Incidence Of Apoptotic Cells Research Articles

Apoptotic cells in mouse blastocysts are eliminated by neighbouring blastomeres

Apoptosis is a physiological process that occurs commonly during the development of the preimplantation embryo. The present work examines the ability of apoptotic embryonic cells to express a signal promoting their phagocytosis, and quantifies the ability of neighbouring, normal embryonic cells to perform that task. Microscopic analysis of mouse blastocysts revealed phosphatidylserine externalization to be 10 times less common than incidence of apoptotic cells (as detected by TUNEL). In spite of the low frequency of phosphatidylserine-flipping (in inner cell mass, no annexin V staining was recorded), fluorescence staining of the plasma membrane showed more than 20% of apoptotic cells to have been engulfed by neighbouring blastomeres. The mean frequency of apoptotic cells escaping phagocytosis by their extrusion into blastocyst cavities did not exceed 10%. Immunochemically visualised RAC1 (an enzyme important in actin cytoskeleton rearrangement) was seen in phagosome-like structures containing a nucleus with a condensed morphology. Gene transcript analysis showed that the embryonic cells expressed 12 receptors likely involved in phagocytic process (Scarf1, Msr1, Cd36, Itgav, Itgb3, Cd14, Scarb1, Cd44, Stab1, Adgrb1, Cd300lf, Cd93). In conclusion, embryonic cells possess all the necessary mechanisms for recognising, engulfing and digesting apoptotic cells, ensuring the clearance of most dying blastomeres.

Follicular development and the expression of BAX and vascular endothelial growth factor in transplanted ovaries in uni- and bilateral ovariectomized mice: An experimental study.

BackgroundSeveral conflicting results have been reported on the survival and function of transplanted ovaries.ObjectiveEvaluation of the follicular development and the expression of vascular endothelial growth factor (VEGF) and Bcl-2-associated X protein (BAX) in ovaries transplanted into uni- and bilaterally ovariectomized mice.Materials and Methods In this experimental study, 40 female NMRI mice (21-days-old, 12-15 gr) were ovariectomized uni- and bilaterally (n = 20/ group), while the 8-wk-old mice were considered as intact control group (n = 6). 5 weeks after transplantation at the proestrus stage, the morphology of recovered transplanted ovaries and the proportion of follicles were studied at different developmental stages. The apoptosis cell death by pro-apoptotic protein BAX and the expression of VEGF were evaluated using immunohistochemistry.Results In the bilaterally ovariectomized mice, among the 455 counted normal follicles, a lower rate of primordial and primary follicles and a higher rate of preantral and antral follicles were observed (p = 0.002). However, the percentages of preantral and antral follicles, and the corpus luteum were significantly lower in the intact control group (among the 508 counted normal follicles in this group) compared to other transplanted groups (p = 0.002). The number of BAX-positive cells in all groups was not significantly different. The VEGF expression was prominent in vessels of the corpus luteum, and also in the theca layer of large follicles of studied groups.ConclusionEarly discharge of ovarian reserve was prominent in the bilaterally ovariectomized group but the incidence of apoptotic cells and VEGF expression as angiogenic factor did not differ in both ovariectomized mice. Thus, unilaterally ovariectomy has less side effects on the ovarian reserve compared to bilateral ovariectomy.

Apoptosis of endplate chondrocytes in cervical kyphosis is associated with chronic forward flexed neck: an in vivo rat bipedal walking model

BackgroundThis study was undertaken to establish a rat bipedal walking model of cervical kyphosis (CK) associated with chronic forward flexed neck and assess the effects of chronic forward flexed neck on endplate chondrocytes.MethodsForty-eight 1-month-old Sprague-Dawley rats were randomly divided into 3 groups: forward flexed neck group (n = 16), bipedal group (n = 16), and normal group (n = 16). Cervical curves were analyzed on a lateral cervical spine X-ray using Harrison’s posterior tangent method before the experiment and at 2-week intervals for a 6-week period. Histologic changes in cartilaginous endplate chondrocytes were observed using hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), and terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labeling.ResultsRadiographic findings suggested a significantly decreased cervical physiological curvature in the forward flexed neck group over the 6-week follow-up; normal cervical curves were maintained in other groups. The average cervical curvature (C2–C7) was − 7.6 ± 0.9° in the forward flexed neck group before the experiment, − 3.9 ± 0.8° at 2 weeks post-experiment, 10.7 ± 1.0° at 4 weeks post-experiment, and 20.5 ± 2.1° at the last follow-up post-experiment. Histologically, results of H&E staining unveiled that cartilaginous endplate chondrocytes were arranged in an irregular fashion, with the decreased number at the observation period; the incidence of apoptotic cells in the forward flexed neck group was noticeably higher at the 6-week follow-up than that in other groups.ConclusionsCK developed as the result of chronic forward flexed neck. Histologic changes suggested that chondrocyte apoptosis may play a critical role in the development of cervical kyphotic deformity associated with chronic forward flexed neck.

