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.

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

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.

Crystals that count! Physical principles and experimental investigations of DNA tile self-assembly

Algorithmic DNA tiles systems are fascinating. From a theoretical perspective, they can result in simple systems that assemble themselves into beautiful, complex structures through fundamental interactions and logical rules. As an experimental technique, they provide a promising method for programmably assembling complex, precise crystals that can grow to considerable size while retaining nanoscale resolution. In the journey from theoretical abstractions to experimental demonstrations, however, lie numerous challenges and complications.

In this thesis, to examine these challenges, we consider the physical principles behind DNA tile self-assembly. We survey recent progress in experimental algorithmic self-assembly, and explain the simple physical models behind this progress. Using direct observation of individual tile attachments and detachments with an atomic force microscope, we test some of the fundamental assumptions of the widely-used kinetic Tile Assembly Model, obtaining results that fit the model to within error. We then depart from the simplest form of that model, examining the effects of DNA sticky end sequence energetics on tile system behavior. We develop theoretical models, sequence assignment algorithms, and a software package, StickyDesign, for sticky end sequence design.

As a demonstration of a specific tile system, we design a binary counting ribbon that can accurately count from a programmable starting value and stop growing after overflowing, resulting in a single system that can construct ribbons of precise and programmable length. In the process of designing the system, we explain numerous considerations that provide insight into more general tile system design, particularly with regards to tile concentrations, facet nucleation, the construction of finite assemblies, and design beyond the abstract Tile Assembly Model.

Finally, we present our crystals that count: experimental results with our binary counting system that represent a significant improvement in the accuracy of experimental algorithmic self-assembly, including crystals that count perfectly with 5 bits from 0 to 31. We show some preliminary experimental results on the construction of our capping system to stop growth after counters overflow, and offer some speculation on potential future directions of the field.

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.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p

Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Dual modulation of cell survival and cell death by β2-adrenergic signaling in adult mouse cardiac myocytes

The goal of this study was to determine whether β1-adrenergic receptor (AR) and β2-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac β2-AR can activate both Gs and Gi proteins, whereas cardiac β1-AR couples only to Gs. To avoid complicated crosstalk between β-AR subtypes, we expressed β1-AR or β2-AR individually in adult β1/β2-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of β1-AR, but not β2-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, β2-AR (but not β1-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting Gi, Gβγ, or phosphoinositide 3 kinase (PI3K) with pertussis toxin, βARK-ct (a peptide inhibitor of Gβγ), or LY294002, respectively. This indicates that β2-AR activates Akt via a Gi-Gβγ-PI3K pathway. More importantly, inhibition of the Gi-Gβγ-PI3K-Akt pathway converts β2-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, β2-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the Gi-Gβγ-PI3K-Akt signaling pathway.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

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.

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.

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.