THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH*

The initial effect of an injection of TdR-5-³H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G₂-cells at the time of injection. The main effect is a shortening of t_g2. No effect is observed after injection of non-radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with ³H-TdR, as well as the risks of using the PLM-method in a partially synchronized system.     

Maturation promoting factor destabilization facilitates postovulatory aging‐mediated abortive spontaneous egg activation in rat

The present study was designed to investigate whether destabilization of maturation promoting factor (MPF) leads to postovulatory aging‐mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee‐1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging‐mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post‐hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr‐14/Tyr‐15 and dephosphorylation of Thr‐161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging‐mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging‐mediated abortive SEA in rat eggs.     

Analyses for in vitro growth conditions,cytokinetics, and sister chromatid exchanges in lymphocytes cultured from the Sprague-Dawley rat

Summary Peripheral blood samples from Sprague-Dawley rats gave successful lymphocyte growth in GIBCO: IA, RPMI 1640, and Eagle's minimum essential medium (MEM) culture media. Various growth conditions, cytokinetics, and sister chromatic exchange (SCE) induction were studied using reconstituted GIBCO 1A only. Neither methoxyflurane anesthesia of the rats before sampling nor washing of the cells with phosphate buffered saline affected the mitotic index. Cultures treated with [³H]thymidine showed the lymphocytes entering into DNA synthesis after approximately 24 h. The time at which BUdR (5-bromo-2′ deoxyuridine) was added, i.e. 0 vs. 24 h incubation, had minimal effect on the mitotic index of cultures harvested at 48 h. However, when harvest was extended to 72 h, mitotic activity was greater in the cultures treated with BUdR at 24 h. No significant differences in mitotic index and the number of average lymphocyte division were detected in cultures exposed to 0.3 to 0.5 μg/ml BUdR at 24 h and harvested at 72 h. Although SCE frequencies increased in the presence of BUdR, the baseline level of SCEs was estimated to be 5 to 6/cell. Average generation time of the lymphocytes dividing between 48 and 72 h was 16.5 h. Because of its simplicity of culture and the reproducible nature of its in vitro growth kinetics, the Sprague-Dawley rat lymphocyte is a suitable model for cytogenetic investigations.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Control of the carcinogenic potential of 99mTechnetium by the immunologic hormone lymphotoxin

Summary Immunologic prevention of the carcinogenicity of the diagnostic gamma-emitting radionuclide ^99mTechnetium (^99mTc) by lymphotoxin was evaluated using an in vivo-in vitro assay of carcinogenesis. Pregnant Syrian golden hamsters received 125–2,300 Ci ^99mTc/kg body weight by injection, and 7 days later colonies of morphologically transformed cells were quantitated in vitro. The transformation frequency increased directly with the radionuclide concentration, and cells derived from transformed colonies produced tumors in athymic nude mice. The total absorbed ^99mTc dose was 0.20 rad following injection of 250 Ci ^99mTc/kg hamster body weight; this compares with an exposure of 0.13 rad following injection of 143 Ci ^99mTc/kg body weight in humans. Intravenous injection of purified hamster lymphotoxin immediately after ^99mTc caused a dose-dependent reduction in the transformation frequency. Transformation was essentially completely prevented (97%) by injection of 8,000 U of lymphotoxin. Thus, the immune system, through the action of lymphotoxin, has the potential to prevent carcinogenesis induced by gamma-radiation from ^99mTc. This emphasizes the importance of considering the recipient's immune and other homeostatic mechanisms as part of a complete diagnostic or therapeutic gamma-radiation regimen.     

The Induction of Chromosomal Aberrations by Tetra Antibiotic in Bone Marrow Cells of Rats in Vivo

The aim of this study was to investigate the in vivo effects of tetra (tetralet) antibiotic on chromosomal aberrations (CA) in bone marrow cells of rats (Rattus norvegicus var. albinos). The tetra antibiotic significantly increased the percentage of abnormal cells and the chromosomal aberrations per cell (CA/cell) in bone marrow cells of rats at concentrations of 100 and 200 mg/kg body weight for 12 and 24 h treatment periods for each. In addition, the percentage of abnormal cells and the CA/cell increased dose-dependently for 12 h treatment period; in contrast, mitotic index was decreased when compared with negative control and solvent controls for 12-h treatment period. However, mitotic index increased depending on tetra antibiotic dose for 24-h treatment period.     