Genetic Increases in Olfactory Bulb BDNF Do Not Enhance Survival of Adult-Born Granule Cells.

Adult-born neurons produced in the dentate gyrus subgranular zone (SGZ) develop as excitatory hippocampal granule cells (GCs), while those from the subventricular zone (SVZ) migrate to the olfactory bulb (OB), where most develop as GABAergic olfactory GCs. Both types of neurons express TrkB as they mature. Normally ~50% of new olfactory GCs survive, but survival declines if sensory drive is reduced. Increases in endogenous brain-derived neurotrophic factor (BDNF) in hippocampus, particularly with wheel running, enhance dentate GC survival. Whether survival of new olfactory GCs is impacted by augmenting BDNF in the OB, where they mature and integrate, is not known. Here, we determined if increasing OB BDNF expression enhances survival of new GCs, and if it counters their loss under conditions of reduced sensory activity. Neurogenesis was assessed under normal conditions, and following unilateral naris occlusion, in mice overexpressing BDNF in the granule cell layer (GCL). OB BDNF levels were significantly higher in transgenic mice compared to controls, and this was maintained following sensory deprivation. Bromodeoxyuridine (BrdU) cell birth dating showed that at 12-14 days post-BrdU, numbers of new GCs did not differ between genotypes, indicating normal recruitment to the OB. At later intervals, transgenic and control mice showed levels of GC loss in deprived and nondeprived animals that were indistinguishable, as was the incidence of apoptotic cells in the GCL. These results demonstrate that, in contrast to new dentate GCs, elevations in endogenous BDNF do not enhance survival of adult-born olfactory GCs.

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Abstract: Although vitrification and follicular survival subsequent to xenotransplantation of canine ovarian tissues have been reported, the degree of cryoinjury after the vitrification has not yet been fully examined. Since cryoinjury after vitrification of ovaries has been reported in bovine and humans, this study evaluated canine ovarian tissues after vitrification by immunohistochemistry and TUNEL assay. Ovaries were collected from immature bitches. The ovarian tissue cubes were pretreated with 1 M DMSO. Subsequently, DAP 213 solution (2 M DMSO, 1 M acetamide, 3 M propylene glycol) was added. The cryotubes containing the ovarian tissues were placed on ice for 5 min, then plunged directly into liquid nitrogen. After warming with 0.25 M sucrose, cryopreserved ovarian tissues were fixed and subsequently stained with hematoxylin-eosin, proliferation cell nuclear antigen (PCNA) antibody, or active caspase-3 (AC-3) antibody, and evaluated by the TUNEL assay. There were no differences in the numbers of primo...

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Sinodielide A exerts thermosensitizing effects and induces apoptosis and G2/M cell cycle arrest in DU145 human prostate cancer cells via the Ras/Raf/MAPK and PI3K/Akt signaling pathways

