Effect of Various Elicitors on Lignan Biosynthesis in Callus Cultures of Linum austriacum

Effects of elicitors (mannan, -1,3-glucan, and ancymidol) on the activity of several key enzymes participating in lignan biosynthesis were studied in Linum austriacum L. cell cultures. The activities of L-phenylalanine ammonia-lyase, polyphenoloxydase, tyrosine ammonia-lyase, soluble phenoloxidase, and membrane-bound and soluble oxidases were assayed. The elicitors under study affected various steps in the metabolic pathway of lignan biosynthesis. Elevated enzyme activity accompanied an elicitor-enhanced synthesis of podophyllotoxins and peltatins.     

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

Mutagenicity of ground water (in the bore-holes) in Ararat Valley (Armenia) detected by Trad-SHM bioassay

The genotoxicity of ground water from four bore-holes of different depths (40-120m) in the Ararat valley (Armenia) used both for drinking and irrigation was investigated. The frequency of recessive somatic mutations was determined using the Tradescantia-stamen-hair-mutation (Trad-SHM) test. The Tradescantia clone 02 was used. The pink mutation events (PMEs) were increased by 3.18-6.81-fold in comparison with the control depending both on the depth of subterranean water location and the increase of Na(+) ion concentration in these water samples. The peak frequency was found in water from the 40-45m depth. The deeper the bore-holes, the lower the mutagenicity of water and the concentration of Na(+) ions. Different types of mutant sector arrangements and their frequencies changed depending on the subterranean water depth.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.     

Spinach SoHXK1 is a mitochondria-associated hexokinase

Hexokinase, a hexose-phosphorylating enzyme, has emerged as a central enzyme in sugar-sensing processes. A few HXK isozymes have been identified in various plant species. These isozymes have been classified into two major groups; plastidic (type A) isozymes located in the plastid stroma and those containing a membrane anchor domain (type B) located mainly adjacent to the mitochondria, but also found in the nucleus. Of all the hexokinases that have been characterized to date, the only exception to this rule is a spinach type B HXK (SoHXK1) that, by means of subcellular fractionation, has been localized to the outer membrane of plastids. However, SoHXK1 has a membrane anchor domain that is almost identical to that of the other type B HXKs. To determine the localization of SoHXK1 enzyme by other means, we expressed SoHXK1::GFP fusion protein in tobacco and Arabidopsis protoplasts and compared its localization with that of the Arabidopsis AtHXK1::GFP fusion protein that shares a similar N-terminal membrane anchor domain. SoHXK1::GFP is localized adjacent to the mitochondria, similar to AtHXK1::GFP and all other previously examined type B HXKs. Proteomic analysis had previously identified AtHXK1 on the outside of the mitochondrial membrane. We, therefore, suggest that SoHXK1 enzyme is located adjacent to the mitochondria like the other type B HXKs that share the same N-terminal membrane anchor domain.     

Immunostimulant adjuvant patch enhances humoral and cellular immune responses to DNA immunization

The focus of this report is on the development of an improved DNA immunization protocol, which takes advantage of the strengths of DNA immunization, as well as those associated with adjuvant delivered by transcutaneous immunostimulatory (IS) patches. Because transcutaneous delivery of adjuvants to the skin at the vaccination site has been shown to amplify the immune response to protein antigens, we hypothesized that the same IS patch when placed on the skin at the site of DNA injection could further enhance the immune response to a DNA influenza vaccine. We have combined an influenza DNA vaccine, hemagglutinin fused with three copies of complement C3d, to enhance uptake and antigen presentation, with an IS patch containing heat-labile enterotoxin from Escherichia coli. Coadministration of a potent adjuvant in IS patches placed on the skin at the site of DNA vaccination dramatically amplifies anti-influenza antibody immune response. Supplementing DNA vaccines with IS patches may be a particularly valuable strategy because DNA vaccines can be rapidly modified in response to mutations in pathogens, and individuals with compromised immune systems such as transplant patients and the elderly will benefit from the enhanced antibody response induced by the IS patches.