Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K_is were in the range of 0.061–1.822 μM for CA I, 1.47–2.94 nM for CA II, 2.25–3.34 μM for CA VA, 0.041–0.37 μM for CA IX, 0.021–1.52 μM for CA XII, 0.007–0.219 μM for CA XIV, 0.35–5.31 μM for CgCA and 0.465–4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.     

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO₂ uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.Energy security and climate change as global problems urge increased efforts to use plants as renewable energy sources both for power generation and transportation fuel production. Selected wood species, such as willows (Salix spp.), can be cultivated as short-rotation coppice for the rapid accumulation of biomass and reduction of CO₂ emission. Coppicing reinvigorates shoot growth, resulting in a special woody plant life cycle that differs from natural tree development, which takes decades. In this cultivation system, small stem cuttings are planted at high densities (15,000–25,000 ha⁻¹). In the soil, these dormant wood cuttings first produce roots and shoots that emerge from reactivated buds. During the first year, the growing shoots mature to woody stems. In the winter, these stems are cut back, and in the following spring, the cut stumps develop multiple shoots. The short-rotation coppice plantations are characterized by a very short, 2- to 3-year rotation, and the most productive varieties can produce up to 15 tons of oven-dried wood per hectare per year (Cunniff and Cerasuolo, 2011). The high-density willow plantations can also be efficiently used for heavy metal or organic phytoremediation, as reviewed by Marmiroli et al. (2011).The biomass productivity of shrub willows is largely dependent on coppicing capability, early vigorous growth, shoot growth rate and final stem height, root system size, photosynthetic efficiency, formation and composition of woody stems, water and nutrient use, as well as abiotic and biotic stress tolerance. Genetic improvement of all these traits can be based on broad natural genetic resources represented by more than 400 species in the genus Salix. More than 200 species have hybrid origins, and ploidy levels vary from diploid up to dodecaploid (Suda and Argus, 1968; Newsholme, 1992). In addition to molecular marker-assisted clone selection, intraspecific and interspecific crosses have been shown to further extend genetic variability in breeding programs for biomass yield (Karp et al., 2011).During natural diversification and artificial crossings of Salix spp., the willow genomes frequently undergo polyploidization, resulting in triploid or tetraploid allopolyploids. In triploid hybrids, both heterosis and ploidy can contribute to the improved biomass yield (Serapiglia et al., 2014). While the alloploid triploids have attracted considerable attention in willow improvement, the potentials of autotetraploid willow genotypes have not been exploited so far. As shown for other short-rotation wood species (poplar [Populus spp.], black locust [Robinia pseudoacacia], Paulownia spp., and birch [Betula spp.]), doubling the chromosome set by colchicine treatment can cause significant changes in organ morphology or growth parameters (Tang et al., 2010; Cai and Kang, 2011; Harbard et al., 2012; Mu et al., 2012; Wang et al., 2013a, 2013b). In several polyploidization protocols, the in vitro cultured tissues are exposed to different doses of colchicine or other inhibitors of mitotic microtubule function, and plantlets are differentiated from polyploid somatic cells (Tang et al., 2010; Cai and Kang, 2011). Alternatively, seeds or apical meristems of germinating seedlings can be treated with a colchicine solution (Harbard et al., 2012). Allotetraploids of poplar were produced by zygotic chromosome doubling that was induced by colchicine and high-temperature treatment (Wang et al., 2013a).Since tetraploid willow plants with 2n = 4x = 76 chromosomes are expected to represent novel genetic variability, especially for organ development and physiological parameters, a polyploidization project was initiated that was based on a highly productive diploid energy willow (S. viminalis var. Energo). Colchicine treatment of reactivated axillary buds of the in vitro-grown energy willow plantlets resulted in autotetraploid shoots and, subsequently, plants. For comparison of diploid and tetraploid variants of willow plants, digital imaging of green organs and roots was used for phenotyping. Among the tetraploid lines, genotypes were identified with improved biomass production, better photosynthetic parameters, and altered organ structure and hormone composition. The new tetraploid willow variants produced can serve as a unique experimental material to uncover key factors in biomass production in this short-rotation energy plant. In the future, these plants can also serve as crossing partners of diploid lines for the production of novel triploid energy willow genotypes.     