Sinodielide A (SA) is a naturally occurring guaianolide, which is isolated from the root of Sinodielsia yunnanensis. This root, commonly found in Yunnan province, is used in traditional Chinese medicine as an antipyretic, analgesic and diaphoretic agent. A number of studies have reported that agents isolated from a species of Umbelliferae (Apiaceae) have antitumor activities. We previously reported, using combined treatments with this medicinal herb and hyperthermia at various temperatures, an enhanced cytotoxicity in the human prostate cancer androgen‑independent cell lines, PC3 and DU145, and analyzed the related mechanisms. In the present study, we investigated the effects of treatment with SA prior to hyperthermia on the thermosensitivity of DU145 cells, and the mechanisms related to the induction of apoptosis and G(2)/M cell cycle arrest via the activation of extracellular-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathways, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. Cells were exposed to hyperthermia alone (40-44˚C) or hyperthermia in combination with SA. Lethal damage to cells treated with mild hyperthermia (40 or 42˚C) for up to 6 h was slight; however, hyperthermia in combination with SA synergistically enhanced thermosensivity. Lethal damage to cells treated with acute hyperthermia (43 or 44˚C) was more severe, but these effects were also enhanced and were more significant by the combined treatment with SA. The kinetics of apoptosis induction and cell cycle distribution were analyzed by flow cytometry. In addition, the levels of ERK1/2, JNK and Akt were determined by western blot analysis. The incidence of apoptotic cells after treatment with SA (20.0 µM) at 37˚C for 4 h, hyperthermia (44˚C) alone for 30 min, and the combination in sequence were examined. The sub-G1 division (%) in the diagram obtained by flow cytometry was applied to that assay. The percentage of apoptotic cells (10.53±5.02%) was higher at 48 h as compared to 0, 12 and 24 h after treatment. The distribution of DU145 cells in the G2/M cell cycle phase was markedly increased after 24 h of heating at 44˚C and after the combined treatment with heating and SA. The phosphorylation of ERK1/2 was reduced following treatment with heating and SA, while the levels of phosphorylated JNK (p-JNK) were markedly increased immediately after heating at 44˚C and when heating was combined with SA. By contrast, the levels of phosphorylated Akt (p-Akt) were immediately increased only after heating at 44˚C. Thus, we concluded that SA exerts its thermosensitizing effects on DU145 cells by inhibiting the activation of the MAPK/ERK1/2 and PI3K/Akt signaling pathways.

Effect ofin vitroManipulation on Development of Pre-implantation Embryos: Lecture of Light Effects

Under normal milieu, mammalian oocytes are fertilized, and the zygotes develop into blastocysts in the female reproductive tract. After pick-up of oocytes, exposure to light during in vitro manipulation is unavoidable. Development of mouse zygotes to blastocysts is unaffected by exposure to cool white fluorescent light for 15 min, but the incidence of apoptotic cells in blastocysts is significantly increased by the light exposure. The blastocysts that develop from zygotes exposed to light develop to term fetuses at a lower rate than those developed from zygotes shielded from light. The incidence of apoptoic cells in blastocysts is significantly decreased by addition of SB20358, p38MAPK inhibitor, or Q-VD-Oph, caspase inhibitor. Furthermore, when HB-EGF is added to the culture medium, the total cell number of blastocysts developing from zygotes exposed to light increases, and the incidence of apoptotic cells in blastocysts decreases. These blastocysts develop to term fetuses at the same rate as those developed from zygotes shielded from light. In vitro conditions are stressful, and are not the best environment for eggs. Light is one of the physical factors affecting the function of mammalian embryos and extreme care should be taken concerning its effects.

Apoptosis of endplate chondrocytes in post-laminectomy cervical kyphotic deformity

The present study was performed to establish an animal model of cervical kyphosis after laminectomy (C2-C5), and to determine the role of endplate chondrocytes apoptosis in cervical kyphosis after laminectomy. Twenty-four 3-month-old sheep were randomly divided into two groups: the laminectomy group (n = 12), and the control group (n = 12). The cervical spine alignment was evaluated on a lateral cervical spine X-ray using Harrison's posterior tangent method before surgery and at follow-up. Cartilaginous endplate chondrocyte apoptosis was confirmed using transmission electron microscopy and terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling. The mean preoperative cervical curvature (C2-5) in the surgery group was -15.8°. The cervical curvature was 19.1° at 3 months post-operation and decreased to 20.2° at the final follow-up postoperatively. The cervical curvature was significantly decreased in the laminectomy group compared with the control group at the last follow-up (P < 0.001), which was a direct indication of kyphotic change. The incidence of apoptotic cells in the surgery group was significantly higher at the 3- and 6-month follow-up than the incidence in the control group. The frequency of endplate chondrocyte apoptosis in the laminectomy group was significantly higher than in the control group, indicating that chondrocyte apoptosis may play a pivotal role in the progress of post-laminectomy cervical kyphosis.

