Transferase-mediated dUTP-X Nick End Labeling Research Articles

Efficacy of electroacupuncture stimulating Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6) on spatial learning and memory deficits in rats with insomnia induced by para-chlorophenylalanine: a single acupoint combined acupoints.

To investiage the effect of electroacupuncture (EA) at a single acupoint of Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6) and at combined acupoints of Shenmen (HT7) and Baihui (GV20) and Sanyinjiao (SP6) on the PKA/CREB and BDNF/TrkB signaling, as well as neuroapoptosis and neurogenesis in hippocampus and elucidate the underlying mechanism of single and combined acupoints on ameliorating spatial learning and memory deficits in a rat model of primary insomnia. Primary insomnia was modeled by intraperitoneal injection of para-chlorophenylalanine (PCPA) once daily for 2 d. EA was applied at Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6), or Shenmen (HT7) + Baihui (GV20) + Sanyinjiao (SP6) (combined) for 30 min daily for 4 d. Spatial learning and memory function was evaluated by the Morris water maze (MWM) test. Protein expressions of hippocampal cAMP-dependent protein kinase (PKA)-Cβ, phosphorylated cAMP-responsive element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), and tyrosine kinase receptor B (TrkB) were evaluated by Western blotting. Neuronal apoptosis in the hippocampus was detected with the transferase-mediated dUTP-X nick end labeling assay. Endogenous neurogenesis was examined with bromodeoxyuridine staining. The MWM test and hippocampal p-CREB, BDNF, and TrkB protein levels in the combined acupoints group were evaluated after the administration of a PKA-selective inhibitor (H89). Spatial learning and memory were significantly impaired in rats with insomnia. The spatial learning deficits were ameliorated in the Shenmen (HT7), Baihui (GV20), Sanyinjiao (SP6), and combined groups; this improvement was significantly greater in the combined group than the single acupoint groups. The spatial memory impairment was improved in the combined, Baihui (GV20), and Shenmen (HT7) groups, but not the Sanyinjiao (SP6) group. The expressions of PKA-Cβ, p-CREB, BDNF, and TrkB were decreased in rats with insomnia. All these proteins were significantly upregulated in the combined group. PKA/p-CREB protein levels were elevated in the Baihui (GV20) and Shenmen (HT7) groups, whereas BDNF/TrkB expression was upregulated in the Sanyinjiao (SP6) group. The staining results showed significant attenuation of hippocampal cell apoptosis and increased numbers of proliferating cells in the combined group, whereas the single acupoint groups only showed decreased numbers of apoptotic cells. In the combined group, the PKA inhibitor reversed the improvement of spatial memory and upregulation of p-CREB expression caused by EA, but did not affect its activation of BDNF/TrkB signaling. EA at the single acupoints Baihui (GV20), Shenmen (HT7), or Sanyinjiao (SP6) had an ameliorating effect on the spatial learning and memory deficits induced by insomnia. EA at combined acupoints exerted a synergistic effect on the improvements in spatial learning and memory impairment in rats with insomnia by upregulating the hippocampal PKA/CREB and BDNF/TrkB signaling, facilitating neurogenesis, and inhibiting neuronal apoptosis. These findings indicate that EA at combined acupoints [(Baihui (GV20), Shenmen (HT7), and Sanyinjiao (SP6)] achieves a more pronounced regulation of hippocampal neuroplasticity than EA at single acupoints, which may partly explain the underlying mechanisms by which EA at combined acupoints exerts a better ameliorative effect on the cognitive dysfunction caused by insomnia.

Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus.

PurposeWe aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus.MethodsThe study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase–mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment.ResultsWe demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment.ConclusionsThis chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment.Translational RelevanceThe cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.

KCa3.1 Transgene Induction in Murine Intestinal Epithelium Causes Duodenal Chyme Accumulation and Impairs Duodenal Contractility.

