Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Simulation of creep in non-homogenous samples of human cortical bone

Characterising the mechanisms causing viscoelastic mechanical properties of human cortical bone, as well as understanding sources of variation, is important in predicting response of the bone to creep and fatigue loads. Any better understanding, when incorporated into simulations including finite element analysis, would assist bioengineers, clinicians and biomedical scientists. In this study, we used an empirically verified model of creep strain accumulation, in a simulation of 10 non-homogeneous samples, which were created from micro-CT scans of human cortical bone of the femur midshaft obtained from a 74-year-old female cadaver. These non-homogeneous samples incorporate the presence of Haversian canals and resorption cavities. The influence of inhomogeneity on the response and variation in the samples in both creep and stress relaxation tests are examined. The relationship between steady-state creep rate, applied loads (stress relaxation and creep tests) and microstructure, that is bone apparent porosity, is obtained. These relations may provide insight into damage accumulation of whole human bones and be relevant to studies on osteoporosis.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Oxidative Inactivation of Paraoxonase1, an Antioxidant Protein and its Effect on Antioxidant Action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu²⁺ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe²⁺ (2.0?μM), the inclusion of Cu²⁺ (0.1–1.0?μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5?mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu²⁺ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu²⁺-catalyzed oxidative inactivation. In addition, Cu²⁺ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu²⁺-induced oxidation of LDL. Noteworthy, ascorbate/Cu²⁺-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu²⁺-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.     

Protection and reactivation of human methylmalonyl-CoA mutase by MMAA protein

Previous studies have reported that some adenosylcobalamin-dependent enzymes suffer inactivation during catalysis due to the oxidation of cobalamin. In addition, the protection or reactivation of their catalytic activities by proteins called “protectases” or reactivases is well known in bacteria. In this study, we examined the influence of human MMAA protein on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase (MCM) by testing both purified recombinant proteins in vitro. Our results showed that MMAA plays dual roles in MCM activity. When it was added at the beginning of the reaction, it prevents inactivation by guarding MCM. After 60 min of reaction, when MCM is inactive, the addition of MMAA increases the enzymatic activity through GTP hydrolysis, indicating reactivation of MCM by exchange of the damaged cofactor. Interaction between MCM and MMAA observed in vitro was confirmed in vivo by yeast two-hybrid system.     

牛肉中单增李斯特菌的热失活模型

【目的】建立牛肉中单增李斯特菌的热失活动力学模型。【方法】将接种了3种不同血清型的单增李斯特菌混合菌液的牛肉分别在55℃、57.5℃、60℃、63℃、66℃和70℃进行热处理,在不同温度条件下单增李斯特菌数从109CFU/g下降至103CFU/g,其热失活曲线用修正的Gompertz模型进行了拟合;利用线性模型对单增李斯特菌的相对失活率(μ)和所持续时间(M)的自然对数值与温度(55℃-70℃)进行拟合;通过在59℃和64℃对牛肉中单增李斯特菌热处理,对所建的模型进行了验证。【结果】建立了牛肉中单增李斯特菌热失活动力学的一级模型和二级模型,经验证其准确因子和偏差因子均在可接受范围内。【结论】本研究所建立的模型能较好的模拟不同温度(55℃-70℃)对牛肉中单增李斯特菌热失活的影响。     

Exploring the molecular basis of human manganese superoxide dismutase inactivation mediated by tyrosine 34 nitration

Manganese Superoxide Dismutase (MnSOD) is an essential mitochondrial antioxidant enzyme that protects organisms against oxidative damage, dismutating superoxide radical () into H₂O₂ and O₂. The active site of the protein presents a Mn ion in a distorted trigonal–bipyramidal environment, coordinated by H26, H74, H163, D159 and one ⁻OH ion or H₂O molecule. The catalytic cycle of the enzyme is a “ping-pong” mechanism involving Mn³⁺/Mn²⁺. It is known that nitration of Y34 is responsible for enzyme inactivation, and that this protein oxidative modification is found in tissues undergoing inflammatory and degenerative processes. However, the molecular basis about MnSOD tyrosine nitration affects the protein catalytic function is mostly unknown.In this work we strongly suggest, using computer simulation tools, that Y34 nitration affects protein function by restricting ligand access to the active site. In particular, deprotonation of 3-nitrotyrosine increases drastically the energetic barrier for ligand entry due to the absence of the proton.Our results for the WT and selected mutant proteins confirm that the phenolic moiety of Y34 plays a key role in assisting superoxide migration.     

Helicobacter pylori chromosomal DNA replication: current status and future perspectives

Helicobacter pylori causes gastritis, gastric ulcer and gastric cancer. Though DNA replication and its control are central to bacterial proliferation, pathogenesis, virulence and/or dormancy, our knowledge of DNA synthesis in slow growing pathogenic bacteria like H. pylori is still preliminary. Here, we review the current understanding of DNA replication, replication restart and recombinational repair in H. pylori. Several differences have been identified between the H. pylori and Escherichia coli replication machineries including the absence of DnaC, the helicase loader usually conserved in gram-negative bacteria. These differences suggest different mechanisms of DNA replication at initiation and restart of stalled forks in H. pylori.     

Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons

Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 μM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D(1) DR antagonist), but not a D(2)DR antagonist sulpiride, suggesting a regulatory role of D(1)DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D(1)DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D(2)DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D(1)DRs via the signal transduction linked to D(1)DRs.