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The pool of ovarian primordial follicles is established during embryonic development or at birth. During the development from primordial to primary, secondary, and antral follicles, only a small portion of follicles can mature and successfully ovulate; the others are destined to degenerate through apoptotic or atretic loss. As aging advances, females ultimately enter the cessation phase of the estrous cycle and are no longer capable of fertilization. The presumption is that if we can slow down the process of folliculogenesis or decrease follicle loss, females may have a larger ovarian follicular reserve and a longer reproductive lifespan. In our study, rats underwent intragastric administration with tea polyphenols, quercetin (meletin), genistein, or resveratrol, once a day for 4 months (from age 12 to 15 months), to test whether they have positive effects on follicular reserve or ovarian functions. The results showed that rats treated with tea polyphenols (27.8 ± 3.2) and quercetin (36.5 ± 4.1) had a comparable number of healthy follicles to those of controls (26.9 ± 3.8), although significantly fewer atretic follicles were observed in the tea polyphenol group (43.4 ± 5.9 vs 79.7 ± 7.5; p < 0.001). Remarkably, both genistein- and resveratrol-treated rats had more healthy follicles (respectively, 42.8 ± 3.9, p < 0.05; and 51.9 ± 6.4, p < 0.001) and fewer atretic follicles (respectively, 58.4 ± 8.0, p < 0.05; and 51.0 ± 6.2, p < 0.01) than controls. These results indicate that genistein and resveratrol can increase the ovarian follicular reserve and prolong the ovarian lifespan in rats, and their positive effects may be not only due to their intervention in the transition from primordial to primary follicle, but also due to the inhibiting effect on follicular atresia. Key words: early aged rats, ovarian follicular reserve, polyphenols, estrous cycle, reproductive lifespan. La reserve de follicules ovariens primordiaux est etablie au cours du developpement embryonnaire ou a la naissance. Lors du developpement des follicules primordiaux en follicules primaires, puis secondaires et antraux, seule un petit nombre d'entre eux parviendront a maturite pour conduire avec succesa l'ovulation, alors que les autres seront destines a degenerer par apoptose ou atresie. Avec l'age, le cycle oestral cesse et les femelles ne peuvent plus etre fecondees. L'on peut presumer que si le processus de folliculogenese est ralenti et que le nombre de follicules degeneres est diminue, les femelles pourraient avoir une reserve folliculaire plus grande et une vie reproductive plus longue. Dans notre etudes, des rates ont ete soumises a une administration intra-gastrique quotidienne de polyphenols du the, de quercetine (meletine), de genisteine et de resveratrol, respectivement, pendant 4mois (de 12 mois a 15 mois d'age) afin de tester si ces traitements exercaient des effets positifs sur la reserve folliculaire ainsi que sur les fonctions ovariennes. Les resultats ont montre que les rates traitees aux polyphenols du the (27,82 ± 3,2) ou a la quercetine (36,5 ± 4,1) possedaient un nombre total de follicules sains comparable a celui du groupe controle (26,9 ± 3,8), alors que la perte de follicules par atresie etait significativement moins elevee dans le groupe traite aux polyphenols du the (43,4 ± 5,9 vs 79,7 ± 7,5) (p < 0,001). Fait remarquable, les rates traitees a la genisteine et au resveratrol possedaient davantage de follicules sains (42,8 ± 3,9, 51,9 ± 6,4) (p < 0,05, p < 0,001) et moins de follicules en atresie (58,4 ± 8,0, 51,0 ± 6,2) (p < 0.05, p < 0,01) que le groupe controle. Ces resultats indiquent que la genisteine et le resveratrol pourraient augmenter la reserve folliculaire et prolonger la duree de vie ovarienne chez la rate, et que leurs effets positifs pourraient etre dus non seulement a leur intervention lors de la maturation des follicules primordiaux en follicules primaires, mais aussi a leur effet inhibiteur de l'atresie folliculaire. Mots-cles: premiers stades de vieillissement chez la rate, reserve de follicules ovariens, polyphenols, cycle oestral, duree de vie reproductive. [Traduit par la Redaction], Introduction The pool of ovarian primordial follicles is established during embryonic development in some mammals (e.g., cows, sheep, humans) and after birth in others (e.g., rodents). This finite reserve of [...]