The epithelial intermediate-conductance calcium/calmodulin-regulated KCa3.1 channel is considered to be a regulator of intestine function by controlling chloride secretion and water/salt balance. Yet, little is known about the functional importance of KCa3.1 in the intestinal epithelium in vivo. Our objective was to determine the impact of epithelial-specific inducible overexpression of a KCa3.1 transgene (KCa3.1+) and of inducible suppression (KCa3.1−) on intestinal homeostasis and function in mice. KCa3.1 overexpression in the duodenal epithelium of doxycycline (DOX)-treated KCa3.1+ mice was 40-fold above the control levels. Overexpression caused an inflated duodenum and doubling of the chyme content. Histology showed conserved architecture of crypts, villi, and smooth muscle. Unaltered proliferating cell nuclear antigen (PCNA) immune reactivity and reduced amounts of terminal deoxynucleotide transferase mediated X-dUTP nick end labeling (TUNEL)-positive apoptotic cells in villi indicated lower epithelial turnover. Myography showed a reduction in the frequency of spontaneous propulsive muscle contractions with no change in amplitude. The amount of stool in the colon was increased and the frequency of colonic contractions was reduced in KCa3.1+ animals. Senicapoc treatment prevented the phenotype. Suppression of KCa3.1 in DOX-treated KCa3.1− mice caused no overt intestinal phenotype. In conclusion, inducible KCa3.1 overexpression alters intestinal functions by increasing the chyme content and reducing spontaneous contractions and epithelial apoptosis. Induction of epithelial KCa3.1 can play a mechanistic role in the process of adaptation of the intestine.

Taxifolin protects rat against myocardial ischemia/reperfusion injury by modulating the mitochondrial apoptosis pathway.

BackgroundTaxifolin (TAX), is an active flavonoid, that plays an underlying protective role on the cardiovascular system. This study aimed to evaluate its effect and potential mechanisms on myocardial ischemia/reperfusion (I/R) injury.MethodsHealthy rat heart was subjected to I/R using the Langendorff apparatus. Hemodynamic parameters, including heart rate, left ventricular developed pressure (LVDP), maximum/minimum rate of the left ventricular pressure rise (+dp/dtmax and −dp/dtmin) and rate pressure product (RPP) were recorded during the perfusion. Histopathological examination of left ventricular was measured by hematoxylin-eosin (H&E) staining. Creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities in the effluent perfusion, and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) in the tissue were assayed. Apoptosis related proteins, such as B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax), and cytochrome c (Cyt-c) were also assayed by ELISA. Western blot was employed to determine apoptosis-executive proteins, including caspase 3 and 9. Transferase-mediated dUTP-X nick end labeling assay was performed to evaluate the effect TAX on myocardial apoptosis.ResultsTaxifolin significantly improved the ventricular functional recovery, as evident by the increase in LVDP, +dp/dtmax, −dp/dtmin and RPP, the levels of SOD, GSH-PX were also increased, but those of LDH, CK-MB, and MDA were decreased. Furthermore, TAX up-regulated the Bcl-2 protein level but down-regulated the levels of Bax, Cyt-c, caspase 3 and 9 protein, thereby inhibits the myocardial apoptosis.DiscussionTaxifolin treatment remarkably improved the cardiac function, regulated oxidative stress and attenuated apoptosis. Hence, TAX has a cardioprotective effect against I/R injury by modulating mitochondrial apoptosis pathway.

Regulation of p14(ARF) expression and induction of cell apoptosis with c-myc in a p53-independent pathway

To explore the regulation of p14(ARF) expression and induction of cell apoptosis with the mutant and wild-type c-myc genes in a p53-independent pathway of signal transduction. The mutant and wild-type c-myc genes were transfected by lentivirus into HCC1937 to form the stable over-expression cell lines. Uninfected cells and lentivirus-infected ones carrying no c-myc gene acted as blank and infection controls respectively. And c-myc and p14(ARF) mRNA and protein, proliferation and apoptosis in HCC1937 with mutant and wild-type c-myc were detected by reverse transcription (RT)-PCR, Western blotting, thiazolyl blue tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) respectively. After the lentivirus-mediated gene transfer, c-myc mRNA and protein expression increased in the mutant and wild-type groups. p14(ARF) mRNA and protein increased in the wild-type group and the mutant group and there were significant difference between them with blank and infection controls (mutant groups: 0.560 ± 0.010, 0.154 ± 0.011, wild-type groups: 0.651 ± 0.010, 0.382 ± 0.013, both P < 0.05). The group of mutant and wild-type c-myc could promote the proliferation of cell growth. And c-myc was more effective to induce apoptosis in the wild-type group as compared with the mutant group (7.1% ± 0.7% vs 3.2% ± 0.4%, P < 0.05). In a p53-independent pathway, the over-expression of wild-type c-myc obviously up-regulates the expression of p14(ARF). And cell apoptosis may be induced through the regulation of p14(ARF)-related gene, keep balance of proliferative promotion and apoptosis induction. When there is a loss-of-function of mutant c-myc, tumorigenicity increases via a disturbed balance of proliferative promotion and apoptosis induction.

