AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

In vitro efficacy of some cattle drugs on bovine serum paraoxonase 1 (PON1) activity

Serum paraoxonase 1 (EC 3.1.8.1, PON1), a calcium-associated enzyme, has an ability to hydrolyze organophosphate compounds. Related to this property, PON1 has a critical role in antioxidant mechanisms. It is well-known that the enzyme protects LDL from oxidation. In this study we investigated the in vitro inhibitory effects of some drugs. These drugs are oxytocin, dexamethasone, atropine sulphate, gentamicin sulphate, sulfadoxine-trimethoprim, furosemid, metamizole sodium and toldimfos sodium. The IC₅₀ values obtained varied markedly from 0.014 to 507.72?mg/mL. According to our findings, most potent and significant inhibition was displayed by dexamethasone, atropine sulphate and furosemid.     

FMO3 allelic variants in Sicilian and Sardinian populations: Trimethylaminuria and absence of fish-like body odor

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria.     

Gerbera micropropagation

Gerbera jamesonii (gerbera) is an important cut-flower in the global floricultural industry. Micropropagation is the main system used to clonally propagate gerbera in vitro resulting in the production of millions of plantlets each year. Numerous types of explants and protocols for micropropagation have been established and used for gerbera. Shoot tips are the commonly used explant while adventitious shoot induction from the capitulum is also a popular method. Most papers in the literature have focused on testing the influence of different types and combinations of plant growth regulators with the aim of improving the regeneration and multiplication stage of one or few cultivars. Genotype is one of the most influential factors on the response of gerbera in vitro. Despite this, no successful universal protocol has yet been developed for multiple cultivars, limiting the usefulness of current protocols for commercial biotechnology labs. Slow-growing endogenous bacteria are one of the most important problems in gerbera micropropagation but require more studies on control and prevention. Individual shoots are normally easy to root, usually in excess of 90% of plantlets, but the acclimatization stage requires improvements and new technologies to increase the survival of plants. Epigenetic variations in micropropagated gerbera are frequently observed only with high concentrations of cytokinins in the culture medium but somaclonal variation is rare.     

Differentiation of adenine non-planarity in valence molecular orbitals

Two molecular orbitals: MO7 (29a) and MO13 (23a) have been identified using dual space analysis (DSA) as the signatures of adenine non-planarity (C₁ point group symmetry). The non-planarity of adenine has been demonstrated to be from the attachment of the amino group (NH₂) to purine rings as well as the non-rigid deformability of the purine ring of adenine. Orbital 29a (3a″ in the planar case), a π-like orbital, is the direct result of the attachment of the amino group to the purine ring. Orbital 23a (23a′ in the planar case) is the result of the deformability of the purine ring in non-planar adenine (NP) and will be experimentally challenging to resolve.     

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5–4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0–5.5-times higher compared to the immobilized form mainly due to the lower K_m values. With constant 55–60% humidity the biocatalyst retained 77% and 67–70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.     

Adenine-induced inhibition of Na(+)-ATPase activity: Evidence for involvement of the Gi protein-coupled receptor in the cAMP signaling pathway

In the present work, we demonstrate that adenine reduced Na⁺-ATPase activity in isolated basolateral membrane (BLM) of proximal tubule in a dose-dependent manner. Adenine metabolism was ruled out by TLC analysis of the potential [³H]adenine derived-metabolites. Specific binding of [³H]adenine to isolated BLM was observed in a dose-dependent manner with K_d and B_max of 242.6 ± 27.6 nM and 2749.9 ± 104.9 fmol mg⁻¹, respectively. Adenine increased the [³⁵S]GTPγS specific binding and it was completely abolished by 10⁻⁶ M GDPβS (G protein inhibitor) but it was not modified by DPCPX, DMPX and MRS1523, selective antagonists for A₁, A₂ and A₃ receptors, respectively. Furthermore, the inhibitory effect of adenine on the Na⁺-ATPase activity was blocked by 10⁻⁶ M GDPβS, 1 μg/ml pertussis toxin (Gi protein inhibitor), 10⁻⁶ M foskolin (adenylyl cyclase activator) and 10⁻⁸ M cAMP. These data demonstrate that adenine inhibits the proximal tubule Na⁺-ATPase activity through the Gi protein-coupled receptor.     

Comparison of gene targeting efficiencies in two mosses suggests that it is a conserved feature of Bryophyte transformation

The moss, Physcomitrella patens, is a novel tool in plant functional genomics due to its exceptionally high gene targeting efficiency that is so far unique for plants. To determine if this high gene targeting efficiency is exclusive to P. patens or if it is a common feature to mosses, we estimated gene-targeting efficiency in another moss, Ceratodon purpureus. We transformed both mosses with replacement vectors corresponding to the adenine phosphoribosyl transferase (APT) reporter gene. We achieved a gene targeting efficiency of 20.8% for P. patens and 1.05% for C. purpureus. Our findings support the hypothesis that efficient gene targeting could be a general mechanism of Bryophyte transformation.