TdT-mediated dUTP-digoxigenin Nick End Labeling Research Articles

Sublethal Effects of Imidacloprid on Fecundity, Apoptosis and Virus Transmission in the Small Brown Planthopper Laodelphax striatellus.

Simple SummaryThe small brown planthopper (SBPH) Laodelphax striatellus is an economically important pest in Asia, especially in China. Imidacloprid, a neonicotinoid insecticide, is commonly applied in rice fields to control the planthoppers. However, the widespread application of imidacloprid also has led to the development of resistance and to other potentially negative effects on crop protection. The sublethal effects of imidacloprid have been reported in many insects. Here, we investigated the potential effects of different sublethal concentrations of imidacloprid on SBPH and found that imidacloprid could affect the fecundity, apoptosis and virus transmission in the viruliferous SBPH. The results indicated that sublethal concentrations of imidacloprid may increase the fecundity of SBPH and the impact of insecticides on the transmission of plant viruses by insects should be considered when insecticides are applied to manage insect pests.Laodelphax striatellus damages plants directly through sucking plant sap and indirectly as a vector of rice stripe virus (RSV), resulting in serious losses of rice yield. It is one of the most destructive insects of rice in East Asia. Insecticides are primarily used for pest management, but the sublethal concentrations of insecticides may benefit several insects. The present research attempted to explore the effects of sublethal concentrations of imidacloprid on the fecundity, apoptosis and RSV transmission in the viruliferous SBPH. The results showed that the fecundity of SBPH was significantly increased after treatment with the LC10 dose of imidacloprid, while the LC30 dose of imidacloprid reduced the fecundity compared with the control. To further investigate the underlying mechanism of increased fecundity after exposure to the LC10 dose of imidacloprid, we examined the expression levels of vitellogenin (Vg), Vg receptor (VgR) and caspases in the ovaries of SBPH, and observed the apoptosis by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). qRT-PCR results indicated that the expression levels of Vg, VgR and four caspase genes were all significantly increased by the LC10 dose of imidacloprid, and TUNEL assays suggested that the frequency of apoptosis was significantly higher in the SBPH treated by the LC10 dose of imidacloprid, suggesting a potential correlation between the increased fecundity and the apoptosis of SBPH ovarioles. Additionally, the expression levels of RNA3 and capsid protein (CP) were both increased significantly by the LC10 dose of imidacloprid, whereas were decreased by the LC30 dose of imidacloprid compared to the control. Therefore, this study clarifies the mechanisms of sublethal effects of imidacloprid on viruliferous SBPH and could be used to optimize pest control strategies.

Impacts of CAD/CAM Metallic Materials on Trace Metals and Biocompatibilities: An in vivo Study in Beagle Dogs

CAD/CAM (computer-aided design and computer-aided manufacturing) technology has been widely applied in clinical dentistry, but the material safety remains a concern. To investigate the impacts of CAD/CAM metallic materials on trace metals and biocompatibilities, selective laser melted (SLM) cobalt-chromium (Co-Cr) alloys and computer numeric controlled milled (CNC milled) commercially pure titanium (CP-Ti) were placed on the maxilla of beagle dogs for 6 months. The trace metals in the oral mucosa, blood, liver, kidney, and hair were then determined by inductively coupled plasma mass spectrometry (ICP-MS). The histopathologic changes and biocompatibilities of tissues were examined by hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) method, immunohistochemistry (IHC) assay, Western blot analysis, and liver and kidney function tests. Our results showed that trace metals released from these two CAD/CAM metallic materials accumulated transiently in the oral mucosa and whole blood. The trace metals released from SLM Co-Cr alloys could also transiently accumulate in the plasm and hair. In addition, these two CAD/CAM metallic materials both induced apoptosis and histopathologic changes in the oral mucosa, with SLM Co-Cr alloys inducing a higher level of apoptosis. In contrast, both materials did not exert autophagic effects on the oral mucosa or affect the trace metals, functions, or biocompatibilities of the liver and kidney. Therefore, this study suggests that CAD/CAM metallic materials should be selected carefully, especially in patients with metal- and apoptosis-related diseases, and CNC-milled CP-Ti can be recommended to patients on account of its better biocompatibility and safety.