Formation of duodenal atresias in fibroblast growth factor receptor 2IIIb−/− mouse embryos occurs in the absence of an endodermal plug

PurposeDuodenal atresia in humans has been hypothesized to arise from a failure of the duodenal lumen to recanalize after formation of an endodermal plug. Recently, mutations in the fibroblast growth factor receptor 2 gene (Fgfr2IIIb) have been shown to cause atretic defects of the duodenum in mice. However, work in rats suggests that murine species do not form an endodermal plug during normal duodenal development. These lines of data led us to hypothesize that mice are able to form a duodenal atresia in the absence of an endodermal plug. To test this hypothesis, we examined duodenal development in wild-type and Fgfr2IIIb−/− embryos. MethodsParaffin sections were generated for H&E, E-cadherin, or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling staining from Fgfr2IIIb−/− and wild-type embryos between embryonic days (E) 10.5 and E14.5. Sections were photographed and reconstructed into 3-dimensional display using Adobe Photoshop and Amira Visage software. ResultsNormal mouse duodenum does not form an endodermal plug, although a plug does form in the pyloric region of the stomach at E14.5. Fgfr2IIIb−/− embryos experience significant apoptosis in the duodenal region at E10.5, followed by the disappearance of the endoderm in the atretic precursor by E11.5. Thereafter, the mesoderm of the atretic precursor involutes over the next 2 days in the absence of further apoptosis. Interestingly, an endodermal plug was not observed at any point during the formation of a duodenal atresia. ConclusionsThese results suggest that duodenal atresia in the Fgfr2IIIb−/− model does not arise from persistence of an epithelial plug. Rather it appears to result from the loss of the endoderm because of apoptosis very early in development.

Simultaneous folate intake may prevent advers effect of valproic acid on neurulating nervous system

The aim of this study is to elucidate the preventive effect of folic acid (FA) on teratogenic effects of valporic acid (VA) in early stage chick embryos on neural tube development. One hundred and fifty specific pathogen-free (SPF) chick eggs were used to investigate the neurulation in five groups. Group A was the control group. Group B was injected 0.02 ml of saline (0.9% NaCl) and was used for sham group. VA (0.72 mg) in 0.02 ml saline was injected in Group C, and 0.342 mcg of FA in 0.02 ml NaCl were administered to the embryos in Group D. VA (0.72 mg) + 0.342 mcg of FA in 0.02 ml saline were administered simultaneously to the eggs in Group E. At the end of 72 h, all embryos were extracted from eggs and were fixed, and for histological analyses hematoxylin and eosine was used, for detection of apoptotic cells terminal deoxyribonucleotide transferase-mediated dUTP-X nick end labeling (TUNEL) was used and for distribution of P53, bcl-2 and caspase-3, caspase-6, caspase-8 and caspase-9 immunoperoxidase techniques were used. While there were no neural tube defects in the embryos of groups A, B and D, eight embryos died in group C and there were 12 embryos with retarded embryological development. In contrast to that, no death was observed in group E, but only eight embryos were detected with maldevelopmental delay stage. These results suggested that VA may induce apoptotic mechanisms but not through the p53 pathway. In addition, FA effectively prevents the teratogenic influence of VA on chick embryo at neurulation stages by stopping cascade of apoptosis before caspase 3 expression.

