Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10⁻² cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.     

A metabolic flux analysis to study the role of sucrose synthase in the regulation of the carbon partitioning in central metabolism in maize root tips

In order to understand the role of sucrose synthase (SuSy) in carbon partitioning, metabolic fluxes were analyzed in maize root tips of a double mutant of SuSy genes, sh1 sus1 and the corresponding wild type, W22. [U-¹⁴C]-glucose pulse labeling experiments permitted the quantification of unidirectional fluxes into sucrose, starch and cell wall polysaccharides. Isotopic steady-state labeling with [1-¹³C]-, [2-¹³C]- or [U-¹³C]-glucose followed by the quantification by ¹H-NMR and ¹³C-NMR of enrichments in carbohydrates and amino acids was also performed to determine 29 fluxes through central metabolism using computer-aided modeling. As a consequence of the suppression of SUS1 and SH1 isozymes, maize root tips diameter was significantly decreased and respiratory metabolism reduced by 30%. Our result clearly established that, in maize root tips, starch is produced from ADP-Glc synthesized in the plastid and not in the cytosol by sucrose synthase. Unexpectedly, the flux of cell wall synthesis was increased in the double mutant. This observation indicates that, in maize root tips, SH1 and SUS1 are not specific providers for cellulose biosynthesis.     

Distribution and submicroscopic immunogold localization of cellular prion protein (PrPc) in extracerebral tissues

In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3

Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause Rett syndrome (RTT), a neurodevelopmental disorder characterized by the loss of language and motor skills during early childhood. We generated mice with a truncating mutation similar to those found in RTT patients. These mice appeared normal and exhibited normal motor function for about 6 weeks, but then developed a progressive neurological disease that includes many features of RTT: tremors, motor impairments, hypoactivity, increased anxiety-related behavior, seizures, kyphosis, and stereotypic forelimb motions. Additionally, we show that although the truncated MeCP2 protein in these mice localizes normally to heterochromatic domains in vivo, histone H3 is hyperacetylated, providing evidence that the chromatin architecture is abnormal and that gene expression may be misregulated in this model of Rett syndrome.     

Production ofAgrobacterium-mediated transgenic fertile plants by direct somatic embryogenesis from immature zygotic embryos ofDatura innoxia

This work describes a new method to obtain transgenic somatic embryos fromAgrobacterium-infected immature zygotic embryos ofDatura innoxia. It has several advantages over previous transformation methods such as the absence of a callus phase, an average transformation rate of 76% and a high regeneration frequency. Critical steps for optimal transformation were the embryo stage and a short preculture treatment. The marker gene -glucuronidase and light microscopy were used to identify the competent embryogenic cells which, after transformation, passed through the classical stages of embryo development. The transgenes were transmitted to the progeny in a Mendelian fashion. The plants regenerated via direct somatic embryogenesis were cytologically and morphologically uniform. We also observed that: (1) wounding or wound-induced divisions were not required for zygotic embryo transformation; (2) epidermal cells were competent for both transformation and regeneration; and (3) competency forAgrobacterium infection was developmental stage-specific. This new method should facilitate the development of new strategies to routinely transform recalcitrant plant species.     

Model for Heterogeneous Random Networks Using Continuous Latent Variables and an Application to a Tree–Fungus Network

Summary The mixture model is a method of choice for modeling heterogeneous random graphs, because it contains most of the known structures of heterogeneity: hubs, hierarchical structures, or community structure. One of the weaknesses of mixture models on random graphs is that, at the present time, there is no computationally feasible estimation method that is completely satisfying from a theoretical point of view. Moreover, mixture models assume that each vertex pertains to one group, so there is no place for vertices being at intermediate positions. The model proposed in this article is a grade of membership model for heterogeneous random graphs, which assumes that each vertex is a mixture of extremal hypothetical vertices. The connectivity properties of each vertex are deduced from those of the extreme vertices. In this new model, the vector of weights of each vertex are fixed continuous parameters. A model with a vector of parameters for each vertex is tractable because the number of observations is proportional to the square of the number of vertices of the network. The estimation of the parameters is given by the maximum likelihood procedure. The model is used to elucidate some of the processes shaping the heterogeneous structure of a well‐resolved network of host/parasite interactions.     

Nutritional n-3 polyunsaturated fatty acids deficiency alters cannabinoid receptor signaling pathway in the brain and associated anxiety-like behavior in mice

N-3 polyunsaturated fatty acids (PUFAs) cannot be synthesized de novo in mammals and need to be provided by dietary means. In the brain, the main n-3 PUFA is docosahexaenoic acid (DHA), which is a key component of neuronal membranes. A low dietary level of DHA has been associated with increased risk of developing neuropsychiatric diseases; however, the mechanisms involved remain to be determined. In this study, we found that long-term exposure to an n-3 deficient diet decreases the level of DHA in the brain and impairs the cannabinoid receptor signaling pathway in mood-controlling structures. In n-3 deficient mice, the effect of the cannabinoid agonist WIN55,212-2 in an anxiety-like behavior test was abolished. In addition, the cannabinoid receptor signaling pathways were altered in the prefrontal cortex and the hypothalamus. Consequently, our data suggest that behavioral changes linked to an n-3 dietary deficiency are due to an alteration in the endocannabinoid system in specific brain areas.     

DNA barcoding in a biodiversity hot spot: potential value for the identification of Malagasy Euphorbia L. listed in CITES Appendices I and II

The island of Madagascar is a key hot spot for the genus Euphorbia, with at least 170 native species, almost all endemic. Threatened by habitat loss and illegal collection of wild plants, nearly all Malagasy Euphorbia are listed in CITES Appendices I and II. The absence of a reliable taxonomic revision makes it particularly difficult to identify these plants, even when fertile, and thereby compromises the application of CITES regulations. DNA barcoding, which can facilitate species‐level identification irrespective of developmental stage and the presence of flowers or fruits, may be a promising tool for monitoring and controlling trade involving threatened species. In this study, we test the potential value of barcoding on 41 Euphorbia species representative of the genus in Madagascar, using the two widely adopted core barcode markers (matK and rbcL), along with two additional DNA regions, nuclear internal transcribed spacer (ITS) and the chloroplastic intergenic spacer psbA‐trnH. For each marker and for selected marker combinations, inter‐ and intraspecific distance estimates and species discrimination rates are calculated. Results using just the ‘official’ barcoding markers yield overlapping inter‐ and intraspecific ranges and species discrimination rates below 60%. When ITS is used, whether alone or in combination with the core markers, species discrimination increases to nearly 100%, whereas the addition of psbA‐trnH produces less satisfactory results. This study, the first ever to test barcoding on the large, commercially important genus Euphorbia shows that this method could be developed into a powerful identification tool and thereby contribute to more effective application of CITES regulations.