Activation of the cGMP signaling pathway is essential in delaying oocyte aging in diabetes mellitus

Uncontrolled diabetes mellitus (DM) adversely affects oocyte maturation and embryo development via mechanisms that are yet unclear. Nonetheless, DM may cause uncoupling of nitric oxide synthases (NOSs) with reduction in the bioavailability of nitric oxide (NO), which is critical to maintain oocyte viability and prevent aging. The current study investigates the role of NO-mediated signaling related to oocyte aging in diabetic and nondiabetic mice. Age-related alterations in the oocytes, including ooplasmic microtubule dynamics (OMD), cortical granule (CG) status, and zona pellucida (ZP) hardening as well as the integrity of the spindle/chromatin were studied using confocal microscopy. Oocytes obtained from diabetic mice exhibited accelerated aging compared to that from nondiabetic mice. Moreover, oocytes from diabetic animals were exquisitely sensitive to NOS and guanylate cyclase (GC) inhibitors (L-NAME, ODQ), which induced aging and relatively resistant to its delay by the cGMP derivative (8-Br-cGMP). Oocytes from nondiabetic control mice displayed similar sensitivity to L-NAME in older oocytes, although to a significantly lower extent than that of DM (P < 0.04-0.0001). Despite the differences in response between DM and nonDM mice, the activation of cGMP pathway is essential to maintain the integrity of oocytes and delay oocyte aging. These findings not only indicate the role of NO signaling in the prevention of oocyte aging but also suggest enhanced aging and NO insufficiency in oocytes from diabetic mice. A comprehensive model incorporating our current findings with NOS, GC, and G kinase cycles is presented.     

Evaluation of cytotoxic properties of organometallic ferrocifens on melanocytes, primary and metastatic melanoma cell lines

Malignant melanoma is one of the most severe forms of skin cancer, and chemotherapeutic agents currently in use are poorly effective in curing the disease. Here we describe the properties of two organometallic ferrocenyl derivatives, ferrocifen (Fc-OH-Tam) and ferrociphenol (Fc-diOH) that show a specific antiproliferative effect on melanoma cells. After a short incubation period, Fc-OH-Tam is highly cytotoxic on melanoma cells but less toxic on melanocytes. Fc-diOH is slightly toxic at a high concentration but no discrepancy is observed between malignant and normal cells. After a long incubation time the latter is highly toxic for malignant cells but not for normal cells while the former was very highly toxic for primary malignant cells and significantly less toxic for normal cells. We also found that oxidative stress is not implicated in the mechanism of cytotoxicity, since both derivatives neither induce reactive oxygen species (ROS) level in melanocytes nor in melanoma cells. Finally, investigation on hair follicle growth revealed that the two organometallic derivatives induced an irreversible ejection of the hair shaft, thus predicting a potential hair loss side effect if used as a chemotherapeutic treatment.     

Genetic Study of Suppressor-Sensitive Mutants of the Bacillus subtilis Bacteriophage φ29

With bacteriophage phi29 of Bacillus subtilis 133, suppressor-sensitive (sus) hydroxylamine mutants have been isolated. Intracistronic and intercistronic quantitative complementation placed the mutants in 13 cistrons, and three-factor crosses have been used to assign an unambiguous order for 10 cistrons. Recombination frequencies have been presented for several regions of the genome to facilitate comparison of the sus system with the previously published temperature-sensitive mapping systems.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

Diversity of plants in cocoa agroforests in the humid forest zone of Southern Cameroon

In the humid forest zone of Southern Cameroon, farmers generally associate cocoa with native and exotic trees in complex agroforestry systems. Despite the socio-economic and ecological importance of these systems, few studies have investigated their plant composition. We investigated tree composition of cocoa agroforests along a gradient of market access, population density and resource use intensity in the humid forest zone of southern Cameroon, comprising (i) the sub-region of Yaoundé, (ii) the sub-region of Mbalmayo, and (iii) the sub-region of Ebolowa. Market access, population density and resource use intensity all decreased from the first to the third sub-region. We quantified the diversity of tree species associated with cocoa within individual agroforests, among agroforests in the same region, and among the three sub-regions, and classified the tree species according to their main uses. A total of 9.1 ha belonging to 60 cocoa agroforests were inventoried in 12 villages. We encountered a total of 206 tree species with an average of 21 tree species per agroforest. In the more urbanized area around Yaoundé, agroforests were less diverse than in the other sub-regions. In all of the agroforests, food producing tree species tended to be more frequent than other species. Two thirds of the food trees were native forest species and one third was introduced. From Ebolowa to Yaoundé, the density of food producing trees doubled and the density of exotic food-producing species increased relative to native species. Some local species producing high-value non-timber forest products were found in the agroforests, but their density was far lower than that of exotic tree species. The agroforests also provide medicine, charcoal and other products for household consumption and sale. We conclude that unless there are specific efforts to promote local forest tree species in cocoa agroforests, these will progressively lose importance with increasing market access, population pressure and land use intensity.     

Purification of an N-acetyl-d-glucosamine specific lectin (P. B. A.) from epidermal cell membranes of Pieris brassicae L

We report the isolation and the purification of an N-acetyl-d-glucosamine specific lectin capable of agglutinating either fixed trypsinized rabbit erythrocytes or chitin particles. An agglutinin assay based on the affinity of this lectin for the chitin was devised with fluorescent particles of scorpion cuticle to measure lectin activity during purification steps.Lectin was isolated from epidermal cell membranes; its molecular weight was determined by gel filtration and polyacrylamide electrophoresis in sodium dodecyl sulfate. Mr was estimated to be 43,000. Lectin could be constituted by two subunits, Mr of which was estimated to be 23,000. The specificity of this lectin against N-acetyl-d-glucosamine and its oligomers suggests a possible role in the dynamics of these saccharides during the cuticle cycle.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Exposure of bovine sperm to pro-oxidants impairs the developmental competence of the embryo after the first cleavage

The present study describes the effects of exposure of bovine sperm to mild and more intense ROS generating conditions. The membrane integrity of the incubated sperm was assessed and the incubated sperm were used for IVF after which the percentages of cleavage and blastocyst formation were determined for a period up to 9 days. The incubated sperm samples showed increased levels of molecular oxidation in the plasma membrane, the mitochondria, the cytosol and to a lesser extent in the sperm's DNA. The sperm membrane integrity as well as the first cleavage rates obtained with sperm from mild ROS generating conditions (100 microM H2O2) were not different from sperm incubated without pro-oxidants. However, exposure of sperm to more severe oxidative stress (500 mM H2O2 or a combination of 100 microM ascorbic acid, 20 microM FeSO4 and 500 microM H2O2) led to plasma membrane oxidation, reduced percentages of cleaved embryos and a reduction in the percentages of cleaved embryos that developed to the blastocyst stage. From these results, we conclude that the impact of oxidative stress to sperm becomes primarily manifest after the first cleavage of the formed zygote. Importantly, the level of lipid peroxidation in the sperm plasma membrane significantly correlates with blastocyst formation when the corresponding sperm is used for in vitro fertilization of oocytes.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at