Participation of glucocorticoid receptors in morphine tolerance development through the signal pathway of extracellular signal-regulated kinase

To investigate the mechanism of glucocorticoid receptors (GR) participating in morphine tolerance development via the extracellular signal-regulated kinase (ERK) signal pathway. Forty healthy male SD rats were implanted with intrathecal catheters and then randomized into 4 groups: Group C received an intrathecal injection of 10 µl saline, Group M 10 µg morphine, Group MR 10 µg morphine followed by 2 µg GR antagonist RU38486 and Group MD 10 µg morphine followed by 4 µg GR agonist dexamethasone (DEX) respectively. Each intrathecal drug was administered twice daily for 7 days. Tail-flick test was employed to evaluate the thermal hyperalgesia. After tail-flick test at Day 8, the lumbar intamescentias were isolated by Western blot to examine the protein expressions of Mu opioid receptor (MOR), GR and pERK. And terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling (TUNEL) stain was performed. DEX and RU38486 enhanced and inhibited morphine induced hyperalgesia to heat respectively. Compared with Group M (count of spinal dorsal horn apoptotic cells 5.4 ± 1.1, protein expression: MOR 37 ± 5, GR 20 ± 6, pERK(1) 39 ± 4, pERK(2) 41 ± 5), apoptotic cells decreased in Group MR (3.2 ± 0.4) (P < 0.01) and increased in Group MD (16.0 ± 1.6) (P < 0.01); the protein expressions of MOR (28 ± 5), GR (12 ± 6), pERK(1)(33 ± 4) and pERK(2)(34 ± 5) were down-regulated in Group MR (P < 0.01) while up-regulated in Group MD (55 ± 6, 28 ± 9, 49 ± 6, 59 ± 5) (P < 0.01). The activity of glucocorticoid receptors affects the morphine-induced spinal neural apoptosis and the expression of spinal pERK in morphine tolerance.

Sustained hypoxia-induced proliferation of carotid body type I cells in rats

Sustained hypoxia (SH) has been shown to cause profound morphological and cellular changes in carotid body (CB). However, results regarding whether SH causes CB type I cell proliferation are conflicting. By using bromodeoxyuridine, a uridine analog that is stably incorporated into cells undergoing DNA synthesis, we have found that SH causes the type I cell proliferation in the CB; the proliferation occurs mainly during the first 1-3 days of hypoxic exposure. Moreover, the new cells survive for at least 1 mo after the return to normoxia. Also, SH does not cause any cell death in CB as examined by the terminal deoxynucleotidyl transferase-mediated dUTP-X nick-end labeling assay. Taken together, our results suggest that SH stimulates CB type I cell proliferation, which may produce long-lasting changes in CB morphology and function.

Study of apoptosis in normal skin tissues

The aim of the study was to investigate the apoptosis in normal human skin by examination of all epidermal layers. The normal skin epidermis of 15 healthy subjects was investigated by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling) technique. Apoptotic cells were evaluated in the germinative and differential compartments and stratum corneum. Only highly TUNEL-positive cells with typical morphological DNA fragmentation signs were calculated. In vital strata (except stratum corneum) of normal skin epidermis, 37.5% of all TUNEL-positive cells were observed in the germinative compartment and 41.7% in the granular layer of differential compartment. The study showed that apoptosis occurs in all layers of normal skin epidermis. It demonstrates that apoptosis is highly important in the renewal of cells and formation of epidermal structure within all compartments.

Comparison of techniques for determination of chondrocyte viability after thermal injury

To compare 2 methods of quantitating chondrocyte viability and to determine chondrocyte response to thermal injury over time. 108 stifle joints from 54 adult rats. Cartilage from the distal aspect of the femur was treated ex vivo with radiofrequency energy at a probe setting that would result in immediate partial-thickness chondrocyte death; untreated sections served as controls. Explants were cultured, and cell viability was compared by use of lactate dehydrogenase (LDH) histochemical staining and calcein AM and ethidium homodimer-1 confocal laser microscopy (CLM) cell viability staining. Terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling (TUNEL) was used to detect apoptosis. All labeling studies were performed 0, 1, 3, 7, 14, and 21 days after treatment. In the treated tissues, a greater percentage of viable cells were found with CLM, compared with LDH staining. This result contrasted that of control tissues in which LDH staining indicated a greater percentage of live cells than CLM. The greatest number of TUNEL-positive chondrocytes was present at day 3, declining at later time intervals. CLM and LDH histochemistry techniques yield different absolute numbers of live and dead cells, resulting in differing percentages of live or dead cells with each technique. These differences may be related to the enzymes responsible for activation in each technique and the susceptibility of these enzymes to thermal injury. Results of TUNEL indicate that apoptosis contributes to chondrocyte death after thermal injury, with a peak signal identified 3 days after insult.