Mitotic cycle kinetics of root meristems of isolated barley embryos and intact seedlings. Labelling of nuclei by3H- thymidine and its cytogenetic consequences

The duration of the mitotic cycle and its individual phases was estimated in root meristems of isolated barley embryos and intact barley seedlings by means of pulse labelling with³H-thymidine and construction of labelled mitoses curve. The duration of the whole mitotic cycle in the cell population of root meristems of isolated barley embryos cultivated in the aerated liquid complete medium is 12.2 h. The mitotic cycle time of root meristems of intact barley seedlings, oultived in Petri dishes on wet blotting paper is 9.2 h. Most of root meristem cells belong to the fraction of rapidly proliferating cells, but this fraction exerts a high degree of variability by itself. Pulse treatment by³H-thymidine in our experimental conditions (74 kBq ml^-1 - or 2 μCi ml^-1, exposure 0.5 h) did not induoe any chromosomal aberrations in unlabelled cells and only a very low frequency of chromosomal aberrations in labelled cells. Measuring the cell population kinetics by pulse labelling with³H-thymidine can be used simultaneously with the study of induction of ohromosomal aberrations by mutagens.     

Calcitonin increases hepatic hepcidin expression through the BMP6 of kidney in mice

BackgroundOsteoporosis is frequently accompanied by iron disorders. Calcitonin (CT) was approved as a clinical drug to treat osteoporosis. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin deficiency leads to iron overload diseases. This study was aimed at investigating the effect of CT on hepatic hepcidin and the mechanism by which CT modulates hepatic hepcidin pathways and iron metabolism.MethodRT-PCR, Western blot, ELISA and siRNA were used to detect the effect of CT on iron metabolism in vivo and in vitro. In addition, the regulatory signal molecules of hepcidin were measured to explore the molecular mechanism of its regulation.ResultsThe results showed that CT strongly increased hepcidin expression and altered iron homeostasis, after mice were intraperitoneal injection of CT. In response to CT administration, BMP6 level in kidney and the serum BMP6 was increased significantly. The phosphorylation of Smad1/5/8 proteins in liver was increased at 3 h and 6 h. Moreover, the Bmp inhibitor LDN-193,189 pretreatment significantly attenuated the CT-mediated increases in phosphorylated Smad1/5/8 and Hamp1 mRNA levels. Calcitonin receptor (CTR) siRNA transfection significant suppressed the role of CT on BMP6 expression in Caki-1 cells.ConclusionOur results suggest that CT strongly induces hepcidin expression and affected iron metabolism. It will provide a new strategy for the treatment of calcium iron related diseases.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

In vitro differential effects of sodium selenite on the growth of human hepatoma cells and human embryonic liver cells

The effects of various concentrations of Na₂SeO₃ on human hepatoma cells and human embryonic liver cells was investigated in vitro. For human hepatoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL Na₂SeO₃, mitotic activity of human hepatoma cells were partially arrested. In human embryonic liver cells continuously treated with Na₂SeO₃, (1 μg/mL) cell count of the treated group decreased only by d 7; mitotic index, labeled index, and mean silver grain number per 50 labeled nuclei were the same as in the control group on exposure to 1, 3, and 5 μg/mL for up to 72 h. In mixed cultures of human hepatoma and embryonic liver cells treated with 3 and 5 μg/mL of Na₂SeO₃ for 24 h, hepatoma cells showed vacuolated cytoplasms, distorted nuclei, condensed chromatin, and even pyknosis, whereas the embryonic liver cells retained a normal morphology under the same treatment.     

Effects of galanin on the secretion and proliferative activity of the immature and regenerating adrenal glands of rats

The effects of galanin and the galanin-receptor antagonist (galanin-A) [D-Thr(6),D-Trp(8,9),15-ol]-galanin(1-15) on the immature and regenerating rat adrenal glands have been investigated in vivo. Adult female rats with adrenal regeneration and their offpring (20-day-old) were given three subcutaneous injections (28, 16, and 4 h before being killed) of 2 nmol/100 g galanin and/or galanin-A, and 0.1 mg/100 g vincristin 3 h before being killed. Plasma corticosterone concentration was measured by radioimmunoassay, and the mitotic index ( per thousand of metaphase-arrested cells) was evaluated. In immature rats, galanin increased plasma corticosterone concentration, without affecting mitotic index; the secretagogue effect was reversed by galanin-A, which alone was ineffective. In rats with regenerating adrenal, galanin-A increased both blood level of corticosterone and mitotic index; galanin was ineffective, but blocked the effects of galanin-A. These findings allowed us to draw the following conclusions: 1) galanin exerts a moderate glucocorticoid secretagogue action on immature rat adrenals, but endogenous galanin does not play a major physiological role in the functional control of the gland; and 2) endogenous galanin exerts a maximal tonic inhibitory control on both glucocorticoid secretion and proliferative activity of regenerating rat adrenals, whose physiological relevance remains to be investigated.     