Chronic, low concentration exposure to pharmaceuticals impacts multiple organ systems in zebrafish

Pharmaceuticals are found in both receiving and drinking water due to their persistent release in waste-water effluents, raising concerns for environmental and human health. Chronic, aqueous exposure of zebrafish (Danio rerio) to environmentally relevant concentrations of acetaminophen (ACE), venlafaxaine (VEN) (10μgL−1), carbamazepine (CBZ) and gemfibrozil (GEM) (0.5 and 10μgL−1) decreased reproductive output. Atretic oocytes and altered ovarian histology were seen in female zebrafish exposed to CBZ and GEM, suggesting a direct effect on oocyte development that may account for the reduced fecundity. Apoptosis within the theca and granulosa cells was identified in exposed female zebrafish with atretic oocytes by TUNEL positive staining. The incidence of follicular apoptosis was nearly 2-fold higher in exposed females than the controls. All compounds significantly altered kidney proximal tubule morphology but there was no difference in the incidence of apoptotic cells within the kidney between control and exposed in either males or females. Liver histology was altered by ACE and GEM exposure. Parental exposure to pharmaceuticals did not increase developmental abnormalities, hatching success, or mortality in embryos. Yet, direct exposure of embryos to ACE increased developmental abnormalities and mortality; exposure to 0.5μgL−1 of all pharmaceuticals increased mortality. CBZ decreased plasma 11-ketotestosterone concentrations in males and females. Overall, these data suggest that low concentration, chronic exposure of fish to pharmaceuticals impacts fish development as well as multiple organ systems in adult fish, leading to effects on reproduction and histology of liver and kidney. These results are significant in understanding the consequences of chronic, low concentration pharmaceutical exposure to fish and suggest that exposed populations are at risk of negative impacts to reproduction and health.

Prognostic relevance of induced and spontaneous apoptosis of disseminated tumor cells in primary breast cancer patients.

e21003 Background: An imbalance between cell proliferation and programmed cell death leads to tumor growth. Although most cytostatic agents used in systemic therapy of breast cancer induce an apoptosis in tumor cells leading to their destruction, a high ratio of apoptotic cells in primary breast cancer is correlated with poor prognosis. The purpose of this study was to investigate the incidence and prognostic significance of apoptotic disseminated tumor cells (DTC) in bone marrow (BM) of primary breast cancer (PBC) patients. Methods: PBC patients who underwent primary surgery (PS) or were treated with neoadjuvant chemotherapy (NACT) between 01/2003 and 12/2007 were included into this prospective study. BM aspirates were analyzed by immunocytochemistry (M30 antibody) using ACIS system (Chromavision) according to the ISHAGE evaluation criteria. Patients without DTC in the BM were excluded from the study. Survival data were evaluated after a median follow up of 44 months. Results: BM aspirates of 383 PBC patients were analyzed. DTC were M30 positive in 82 of 298 (27%) patients who underwent PS and 41 of 85 (48%) patients treated with NACT. After NACT, the incidence of apoptotic cells in patients with complete/partial remission as compared to patients with stable/progressive disease was 63%/53% vs. 31%/0% (p=0.034). In the NACT group, M30-positive patients suffered less likely of a relapse than those without apoptotic DTC (7% vs. 23% of the events, p = 0.049). Inversely, overall survival in the PS group was significantly shorter in M30-positive as compared to M30-negative patients (75 months, 95% CI: 68-81 months vs. 84 months, 95% CI: 81-86 months; p = 0.008). Conclusions: Apoptotic DTC can be detected in breast cancer patients before and after systemic treatment. The presence of apoptotic DTC in BM of breast cancer patients can reflect the effects of neoadjuvant treatment. Spontaneous and chemotherapy-induced apoptosis may have different prognostic implications.

MiR-143 over-expression reduces the growth of xenograft tumors from colon carcinoma cells

We have previously shown that miR-143 is down-regulated in colorectal cancer and that miR-143 over-expression in HCT116 cells increases sensitivity to 5-fluorouracil, reduces cell viability and increases apoptosis in vitro. In the present study, we evaluated the role of miR-143 over-expression on HCT116 xenograft tumor growth in nude mice. HCT116 cells with stable miR-143 over-expression (over-143) and control (empty) cells were injected s.c. into the backs of nude mice, and tumor growth was evaluated. Tumors arose approximately 14 days later, and the experiment was ended 40 days after injection. miR-143 was confirmed to be significantly over-expressed in over-143 versus empty xenografts, by Taqman real-time PCR. Over-143 xenografts displayed slower tumor growth compared to empty xenografts, with significantly smaller tumor volumes, from 23 until 40 days in vivo (p < 0.05), with final volumes of 928 ± 338 and 2312 ± 387 mm3, respectively. Evaluation of apoptotic proteins showed that over-143 versus empty xenografts, display reduced Bcl-2 expression, and increased caspase-3 activation and PARP cleavage (p < 0.05). In addition, the incidence of apoptotic cells, assessed by TUNEL, was increased in over-143 versus empty xenografts. Therefore, our results suggest that the reduced tumor volume may, in part, be due to increased miR-143-induced apoptosis. Collectively, our results reinforce the relevance of miR-143 in colorectal cancer, suggesting an important role in the control of in vivo tumor progression. This further expands its anti-proliferative, pro-apoptotic and chemosensitizer role that we have previously demonstrated in vitro.