Long non-coding RNA SNHG5 regulates ulcerative colitis via microRNA-375 / Janus kinase-2 axis

ABSTRACT Ulcerative colitis (UC) is an intestinal inflammatory disorder. Long non-coding RNAs (lncRNAs) are collectively involved in UC. This study is designed to explore the roles of lncRNA (small nucleolar RNA host gene 5) SNHG5 in UC. Gene or microRNA (miRNA) expression was detected using RT-qPCR and western blot, respectively. Cellular functions were analyzed by cell counting kit 8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, and the terminal deoxyribonucleotidyl transferase (TDT)‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) assays. Lactate dehydrogenase (LDH) content was determined by a cell cytotoxicity assay. The interactions between miR-375 and SNHG5 or Janus kinase-2 (JAK2) were verified by a luciferase reporter assay. SNHG5 was up-regulated in intestinal mucosa tissues of UC patients as well as tumor necrosis factor alpha-treated (TNF-α-treated) young adult mouse colon (YAMC) cells. Down-regulated SNHG5 promoted cell proliferation and inhibited apoptosis of YAMC cells. miR-375 was verified to be a target of SNHG5 and was suppressed by TNF-α treatment in YAMC cells. Over-expression of miR-375 restored YAMC cellular functions. Additionally, miR-375 targeted JAK2, which was up-regulated by TNF-α treated YAMC cells. Up-regulation of JAK2 induced the dysfunction of YAMC cells. Knockdown of SNHG5 promoted the proliferation and suppressed the apoptosis of YAMC cells via regulating miR-375/JAK2 axis. Therefore, knockdown of SNHG5 may be a promising therapy for UC.

The P20R mutation of αB-crystallin diminishes its anti-apoptotic activity in human lens epithelial cells

αB-crystallin acts as an anti-apoptosis protein in human lens epithelial (HLE) cells. We recently identified a missense mutation in αB-crystallin that changes proline 20 to an arginine (P20R) in a Chinese family with autosomal dominant congenital posterior polar cataract. The impact of the P20R mutation on the anti-apoptosis function remains unclear. To explore the anti-apoptotic activity of αB-crystallin wild type (αB-wt) and its P20R mutant under oxidative stress, HLE cells were transfected with αB-wt and αB-P20R constructs and expression was measured by western blotting. Flow cytometry and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL) staining were performed to investigate apoptosis. We found that αB-wt performed a dominant role in inhibiting stress-induced apoptosis, but this function was impeded in cells expressing αB-P20R. The P20R mutant of αB-crystallin exhibits diminished anti-apoptotic activity compared with the native protein.

Nitroxoline induces apoptosis and slows glioma growth in vivo.

Nitroxoline is an FDA-approved antibiotic with potential antitumor activity. Here we evaluated whether nitroxoline has antiproliferative properties on glioma cell growth in vitro and in vivo using glioma cell lines and a genetically engineered PTEN/KRAS mouse glioma model. The effect of nitroxoline treatment on U87 and/or U251 glioma cell proliferation, cell-cycle arrest, invasion, and ability to induce an apoptotic cascade was determined in vitro. Magnetic resonance imaging was used to measure glioma volumes in genetically engineered PTEN/KRAS mice prior to and after nitroxoline therapy. Induction of apoptosis by nitroxoline was evaluated at the end of treatment using terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL). Nitroxoline inhibited the proliferation and invasion of glioblastoma cells in a time- and dose-dependent manner in vitro. Growth inhibition was associated with cell-cycle arrest in G1/G0 phase and induction of apoptosis via caspase 3 and cleaved poly(ADP-ribose) polymerase. In vivo, nitroxoline-treated mice had no increase in tumor volume after 14 days of treatment, whereas tumor volumes doubled in control mice. Histological examination revealed 15%-20% TUNEL-positive cells in nitroxoline-treated mice, compared with ∼5% in the control group. Nitroxoline induces apoptosis and inhibits glioma growth in vivo and in vitro. As an already FDA-approved treatment for urinary tract infections with a known safety profile, nitroxoline could move quickly into clinical trials pending confirmatory studies.

Green tea extract suppresses N-methyl-N-nitrosourea-induced photoreceptor apoptosis in Sprague-Dawley rats.