The sirtuin proteins are nicotinamide adenine dinucleotide dependent deacetylases and adenosine diphosphate (ADP)-ribosyl transferases associated with metabolic balance and lifespan extension. Sirtuin 1 (SIRT1) and sirtuin 4 (SIRT4) have been reported to regulate insulin secretion, but their association with the development of insulin resistance and nonalcoholic fatty liver disease remain undefined. The aim of this study was to determine the expression of SIRT1 and SIRT4 in the liver and pancreas of rats fed with different diets and analyze the association of these proteins with insulin resistance and nonalcoholic fatty liver disease. Male Sprague-Dawley rats were randomly divided into the following 4 diet treatment groups: normal control (NC), calorie restriction (CR), high-fat (HFa), and high-fructose (HFr), and these groups were maintained for 12 weeks. Blood biochemical analysis and histopathology indicated that HFa and HFr groups were insulin resistant and developed nonalcoholic fatty livers. SIRT1 was present in the nucleus and cytoplasm of the pancreatic β-cells, while SIRT4 was located in the cytoplasm. Treatment with the CR diet increased the expression of SIRT1 in both the pancreas and liver, while treatment with the HFa and HFr diets caused a decrease. SIRT4 was upregulated in the liver of rats treated with the HFa diet, but did not change with the CR diet treatment. These data suggest that SIRT1 and SIRT4 were both involved in the development of insulin resistance and nonalcoholic fatty liver disease. Key words: SIRT1 and SIRT4 expression, caloric restriction, insulin resistance, nonalcoholic fatty liver disease in rats. Les sirtuines sont des deacetylases dependantes du nicotinamide adenine dinucleotide, et des adenosinediphosphate (ADP)-ribosyl transferases associees a la balance metabolique et a l'allongement de la duree de vie. La sirtuine 1 (SIRT1) et la sirtuine 4 (SIRT4) regulent la secretion d'insuline, mais leur association au developpement de la resistance a l'insuline et de la steatose hepatique non alcoolique demeure mal definie. Cette etude visait a determiner les niveaux d'expression de SIRT1 et SIRT4 dans le foie et le pancreas de rats soumis a differentes dietes, et a analyser l'association de ces proteines a la resistance a l'insuline et a la steatose hepatique non alcoolique. Des rats males Sprague-Dawley ont ete divises aleatoirement en quatre groupes selon la diete: controle normal (CN), pauvre en calories (PC), riche en gras (RG) et riche en fructose (RF), et ces groupes ont ete traites pendant 12 semaines. Des analyses sanguines biochimiques et l'histopathologie ont indique que les groupes RG et RF etaient devenus resistants a l'insuline et avaient developpee une steatose hepatique non alcoolique. SIRT1 etait presente dans le noyau et le cytoplasme des cellules β du pancreas, alors que SIRT4 etait localisee dans le cytoplasme. Une diete PC augmentait l'expression de SIRT1 dans le pancreas et le foie, alors que les dietes RG et RF la diminuaient. L'expression de SIRT4 etait stimulee dans le foie des rats traiteesa la diete RG, mais ne changeait pas apres une diete PC. Ces resultats suggerent que SIRT1 et SIRT4 sont toutes deux impliqueees dans le developpement de la resistance a l'insuline et de la steatose hepatique non alcoolique. Mots-cles: expression de SIRT1 et SIRT4, restriction calorique, resistance a l'insuline, steatose hepatique non alcoolique chez le rat. [Traduit par la Redaction], Introduction The increasing incidence of diabetes mellitus is primarily the result of more sedentary lifestyles and high-calorie intakes as Insulin resistance (IR), the core of diabetes mellitus, is characterized by [...]

Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a. Key words: FOXO3a, oocyte apoptosis, neonatal rat ovary. Des etudes precedentes ont propose; que le facteur de transcription membre de la famille << forkhead >> FOXO3a soit implique dans l'arret du cycle cellulaire et l'apoptose, et qu'il puisse aussi reprimer le developpement folliculaire en induisant l'arret du cycle cellulaire dans l'ovaire. Nous avons recemment demontre que FOXO3a induit l'apoptose des ovocytes de rates neonatales in vitro. Dans cette presente etude, nous avons evalue le role de FOXO3a dans l'apoptose des ovocytes in vivo. Les ovaires de rates ont ete obtenus de nouveau-nes aux jours postnataux (PN) 1, 2, 3 et 4. Les resultats de l'essai TUNEL ont montre que l'apoptose des ovocytes survenait principalement aux jours PN1 et 2. Une coloration immunohistochimique de FOXO3a, de Bim, du ligand de Fas (FasL), de p27KIP1, de la casapase-8 et de la caspase-3 a montre qu'ils etaient tous exprimes principalement dans les ovocytes nus aux jours PN 1 et 2. Le pourcentage d'ovocytes colores positivement pour FOXO3a atteignait un pic dans les ovaires des rates de 2 jours, ce qui concordait avec le taux d'apoptose determine par TUNEL. Les pourcentages de cellules positives en TUNEL et a FOXO3a nucleaire n'etaient pas significativement differents dans les ovaires des rates de 4 jours. De plus, le pourcentage d'ovocytes positifs aux facteurs cibles de FOXO3a (Bim, p27KIP1 et FasL) et aux proteines pro-apoptose (caspase-2 et caspase-8) atteignait aussi un pic dans les ovaires de rates de 2 jours, similairement aux taux d'ovocytes positifs a FOXO3a. Ces resultats suggerent que FOXO3a present dans le noyau des ovocytes est implique dans l'apoptose des ovocytes, et ce faisant, que les ovocytes positifs a FOXO3a peuvent etre en apoptose. Afin de verifier ceci, les ovocytes de rates ont ete soumis aux essais TUNEL et a un double marquage en immunofluorescence. Nous avons trouve que les cellules positives en TUNEL sont aussi positives a FOXO3a, Bim, et FasL. Afin d'identifier les cibles de FOXO3a, une double coloration en immunofluorescence a l'aide d'anticorps diriges contre Bim et FasL a ete realisee. Nous avons trouve que les cellules positives a FOXO3a etaient aussi positives a Bim et a FasL. Nous concluons donc que la surexpression de FOXO3a dans le noyau des ovocytes des ovaires des rates neonatales peut jouer un role important dans l'apoptose des ovocytes nus, et que Bim, FasL et p27KIP1 sont des facteurs cles en aval de FOXO3a. Mots-cles: FOXO3a, apoptose des ovocytes, ovaire de rate neonatale. [Traduit par la Redaction], Introduction It is widely accepted that the wave of oocyte degeneration, which leads to the formation of a fixed reserve pool of primordial follicles, occurs during the first 3 days [...]

Abstract: The Ras subfamily proteins are small, monomeric GTP-binding proteins with vital roles in regulating eukaryotic signal transduction pathways. Gene duplication and divergence have been postulated as the mechanism by [...]

Abstract: Spliceosomal introns play a key role in eukaryotic genome evolution and protein diversity. A large Rab GTPase family has been identified in a unicellular eukaryote Trichomonas vaginalis. However, the characteristics of introns in Rab genes of T. vaginalis have not been investigated previously. In this study, we identified a 25-bp spliceosomal intron in the T. vaginalis Rab1a (TvRab1a) gene, the smallest intron in T. vaginalis to be characterized to date. This intron contains a canonical splice site at both 5′ (GT) and 3′ (AG) ends, and a putative branch-point sequence (TCTAAC) that matches the Trichomonad consensus sequence of ACTAAC except for the first nucleotide. The position and phase of the TvRab1a intron are evolutionarily conserved in Rab1 homologous genes across at least five eukaryotic supergroups, including Opisthokonta, Amoebozoa, Excavata, Chromalveolata, and Plantae. These results strongly suggest that the TvRab1a intron is likely to be an ancient spliceosomal intron, and it can therefore be used as a phylogenetic marker to evaluate particular eukaryotic groupings. Identification and characterization of the TvRabla intron may provide an insight into the evolution of the large Rab repertoire in T. vaginalis. [Copyright &y& Elsevier]

