Cheating death at the dawn of life: developmental control of apoptotic repression in the preimplantation embryo

During early development, the mammalian embryo is resistant to pro-apoptotic signals because of biochemical properties of the mitochondrion and nucleus. Mitochondria of the bovine two-cell embryo are resistant to depolarization because of low amounts of the proapoptotic protein BAX and high concentrations of the anti-apoptotic protein BCL2. As development proceeds, BAX content increases, BCL2 content declines, and mitochondria becomes capable of pore formation and depolarization in response to pro-apoptotic signals. The nucleus of the two-cell embryo is resistant to degradation by the DNase DFFB because epigenetic modifications, including DNA methylation and histone deacetylation, mask internucleosomal sites for DNA cleavage. Blastomere DNA becomes progressively less methylated during development so that DNA becomes accessible to cleavage by DFFB.     

Rapid enumeration of respiratory active mycobacteria with fluorescent double staining

A new method for rapid enumeration of physiologically active mycobacteria was developed by acid-fast bacilli staining (Auramine O) following formazan reduction. Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the widely used culture-dependent approach.     

Amount of maternal body fat significantly affected the quality of isolated mouse preimplantation embryos and slowed down their development

The aim of our study was to investigate the effect of maternal obesity on the quality and developmental capabilities of in vivo-derived preimplantation embryos. A two-generation dietary model, based on mice overfeeding during intrauterine and early postnatal development, was used to produce four types of female animals: with physiological (7%–8%), slightly elevated (8%–11%), highly elevated (>11%), and low (<7%) amounts of body fat. Spontaneously ovulating females (5–6 weeks old) were mated with male animals and subjected to embryo isolation at Day 4. Stereomicroscopical evaluation of collected embryos showed that the amount of maternal body fat did not affect the average number of collected embryos per dam. However, significant differences were found in the stage-distribution of isolated embryos: dams with highly elevated body fat and dams with low fat delivered decreased numbers of blastocysts and increased numbers of lower-stage or degenerated embryos compared with dams with physiological or slightly elevated fat value. Fluorescence staining showed that blastocysts isolated from dams with high and low percentage of body fat contained significantly higher numbers of dead cells. Most of such dead cells were of apoptotic origin. In contrast, the amount of maternal body fat did not affect blastocyst growth—the average numbers of cells per blastocyst were comparable in all groups. In conclusion, highly elevated or decreased amount of maternal body fat slowed down the development and negatively affected the quality of naturally in vivo-derived preimplantation embryos. No negative effect of slightly elevated fat was observed.     

Differential staining and microchromosomal variation in karyotypes of four Brazilian species of Tupinambinae lizards (Squamata: Teiidae)

Kayotypes of four neotropical teiid lizard species (Tupinambinae) were herein studied after conventional as well as silver staining and CBG-banding: Crocodilurus amazonicus (2n = 34), Tupinambis teguixin (2n = 36), Tupinambis merianae and Tupinambis quadrilineatus (2n = 38). The karyological data for T. quadrilineatus as well as those obtained using differential staining for all species were unknown until now. The karyotypes of all species presented 12 macrochromosomes identical in morphology, but differed in the number of microchromosomes: 22 in C. amazonicus, 24 in T. teguixin and 26 in T. quadrilineatus and T. merianae. The Ag-NOR located at the secondary constriction at the distal end of pair 2 is shared by all species, contrasting with the variability observed for this character in species of the related Teiinae. CBG-banding revealed a species-specific pattern in T. quadrilineatus with conspicuous interstitial C-blocks at the proximal region of the long arm of pair 4 and the whole heterochromatic short arm of pair 6. The karyological data reported here corroborates the relationship hypothesis obtained for Tupinambis based on molecular characters. T. teguixin presents the putative ancestral karyotype for the genus with 2n = 36 whereas T. merianae and T. quadrilineatus exhibit 2n = 38, due to an additional pair of microchromosomes.     