Cytotoxic Effect of Oyster Mushroom Pleurotus ostreatus on Human Androgen-Independent Prostate Cancer PC-3 Cells

Twenty species of edible mushrooms and three purified mushroom polysaccharides were screened for their antitumor potential on human androgen-independent cancer PC-3 cells. A water-soluble extract (POE) prepared from the fresh oyster mushroom Pleurotus ostreatus produced the most significant cytotoxicity on PC-3 cells among the mushroom species tested. At the same time, POE induced a rapid apoptosis on PC-3 cells detected with annexin V-fluorescein isothiocyanate flow cytometry when the cells were exposed to POE (150 microg/mL) for 2 hours. Induced apoptosis was also confirmed by DNA fragment terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling staining while POE (200 microg/mL) was added to PC-3 cells for 6 hours. Both cytotoxicity and induced apoptosis mediated by POE in PC-3 cells are dose-dependent. Interestingly, PC-3 cells appeared to be more sensitive to POE in anchorage-independent growth condition. Tumor colony-forming efficiency was dramatically reduced to 4.5% or 0.5% in POE (60 or 120 microg/mL)-supplemented soft agar medium compared with that of POE-free medium (defined as 100%). Temperature in POE processing plays a decisive role for the cytotoxic activity. Bioactivity of POE was eliminated by exposure to high temperature (80 degrees C) for 2 hours; however, it remained stable at a series temperatures of below 40 degrees C. The active fraction POE-F2 was analyzed and identified by size exclusion of high performance liquid chromatography and the CellTiter 96 AQueous Cell Proliferation Assay (Promega, Madison, WI). Since POE-F2 is also sensitive to heat and has strong 280 nm absorption, the results imply that active compounds recovered from P. ostreatus are water-soluble proteins or polypeptides.

Rapid Induction of Apoptosis in Gastrulating Mouse Embryos by Ethanol and Its Prevention by HB‐EGF

Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol-induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis. Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation-stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos. Both annexin V binding and TUNEL were elevated (p < 0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p < 0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p < 0.05); however, exogenous HB-EGF prevented apoptosis. Ethanol rapidly produced apoptosis in gastrulation-stage embryos, consistent with induction by intracellular signaling. The ethanol-induced apoptotic pathway was blocked by the endogenous survival factor, HB-EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally.

Caspase-1α Is Down-regulated in Human Ovarian Cancer Cells and the Overexpression of Caspase-1α Induces Apoptosis

Caspase-1 plays a key role in the processing of cytokines and in the apoptosis of neurons and macrophages. Whether it also causes apoptosis of cancer cells has been unclear. In this study, we screened an array of apoptosis-related proteins in ovarian carcinoma cell lines and their tissue of origin, ovarian surface epithelium (OSE). Caspase-1alpha protein was abundant in OSE and in nontumorigenic OSE with extended but limited life spans (immortalized OSE), but was reduced in the cancer lines A2780 and OVCAR10. By Western blot and immunofluorescence, caspase-1alpha levels were greatly reduced in six of eight ovarian carcinoma lines compared with OSE. By real-time reverse transcription-PCR, steady-state transcripts of the CASP1 gene were proportional to protein levels. Caspase-1alpha overexpression caused significant apoptosis, but overexpression of a caspase-1alpha mutant without catalytic activity did not, confirming that the effect was caspase-1alpha-specific. Immunofluorescence of caspase-1alpha and terminal nucleotidyl transferase-mediated dUTP-X nick end labeling colocalization clearly established a link between apoptosis and caspase-1alpha expression. Caspase-9 and caspase-3 were activated in caspase-1alpha overexpressing A2780 cells, suggesting involvement of an intrinsic apoptotic pathway. Caspase-1alpha overexpression did not change the apoptotic effect of cisplatin in A2780 and OVCAR10 cells, suggesting that this agent activates a different pathway. Immunohistochemically, caspase-1 was lower in ovarian serous carcinomas than in OSE. Our study indicates, for the first time, that caspase-1alpha is proapoptotic in ovarian cancer cells, and raises the possibility that its down-regulation is one of the mechanisms which increase resistance to apoptosis in cancer cells.