Atherosclerosis is a chronic immunoinflammatory disease associated with blood lipid disorders. Previous studies in mice have demonstrated that liver X receptor (LXR)‑ATP‑binding cassette (ABC) A1/ABCG1/C‑C chemokine receptor type 7 (CCR7) and nuclear factor κB (NF‑κB) signaling pathways are important for atherosclerotic plaque formation. In addition, Sirtuin 1 (SIRT1) has been reported as a key regulator in the protection from risk of atherosclerosis. However, the exact mechanism by which SIRT1 prevents atherosclerosis remains largely unknown. To explore the possible mechanisms, the expression of SIRT1 and the association between SIRT1, LXR and NF‑κB in the process of foam cell formation was investigated in an in vitro human mononuclear U937 cell line. Monocyte‑derived foam cells were induced by palmitate and Ox‑LDL treatment. Oil Red O staining revealed an accumulation of a large number of lipid droplets in foam cells. Results of reverse transcription polymerase chain reaction (RT-PCR) revealed that SIRT1 expression was downregulated during foam cell formation. In addition, the expression of LXRα and its targets, ABCA1, ABCG1 and CCR7, were downregulated. However, NF‑κB and its targets, tumor necrosis factor α (TNFα) and interleukin (IL)‑1β, were upregulated in foam cells. Following activation of SIRT1 by SRT1720, the expression of LXRα and its targets increased, whereas expression of NF‑κB and its targets decreased. Furthermore, the formation of foam cells was blocked. The SIRT1 inhibitor, nicotinamide, was found to eliminate the effects of SRT1720. Results of the present study indicate that SIRT1 may prevent the formation and progression of atherosclerosis by enhancing the LXR‑ABCA1/ABCG1/CCR7 and inhibiting the NF‑κB pathways.

Objective: To study the regulation effects of CR (caloric restriction) and SIRT3 in the H2O2-induced oxidative stress injury of PC12 cell., Methods: The cells were divided into four groups: H2O2, H2O2 + CR, CR and control (high glucose). For control and H2O2 group, cells were cultured in DMEM containing 0.45% glucose; for group in CR condition, cells were treated with the medium containing 0.1% glucose. For groups with H2O2, the H2O2 was diluted in EMEM medium to obtain the final concentration containing 60 µmol/L. Viability of PC12 cells were measured by MTT assay. The medium was refreshed with different concentration of H2O2 (from 10 to 120 µmol/L). The absorbance of the samples was measured at 492 nm using a microtiter plate reader. We detected TUNEL-positive cells using the In Situ Cell Apoptosis Detection kit pretreated with CR and H2O2. Immunofluorescence double staining detected the expression and localization of SIRT3. RT-PCR and Western-blot methods detected the expression of SIRT3, Caspase-3., Results: After pretreating with 60 µmol/L H2O2 for 6 h, the viability of PC12 cells in H2O2 group (74.01 ± 2.21)% retained above 70%, and have statistical significance contrasted with control group (P < 0.05); After pretreating with 120 µmol/L H2O2, the viability of PC12 cells declined significantly (38.22 ± 3.34)%. So 60 µmol/L H2O2 is our experiment concentration. The viability of H2O2 group (74.01 ± 2.21)% was much lower than CR + H2O2 group (97.26 ± 1.92)% (P < 0.05). After pretreating with H2O2, TUNEL staining showed the apoptosis cells of CR + H2O2 group decreased significantly contrasted with H2O2 group. The immunofluorescence double staining results showed that SIRT3 was a mitochondria protein. Western-blot showed the expression of SIRT3 in CR group (6857 ± 157) (P < 0.05) increased and decreased in H2O2 group (3786 ± 160) (P < 0.05) contrasted with control group (5256 ± 143). The expression of SIRT3 in CR + H2O2 group (5056 ± 121) (P < 0.05) increased contrasted with H2O2 group (3786 ± 160). We also detected that Caspase-3 in H2O2 group (8499 ± 426) (P < 0.001) was much higher than control group than (5342 ± 420), but in the CR + H2O2 group (5750 ± 438) the expression of Caspase-3 was much lower than H2O2 group (8499 ± 426) (P < 0.001). RT-PCR also showed that the expression of SIRT3 in CR group (7214 ± 148) increased and decreased in H2O2 group (4807 ± 143) (P < 0.05) contrasted with control group (6204 ± 134). The expression of SIRT3 in CR + H2O2 group (6195 ± 166) increased contrasted with H2O2 group (4807 ± 143) (P < 0.05)., Conclusion: CR causes anti-oxidative injury and has apoptotic effects in PC12 cell. It up-regulates the expression of SIRT3 and the effects of CR-SIRT3 can prevent PC12 cell from H2O2-induced apoptosis. And SIRT3 may be a novel molecule of regulating target in the delay of neuronal senescence.

