Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.     

Evaluation of the Metabolic Response to Cyclopamine Therapy in Pancreatic Cancer Xenografts Using a Clinical PET-CT System

OBJECTIVES: We analyzed the effects of anti-hedgehog signaling on the ¹⁸F-FDG uptake of pancreatic cancer xenografts (PCXs) using a clinically implemented positron emission tomography (PET)-computer tomography (CT) scanner with high-resolution reconstruction. METHODS: PCXs from two pancreatic cancer cell lines were developed subcutaneously in nude mice and injected intraperitoneally with a low dose of cyclopamine for 1 week. ¹⁸F-FDG PET-CT was performed using a new-generation clinical PET-CT scanner with minor modifications of the scanning protocol to adapt for small-animal imaging. The data set was reconstructed and quantified using a three-dimensional workstation. RESULTS: MiaPaCa-2 cells, which respond to cyclopamine, showed decreased ¹⁸F-FDG uptake without a change in tumor size. For hip tumors, the maximum standardized uptake value (SUV_max) was reduced by -24.5 ± 9.2%, the average SUV (SUV_avg) by -33.5 ± 7.0%, and the minimum SUV (SUV_min) by -54.4 ± 11.5% (P < .05). For shoulder tumors, SUV_max was reduced by -14.7 ± 7.5%, SUV_avg by -12.6 ± 6.3, and SUV_min by -30.3 ± 16.7% (P < .05). Capan-1 cells, which do not respond to cyclopamine, did not show significant SUV changes. CONCLUSIONS: The new generations of clinically implemented PET-CT scanners with high-resolution reconstruction detect a minimal response of PCX to low-dose short-term cyclopamine therapy without changes in tumor size and offer potential for preclinical translational imaging.     

Lipid-specific membrane activity of human beta-defensin-3

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.     

Inhibition of some hepatic glycosidases by the diseco nucleoside, 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analog

The in vivo and in vitro effects of 4-amino-3-(D-glucopentitol-1-yl)-5-mercapto-1,2,4-triazole and its 3-methyl analogue on alpha- and beta-glucosidases, beta-glucuronidase as well as alpha-amylase have been investigated. alpha-Glucosidase is the enzyme that is markedly affected in vivo and in vitro in a dose-dependent manner. The compounds showed a reversible inhibition of a competitive type for alpha-glucosidase. Moreover, they exert a relatively potent inhibition on alpha-glucosidase with a Ki magnitude of 3.6 x 10(-4), 9.5 x 10(-5) M.     

Autophagy-independent function of MAP-LC3 during intracellular propagation of Chlamydia trachomatis

Microtubule-associated protein 1 (MAP1) light chain 3 (LC3) has proven useful as autophagosomal marker in studies on the interaction between pathogens and the host autophagic machinery. However, the function of LC3 is known to extend above and beyond its role in autophagosome formation. We previously reported that intrinsic LC3 is associated with the intracellular Chlamydia trachomatis inclusion in human epithelial cells. Here we show that LC3, most likely the cytoplasmic nonlipidated form, interacts with the C. trachomatis inclusion as a microtubule-associated protein rather than an autophagosome-associated component. In contrast, N-terminally GFP-tagged LC3 exclusively targets autophagosomes rather than chlamydial inclusions. Immunofluorescence analysis revealed an association of LC3 and MAP1 subunits A and B with the inclusion as early as 18 h post infection. Inclusion-bound LC3 was connected with the microtubular network. Depolymerization of the microtubular architecture disrupted the association of LC3/MAP1s with the inclusion. Furthermore, siRNA-mediated silencing of the MAP1 and LC3 proteins revealed their essential function in the intracellular growth of C. trachomatis. Interestingly, defective autophagy remarkably enhanced chlamydial growth, suggesting a suppressive effect of the autophagic machinery on bacterial development. However, depletion of LC3 in autophagy-deficient cells noticeably reduced chlamydial propagation. Thus, our findings demonstrate a new function for LC3, distinct from autophagy, in intracellular bacterial pathogenesis.