Physiological basis of salt stress tolerance in rice expressing the antiapoptotic gene SfIAP

Programmed cell death-associated genes, especially antiapoptosis-related genes have been reported to confer tolerance to a wide range of biotic and abiotic stresses in dicotyledonous plants such as tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.). This is the first time the antiapoptotic gene SfIAP was transformed into a monocotyledonous representative: rice (Oryza sativa L.). Transgenic rice strains expressing SfIAP were generated by the Agrobacterium-mediated transformation method and rice embryogenic calli, and assessed for their ability to confer tolerance to salt stress at both the seedling and reproductive stages using a combination of molecular, agronomical, physiological and biochemical techniques. The results show that plants expressing SfIAP have higher salt tolerance levels in comparison to the wild-type and vector controls. By preventing cell death at the onset of salt stress and maintaining the cell membrane’s integrity, SfIAP transgenic rice plants can retain plant water status, ion homeostasis, photosynthetic efficiency and growth to combat salinity successfully.

A Novel Anticancer Agent, 8-Methoxypyrimido4 `,5 `: 4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and Independent Apoptotic Pathways

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido 4 `,5 `: 4,5] thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly ( ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N-1,N-1-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb3+ (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (A,. = 548 nm and A,. = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb3+ chelate at the T-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb3+ chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb3+ chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb3+ chelate at 325 run, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.

Fasciola hepatica:Histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4. days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1-4. days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis. © 2012 Elsevier B.V.

Réduction des dommages myocardiques par le célécoxib suite à une ischémie transitoire chez le rat

Cette étude a été conçue afin d’évaluer l’effet d’un pré-traitement à long terme au célécoxib sur la taille d’infarctus suite à un infarctus du myocarde. Sachant que le célécoxib est un anti-inflammatoire et que des dommages myocardiques peuvent découler des processus inflammatoires, l’inhibition de l’inflammation devrait hypothétiquement réduire la taille d’un éventuel infarctus. Pour ce faire, un traitement au célécoxib (3 mg/kg/jour i.p.) ou au véhicule (DMSO 50% ; EtOH 15% ; eau distillée) a été administré chroniquement pendant 28 jours à des rats mâles Sprague-Dawley (n=18 par groupe) par pompes osmotiques ALZET. Après avoir été anesthésiés, les animaux ont été sujets à l’occlusion de l’artère coronaire gauche descendante, suivie d’une période de reperfusion de 24 heures. Les résultats démontrent que la taille de l’infarctus des animaux traités au célécoxib est significativement réduite comparativement à celle du groupe témoin (37,5±2,5% versus 48,0±2,6% de la zone à risque, p < 0,05). Par la suite, l’accumulation de neutrophiles indique une hausse de ces leucocytes pour la zone ischémique, sans toutefois discriminer entre les groupes traité et non-traité, qui contenaient aussi les couches sub-endocardique et sous-épicardique. Cependant, aucune différence significative est notée entre les groupes traité et témoin au niveau de l’expression de la prostaglandine E2 plasmatique et du facteur de nécrose tumorale alpha. D’un autre côté, l’apoptose, déterminée par le ratio de Bax/Bcl2 et par un essai TUNEL est significativement réduite pour la couche sub-endocardique de la zone à risque des animaux traités au célécoxib. Enfin, l’agrégation plaquettaire, induite à l’adénosine diphosphate et analysée dans le sang complet, suggère que le célécoxib diminue l’agrégation plaquettaire. Cette étude indique alors qu’un pré-traitement au célécoxib peut réduire la taille d’infarctus par un mécanisme impliquant l’apoptose.

Atténuation de la dépression post-infarctus par une diète riche en oméga-3 ou par la prise de probiotiques

L'Infarctus du myocarde (IM) provoque, chez le rat, une augmentation de l'apoptose dans le système limbique en plus d'induire des symptômes qui s'apparentent à la dépression chez l'humain. Nous avons démontré qu'une diète élevée en oméga-3 ou la prise de probiotiques pouvaient être efficaces pour réduire ces effets si ces interventions débutaient avant l'induction de l'ischémie myocardique. Cette étude a pour objectif de déterminer l'efficacité de ces interventions si elles débutent après l'ischémie myocardique. L’IM a été induit chez le rat mâle Sprague-Dawley par l’occlusion de l’artère coronaire descendante antérieure gauche pendant 40 minutes. À la suite de l’ischémie, les rats ont reçu des probiotiques (1 billion de bactéries vivantes de L. helveticus R0052 et de B. longum R0175) ou un véhicule dans leur eau de boisson en présence d'une diète élevée ou faible en oméga-3. À 3 jours post-IM, l’activité enzymatique de la caspase-3 et le nombre de cellules dUTP nick-end labelling (TUNEL) positives sont diminués dans les régions CA1 et le corps godronné de l’hippocampe ainsi que dans l’amygdale en présence de la diète élevée en oméga-3. La prise de probiotiques atténue également l’activité de la caspase-3 et le nombre de cellules TUNEL positives dans le corps godronné et l’amygdale médiane. À 2 semaines post-IM, le comportement dépressif évalué par 3 tests comportementaux (test d’interaction sociale, test de nage forcée et test d’évitement passif) a été observé chez le groupe recevant la diète faible en oméga-3 sans probiotiques et le comportement dépressif a été atténué avec la diète élevée en oméga-3 et/ou la prise de probiotiques. Les probiotiques ont augmenté les niveaux plasmatiques d’interleukine-4 (IL- 4) tandis que la diète élevée en oméga-3 a montré une diminution de la protéine chimiotactique monocytaire 1 (MCP-1). Ces résultats indiquent qu’une diète élevée en oméga-3 ou la prise de probiotiques, débutant à la suite de l’IM, s’avèrent bénéfiques pour atténuer la dépression et l’apoptose dans le système limbique.

