Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.     

Highly compact folding of chromatin induced by cellular cation concentrations. Evidence from atomic force microscopy studies in aqueous solution

We have performed a very extensive investigation of chromatin folding in different buffers over a wide range of ionic conditions similar to those found in eukaryotic cells. Our results show that in the presence of physiological concentrations of monovalent cations and/or low concentrations of divalent cations, small chicken erythrocyte chromatin fragments and chromatin from HeLa cells observed by transmission electron microscopy (TEM) show a compact folding, forming circular bodies of approximately 35 nm in diameter that were found previously in our laboratory in studies performed under very limited conditions. Since TEM images are obtained with dehydrated samples, we have performed atomic force microscopy (AFM) experiments to analyze chromatin structure in the presence of solutions containing different cation concentrations. The highly compact circular structures (in which individual nucleosomes are not visible as separated units) produced by small chromatin fragments in interphase ionic conditions observed by AFM are equivalent to the structures observed by TEM with chromatin samples prepared under the same ionic conditions. We have also carried out experiments of sedimentation and trypsin digestion of chromatin fragments; the results obtained confirm our AFM observations. Our results suggest that the compaction of bulk interphase chromatin in solution at room temperature is considerably higher than that generally considered in current literature. The dense chromatin folding observed in this study is consistent with the requirement of compact chromatin structures as starting elements for the building of metaphase chromosomes, but poses a difficult physical problem for gene expression during interphase.     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

Examining the Role of Collective Action in an Informal Seed System: A Case Study from the Central Valleys of Oaxaca, Mexico

This paper explores social arrangements associated with seed transactions among small-scale maize farmers in the Central Valleys of Oaxaca, Mexico, where no formal seed supply system exists. We test the hypothesis that individual farmers have strong incentives to participate in some form of collective action to ensure their access to seed. Six communities were studied, three of them in detail, using in-depth, semistructured interviews with key informants; focus group discussions; and a tracer study that followed seed flows among farmers. Farmers mostly saved seed and only occasionally acquired seed from outside sources. We found no evidence of a specialized social organization based on collective action to mediate seed flows. Seed transactions are infrequent, bilateral, and ad hoc, although trust is an important component, as it ensures reliable information about the seed is provided. Implications of these findings are discussed, especially for genetic diversity if the current supply system breaks down.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Evoked response potential markers for anesthetic and behavioral states

The rodent whisker sensory system is a commonly used model of cortical processing; however, anesthetics cause profound differences in the shape and timing of evoked responses. Evoked response studies, especially those that use spatial mapping techniques, such as fMRI or optical imaging, will thus show significantly different results depending on the anesthesia used. To describe the effect of behavioral states and commonly used anesthetics, we characterized the early surface-evoked response potentials (ERPs) components (first ERP peak: gamma band 25-45 Hz; fast oscillation: 200-400 Hz; and very fast oscillation: 400-600 Hz) using a 25-channel electrode array on the somatosensory cortex during whisker stimulation. We found significant differences in the ERP shape when ketamine/xylazine, urethane, propofol, isoflurane, and pentobarbital sodium were administered and during sleep and wake states. The highest ERP amplitudes were observed under propofol anesthesia and during quiet sleep. Under isoflurane, the ERP was nearly absent, except for a very late component, which was concombinant with burst synchronization. The slowest responses were seen under urethane and propofol anesthesia. Spatial mapping experiments that use electrical, NMR, or optical techniques must consider the anesthetic dependency of these signals, especially when stimulation protocols or electrical and metabolic responses are compared.     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

Study on mutagenic effects of bisphenol A diglycidyl ether (BADGE) and its derivatives in the Escherichia coli tryptophan reverse mutation assay

The di-epoxy compound bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (BADGE.H2O and BADGE.2H2O, respectively) and its bis-chlorohydrin derivative (BADGE.2HCl) were examined for their mutagenicity in the Escherichia coli tryptophan reverse mutation test with strains WP2, WP2uvrA and IC3327. The assays were performed in the presence and absence of exogenous metabolic activation (S9 fraction from rat liver). The di-epoxy compound BADGE was able to induce mutagenic effects in strains WP2uvrA and IC3327 and the epoxy-diol BADGE.H2O also showed a positive response with these strains, although the latter was less potent than the former. On the other hand, the lack of mutagenic activity of BADGE.2H2O and BADGE.2HCl was also demonstrated.