Developmental patterns of caspase-3, bax and bcl-2 proteins expression in the human spinal ganglia

The distribution of the bcl-2, bax and caspase-3 proteins was investigated in the cells of developing human spinal ganglia. Paraffin sections of 10 human conceptuses between 5th and 9th gestational weeks were analysed morphologically, immunohistochemically and by TUNEL-method. Cells positive to caspase-3 had brown stained nuclei or nuclear fragmentations. At earliest stages, 6% of ganglion population were caspase-3 positive cells. Later on, a significant increase in number of caspase-3 positive cells appeared, particularly in the ventral part of ganglia (12%), and subsequently decreased to 6%. TUNEL-positive cells had the same distribution pattern as caspase-3 positive cells. Bax-positive cells followed the developmental pattern similar to caspase-3 cells, changing in range between 20% and 32%. There were 8% of bcl-2 positive cells at earliest stages. They increased significantly in dorsal part of the ganglion during the 7th week (28%), and than dropped to 15% by the end of the 8th week. These findings suggest a ventro-dorsal course of development in human spinal ganglia. Number of bcl-2, bax and caspase-3 positive cells changed in a temporally and spatially restricted manner, coincidently with ganglion differentiation. While apoptosis might control cell number, bcl-2 could act in suppression of apoptosis and enhancement of cell differentiation.     

Differential staining for live and dead sperm of honey bees

ABSTRACT Seven staining techniques were modified and tested for differentially staining the live and the dead sperm cells for the honey bee (Apis mellifera L.). The eosin Y staining method was found to be a simple technique by which the live cells stain bluish purple whereas the dead cells stain bright yellow to greenish yellow. Therefore, it produces a strong contrast between the dead and live sperm cells, and appears to be the most suitable supravital staining method for evaluating the viability of honey bee sperm cells. The significance of supravital staining techniques in assessing the quality of sperm cells during cryopreserving sperm cells is discussed.     

Tandem use of immunofluorescent and DNA staining assays to validate nested PCR detection of mycoplasma

Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.     

Caspase-12 compensates for lack of caspase-2 and caspase-3 in female germ cells

Previously, we analyzed mice lacking either caspase-2 or caspase-3 and documented a role for caspase-2 in developmental and chemotherapy-induced apoptosis of oocytes. Those data also revealed dispensability of caspase-3, although we found this caspase critical for ovarian granulosa cell death. Because of the mutual interdependence of germ cells and granulosa cells, herein we generated caspase-2 and -3 double-mutant (DKO) mice to evaluate how these two caspases functionally relate to each other in orchestrating oocyte apoptosis. No difference was observed in the rate of spontaneous oocyte apoptosis between DKO and wildtype (WT) females. In contrast, the oocytes from DKO females were more susceptible to apoptosis induced by DNA damaging agents, compared with oocytes from WT females. This increased sensitivity to death of DKO oocytes appears to be a specific response to DNA damage, and it was associated with a compensatory upregulation of caspase-12. Interestingly, DKO oocytes were more resistant to apoptosis induced by methotrexate (MTX) than WT oocytes. These results revealed that in female germ cells, insults that directly interfere with their metabolic status (e.g. MTX) require caspase-2 and caspase-3 as obligatory executioners of the ensuing cell death cascade. However, when DNA damage is involved, and in the absence of caspase-2 and -3, caspase-12 becomes upregulated and mediates apoptosis in oocytes. Takai and Matikainen contributed equally to this work.     

Influence of co-down-regulation of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic cell death of Chinese hamster ovary cells producing thrombopoietin

Previously, the expression of caspase-3 siRNA could not effectively inhibit sodium butyrate (NaBu)-induced apoptotic cell death of recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Caspase-3 siRNA expressing cells appeared to compensate for the lack of caspase-3 by increasing active caspase-7 levels. For the successful inhibition of NaBu-induced apoptosis of rCHO cells, both caspase-3 and caspase-7 were down-regulated using the siRNA expression vector system. Co-down-regulation of caspase-3 and caspase-7 increased cell viability and extended culture longevity in serum-free culture in the presence or absence of 1mM NaBu addition. In the cultures with 1mM NaBu addition, the maximum hTPO concentration in rCHO cells with down-regulation of both caspases was approximately 55% higher than that in rCHO cells without down-regulation of caspases and approximately 16% higher than rCHO cells with down-regulation of only caspase-3. However, in the culture with 3mM NaBu, this strategy could not dramatically enhance the culture longevity and hTPO production, compared to Bcl-2 overexpression. The different result in hTPO production between down-regulation of caspases and Bcl-2 overexpression may be because the down-regulation of caspase-3 and caspase-7, unlike Bcl-2 overexpression, could not maintain mitochondrial membrane potential in the presence of 3mM NaBu. Taken together, co-down-regulation of caspase-3 and caspase-7 is effective in regard to extension of culture longevity and enhancement of hTPO production in a serum-free culture in the presence or absence of 1mM NaBu addition.     