Relationship between DNA damage and sperm head birefringence

Birefringence or double refraction is the decomposition of a ray of light into two rays when it passes through an anisotropic material such as quartz. Sperm cells have been demonstrated to be optically anisotropic. The objective of this study was to evaluate the relationship between the pattern of human sperm head birefringence (SHBF) and DNA damage. A total of 26 patients with normal semen were included. DNA damage (fragmentation and denaturation) was evaluated in the sperm head in the context of birefringence, both total (SHBF-T) and partial (SHBF-P), by terminal deoxyribonucleotidyl transferase (TdT)-mediated dUDP nick-end labelling assay and acridine orange fluorescence, respectively. Positive DNA fragmentation in spermatozoa with SHBF-T (205/1053; 19.5%) was significantly higher (P < 0.0001) than in spermatozoa that presented SHBF-P (60/820; 7.3%). However, the percentage of denatured DNA in spermatozoa with SHBF-T (824/1256; 65.6%) was not significantly different from the ones with SHBF-P (666/1009; 66.0%). In conclusion, the data support a positive relationship between spermatozoa with total SHBF in their head and increased DNA fragmentation. (C) 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Apoptosis during the seasonal spermatogenic cycle of Rana catesbeiana

In the bullfrog Rana catesbeiana, testicular weight is constant throughout the year, but the volume densities of germinative and interstitial compartments undergo inverse changes from winter (non-breeding) to summer (breeding). The occurrence of apoptosis in the seminiferous lobules of bullfrogs was investigated in these two periods using sections stained with haematoxylin and eosin (H&E), the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) method and transmission electron microscopy. TUNEL-positive cells were observed in the seminiferous lobules, and ultrastructural morphological details confirmed the occurrence of cell death by apoptosis. In summer, the occurrence of several spermatogenic processes (in addition to spermiogenesis and spermiation), and then the overconsumption of Sertoli cell-derived pro-survival factors, could be responsible for the increased density of apoptotic cells. Alternatively, the low apoptotic frequency in winter could be related to the constant homeostasis in the germinative compartment given that most lobules are filled with primary spermatocytes. As volume densities of interstitial and germinative compartments undergo inverse seasonal variations through the year, the incidence of apoptosis (in summer) could play a part in controlling the spermatogenic process, maintaining the lobular size when interstitial tissue is maximally developed. In winter, the low apoptotic cell density leads to spermatogenic recrudescence and, thereby, the production of an adequate quantity of spermatozoa for the next breeding period. Thus, apoptosis may participate not only in the maintenance of spermatogenic homeostasis, but also in the cyclical control of the different spermatogenic processes according to seasonal changes of the testicular compartments as a whole.

Immunohistochemical detection of estrogen receptor beta in alveolar bone cells of estradiol-treated female rats: possible direct action of estrogen on osteoclast life span

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da apoptose em tecido ovariano em gatas

Há um crescente interesse nas técnicas reprodutivas, por agilizar a procriação de populações animais de interesse econômico, a preservação de espécies em extinção ou a utilização como modelo experimental em outras espécies. A reprodução de felinos em cativeiro é dificultada devido ao ambiente estranho, o que os leva a apresentarem alterações na própria fisiologia. Torna-se importante o estudo de fenômenos reprodutivos destes animais com a finalidade de melhor compreensão e, posteriormente, adaptação adequada das biotécnicas reprodutivas. A atuação da apoptose na fisiologia ovariana ainda não é bem compreendida, havendo a necessidade da comunidade científica elucidá-la melhor. O objetivo deste trabalho foi testar a hipótese de que os índices de apoptose no tecido ovariano são diferentes em gatas jovens, adultas e idosas. Foram utilizadas 18 gatas distribuídas em 3 grupos contendo 6 animais cada, de acordo com a idade (jovens, adultos e idosos). As amostras do tecido ovariano destinadas a avaliação de apoptose, foram submetidas ao método terminal deoxynucleotidyltransferase-biotin nick end-labelling (TUNEL). Os dados referentes aos diferentes grupos foram submetidos à análise de variância e as médias foram comparadas pelo Teste de Tukey. Não houve diferença significativa quanto ao número de folículos e número de células positivas das gatas jovens, adultas e idosas, sendo P < 0,05. O resultado do trabalho sugere que o fenômeno da apoptose em tecido ovariano em gatas não possui relação com a faixa etária, ou seja, a apoptose ocorre de forma contínua nos folículos não-dominantes envolvidos em cada ciclo estral apresentado

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.