Molecular basis of alpha-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.

A mutant of Arabidopsis thaliana, amt-1, was previously selected for resistance to growth inhibition by the tryptophan analog alpha-methyltryptophan. This mutant had elevated tryptophan levels and exhibited higher anthranilate synthase (AS) activity that showed increased resistance to feedback inhibition by tryptophan. In this study, extracts of the mutant callus exhibited higher AS activity than wild-type callus when assayed with either glutamine or ammonium sulfate as amino donor, thus suggesting that elevated AS activity in the mutant was due to an alteration in the alpha subunit of the enzyme. The mutant also showed cross-resistance to 5-methylanthranilate and 6-methylanthranilate and mapped to chromosome V at or close to ASA1 (a gene encoding the AS alpha subunit). ASA1 mRNA and protein levels were similar in mutant and wild-type leaf extracts. Levels of ASA1 mRNA and protein were also similar in callus cultures of mutant and wild type, although the levels in callus were higher than in leaf tissue. Sequencing of the ASA1 gene from amt-1 revealed a G to A transition relative to the wild-type gene that would result in the substitution of an asparagine residue in place of aspartic acid at position 341 in the predicted amino acid sequence of the ASA1 protein. The mutant allele in strain amt-1 has been renamed trp5-1.     

Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates.

Two mutants of Arabidopsis thaliana that are resistant to growth inhibition by indole-3-acetic acid (IAA)-phenylalanine have been isolated. Both mutants were 2- to 3-fold more resistant than wild type to inhibition by IAA-phenylalanine, IAA-alanine, and IAA-glycine in root growth assays. The mutant icr1 (but not icr2) also shows some resistance to IAA-aspartate. Studies using 3H-labeled IAA-phenylalanine showed that the uptake of conjugate from the medium by icr1 was the same as wild type and was reduced by about 25% in icr2. No differences in hydrolysis of the exogenous conjugate were detected between the mutants and their wild-type parents. There was no significant metabolism of the IAA released from the [3H]IAA-phenylalanine, whereas exogenous [3H]IAA was rapidly metabolized to two unidentified products considerably more polar than IAA. Analysis of a cross between icr1 and icr2 indicated that these mutations were at distinct loci and that their effects were additive, and preliminary mapping data indicated that icr1 and icr2 were located at the top and bottom of chromosome V, respectively.     

3q26.31–q29 duplication and 9q34.3 microdeletion associated with omphalocele,ventricular septal defect,abnormal first-trimester maternal serum screening and increased nuchal translucency: Prenatal diagnosis and aCGH characterization

We present prenatal diagnosis and array comparative genomic hybridization characterization of 3q26.31–q29 duplication and 9q34.3 microdeletion in a fetus with omphalocele, ventricular septal defect, increased nuchal translucency, abnormal first-trimester maternal screening and facial dysmorphism with distinct features of the 3q duplication syndrome and Kleefstra syndrome. The 26.61-Mb duplication of 3q26.31–q29 encompasses EPHB3, CLDN1 and CLDN16, and the 972-kb deletion of 9q34.3 encompasses EHMT1. We review the literature of partial trisomy 3q associated with omphalocele and discuss the genotype–phenotype correlation in this case.     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate Nα-(indol-3-ylacetyl)-glutamine and initiation of a mutant screen

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μ M IAA. In addition, a new metabolite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole-3-acetyl-aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-acetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is allelic to an existing class of photorespiration mutants ( gluS ) deficient in chloroplastic glutamate synthase.     

Consequences of Whole-Genome Triplication as Revealed by Comparative Genomic Analyses of the Wild Radish Raphanus raphanistrum and Three Other Brassicaceae Species