Evidence of Programmed Cell Death in Post-Phloem Transport Cells of the Maternal Pedicel Tissue in Developing Caryopsis of Maize

We present cellular- and ultracellular-level studies here to show developmental programmed cell death (PCD) of placento-chalazal (P-C) cell layers in maternal pedicel tissue in developing caryopses of normal seed (Mn1) and in the invertase-deficient miniature (mn1) seed mutant in maize (Zea mays). PCD was evidenced by loss of nuclei and all subcellular membranous organizations in many P-C layers. The terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) stain that is diagnostic of apoptotic-like PCD identified spatially and temporally two distinctive subdomains, which coincided with nucellar and integumental P-C layers based on their developmental origins. The early phase of PCD in the nucellar P-C was TUNEL negative and was specific to only the fertilized caryopses, indicating that the signaling for PCD in these maternal cells originated in the zygotic tissues. In fact, the initiation of PCD coincided with endosperm cellularization and was rapidly and coordinately completed prior to the beginning of the major storage phase in endosperm. Cell shape in these cell layers was also influenced by the genotype of filial endosperm. The later phase of PCD was restricted to the integumental P-C layers underneath the nucellar cells and was TUNEL positive in both genotypes. The two subdomains of the P-C layers were also distinguishable by unique cell wall-associated phenolic compounds. Based on collective evidence, we infer that the nucellar PCD may have osmolytic etiology and may lead to activation of the post-phloem transport function of the P-C layer, whereas the integumental PCD was senescent related, in particular, protecting the maturing seed against microbes that may be transported from the maternal tissue.

Preterm as Compared with Full-Term Neonatal Calves Are Characterized by Morphological and Functional Immaturity of the Small Intestine

Intestinal diseases in neonatal calves may be due to morphological and functional immaturity. We have studied histomorphology, crypt cell proliferation rates (based on incorporation of 5-bromo-2′-deoxyuridine into DNA), presence of apoptotic cells (based on terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling), and brush border enzyme activities in preterm calves (277 d of gestation), euthanized on d 1 (P0) or 8 (P8), and in full-term calves (290 d of gestation), euthanized on d 1 (F0) or 8 (F8). Vacuolated epithelial cells were present in ileum of P0 and F0 but not in P8 and F8. During the first 8 d, villus sizes, crypt depths, and proliferation rates of crypt cells in the small intestine of preterm calves did not significantly change. In contrast, in full-term calves during the first 8 d, villus sizes in jejunum decreased, crypt depths increased in small intestine and colon, and crypt cell proliferation increased in duodenum and jejunum. Submucosal thickness in jejunum was highest in P0, but in ileum it increased with gestational age and feeding. Gestational age×feeding interactions indicated increased activities of aminopeptidase N and reduced lactase activities only in F8 and reduced dipeptidylpeptidase IV activities only in P8. In conclusion, in preterm calves the small intestinal epithelium was immature and brush border enzyme activities differed in part from those in full-term calves.

The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells

The purpose of this work was to establish whether exposing cultured rabbit corneal and lens epithelial cells to ultraviolet radiation equivalent to several hours under the sun would damage the cells. Confluent rabbit corneal epithelial cells were irradiated with broadband UV-A or UV-B, and confluent lens epithelial cells were irradiated with broadband UV-A. The maximum dose of UV-A was 6·3 J cm −2 and that of UV-B was 0·60 J cm −2. Damage to corneal epithelial cell was studied using the terminal deoxynucleotidyl transferase mediated dUTP-X nick end labeling (TUNEL) assay and damage to lens epithelial cell was studied using the single cell gel electrophoresis (comet) assay and trypan blue exclusion assay. Lipid peroxidation was assayed using the thiobarbituric acid reaction. Both UV-B and UV-A induced cell death in corneal epithelial cells with different latent periods. UV-A damage included cell death, decreased viability and increased lipid peroxidation of lens epithelial cell. In addition, UV irradiation of the corneal and lens epithelial cells decreased the activity of catalase to thirty to fifty percent of its original value, while the activities of glutathione peroxidase and superoxide dismutase did not decrease within experimental error. Thus, even sub-solar UV radiation can cause irreversible damage to corneal and lens epithelial cells.