This study aimed at investigating the effect of quercetin on neointima hyperplasia in the abdominal aorta of rats after balloon injury and expressions of related growth factors. Fifty-four healthy male Sprague-Dawley rats were randomly divided into five groups: a sham-operation group (sham, n=6), a control group (control, n=12), and three quercetin-treated groups: Q50 group (50mg/kg body weight/day, n=12), Q100 group (100mg/kg body weight/day, n=12), and Q200 group (200mg/kg body weight/day, n=12) 3 days before balloon injury until the end of the experiment. Fourteen days after injury, rats were killed, and the abdominal aortas were harvested. Hematoxylin-eosin staining showed that quercetin significantly reduced the neointimal areas and the intimal to medial ratio in the Q100 and Q200 groups 14 days after injury. Immunohistochemical analysis showed that quercetin significantly inhibited PCNA, PDGF-BB, b-FGF, and TGF-beta1 expressions in the neointima. Masson's trichrome showed that quercetin significantly reduced collagen deposition in the neointima. We concluded that quercetin significantly inhibited neointimal hyperplasia in rat abdominal aorta 14 days after injury in relatively high doses. This effect of quercetin might be partially attributed to the suppression of PDGF-BB, b-FGF, and TGF-beta1 expressions.

Objective: To clone and characterize a RRas-like gene from Trichomonas vaginalis for studying cellular signal transduction pathways in the organism., Methods: A cDNA clone, which showed homology with RRas proteins of human being, was isolated and sequenced from a cDNA expression library of T. vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified using PCR technique and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and Clustal W programs. Phylogenetic tree was constructed and bootstrapped with 1050 replicates using the software MEGA3., Results: The cDNA sequence showed a length of 705 bp with an open reading frame of 615 bp. The deduced amino acid sequence from the open reading frame possesses 205 residuals. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codons was identical to the cDNA sequence. Sequence analysis demonstrated that this gene was most homologous to the RRas members of Homo sapiens and Mus musculus (both having 51% identity and 70% similarity), and the amino acid sequence contains highly conserved GTP-binding domains and a fully conserved effector domain of human RRas member. Phylogenetic analysis showed that TvRRas clustered with RAS oncoprotein branch and RRAS branch of human., Conclusion: The encoding protein probably belongs to a RRas family of T. vaginalis.

Total RNA was isolated from Trichomonas vaginalis and Tv-Sir2-like cDNA was amplified by RT-PCR and cloned into pGEM-T Easy plasmid. A fragment of Tv-Sir2-like cDNA was subcloned into the expression vector pET-41b and expressed in E.coli BL21 with induction of IPTG. The full-length of Tv-Sir2-like cDNA was cloned and sequenced. The prokaryotic expression system of pET-41b/Tv-Sir2-like was constructed. The fusion protein of Tv-Sir2-like was expressed in E. coli BL21, occupying 30% of the total bacterial protein after being induced by IPTG for 5 h. SDS-PAGE analysis showed that the fusion protein was about Mr 59000. The recombinant protein of Tv-Sir2-like is efficiently expressed in E. coli BL21.

Objective: To inhibit the expression of the target genes of peroxiredoxin (Prx) and thioredoxin reductase (TrxR) by RNA interference and evaluate its effect on the growth of Trichomonas vaginalis., Methods: Genomic DNA was extracted from cultured Trichomonas vaginalis with phenol-chloroform method and was transcribed to double stranded RNA (dsRNA). Short interference RNAs (siRNA, 21-23 bp) synthesized by digestion of dsRNA with RNase III and purified through filter cartridge, were transfected into the cells in three groups (A, B and C) to degrade the target genes of Prx, TrxR and Prx+TrxR through siPORT lipid, respectively, and the untransfected was selected as a control (group D). The levels of Prx and TrxR mRNA were determined 24 h and 48 h post-transfection by relative quantitative RT-PCR, and the growth of Trichomonas vaginalis was estimated under microscope 36 h post-transfection., Results: Trichomonas vaginalis mRNA levels of Prx and TrxR decreased. Though the cell activity showed no significant difference (P > 0.05) in four groups as expected, a difference existed (P < 0.01) between the groups in the average of vells (7.2 x 10(7)/L, 14.2 x 10(7)/L, 3.8 x l0(7) L and 20.3 x 10(7)/L in groups A, B, C and D respectively)., Conclusions: RNA interference inhibits the expression of the genes of Prx and TrxR and extended Trichomonas vaginalis cells cycle considerably, but showed no influence on the cell activity.

Objective: To clone and characterize a Rac1 homologue from Trichomonas vaginalis for studying cell cycle of the organism., Methods: A cDNA library derived from T. vaginalis mRNA was constructed into lambda TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs., Results: A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with >60% identity. The conserved sequence elements of Rho GTPases, such as GTP-binding sites, GTPase-activating protein (GAP) interaction motifs, GTP-dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa., Conclusion: The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.