22例子宫卒中性平滑肌瘤的临床病理特征分析

目的:探讨卒中性平滑肌瘤的临床病理特征、免疫表型及预后。方法:回顾性分析22例子宫卒中性平滑肌瘤的临床特点、病理形态学特点及免疫表型,并复习相关文献。结果:卒中性平滑肌瘤患者的临床症状主要为异常子宫出血,腹痛等。肿瘤大体常伴有以下特征,多灶性出血、坏死、囊性变、水肿变性及质地变软等,镜检可见多灶出血区,呈放射状、卵圆形或不规则形,其中央常可伴有坏死、玻璃样变或肿瘤细胞减少,周边肿瘤细胞富集。22例卒中性平滑肌瘤中,15例核分裂增加(最高达13个/10HPF),但细胞均未见病理性核分裂及显著细胞异型性;22例肿瘤组织弥漫强表达Desmin、SMA、H-caldesmon,Ki67指数范围(3%-15%);18例出血及凝固性坏死区及其周围的肿瘤组织不同程度表达CD10。22例患者均获得完整随访资料,平均随访30个月(10~110个月),均无瘤生存。结论:子宫卒中性平滑肌瘤预后较好,但由于其具有一系列异常的病理形态学特征,如多发出血灶,出血灶中央可伴有坏死,其周围富于肿瘤细胞区细胞可见轻度非典型性且核分裂不同程度增加,易被误诊为子宫恶性潜能未定的平滑肌瘤甚至是子宫平滑肌肉瘤。正确认识该类肿瘤宽广的形态学谱系,有助于临床病理医师做出正确诊断。     

Changes in the CO2 Concentrating Mechanism During the Cell Cycle in Dunaliella tertiolecta

Synchronised cells of Dunaliella tertiolecta were used to investigate the expression of the CO₂ concentrating mechanism over the cell cycle during growth in either ambient air (low Ci cells) or air enriched with 5% CO₂ (high Ci cells). The cultures were analysed for extracellular carbonic anhydrase activity, affinity of photosynthesis for inorganic carbon (Ci) and the ability to accumulate Ci. In high Ci cells, carbonic anhydrase activity changed between 2 ? 4 units mg^?1 Chi during the light-dark rhythm showing no clear periodicity. Similarly, the apparent affinity for Ci remained rather constant over the cell cycle. This was judged from the Ci concentrations required for half maximum rate of photosynthesis (K_1/2(Ci)) of 72 ? 80μM. In the same cells the accumulation ratio of internal Ci versus external Ci ranged between 5 and 9.5 without a clear rhythm. In contrast, these parameters showed distinct periodical changes in synchronised low Ci cells. Carbonic anhydrase activity changed from 10 to 350 units mg^?1 Chl with maximum and minimum activities occurring in the middle and at the end of the light period, respectively. The K_1/2(Ci) values showed similar periodicity ranging between 13 ? 36μM. In addition the accumulation ratio increased up to 30 in the middle of illumination and decreased to its lowest level of 12 at the end of the light period. These results indicate the presence of a common step in regulating the induction of the measured parameters and that light is not an absolute requirement for the induction of the CO₂ concentrating mechanism in synchronous low CO₂ grown cells of Dunaliella tertiolecta.     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.     

EFFECTS OF CORTICOSTEROIDS ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: POSTNATAL CELL FORMATION

(1) Treatment with cortisol acetate (0.2 mg daily during the first 4 days after birth) reduced the rate of growth in the rat: at 35 days of age the body weight was reduced by 50 per cent and the brain weight, depending on the region, by up to 30 per cent. (2) In the brain the normal increase in cell number was severely inhibited during the period of cortisol treatment; this resulted in a final deficit in cell number of about 20 per cent in the cerebrum and 30 per cent in the cerebellum. (3) To determine whether cortisol affected primarily cell formation or cell destruction the labelling of brain DNA was studied 1 h after a subcutaneous injection of 20 Ci/100 g [2-¹⁴C]thymidine. In the controls the amount of labelled DNA increased by a factor of two in the cerebrum and seven in the cerebellum during the period 2-13 days, and it decreased to 40 and 27 per cent of the peak values in the cerebrum and cerebellum respectively in the following 7 days. The results indicated that mitotic activity is higher in the cerebellum than in the cerebrum in the 2nd week of life. It would appear that in the cerebrum appreciable cell death accompanies new cell formation, especially during the period 13-35 days of age. (4) Cortisol treatment affected cell division rather than cell destruction in the brain since it strongly inhibited the incorporation of [2-¹⁴C]thymidine into DNA. The inhibition was severe during the period of treatment but it did not result in a lasting fall in mitotic activity. At the age of 13 days the amount of labelled DNA formed approached the normal level and it was twice that in controls at 20 days, indicating a tendency for compensating cell deficit by an accelerated mitotic activity. Nevertheless, massive cell proliferation ceased at about the same age as in normals; the labelling of DNA decreased markedly between 13 and 20 days after birth, and the DNA content did not increase after the age of 20 days. (5) In contrast to the marked effect on cell number, cortisol treatment did not influence significantly the maturational changes related to average cell size (DNA concentration) or the chemical composition of cells (RNA/DNA and protein/DNA).     

