Genetic transformation of Eucalyptus globulus using the vascular-specific EgCCR as an alternative to the constitutive CaMV35S promoter

Strong constitutive promoters, such as CaMV35S, are widely used for plant transformation, but undesirable phenotypic changes have been reported when used to drive biotic stress tolerance and/or for modifying lignin content. The promoter of the eucalyptus cinnamoyl CoA reductase (CCR), a key enzyme of the lignin biosynthetic pathway, was shown to be preferentially expressed in vascular tissues both in herbaceous and woody transgenic plants but not eucalyptus. In this work, we transformed Eucalyptus globulus with the EgCCR promoter governing both β-glucuronidase (GUS) and GFP activity patterns. No statistical differences were found between the survival rate and percentage of GUS positive shoots between eucalyptus transformed with either the constitutive CaMV35S or with the EgCCR promoter. The EgCCR transformed plantlets exhibited high GUS expression levels associated with the vascular tissues opening the possibility of targeting vascular-associated traits such as lignin content or vascular pathogen resistance in adult elite plants of eucalyptus while avoiding the undesirable pleiotropic effects caused by strong constitutive promoters.     

Telomere stability and development of ctc1 mutants are rescued by inhibition of EJ recombination pathways in a telomerase-dependent manner

The telomeres of linear eukaryotic chromosomes are protected by caps consisting of evolutionarily conserved nucleoprotein complexes. Telomere dysfunction leads to recombination of chromosome ends and this can result in fusions which initiate chromosomal breakage–fusion–bridge cycles, causing genomic instability and potentially cell death or cancer. We hypothesize that in the absence of the recombination pathways implicated in these fusions, deprotected chromosome ends will instead be eroded by nucleases, also leading to the loss of genes and cell death. In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis. Telomere protection in Arabidopsis implicates KU and CST and their absence leads to chromosome fusions, severe genomic instability and dramatic developmental defects. We have analysed the involvement of end-joining recombination pathways in telomere fusions and the consequences of this on genomic instability and growth. Strikingly, the absence of the multiple end-joining pathways eliminates chromosome fusion and restores normal growth and development to cst ku80 mutant plants. It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects. This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.     

PSD95 Suppresses Dendritic Arbor Development in Mature Hippocampal Neurons by Occluding the Clustering of NR2B-NMDA Receptors

Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2–25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.     

Basolateral expression of GRP94 in parietal cells of gastric mucosa

GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na₂CO₃ treatment dissociates part of it from the membrane. However, 100 mM Na₂CO₃ treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-lig- and binding experiments. Both the K _D value obtained in saturation experiments with N-ethylcarboxamido-[³H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.     

Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady‐state retention of mutant forms in the proximal golgi

The melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to reddish, poorly photoprotective pheomelanins. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation (the RHC phenotype) and increased skin cancer risk. Three highly penetrant RHC variants, R151C, R160W, and D294H are loss‐of‐function MC1R mutants with altered cell surface expression. In this study, we show that forward trafficking was normal for D294H. Conversely, export traffic was impaired for R151C, which accumulated in the endoplasmic reticulum (ER), and for R160W, which was enriched in the cis‐Golgi. This is the first report of steady‐state retention in a post‐ER secretory compartment of a GPCR mutant found in the human population. Residues R151 and R160 are located in the MC1R second intracellular loop (il2). Two other mutations in il2, T157A preventing T157 phosphorylation and R162P disrupting a ¹⁶⁰RARR¹⁶³ motif, also caused intracellular retention. Moreover, T157 was phosphorylated in wild‐type MC1R and a T157D mutation mimicking constitutive phosphorylation allowed normal traffic, and rescued the retention phenotype of R160W and R162P. Therefore, MC1R export is likely regulated by T157 phosphorylation and the ¹⁶⁰RARR¹⁶³ arginine‐based motif functions as an ER retrieval signal. These elements are conserved in mammalian MC1Rs and in all five types of human melanocortin receptors. Thus, members of this GPCR subfamily might share common mechanisms for regulation of plasma membrane expression. J. Cell. Physiol. 220: 640–654, 2009. © 2009 Wiley‐Liss, Inc.     

Nitrogen compounds in embryogenic and non-embryogenic calluses of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Nitrogen (N) metabolism during embryogenesis may be fundamental in the embryogenic response. We used different explants of Medicago arborea L. subsp. arborea seedlings: cotyledons, petioles and leaves, which form calluses with different embryogenic responses. The endogenous contents of total nitrogen, nitrate, nitrite and ammonia and nitrate reductase activity were determined in embryogenic and non-embryogenic calluses induced from the different explants. The endogenous total N content decreased in the calluses as the culture time progressed, this decrease being more pronounced in the more embryogenic calluses obtained from petioles with the H8 and F0 media. Inorganic N decreased during embryogenesis, coinciding with an increase in organic N. Thus, N metabolism somehow seems to be essential in embryogenesis. The N detected in calluses, at the start of culture, was mainly metabolised to nitrite. This metabolism was very pronounced; especially in embryogenic calluses obtained from cotyledons and petioles. That is, the metabolism of N seemed to be more marked in the calluses in which embryogenesis was greater. The nitrite content decreased in all the calluses, the contents being lower, especially in the last months of culture, in the more embryogenic calluses obtained from petioles. In many calluses, ammonia levels did not follow any general pattern. Neither was it possible to detect changes in ammonia levels between the embryogenic and non-embryogenic calluses. Regarding nitrate reductase activity, no clear differences between embryogenic and non-embryogenic calluses were found.     

Reproductive axis response to repeated lipopolysaccharide administration in peripubertal female rats

Immune system disorders are often accompanied by alterations in the reproductive axis. Several reports have shown that administration of bacterial lipopolysaccharide (LPS) has central inflammatory effects and activates cytokine release in the hypothalamus where the luteinizing hormone releasing hormone (Gn-RH) neurons are located. The present study was designed to investigate the effect of repeated LPS administration on the neuroendocrine mechanisms of control of the reproductive axis in peripubertal female rats (30-day-old rats). With this aim, LPS (50 μg/kg weight) was administered to the animals during 25, 27 and 29 days of age and sacrificed on 30 day of life. Gn-RH, γ?amino butyric acid (GABA) and glutamic acid (GLU), two amino acids involved in the regulation of Gn-RH secretion, hypothalamic content were measured. LH and estradiol serum levels were also determined and the day of vaginal opening examined. The results showed a significant increase in Gn-RH and GLU content (p?<?0.0001), shared by a reduction of GABA one (p?<?0.0001). LH and estradiol serum levels were decreased (p?<?0.01, p?<?0.001) and delay in the day of vaginal opening was also observed in treated animals. Present results show that repeated LPS administration impaired reproductive function, modifying the neuroendocrine mechanisms of control of the axis in peripubertal female rats.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.