158 IN VITRO DEVELOPMENT OF WOOD BISON (BISON BISON ATHABASCAE) EMBRYOS BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER

Wood bison (Bison bison athabascae) are currently classified as threatened in Canada. Interspecies somatic cell nuclear transfer (iSCNT) is a valuable tool for embryo production in non-domestic species in which access to gametes is limited. Unlike fertilization, SCNT allows preservation of the entire genome, thus avoiding dilution of valuable alleles, an important factor for the preservation of genetic diversity. The present study compared the developmental competence of iSCNT embryos reconstructed from adult female wood bison ear fibroblasts (bison NT) with development of embryos reconstructed from adult female cattle ear fibroblasts (cattle NT). Domestic cattle (Bos taurus) oocytes were used as recipient ooplasm for both donor cell types. In vitro fertilized (IVF) and parthenogenetic (PA) cattle embryos were used as controls. Fibroblast cultures at passages 3 to 5 confluent for 5 days were used for SCNT. Mature oocytes were enucleated, reconstructed by transfer of donor cells, and fused with an electrical stimulus of 1.5 kV cm–1 for 40 μs in 0.28 m mannitol containing 100 μm CaCl2 and MgCl2. Oocytes for parthenogenesis and following reconstruction were activated for 5 min in 5 μm ionomycin followed by 5 h in 10 μg mL–1 cycloheximide. Embryos produced by IVF, PA, and SCNT were cultured in modified synthetic oviductal fluid medium at 38.5°C in 5% CO2, 5% O2, 90% N2. Cleavage, blastocyst development to day 8, apoptosis (TUNEL assay, Roche Diagnostics, IN, USA), and total cell number were evaluated. Statistical analyses were carried out using one-way ANOVA, followed by Tukey post hoc analysis or the equivalent nonparametrical Kruskal-Wallis test. Cleavage rate was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (86.9 ± 2.9% v. 71.6 ± 4.5% to 78.1 ± 5.1%). Blastocyst rates, expressed as a percentage of cleaved embryos, were similar among all treatment groups (33.4 ± 3.3% to 39.8 ± 5.7%) except for bison NT which had significantly (P &lt; 0.05) lower development to blastocyst (19.2 ± 5.5%). The percentages of TUNEL-positive cells among PA embryos (6.6 ± 1.5%) and bison NT embryos (6.7 ± 2.4%) were significantly (P &lt; 0.05) higher than in IVF embryos (4.2 ± 1.0%), but similar to cattle NT embryos (5.4 ± 1.7%), which did not differ from the IVF group. Total cell number was significantly (P &lt; 0.05) higher in the IVF group than in all other groups (133.2 ± 10.2 v. 91.2 ± 7.8 to 100.1 ± 12.9). These results confirm that in vitro-matured domestic cattle oocytes can serve as suitable recipients of wood bison somatic cells and that iSCNT may provide a possible alternative for embryo production and genetic preservation of endangered cattle species. Both the incidence of apoptotic cells and total cell number did not differ between cattle and bison NT embryos; thus other factors must play a role in the significantly decreased blastocyst development observed in bison NT embryos. This work was supported by Endangered Species Reserve Fund, Toronto Zoo, and the Canada Research Chairs program.