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and eventual blindness. A single intraperitoneal (ip) injection of N-methyl-N-nitrosourea (MNU) causes photoreceptor cell apoptosis within 7 days in rats. Green tea extract (THEA-FLAN 90S; GTE) is a common herbal supplement with pluripotent properties including antioxidant activity. The purpose of the present study was to evaluate the efficacy of GTE against photoreceptor apoptosis in 7-week-old female Sprague-Dawley rats that received a single ip injection of 40 mg/kg MNU. The oral administration of 250 mg/kg/day GTE was initiated 3 days prior to MNU injection and continued once daily throughout the experiment. Rats were sacrificed at 12, 24, and 72 h and 7 days after MNU injection, and the eyes were examined morphologically and morphometrically. The photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio were used to determine the structural and functional alterations. The number of apoptotic photoreceptor cells per mm(2) was determined in situ by TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL). Our results indicated that oral administration of GTE significantly suppressed the loss of photoreceptor cells morphometrically 7 days after MNU injection. The number of TUNEL-positive cells per mm(2) in MNU-exposed rat central retina with or without GTE administration was 981 vs. 2056 at 24 h after MNU injection. GTE structurally and functionally suppressed MNU-induced photoreceptor cell apoptosis. These findings indicate that GTE may help to ameliorate the onset and progression of human RP.

Apoptosis, senescence, and autophagy in rat nucleus pulposus cells: Implications for diabetic intervertebral disc degeneration

This research was aimed to study the mechanisms by which diabetes aggravates intervertebral disc degeneration (IDD) and to discuss the relationship between autophagy and IDD in nucleus pulposus (NP) cells. Sixteen weeks after injecting streptozotocin (STZ), the intervertebral discs (IVDs) were studied by histology, Alcian blue, 1,9-dimethylmethylene blue (DMMB), immunohistochemistry, and RT-PCR to explore the IDD. The apoptosis and senescence of NP cells was investigated by terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay, immunohistochemistry, and Western blot for caspase3, caspase8, caspase9, and p16lnk4A (increased in cellular senescence). The level of autophagy in NP cells was detected by Western blot, immunohistochemistry, and transmission electron microscopy (TEM). The proteoglycan and collagen II in the extracellular matrix and the aggrecan and collagen II mRNA expression in NP cells of diabetic rats were decreased compared with the control group. Diabetes increased apoptosis of NP cells and led to activations of initiators of intrinsic (caspases-9) and extrinsic (caspase-8) pathways as well as their common executioner (caspase-3). Cellular senescence was increased about twofold in NP of diabetic rats. In addition, the Western blot, immunohistochemistry, and TEM demonstrated higher level of autophagy in NP cells of diabetic rats than control rats to a statistically significant extent. These findings support that diabetes induced by STZ can cause IDD by accelerating the apoptosis and senescence of NP cells excluding the overweight influence. And the results suggest that the autophagy may be a response mechanism to the change of NP cells in diabetic rats.

Apoptosis in Normal and Coxiella burnetii–Infected Placentas From Alaskan Northern Fur Seals (Callorhinus ursinus)

In 2010, Coxiella burnetii was identified in 75% of northern fur seal placentas from a single rookery in Alaska, but nothing was known about the significance of this organism in the population. Although many infectious organisms cause increased cell death, C. burnetii has been shown to suppress apoptosis of the host macrophages as an intracellular survival mechanism. To determine if infection induces a similar functional change in the placenta, immunohistochemistry for antibodies to cleaved caspase-3 (activated caspase-3) and the (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) technique were used to compare the amount of placental apoptosis in infected and noninfected placentas. There was a statistically significant difference in the frequency of apoptotic cells between infected and uninfected placentas, with more apoptosis identified in the uninfected placentas. This finding suggests that the survival mechanism of C. burnetii in host macrophages to reduce apoptosis may also be utilized in trophoblasts. The significance of decreased trophoblastic apoptosis for the northern fur seal fetus requires further investigation.

Gonadotropin-releasing hormone-agonist induces apoptosis of human granulosa-luteal cells via caspase-8, -9 and -3, and poly-(ADP-ribose)-polymerase cleavage.