Indirect immunofluorescent staining of the microtubules in interphase and mitotic amoebae of the myxomycetePhysarum polycephalum

Summary The microtubules ofPhysarum amoebae have been decorated with rat antibodies against yeast tubulin. The indirect fluorescent staining observed in interphase amoebae and in flagellated amoebae is consistent with the three-dimensional reconstructions previously deduced from electron microscopic studies. Mitotic amoebae exhibit a pattern of fluorescence which is similar to that exhibited by mammalian cells and is consistent with the previous electron microscopic studies, except that we also observe pole-pole microtubule fibers during metaphase and anaphase and the presence of a typical midbody during cytokinesis. The various types of tripolar mitosis which are observed suggest that there is a regulatory mechanism allowing the formation of pseudo-bipolar mitotic apparatuses in amoebae possessing more than two mitotic centers during mitosis. The mitotic center, located in the middle of the centrosphere, is not fluorescent after staining of the monoasters induced with taxol suggesting the absence of tubulin in the mitotic center.     

Establishment of the expression system for studying the function of active caspase-3 in zebrafish

Caspase-3, a key molecule in apoptosis, has been extensively studied in cell culture system; however, it has been less well characterized in vivo because certain mediators are required for the proteolytic activation of effector caspases, including caspase-3. In this study, various forms of caspase-3 with the C-terminal GFP tag were inserted into the pCS2+ plasmid, and the expression patterns of caspase-3 proteins were characterized in a zebrafish model system using microinjection of nucleic acids into zebrafish embryos. We have verified that active caspase-3 was generated by its autocatalytic activity under the condition of caspase-2 prodomain (C2P)-caspase-3-GFP overexpression, indicating that the C2P domain is crucial for the activation of caspase-3. We also confirmed that the C2P domain plays an important role in regulating the nuclear localization of the C2P-caspase-3 chimeric protein. We used this expression system to establish an animal model system suitable for the investigation of the functional characteristics of caspase-3 in vivo. Thus, our study provides a useful and specific tool for investigating the molecular mechanisms by which active caspase-3 regulates apoptosis during embryonic development.     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

Purification of catalytically active caspase-12 and its biochemical characterization

Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER-mediated apoptosis and inflammatory caspase activation pathway. Cleavage of the prodomain by caspase-3/-7 at the carboxyl terminus of Asp94 or m-calpain at the carboxyl terminus of Lys158 was reported to be a part of caspase-12-involved apoptosis. We biochemically characterized the prodomain-free forms of caspase-12 and the equivalent enzymes; Δpro1(G95-D419), rev-Δpro1[(T319-N419)-(G95-D318), a reverse form of Δpro1] and rev-Δpro2[(T319-N419)-(T159-D318)]. The three variants showed comparable activities which were dependent on salt concentration and pH. Auto-proteolytic cleavage was observed at two sites (carboxyl termini of Asp318 and Asp320) in Δpro1. Constitutively active forms of caspase-12 (rev-Δpro1 and rev-Δpro2) could induce cell death in cells transfected with the corresponding expression vectors, but no cleavage of caspase-3, DFF45 or Bid was observed, indicating caspase-12 may mediate a distinct apoptotic pathway rather than caspase-8 or -9-mediated cell death.     

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-beta-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3+/-1.8%, that of expressing embryos was only 43.9+/-1.6% (P<0.0001). Developmental competence of embryos negatively correlated (r=-0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1+/-5.3%, 50+/-5%, and 9.5+/-3.9% blastocysts, respectively (P<0.002). Interestingly, blastocysts expressing faint-moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic 'green' mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint-moderate level of its expression is compatible with normal embryogenesis in the mouse.     

Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.     