Polyploidization events are frequent among flowering plants, and the duplicate genes produced via such events contribute significantly to plant evolution. We sequenced the genome of wild radish (Raphanus raphanistrum), a Brassicaceae species that experienced a whole-genome triplication event prior to diverging from Brassica rapa. Despite substantial gene gains in these two species compared with Arabidopsis thaliana and Arabidopsis lyrata, ∼70% of the orthologous groups experienced gene losses in R. raphanistrum and B. rapa, with most of the losses occurring prior to their divergence. The retained duplicates show substantial divergence in sequence and expression. Based on comparison of A. thaliana and R. raphanistrum ortholog floral expression levels, retained radish duplicates diverged primarily via maintenance of ancestral expression level in one copy and reduction of expression level in others. In addition, retained duplicates differed significantly from genes that reverted to singleton state in function, sequence composition, expression patterns, network connectivity, and rates of evolution. Using these properties, we established a statistical learning model for predicting whether a duplicate would be retained postpolyploidization. Overall, our study provides new insights into the processes of plant duplicate loss, retention, and functional divergence and highlights the need for further understanding factors controlling duplicate gene fate.     

Integration of Visual Information in Auditory Cortex Promotes Auditory Scene Analysis through Multisensory Binding

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Granzyme M has a critical role in providing innate immune protection in ulcerative colitis

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn''s disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.Inflammatory bowel disease (IBD) is a gut-associated inflammatory disorder, which stems from a dysfunctional mucosal immune response to commensal bacteria.¹ As a multifactorial disease, IBD is the consequence of a complex interplay between environmental triggers, genetic susceptibility, and immunoregulatory defects, resulting in a pathogenesis that is still poorly understood.² These interactions result in the inability of an individual to control the normal inflammatory response to pathogens in the gut, leading to a chronic state of sustained and inappropriate inflammation. IBD underlies disease states such as ulcerative colitis (UC) and Crohn''s disease (CD), with symptoms including weight loss, abdominal pain, diarrhea, and rectal bleeding which often require intensive medical therapy and resective surgery.³ The pathogenesis of IBD, characterized by a defective mucosal immune response to microbial exposure in the gastrointestinal tract, is thought to be caused by a dysfunctional immune response to host microbiota, infection by specific pathogens, and/or a defective mucosal barrier to luminal pathogens.^{1, 2} IBD patients also have a high risk of developing colitis-associated colon cancer (CAC).⁴ Additionally, histological assessment of inflamed ileal and colonic segments from IBD patients typically shows increased infiltration of immune cells, particularly neutrophils, as well as crypt abscesses, mucin depletion, and ulcers—all correlating with the severity of small bowel and colonic tissue damage.⁵Cytotoxic pathways mediated by lymphocytes directly trigger cell death in target cells.⁶ These cytotoxic pathways are mediated by proteins such as perforin, which mediates pore formation in the target cell surface and allows granzyme (Grz)s to enter the intracellular compartment and induce cell death.⁷ To date, five different Grzs have been identified in humans (GrzA, GrzB, GrzH, GrzK, and GrzM), whereas mice express eleven Grzs (GrzA, GrzB, GrzC, GrzD, GrzE, GrzF, GrzG, GrzK, GrzL, GrzM, and GrzN).^{8, 9} Walch et al.¹⁰ recently demonstrated that Grzs (GrzA and GrzB) directly kill bacteria through granulysin-mediated delivery, suggesting that Grzs act as microbial modulating factors. Moreover, recently GrzA was shown to be increased in the colon biopsies of UC patients undergoing treatment with Etrolizumab, a monoclonal antibody targeting the β7 integrin subunit. Higher levels of GrzA could predict which patients were more likely to benefit from the therapy; however, the precise mechanism of action of GrzA in UC remains to be addressed.¹¹ GrzM was initially described as being constitutively expressed by natural killer (NK) cells,^{12, 13} and specifically associated with inflammation.¹⁴ This enzyme has been shown to preferentially cleave methionine and leucine residues in target cells, mediating direct, non-specific cell death.^{15, 16} More recently, GrzM was also shown to be an important mediator for the release of MIP-1α from NK cells, inducing NK cell and neutrophil recruitment during early microbial infection.¹⁷ We now observe that GrzM expression is increased in inflamed colon tissue samples from UC, but not CD patients. Further, GrzM-deficient (GrzM^−/−) mice are more sensitive to a mouse model of IBD and IBD-induced colorectal cancer (CRC). These findings demonstrate, for the first time, that GrzM has a critical role in mediating the early stages of the gut mucosal immune response.