Gonadotropin-releasing hormone-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET) has been known to directly affect apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-8, -9, and -3 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. The prospective study was conducted in the research institute and clinical fertility center of university hospital. Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10-6 M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after treatment. Activity of caspase-8, -9, and -3 in the cells was examined using a fluorogenic substrate. Caspase-8, -9, and -3 activation and PARP cleavage were analyzed by Western blot. DNA fragmentation in the cells increased at concentrations over 10-6 M GnRH-Ag. In TUNEL assays, the rate of apoptotic cell formation in GnRH-Ag treatment increased significantly compared with that of saline treatment (p < 0.05). The activity of caspase-8, -9 and, -3 investigated using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, cells treated with GnRH-Ag revealed an increase in active forms of caspase-8, -9, and -3 compared with saline treatment. In addition, cleavage of PARP also increased in cells treated with GnRH-Ag. These results suggest that activation of caspase-8, -9, and -3 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.

Normal development of hindgut and anorectum in human embryo

The aim of the present analysis is to examine the morphological changes, the spatiotemporal distribution of apoptosis/proliferation in the human embryonic anorectum, to reveal the normal development of human anorectum, and investigate the possible roles of apoptosis/proliferation during anorectal development. The embryos were sectioned serially and sagittally, stained with hematoxylin and eosin (H & E) between the third and eighth week of gestation, TdT-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and proliferative cell-specific nuclear antigen (PCNA) immunohistochemical staining from the sixth to the eighth week. From the fourth to the seventh week, with the growth of the mesenchyme around the cloaca, the cloaca was remolded, subsequently, the cloacal membrane (CM) moved perpendicularly then horizontally. The dorsal cloaca gradually descended to the tail groove, the urorectal septum (URS) and the CM approximated; however, the fusion of URS with the dorsal CM was never observed. During the eighth week, the URS shifted ventrally and finally fused with the ventral CM. Moreover, from the sixth to the eighth week, the apoptotic cells were concentrated in the CM, the mesenchyme of terminal rectum, and the dorsal rectum. Meanwhile, the proliferative cells could be observed in the ventral mesenchyme around the cloaca, the CM, the fused tissue between the URS, and the ventral CM. During the development of human anorectum, it was intriguing to reveal that the URS never fused with the dorsal CM before dorsal CM disintegration, the normal anorectal development may depend on the dorsal cloaca and the dorsal CM; furthermore, the distribution of apoptosis and proliferation in the anorectum and ventral cloacal mesenchyme played a pivotal role in the formation of the anorectum.

Anti-Tissue Transglutaminase Antibodies From Celiac Patients Are Responsible for Trophoblast Damage via Apoptosis In Vitro

The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro. Human primary trophoblastic cells, isolated from term placenta, were exposed to anti-tTG immunoglobulin G (IgG) antibodies, both commercially available and separated from sera of three untreated celiac women. The ability of anti-tTG antibodies to bind to trophoblastic cells, invasiveness of placental cells through a layer of extracellular matrix, and the activity of cellular matrix metalloprotease (MMP) and cellular apoptosis were evaluated, as indicators of trophoblast damage, by TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) and annexin V expression. Anti-tTG IgG showed a specific dose- and time-dependent binding to human trophoblast. In addition, trophoblastic cells, after being exposed to anti-tTG IgG antibodies, both commercially available and separated from sera of celiac women, showed an impaired invasiveness, a decreased activity of cellular MMP, and a greater percentage of TUNEL positivity and annexin V positivity. We showed that the binding of anti-tTG antibodies to trophoblast might represent a key mechanism by which the embryo implantation and pregnancy outcome are impaired in untreated celiac pregnant women. Because healthy trophoblast development is essential for placental and fetal development, these data provide a novel mechanism for CD-induced infertility, early pregnancy loss, and intrauterine growth retardation.

Analysis of gene expression profiles in the liver and spleen of mice infected with Trypanosoma evansi by using a cDNA microarray

Trypanosoma evansi, the cause of the disease Surra in livestock, is the most widely geographically distributed pathogenic trypanosome occurring in Africa, South and Central America, and Asia, where it causes significant economic loss. Although many studies have described the histopathology induced in the organs of mice infected with T. evansi, few studies have been conducted on gene expression in these organs. Here we used complementary DNA microarray to analyze the gene expression profiles in the liver and spleen of mice infected with T. evansi (STIB 806) at the peak parasitemia (7 days after infection). A total of 14,000 sequences including full length and partial complementary DNAs representing novel, known, and control genes of mouse were analyzed. Results from GeneOntology annotation showed that 158 genes in the liver and 73 genes in the spleen were up-regulated in the infected mice and that 178 genes in the liver and 117 genes in the spleen of infected mice were down-regulated compared with control (non-infected) mice. Most of these genes are metabolism, transport, protein biosynthesis, transcription factors, and nucleic acid binding protein-related genes. The changes of some important genes, such as heat shock protein 70 and proliferating cell nuclear antigen, were confirmed by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. TdT-mediated dUTP-digoxigenin nick end labeling analysis results revealed that extensive apoptosis occurred in the liver of infected mice at the peak of parasitemia. Our results provide a comprehensive profile of changes in gene expression in the liver and spleen of mice infected with T. evansi and may be helpful in understanding the pathogenesis of Surra at a molecular level.

