Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.     

Phosphorylation relieves autoinhibition of the kinetochore motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.     

Environmental Response and Genomic Regions Correlated with Rice Root Growth and Yield under Drought in the OryzaSNP Panel across Multiple Study Systems

The rapid progress in rice genotyping must be matched by advances in phenotyping. A better understanding of genetic variation in rice for drought response, root traits, and practical methods for studying them are needed. In this study, the OryzaSNP set (20 diverse genotypes that have been genotyped for SNP markers) was phenotyped in a range of field and container studies to study the diversity of rice root growth and response to drought. Of the root traits measured across more than 20 root experiments, root dry weight showed the most stable genotypic performance across studies. The environment (E) component had the strongest effect on yield and root traits. We identified genomic regions correlated with root dry weight, percent deep roots, maximum root depth, and grain yield based on a correlation analysis with the phenotypes and aus, indica, or japonica introgression regions using the SNP data. Two genomic regions were identified as hot spots in which root traits and grain yield were co-located; on chromosome 1 (39.7–40.7 Mb) and on chromosome 8 (20.3–21.9 Mb). Across experiments, the soil type/ growth medium showed more correlations with plant growth than the container dimensions. Although the correlations among studies and genetic co-location of root traits from a range of study systems points to their potential utility to represent responses in field studies, the best correlations were observed when the two setups had some similar properties. Due to the co-location of the identified genomic regions (from introgression block analysis) with QTL for a number of previously reported root and drought traits, these regions are good candidates for detailed characterization to contribute to understanding rice improvement for response to drought. This study also highlights the utility of characterizing a small set of 20 genotypes for root growth, drought response, and related genomic regions.     

Trait‐based approaches to analyze links between the drivers of change and ecosystem services: Synthesizing existing evidence and future challenges

Understanding the responses of biodiversity to drivers of change and the effects of biodiversity on ecosystem properties and ecosystem services is a key challenge in the context of global environmental change. We performed a systematic review and meta‐analysis of the scientific literature linking direct drivers of change and ecosystem services via functional traits of three taxonomic groups (vegetation, invertebrates, and vertebrates) to: (1) uncover trends and research biases in this field; and (2) synthesize existing empirical evidence. Our results show the existence of important biases in published studies related to ecosystem types, taxonomic groups, direct drivers of change, ecosystem services, geographical range, and the spatial scale of analysis. We found multiple evidence of links between drivers and services mediated by functional traits, particularly between land‐use changes and regulating services in vegetation and invertebrates. Seventy‐five functional traits were recorded in our sample. However, few of these functional traits were repeatedly found to be associated with both the species responses to direct drivers of change (response traits) and the species effects on the provision of ecosystem services (effect traits). Our results highlight the existence of potential “key functional traits,” understood as those that have the capacity to influence the provision of multiple ecosystem services, while responding to specific drivers of change, across a variety of systems and organisms. Identifying “key functional traits” would help to develop robust indicator systems to monitor changes in biodiversity and their effects on ecosystem functioning and ecosystem services supply.     

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.     

Few cultured rat primary sensory neurons express a tolbutamide-sensitive K+ current

The response of dorsal root ganglion (DRG) neurons to metabolic inhibition is known to involve calcium-activated K⁺ channels; in most neuronal types ATP-sensitive K⁺ channels (K_ATP) also contribute, but this is not yet established in the DRG. We have investigated the presence of a K_ATP current using whole-cell recordings from rat DRG neurons, classifying the neurons functionally by their "current signature" (Petruska et al, J Neurophysiol 84: 2365–2379, 2000). We clearly identified a K_ATP current in only 1 out of 62 neurons, probably a nociceptor. The current was activated by cyanide (2 mM NaCN) and was sensitive to 100 μM tolbutamide; the relation between reversal potential and external K⁺ concentration indicated it was a K⁺ current. In a further two neurons, cyanide activated a K⁺ current that was only partially blocked by tolbutamide, which may also be an atypical K_ATP current. We conclude that K_ATP channels are expressed in normal DRG, but in very few neurons and only in nociceptors.     

Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region

Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape. DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway. The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway. In the present study the promoter of the grape dfr gene was cloned. Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs. The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits. The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems. Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits. The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits. Light, calcium and sucrose induced the dfr gene expression. In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light. The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene. The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways.     

Random amplified polymorphic DNA profiles as a tool for the identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates.     

Random Amplified Polymorphic DNA Profiles as a Tool for the Identification of Acanthamoeba divionensis

In the present study, we demonstrated the Random Amplified Polymorphism DNA (RAPD) diagnostic validity. In our study, we have analyzed RAPD profiles searching for characteristic and useful bands for Acanthamoeba diagnosis at the species level. We found a distinctive 370-bp band in A. divionensis RAPD patterns, using the OPC14 primer (TGCGTGCTTG) (Operon Technologies, Inc., Alameda, CA). The band specificity was confirmed by hybridization, using it as a probe, against all OPC14 amplifications from 10 different Acanthamoeba species. Once we sequenced this band, we used it to design a specific primer pair which showed positive amplification only in A. divionensis isolates. RID= ID= <E5>Correspondence to:</E5> A. Ortega-Rivas; <E5>email</E5>: aortega&commat;ull.es Received: 5 August 2002 / Accepted: 23 September 2002