Effect of vitamin C and zinc on osmotic fragility and lipid peroxidation in zinc-deficient haemodialysis patients

Peroxidation of the membrane lipid structure of red blood cell leads to haemolysis and anaemia in haemodialysis patients. Dietary constituents of antioxidant vitamins and trace elements may play an important role in protecting against oxidant damage. In this study, the effects of supplementation of vitamin C and zinc on osmotic fragility and lipid peroxidation of erythrocytes were investigated in 34 zinc-deficient haemodialysis patients. Sixteen sex- and age-matched normal volunteers acted as controls. Patients were randomized to receive vitamin C (250 mg day(-1)), zinc (20 mg day(-1)) or a placebo treatment for 3 months. The levels of vitamin C, zinc, malondialdehyde (MDA) and osmotic fragility were measured initially and 3 months after supplementation. Mean serum concentration of vitamin C and zinc increased significantly in the groups at the end of the respective study periods. Supplementation with vitamin C and zinc improved osmotic fragility, and decreased the level of MDA in the groups, but some side-effects (i.e. nausea, vomiting, fever, muscle pain, weakness) were observed during the zinc treatment. The results showed that the supplementation of both treatments decreased osmotic fragilty and MDA in zinc-deficient haemodialysis patients. However, vitamin C treatment was found to be safer than zinc supplementation.     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

Effects of Ni(2+), Co(2+), and Mn(2+) on desensitized butyrylcholinesterase prepared from human serum

Human serum butyrylcholinesterase (BChE) has been converted into a stable but less active desensitized form when heated at 45°C for 24 h. The desensitized BChE follows Michaelis-Menten kinetics, whereas native enzyme exhibits slightly negative cooperativity with respect to butyrylthiocholine binding. In this study, we investigated the effects of Ni²⁺, Co²⁺, and Mn²⁺ on the desensitized BChE. It is found that all three ions were noncompetitive inhibitors of the desensitized BChE, and K _i values have been determined as 7.816±1.060 mM, 48.722±4.635 mM, and 84.795±5.249 mM for Ni²⁺, Co²⁺, and Mn²⁺, respectively. In our previous study, these ions were linear mixed-type inhibitors of the native BChE. This finding confirms that desensitized BChE changes to a different conformation than native BChE. From the comparison of K _i values of the trace elements, it can be said that Ni²⁺ is a more effective inhibitor of the desensitized BChE than Co²⁺ and Mn²⁺.     

Kinetic analysis of the inhibitory effect of trichloroethylene (TCE) on nitrification in cometabolic degradation

In this study, the inhibitory effect of TCE on nitrification process was investigated with an enriched nitrifier culture. TCE was found to be a competitive inhibitor of ammonia oxidation and the inhibition constant (K _I ) was determined as 666–802 μg/l. The TCE affinity for the AMO enzyme was significantly higher than ammonium. The effect of TCE on ammonium utilization was evaluated with linearized plots of Monod equation (e.g., Lineweaver–Burk, Hanes–Woolf and Eadie–Hofstee plots) and non-linear least square regression (NLSR). No significant differences were found among these data evaluation methods in terms of kinetic parameters obtained.     

Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.     

Immunolocalization of a novel annexin‐like protein encoded by a stress and abscisic acid responsive gene in alfalfa

We report here on the isolation and characterization of a full-length cDNA clone from alfalfa termed AnnMs2 encoding a 333 amino acid long polypeptide that shows 32–37% sequence identity with both mammalian and plant annexins, and has four tandem repeats. While other plant annexins exhibit a high level of sequence similarity to each other (up to 77% identity at amino acid level), AnnMs2 appears to be a distinct type of plant annexins. All the four endonexin folds contain the conserved eukaryotic motif within this alfalfa protein, but this element is considerably different in the second repeat. The AnnMs2 gene is expressed in various tissues of alfalfa with elevated mRNA accumulation in root and flower. This gene is activated in cells or tissues exposed to osmotic stress, abscisic acid (ABA) or water deficiency. The recombinant AnnMs2 protein is able to bind to phospholipid in the presence of Ca²⁺. Indirect immunofluorescence studies using affinity purified rabbit anti-AnnMs2 peptide antibody show mainly nucleolar localization, but the protein sequence lacks the usual nuclear localization signal. The potential role of this novel annexin-like protein in the basic and stress-induced cellular functions is discussed.