Alternative Activation

The calcium/calmodulin (Ca 2+ /CaM)-dependent protein kinase II (CaMKII) is activated through the binding of Ca 2+ /CaM to its regulatory domain and plays important roles in the nervous system and the heart. CaMKII activity is also increased under pro-oxidant conditions, so Erickson et al . investigated whether reactive oxygen species (ROS) might be involved. Exposure of purified CaMKII to hydrogen peroxide (H 2 O 2 ) in the presence, but not absence, of Ca 2+ /CaM resulted in prolonged enzyme activity compared with that of untreated CaMKII. Ca 2+ /CaM-dependent autophosphorylation of Thr 287 is known to prevent the reassociation of the enzyme’s autoinhibitory region with its catalytic domain and so enable prolonged enzyme activity. However, the effect of H 2 O 2 on enzyme activity still occurred with a mutant form of CaMKII in which Thr 287 was mutated to alanine. Mutation of Met 281 and Met 282 in the regulatory domain of CaMKII to valines blocked H 2 O 2 -dependent activation, although Thr 287 autophosphorylation was unaffected. This suggests that Thr 287 phosphorylation and Met 281 /Met 282 oxidation are distinct mechanisms that lead to prolonged CaMKII activation in the presence of Ca 2+ /CaM. The authors developed an antibody against oxidized Met 281 /Met 282 residues and found that the hormone angiotensin II (AngII) increased the abundance of oxidized CaMKII in the hearts of wild-type mice compared with that in untreated mice. AngII induced ROS production and apoptosis in treated cardiomyocytes, which was blocked when CaMKII was knocked down by short hairpin RNA. Finally, the abundance of oxidized CaMKII and the incidence of apoptotic cells were higher in the hearts of AngII-treated mice deficient in MsrA, an enzyme that reverses methionine oxidation, than in AngII-treated wild-type mice. As Griffith notes in commentary, the discovery that ROS directly activate CaMKII may help in our understanding of oxidant-dependent diseases of the heart and nervous system. J. R. Erickson, M.-l. A. Joiner, X. Guan, W. Kutschke, J. Yang, C. V. Oddis, R. K. Bartlett, J. S. Lowe, S. E. O’Donnell, N. Aykin-Burns, M. C. Zimmerman, K. Zimmerman, A.-J. L. Ham, R. M. Weiss, D. R. Spitz, M. A. Shea, R. J. Colbran, P. J. Mohler, M. E. Anderson, A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation. Cell 133 , 462-474 (2008). [PubMed] L. C. Griffith, CaMKII: New tricks for an old dog. Cell 133 , 397-399 (2008). [PubMed]

Perturbation of organogenesis by the herbicide atrazine in the amphibian Xenopus laevis.

BackgroundExposure to anthropogenic chemicals during development can disrupt the morphogenesis of organ systems. Use of the herbicide atrazine has been debated in recent years because of its implicated, but poorly characterized, effects on vertebrates. Previous studies primarily examined the effects of atrazine exposure during metamorphosis or early developmental stages of amphibians.ObjectivesWe sought to identify and characterize the susceptibility during the often-overlooked developmental stage of organ morphogenesis.MethodsWe used a static renewal experimental treatment to investigate the effects of 10, 25, and 35 mg/L atrazine from early organ morphogenesis through the onset of tadpole feeding in the aquatic amphibian model system, Xenopus laevis. We quantified malformations of the body axis, heart, and intestine, as well as apoptosis in the midbrain and pronephric kidney.ResultsWe found a significant dose-dependent increase in the percentage of atrazine-exposed tadpoles with malformations of multiple tissues including the main body axis, circulatory system, kidney, and digestive system. Incidence of apoptotic cells also increased in the both midbrain and kidney of atrazine-exposed tadpoles.ConclusionsOur results demonstrate that acute atrazine exposure (10–35 mg/L for ≤ 48 hr) during early organ morphogenesis disrupts proper organ development in an amphibian model system. The concurrent atrazine-induced apoptosis in the pronephric kidney and midbrain begins to elucidate a mechanism by which atrazine may disrupt developmental processes in nontarget organisms.

Induction of apoptosis in populations of mammalian cells in vitro under the influence of lectins

A study of the cytogenetic action of lectins that differ in terms of origin and hydrocarbon specification is presented. In a culture of Chinese hamster cells it is shown that at high concentrations (20 μg/ml), all lectins are capable of inducing an increase in the level of apoptosis two days after treatment with a high degree of reliability. In the case of perch roe lectin, such an increase occurs both immediately after treatment as well as two days later. It is found that, unlike lentil and perch roe lectin, in a culture of human cells black elder lectin does not have any effect on the incidence of apoptotic cells. A trend towards stimulation of proliferation of human cells under the influence of perch roe lectin at low concentrations (0.2 μg/ml) is discovered.