Detection of Cell Death in Spider Embryos Using TUNEL

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American Wandering Spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. A feature of apoptosis (i.e., cell death) is the cleavage or fragmentation of DNA that occurs in dead or dying cells. This protocol describes the detection of fragmented DNA in whole-mount Cupiennius embryos. The 3'-OH ends of these DNA fragments can be labeled with the terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) technique. This protocol uses a terminal deoxynucleotidyl transferase to add labeled dUTP to the fragmented DNA, and this label is then detected by immunocytochemistry. The TUNEL technique is a relatively easy way to obtain a reliable picture of the cell death pattern during normal and abnormal development.

Collection and Fixation of Spider Embryos

INTRODUCTIONThe spider Cupiennius salei, commonly known as the American wandering spider, is a particularly useful laboratory model for embryological studies because of the availability of tools to study and manipulate its embryonic development. Cupiennius is used to study axis formation, segmentation, appendage development, neurogenesis, and silk production. These studies contribute to our understanding of the evolution of these processes, but they also help us to understand the origin and diversification of evolutionary novelties. Comparisons between spiders and insects can show the degree of conservation and divergence of developmental mechanisms during arthropod evolution. Any embryological feature conserved between spiders and insects is likely to represent an ancestral feature for arthropods. Comparative molecular embryological work in insects and spiders should eventually allow us to define a molecular archetype for the phylum Arthropoda. This in itself will be a necessary cornerstone for comparing the different metazoan phyla, including chordates. This protocol describes the collection and fixation of embryos from C. salei. The fixed embryos can be stored at -20°C for prolonged periods and used for in situ hybridization, in studies of apoptosis using terminal deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and for immunohistochemistry.

High expression of EZH2 is associated with tumor proliferation and prognosis in human oral squamous cell carcinomas

Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes and is important in cell cycle regulation. Overexpression of EZH2 protein has been associated with biological malignancy of prostate cancer and several other cancers. The aim of the present study was to evaluate the expression of EZH2 protein in human oral normal mucosa, dysplasia and oral squamous cell carcinoma (OSCC) with clinicopathological profiles. EZH2 expression was assessed by Western blotting and immunohistochemistry in 3 OSCC cell lines, 10 normal mucosae, 50 dysplasias and 102 OSCCs. The labeling indices (LIs) of EZH2, Ki-67, P53, and the apoptotic index (AI) were evaluated by immunohistochemistry and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method. Western blot analysis detected EZH2 protein as a single band at 91kDa in the 3 OSCC cell lines, but it was almost absent in non-tumoral oral mucosae. The LI of EZH2 was highest in the OSCCs, followed by the dysplasias (p<0.05) and normal mucosae (p<0.05) with significant difference. The LI of EZH2 correlated with the clinical stage, tumor size, lymph node metastasis and LIs of Ki-67 and P53, but not with the AI in OSCCs, and inversely correlated with the histological differentiation of OSCCs. The survival rate calculated by the Kaplan-Meier method revealed that OSCC patients with higher EZH2 expression showed poorer prognosis than those with a lower EZH2 expression (p<0.01). These results suggest that overexpression of EZH2 is correlated with malignant potential and poor prognosis in OSCCs. EZH2 might serve as a novel biomarker for predicting prognosis in patients with OSCCs.

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-microm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-microm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei.

Ectopic germline cells in embryos of Xenopus laevis

Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.

