short days (SD) for 9 d (0–8) or exposed to 7 longdays (LD), or 7 SD with a 5 min exposure at 1700 h of each dayto far-red (FR), red (R) or 5 min FR/5 min R, or 7 dark-interrupted(di = 1700–1720 h) LD. Treatments were followed by twofurther SD. The mitotic index and G1 and G2 proportions weremeasured in the shoot apices of plants sampled at 2000 h ofeach day of each replicated treatment. Exposure to 7 LD (= 100per cent flowering) resulted in significant increases, relativeto the SD controls, in both the G2 proportion and the mitoticindex on d 0 to 3, 7 and 8. Five minute FR (= 0 per cent flowering)resulted in cell cycle responses similar to those in LD onlyfrom d 0 to 2. R and FR/R (both = 0 per cent flowering) didnot result in any increases in the G2 proportion in the apexapart from d 3 of FR/R. However 5 min FR/5 min R, and to a lesserextent 5 min R, did result in significant increases in the mitoticindex on d 0, 1, 7, and 8. diLD (= 8–10 per cent flowering)also prevented any significant increases in the G2 proportionon d 0 to 3, and 5 to 8 but the mitotic index was again higheron these days compared with control data. Thus the transitionto floral growth for 90 per cent of the plants is associatedwith changes in the cell cycle in the shoot apex measured asincreases in the G2 proportion at 2000 h of LD 0 to 3 and 7to 8. Silene coeli-rosa L., cell cycle, flowering, phytochrome, shoot apex     

Cellular responses of leaf explants of Cocos nucifera L. in vitro

Leaf explants of Cocos nucifera L. (coconut palm) were studied in vitro in order to establish whether or not rapid cellular changes contribute to the well known recalcitrance of coconut cells in tissue culture. Segments from the base of immature leaves were cultured on modified Eeuwens' medium at 30°C in darkness. The mitotic index, nuclear DNA amounts, cell and nuclear size were measured both before and during culture (from 0 to 70 days). There was no basipetal gradient of cell division in immature coconut leaves; the mitotic index never exceeded 2% and showed neither a positional nor temporal relationship with leaf development. Moreover the vast majority of cells were in G1 of the cell cycle. This cell cycle pattern was maintained for most of the period in culture although at 70 days there was an increase in the proportion of cells in S- and G2-phases consistent with low rates of callus formation. The nuclear: cell size ratio was constant in cells within the immature leaf irrespective of developmental age. However upon transfer to culture media, cell size but not nuclear size increased. We suggest that this uncoupling of cell and nuclear size disrupts cell co-ordination and is a key contributor to recalcitrant cellular behaviour of this species in vitro.     

Administration of caerulein to rats promotes antral epithelial cell renewal

Summary The growth-promoting effect of caerulein on antral gastric mucosa was explored using Wistar rats. Implanted osmotic minipumps were used to administer submaximal doses of either caerulein or saline to normal rats for up to 4 days. In one group, reflux of bile and pancreatic juice into the stomach was avoided by previous surgical diversion of the distal common bile duct to the jejunum. DNA synthetic and mitotic activity in the antrum epithelium were estimated by ³H-thymidine pulse labelling and autoradiography during the administration of the peptide. The rate of cell migration was determined in animals killed 1, 2 and 3 days after the ³H-thymidine pulse. Administration of caerulein to normal rats provoked significant increases in both labelling and mitotic indices, and a significant acceleration of the upward cell migration in the glandular tubes. In the animals with distal diversion of bile and pancreatic secretions both labelling and mitotic indices were also increased over control values under the effect of the peptide. These data indicate that administration of caerulein stimulates cell proliferation in the antral gastric mucosa. This effect cannot be explained through increased reflux of pancreaticobiliary secretions in the stomach.     

Chromosome preparation from single blastomeres after colcemid treatment and removal from rabbit morulae -- unsuitable for sexing in routine embryo transfer

Cell cycle of blastomeres of 3 days old rabbit morulae were synchronized by exposing the entire embryo to colcemid in BSM-culture-medium. Few cells were removed by micropuncture and prepared for chromosome analysis. Neither colcemid treatment nor removal of cells appears to harm the embryo. Successful diagnosis of embryonic sex was performed in 14 % of all embryos only. The poor success is due to a mitotic rate of less than 10 %. The method appears unsuitable for routine use.     

DDA3 recruits microtubule depolymerase Kif2a to spindle poles and controls spindle dynamics and mitotic chromosome movement

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.     

Tissue expansion: dividend or loan?

Epidermal mitotic activity during tissue expansion has been assessed in the guinea pig using tritiated thymidine and other confirmatory techniques. Implant inflation results in a threefold elevation of epidermal mitotic activity within 24 hours, followed by a gradual return to normal baseline over 2 to 5 days. Implant deflation, conversely, causes a transient decrease in epidermal mitotic activity. Neither of these phenomena has been previously described. It confirms previous animal studies which illustrate the highly responsive nature of the epidermis to physical stimuli and supports the view that tissue expansion is a highly useful manipulation of normal physiologic processes.