Transient expression of endothelins in the amoeboid microglial cells in the developing rat brain

Amoeboid microglial cells (AMC) which transiently exist in the corpus callosum in the postnatal rat brain expressed endothelins (ETs), specifically endothelin-1 (ET-1) and ET3 as revealed by real time RT-PCR. ET immunoreactive AMC occurred in large numbers at birth, but were progressively reduced with age and were undetected in 14 days. In rats subjected to hypoxia exposure, ET immunoexpression in AMC was reduced but the incidence of apoptotic cells was not increased when compared with the control suggesting that this was due to its downregulation that may help regulate the constriction of blood vessels bearing ET-A receptor. AMC were endowed ET-B receptor indicating that ET released by the cells may also act via an autocrine manner. In microglia activated by lipopolysaccharide (LPS), ET-1 mNA expression coupled with that of monocyte chemoattractant protein (MCP-1) and stromal derived factor-1 (SDF-1) was markedly increased; ET-3 mRNA, however, remained unaffected. AMC exposed to oxygen glucose deprivation (OGD) in vitro resulted in increase in both ET-1 and ET-3 mRNA expression. It is suggested that the downregulated ETs expression in vivo of AMC subjected to hypoxia as opposed to its upregulated expression in vitro may be due to the complexity of the brain tissue. Furthermore, the differential ET-1 and ET-3 mRNA expression in LPS and OGD treatments may be due to different signaling pathways independently regulating the two isoforms. The present novel finding has added microglia as a new cellular source of ET that may take part in multiple functions including regulating vascular constriction and chemokines release.

P53- dependent hyperthermic enhancement of tumour growth inhibition by X-ray or carbon-ion beam irradiation

To elucidate p53 -dependency on combined treatment with radiation and hyperthermia, growth inhibition and apoptosis were analysed using transplantable human tumour. Human head and neck squamous cell carcinoma (HNSCC) cells carrying different p53 genes were transplanted into the thigh of nude mice. When the mean diameter of tumour reached 5-6 mm, the tumours were exposed to X-rays (2 Gy) or Carbon-ion (C-) beams (1 Gy) followed by heating at 42°C for 20 min. Tumour growth inhibition was evaluated by measuring the diameters of tumour. The induction of apoptosis and accumulation of apoptosis-related proteins were also analysed by immunohistochemical staining. Synergistic enhancement of tumour growth inhibition by hyperthermia was observed in wild-type p53 tumours treated with X-rays or C-beams but not in mutant p53 tumours. The incidence of apoptotic cells and activated-caspase-3-positive cells after combined treatment with them were significantly high in wild-type p53 tumours compared with that in mutant p53 tumours. The hyperthermic enhancement of tumour growth inhibition by X-ray- or C-beam-irradiation was p53 -dependent, suggesting that it might be highly correlated with p53 -dependent apoptosis.

Fate of hepatocyte and sinusoidal lining cell function and kinetics after extended cold preservation and transplantation of the rat liver.

We investigated the chronological profile of graft damage and recovery after liver cold ischemia-reperfusion (I/R) injury, with particular attention to the role of apoptosis on hepatocyte and sinusoidal endothelial cell (SEC) damage. Male Lewis rats underwent rearterialized orthotopic liver transplantation using grafts subjected to a short (University of Wisconsin [UW] solution for 1 hour [UW1h]) and prolonged period (UW16h) of cold preservation. Experiments were performed immediately after preservation and 4 hours, 24 hours, 3 days, and 7 days after reperfusion. At each time, graft function, incidence of apoptotic cells, expression of the epitope recognized by a monoclonal antibody specific to rat SECs (SE-1), and incidence of proliferating cells were estimated. In the UW16h group, the proportion of apoptotic SECs was markedly elevated at 4 hours. The incidence of hepatocyte apoptosis was very low, although massive hepatocyte necrosis was evident at 24 hours. The incidence of proliferating hepatocytes and SECs peaked at 3 days, then returned to normal by 7 days. SE-1 expression was reduced immediately after preservation, followed by a marked reduction at 4 and 24 hours after reperfusion, and expression returned to normal by 7 days. Although SEC apoptosis was induced in the early phase of cold I/R injury, hepatocyte damage developed without the occurrence of apoptosis. Regeneration of both hepatocytes and SECs after cold I/R injury peaked at 3 days and was complete by 7 days, whereas functional recovery of these cell populations was complete 3 days after reperfusion.

The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes

Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl4)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl4 administration. acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl4 treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl4, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in comparison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover. hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-beta1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this animal model.