Endothelin-1 stimulates mitotic activity in the zona glomerulosa of the rat adrenal cortex.

Subcutaneous infusion with endothelin-1 (ET-1; 30 pM min-1) for 24 h induced a 9-fold increase in the mitotic index (% of metaphase-arrested cells) of rat adrenal zona glomerulosa (ZG). Infusions with adrenocorticotrophic hormone (ACTH) angiotensin-II (ANG-II) or arginine vasopressin (AVP) (30 pM min-1/24 h) raised the ZG mitotic index 13-fold, 9-fold and 10-fold, respectively. Combined infusion with ET-1 and ACTH increased the ZG mitotic index 20-fold, while the effects of ET-1 and ANG-II or AVP were not additive. These findings suggest that ET-1 exerts a strong proliferogenic effect on the rat ZG, by a mechanism probably similar to that underlying the adrenoglomerulotrophic actions of ANG-II and AVP.     

Clastogenic effects of food additive citric acid in human peripheral lymphocytes

Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant.     

Implications of childhood obesity for adult health: findings from thousand families cohort study

ObjectiveTo determine whether being overweight in childhood increases adult obesity and risk of disease.DesignProspective cohort study.SettingCity of Newcastle upon Tyne.Participants932 members of thousand families 1947 birth cohort, of whom 412 attended for clinical examination age 50.ResultsBody mass index at age 9 years was significantly correlated with body mass index age 50 (r=0.24, P<0.001) but not with percentage body fat age 50 (r=0.10, P=0.07). After adult body mass index had been adjusted for, body mass index at age 9 showed a significant inverse association with measures of lipid and glucose metabolism in both sexes and with blood pressure in women. However, after adjustment for adult percentage fat instead of body mass index, only the inverse associations with triglycerides (regression coefficient= −0.21, P<0.01) and total cholesterol (−0.17, P<0.05) in women remained significant.ConclusionsLittle tracking from childhood overweight to adulthood obesity was found when using a measure of fatness that was independent of build. Only children who were obese at 13 showed an increased risk of obesity as adults. No excess adult health risk from childhood or teenage overweight was found. Being thin in childhood offered no protection against adult fatness, and the thinnest children tended to have the highest adult risk at every level of adult obesity.

What is already known on this topic

Many studies have found that body mass index in childhood is significantly correlated with body mass index in adulthoodObese children have been found to have higher all cause mortality as adults

What this study adds

No excess health risk from childhood overweight was foundChildhood body mass index was linked to adulthood body mass index but not percentage body fatOnly children who were obese at 13 showed a significant increased risk of obesity as adultsPeople who were thinnest as children and fattest as adults tended to have the highest adult risk     

RADIOTOXICITY OF INCORPORATED [3H]THYMIDINE AS STUDIED BY AUTORADIOGRAPHY AND FLOW CYTOMETRY CONSEQUENCES FOR THE INTERPRETATION OF FLM DATA

The radiotoxic effects of incorporated [³H]thymidine on proliferation kinetics of flash-labelled (30 min, 0.3 μCi/ml, 40 Ci/mM) L-929 cells in vitro were studied by means of autoradiography and flow cytometry. the flow cytometric results obtained by applying the BUdR-33258 Hoechst technique, using new evaluation procedures, showed that the labelled cells are delayed in their progression through the S and G₂+ M phases, leading to mitotic delay. From autoradiographs, the fraction of labelled mitoses was determined and, in addition, the ratio of labelled and of unlabelled mitotic cells to all cells. the radiotoxic effects are not evident from the FLM curve, even if the ratio of labelled mitotic cells to all cells shows a highly distorted shape. A mathematical model has been developed that describes the perturbed cell kinetics due to radiotoxic effects of the incorporated [³H]thymidine. These findings have considerable consequences for the interpretation of autoradiograhic data, especially of labelled mitoses curves.