特殊染色及免疫组化双重染色在恒河猴糖尿病模型中的应用

目的在链脲佐菌素（STZ）诱导恒河猴糖尿病动物模型基础上,用特殊染色、免疫组化双染方法显示胰腺等组织中的特征性病变。方法健康恒河猴5只,小剂量（30mg／kg）多次静脉注射STZ,濒死状态时将动物安乐死。取胰腺、心脏、肾脏、脾脏、肝脏、眼球、脑等器官制成石蜡切片,用HE、PAS、Masson、天狼星红和甲苯胺蓝等方法进行特殊染色,用免疫组化双重染色法同时显示胰岛A、B细胞。结果模型动物的胰岛萎缩,数量减少。Masson染色见外分泌部间质内纤维增生。局灶性慢性肾炎,病变区肾间质纤维组织增生,部分肾小球及肾小管萎缩。天狼星红染色见脾脏中央动脉管壁增厚。免疫组化双染见胰岛A胰高血糖素表达增多,胞浆呈棕褐色。B胰岛素表达减少,胞浆粉红色。结论HE染色结合特殊染色和免疫组化双重染色可较好地对STZ诱导糖尿病动物模型进行组织学评价。     

Differential regulation of JNK in caspase-3-mediated apoptosis of MPP(+)-treated primary cortical neurons

MPTP (1-methyl-1,2,3,6-tetrahydropyridine), a chemical contaminant of synthetic heroin, induces neuropathological changes with clinical features similar to idiopathic Parkinson's disease. The mechanism by which MPTP and its metabolite MPP(+)(1-methyl-4-phenylpyridinium) induces neuronal cell death remains unclear. We employed primary cortical/telencephalon neuronal cultures to investigate the potential role of caspase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathways in MPP(+)-induced neuronal death. DNA fragmentation and caspase-3 activity analysis showed that cortical neuronal cells underwent apoptosis after MPP(+)treatment. However, a basal level of apoptotic cells was also observed in untreated cultures. Interestingly, JNK activity increased in untreated cultures over time, whereas it was down-regulated after MPP(+)treatment. This indicates that the JNK pathways could be differentially regulated in different apoptotic processes.     

Repression of induced apoptosis in the 2-cell bovine embryo involves DNA methylation and histone deacetylation

Apoptosis in the bovine embryo cannot be induced by activators of the extrinsic apoptosis pathway until the 8-16-cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in 2-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by methylation of DNA and deacetylation of histones. To test this hypothesis, we evaluated whether reducing DNA methylation by 5-aza-2′-deoxycytidine (AZA) or inhibition of histone deacetylation by trichostatin-A (TSA) would make 2-cell embryos susceptible to DNA fragmentation caused by CCCP. The percent of blastomeres positive for TUNEL was affected by a treatment × CCCP interaction (P < 0.0001). CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle but did increase the percent of cells that were TUNEL positive if embryos were pretreated with AZA or TSA. Immunostaining using an antibody against 5-methyl-cytosine antibody revealed that AZA and TSA reduced DNA methylation. In conclusion, disruption of DNA methylation and histone deacetylation removes the block to apoptosis in bovine 2-cell embryos.     

Effect of Bcl—2 and caspase—3 on calcium distribution in apoptosis of HL—60 cells

Apoptosis manifests in two major execution programs downstream of the death signal:the caspase pathway and organelle dysfunction.An important antiapoptosis factor,Bcl-2 protein,contributes in caspase pathway of apoptosis.Calcium,an important intracellular signal element in cells,is also observed to have changes during apoptosis,which maybe affected by Bcl-2 protein.We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells,there‘s change of intracellular calcium distribution,oving from cytoplast especially Golgi‘s apparatus to nucleus and accumulating there with the highest concentration.We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells,which can be inhibited by overexpression of Bcl-2 protein.No sign of apoptosis or intracellular calcium movement from Golgi‘s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO,a specific inhibitor of caspase-3.The results indicate that activated caspase-2 can promote the movement of intracellular calcium from Golgi‘s apparatus to nucleus,and the process is inhibited by Ac-DEVD-CHO(inhibitor of caspase-3),and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3.Calcium relocalization in apoptosis seems to be irreversible,which is different from the intracellular calcium changes caused by growth factor.