In vitro organogenesis and plant formation in Acacia sinuata

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973–25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.     

A mutation in the Cdon gene potentiates congenital nevus development mediated by NRASQ61K

Congenital nevi develop before birth and sometimes cover large areas of the body. They are presumed to arise from the acquisition of a gene mutation in an embryonic melanocyte that becomes trapped in the dermis during development. Mice bearing the Cdk4^R24C::Tyr‐NRAS^Q61K transgenes develop congenital nevus‐like lesions by post‐natal day 10, from melanocytes escaping the confines of hair follicles. We interbred these mice with the collaborative cross (CC), a resource that enables identification of modifier genes for complex diseases (those where multiple genes are involved). We examined variation in nevus cell density in 66 CC strains and mapped a large‐effect quantitative trait locus (QTL) controlling nevus cell density to murine chromosome 9. The best candidate for a gene that exacerbates congenital nevus development in the context of an NRAS mutation is Cdon, a positive regulator of sonic hedgehog (Shh) that is expressed mainly in keratinocytes.     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Tumor-suppressive effects of psoriasin (S100A7) are mediated through the β-catenin/T cell factor 4 protein pathway in estrogen receptor-positive breast cancer cells

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.     

Plant growth promotion and suppression of charcoal‐rot fungus (Macrophomina phaseolina) in velvet bean (Mucuna pruriens L.) by root nodule bacteria

Root‐nodulating bacteria are intimate associates of legumes. From a pool of rhizobia isolated from root nodules of Mucuna pruriens (Velvet bean/Kaunch), RMP66 and BMP17 were found to be capable of promoting siderophore and IAA production and phosphate solubilization (insoluble tri‐calcium). Both symbionts were studied further to determine their abilities to promote plant growth and to control root‐rot in Mucuna pruriens caused by the pathogenic plant fungus Macrophomina phaseolina. RMP66 and BMP17 were selected based on their excellent inhibitory activities against M. phaseolina (by 78% and 71%, respectively) in dual culture and in agar‐well assays using cell‐free culture filtrate (CFCF) (by 76% and 62%, respectively). Both strains inhibited fungal growth to a greater extent in iron‐deficient medium (51% and 69%) than in iron‐supplemented medium (37% and 0%), respectively. CFCFs of RMP66 and BMP17 obtained from Pikovskaya's broth and tryptophan‐amended YEM broth inhibited fungal growth by 80%‐55% and 70%‐43%, respectively, and were identified as Sinorhizobium meliloti RMP66 and Bradyrhizobium diazoefficiens BMP17 by 16S rDNA sequencing. Centrifuged and pelleted cells harvested from exponentially grown cultures of S. meliloti RMP66 and B. diazoefficiens BMP17 were used to bacterize seeds of M. pruriens, which then showed enhanced seed germination (by up to 17% and 12%, respectively), and subsequent increases in other plant growth parameters in field trials. Considerable increases in seedling vigour indices (62%: 53% and 110%: 130%) and biomass (8%: 13% and 25%: 28%) were also observed for bacterial treatments. Tn5‐mediated antibiotic‐resistant marker strains showed enhanced nodule occupancy by up to 72% and 68%, respectively. This study describes a multifunctional legume nodule rhizobia that could be utilized in multicropping systems under different agroclimatic conditions as a bioinoculant and alternative to fertilizers.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease

We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.