Mitomycin-C Concentration in Cornea and Aqueous Humor and Apoptosis in the Stroma After Topical Mitomycin-C Application

To evaluate the effects of the applied mitomycin-C (MMC) concentration and application time on the aqueous MMC concentration and apoptosis in the corneal stroma. New Zealand white rabbits underwent mechanical epithelium debridement of the central 7.5 mm of the cornea. A sponge soaked in MMC solution was placed on the denuded corneal stroma. The effect of the exposure times ranging from 15 to 120 seconds and the different MMC concentrations ranging from 0.005% to 0.04% on the aqueous MMC concentration and the apoptosis in the stromal cells were evaluated. The aqueous concentration of MMC increased linearly with increasing exposure time and MMC concentration. The correlation between the aqueous MMC concentration and the applied concentration (r = 0.809, P < 0.001) was higher than the correlation between the aqueous MMC concentration and the application time (r = 0.693, P < 0.001). Terminal deoxyribonucleotidyltransferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells were detected in the superficial stroma of the central denuded cornea. The numbers of TUNEL-positive cells increased linearly with increasing concentrations, and the increase was statistically significant (P = 0.026). However, the numbers of TUNEL-positive cells increased only slightly with an increasing application time, and the increase was not statistically significant (P = 0.928). Reducing the applied concentration or decreasing the exposure time might be a good modality for reducing the potential MMC toxicity. The applied MMC concentration had greater effects on the aqueous MMC concentration and apoptosis in the stromal cells than the exposure time.

Graft Tumor Necrosis Factor Receptor-1 Protects After Mouse Liver Transplantation Whereas Host Tumor Necrosis Factor Receptor-1 Promotes Injury

Tumor necrosis factor (TNF)-alpha is a cytokine with pleiotropic effects on the liver. The predominant hepatic receptor for TNFalpha is TNF receptor-1 (TNFR1). TNFR1 mediates liver injury after ischemia/reperfusion but is also mitogenic during hepatic regeneration. This study investigated the role of graft and host TNFR1 in early graft injury after liver transplantation in mice. Livers from TNFR1 deficient (TNFR1-/-) and wild type (WT) mice were transplanted into either TNFR1-/- or WT recipients in all four possible combinations after 12 hours of cold storage. After eight hours, alanine transferase (ALT), necrosis, TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining, caspase-3 activation, and myeloperoxidase were determined. When TNFR1-/- livers were transplanted into either WT or TNFR1-/- recipients, ALT was twofold greater than when WT donor livers were used. Necrosis and TUNEL staining also increased twofold and sevenfold, respectively, after transplantation of TNFR1-/- donor livers compared to WT. By contrast, ALT and necrosis decreased when WT or TNFR1-/- livers were transplanted into TNFR1-/- hosts compared to WT, which was associated with decreased neutrophil infiltration. In conclusion, graft and recipient TNFR1 has opposing effects. Graft TNFR1 decreases graft injury, whereas recipient TNFR1 mediates an increase of injury associated with enhanced neutrophil infiltration. Cross-transplanting of knockout and wild-type livers provides a new means to investigate graft-host interactions during hepatic injury.

Cryopreservation of intact human ovary with its vascular pedicle

The aim of this study was to assess the immediate post-thawing injury to the human ovary that was cryopreserved either as a whole with its vascular pedicle or as ovarian cortical strips. Bilateral oophorectomy was performed in two women (46 and 44 years old) undergoing vaginal hysterectomy and laparoscopic hysterectomy, respectively. Both women agreed to donate their ovaries for experimental research. In both patients, one of the harvested ovaries was sectioned and cryopreserved (by slow freezing) as ovarian cortical strips of 1.0 x 1.0 x 5.0 mm(3) each. The other ovary was cryopreserved intact with its vascular pedicle. After thawing 7 days later, follicular viability, histology, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay (to detect apoptosis) and immunoperoxidase staining (to define Bcl-2 and p53 protein expression profiles) of the ovarian tissue were performed. Tissues from non-cryopreserved ovaries served as control specimens (two cases). The overall viability of the primordial follicles was 75 and 78% in intact cryopreserved-thawed (C-T) ovaries and 81 and 83% in ovarian cortical strips in the 46- and 44-year-old patients, respectively. Comparable primordial follicle counts, absence of features of necrosis, mean values of apoptosis and weak Bcl-2 and p53 protein expressions were observed both in the intact C-T ovary and in the C-T ovarian cortical strips. Cryoperfusion and cryopreservation of entire human ovary can be achieved with the maintenance of excellent viability of the superficial and the deeper tissues using a slow-freezing protocol. Cryopreservation injury is associated neither with significant alteration in the expression pattern of Bcl-2 and p53 proteins in the ovarian tissues nor with significant follicular damage.