Cell Proliferation, Apoptosis, and B- and T-Lymphocytes in Peyer's Patches of the Ileum, in Thymus and in Lymph nodes of Preterm Calves, and in Full-Term Calves at Birth and on Day 5 of Life

Peyer's patches, thymus, and lymph nodes contain the majority of lymphocytes. We have studied proliferation rates, apoptosis rates, and numbers of B- and T-lymphocytes in Peyer's patches in ileum, thymus, and mesenterial and prescapular lymph nodes (LM and LP) in unfed preterm calves (GrP; born 13 d before expected normal term after dams were injected with prostaglandin F2α and glucocorticoids) and normal-term calves (GrF) immediately after birth and on d 5 of life after feeding colostrum for 4 d (GrC). Immunohistochemical methods in conjunction with incorporation of 5-bromo-2′-deoxyuridine or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling were used to evaluate cell proliferation rates and apoptosis rates, respectively. The number of T- and B-lymphocytes was determined with monoclonal antibodies directed against CD3 and CD79, respectively. In GrF compared with GrP, there were higher numbers of proliferating and apoptotic cells in LM and LP, of B-lymphocytes in paracortex and follicles of LM and LP, and of proliferating cells in cortex and medulla of thymus. In thymus cortex and medulla, numbers of proliferating cells were higher in GrC than in GrF. Apoptotic rates were generally smaller at all sites of Peyer's patches in GrC than in GrF, and proliferation rates increased from GrP to GrF in intrafollicular areas and from GrF to GrC in all tissues. Numbers of T-lymphocytes in Peyer's patches were higher in GrF than in GrP, but lower in GrC than in GrF, except in the domes. Numbers of B-lymphocytes did not change in Peyer's patches despite high proliferation and low apoptotic rates, suggesting that they leave Peyer's patches during the first days of life. In conclusion, proliferation and apoptosis rates and numbers of B- and T- lymphocytes in Peyer's patches in ileum, thymus, and LM and LP exhibited different developmental changes and were affected by feeding.

Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis

The effect of hyperosmolarity on CD95 membrane targeting and CD95 ligand (CD95L)–induced apoptosis was studied in rat hepatocytes. CD95 showed a predominant intracellular localization in normoosmotically exposed rat hepatocytes, whereas hyperosmotic exposure induced, within 1 hour, CD95 trafficking to the plasma membrane followed by activation of caspase-3 and -8. Hyperosmotic CD95 membrane targeting was sensitive to inhibition of c-Jun-N-terminal kinase (JNK), protein kinase C (PKC), and cyclic adenosine monophosphate, but not to inhibition of extracellular regulated kinases (Erks) or p38 mitogen activated protein kinase (p38 MAPK). Hyperosmotic CD95 targeting to the plasma membrane was dose-dependently diminished by glutamine or taurine, probably caused by an augmentation of volume regulatory increase. Despite CD95 trafficking to the plasma membrane and caspase activation, hyperosmolarity per se did not induce apoptosis. Hyperosmolarity, however, sensitized hepatocytes toward CD95L-induced apoptosis, as assessed by annexin V staining and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) assay. This sensitization was abolished when hyperosmotic CD95 membrane trafficking was prevented by cyclic adenosine monophosphate, PKC, or JNK inhibition, whereas these effectors had no effect on CD95L-induced apoptosis in normoosmotically exposed hepatocytes. CD95L addition under normoosmotic conditions caused CD95 membrane trafficking, which was sensitive to JNK inhibition, but not to cyclic adenosine monophosphate or inhibition of PKC, Erks, and p38 MAPK. In conclusion, multiple signaling pathways are involved in CD95 membrane trafficking. Hyperosmotic hepatocyte shrinkage induces CD95 trafficking to the plasma membrane, which involves JNK-, PKA-, and PKC-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis. (H EPATOLOGY 2002;36:602-614.)

Structure-apoptotic potency evaluations of novel sterols using human leukemic cells.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM-C7-14 human leukemic cells. In addition to testing 15-ketocholestenol (K15), 15-ketocholestenol hydroxyethyl ether (CK15), and 7-ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25-hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC > K15 > CK15 > CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c-myc mRNA was suppressed by all three oxysterols, with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.