Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.     

Methionine in proteins: The Cinderella of the proteinogenic amino acids

Methionine in proteins, apart from its role in the initiation of translation, is assumed to play a simple structural role in the hydrophobic core, in a similar way to other hydrophobic amino acids such as leucine, isoleucine, and valine. However, research from a number of laboratories supports the concept that methionine serves as an important cellular antioxidant, stabilizes the structure of proteins, participates in the sequence‐independent recognition of protein surfaces, and can act as a regulatory switch through reversible oxidation and reduction. Despite all these evidences, the role of methionine in protein structure and function is largely overlooked by most biochemists. Thus, the main aim of the current article is not so much to carry out an exhaustive review of the many and diverse processes in which methionine residues are involved, but to review some illustrative examples that may help the nonspecialized reader to form a richer and more precise insight regarding the role‐played by methionine residues in such processes.     

微生物次生代谢产物生物合成中的拜耳-维立格单加氧酶

拜耳-维立格单加氧酶是一类可以催化酮生成酯以及硫等杂原子氧化的黄素依赖的单加氧酶,在合成化学和生物催化等工业领域有重要的应用前景。本文总结了微生物次生代谢产物生物合成途径中涉及的拜耳-维立格反应,讨论了其反应的特点和催化这些反应的拜耳-维立格单加氧酶的氨基酸序列特征,为拜耳-维立格单加氧酶的蛋白质工程改造提供参考。     

The use of metal-catalyzed oxidation to suppress background staining caused by marker amplification reagents and the effects of this oxidation on the stability of antibody-antigen complexes in immunohistochemistry

Marker amplification is a powerful technique for visualizing immunohistochemically deposited markers that otherwise would be invisible. Amplification usually is achieved with physical developers, which are solutions that contain a source of silver(I) plus a reducing agent. When the marker is present in extremely small quantities, prolonged incubation in the developer is required and unwanted background staining in the form of type III argyrophilia becomes problematic. Suppression of type III argyrophilia can be achieved by metal-catalyzed oxidation using the copper/H₂O₂ system, which normally is applied immediately prior to amplification. Because there is no reason, in principle, why metal-catalyzed oxidation should not be employed at earlier stages in the immunohistochemical staining procedure, we investigated whether earlier oxidation might confer any advantages over the traditional methodology. Immunocolloidal gold combined with two light insensitive physical developers was chosen as the model system, because visualization by light microscopy requires extended periods in the developers. Moreover, the system does not suffer from problems concerning endogenous enzyme- or non-enzyme-catalyzed marker deposition. Applying metal-catalyzed oxidation at each stage of the immunohistochemical procedure revealed that the technique could be employed successfully prior to staining, but not following the primary or secondary antibodies. In the latter cases, specific immunolocalization was lost entirely and only generalized nonspecific staining was seen. A limited investigation into the mechanism of metal-catalyzed oxidation of aldehyde fixed tissue sections suggested that it involved the formation of aldehyde groups. We suggest that the application of metal-catalyzed oxidation prior to immunohistochemical staining would have the advantages of both suppressing type III argyrophilia and inhibiting unwanted endogenous peroxidase activity. We also suggest that metal-catalyzed oxidation might reduce the affinity of tissue for other transition metals, such as copper, whose potential for improving marker amplification techniques has been demonstrated previously in dot-blot model systems.     

Combined inhibiting action of new salicylic acid derivatives and α-tocopherol on oxidation of methyl oleate

The inhibitory effects of compositions of α-tocopherol (α-TP) and salicylic acid derivatives on the process of initiated oxidation of methyl oleate have been investigated. α-TP and the salicylic acid derivatives exhibited the synergistic effect, which was demonstrated by the methods of UV-spectroscopy and high-performance liquid chromatography (HPLC). Kinetics of α-TP utilization during methyl oleate oxidation was investigated under conditions of its independent use as well as using its binary mixture with the synthetic antioxidants.     

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH. Published: May 18, 2007     

Changes in oxidant-antioxidant status in young diabetic patients from clinical onset onwards

Oxidative stress has been implicated as a mechanism underlying hyperglycaemia-associated cellular damage and could play a role in the development of diabetes-related complications. This study aimed to evaluate the significance of changes in oxidant-antioxidant status in 176 child and adolescent diabetic patients at clinical onset, during disease progression and when early microvascular complications appeared. Indicative lipid and protein oxidation markers and antioxidant defence activity were measured in plasma and correlated with clinical data, diabetes duration, long-term glycometabolic control and serum lipids. Compared with their respective age-matched controls, diabetic patients had greater oxidative damage to lipids and proteins, demonstrated through the analysis of hydroperoxides, lipoperoxides and oxidation protein products, all of which were significantly raised at onset, decreased during the first 1.5 years of evolution and rose progressively thereafter. Plasma levels of oxidizable lipids were significantly associated with lipid and protein oxidation products. Overall, plasma antioxidant capacity was significantly and consistently lower from clinical onset onwards. These results suggest that insulin therapy in the first year improved metabolic and oxidant homeostasis and consequently hyperglycaemia-derived biomolecular oxidative damage. Diabetes-associated hyperlipidaemia is related to lipid and protein oxidation processes, which supports the concept of glucose toxicity and lipotoxicity being interrelated. The greatest increase in lipid and protein oxidative damage biomarkers in young diabetic patients with premature microangiopathy points to oxidative stress as a possible contributing mechanism of microvascular dysfunction. Consequently, tight lipid and glycometabolic control may have therapeutic potential by diminishing oxidative tissue-damaging effects of hyperglycaemia.     

Progressive oxidation of cytoskeletal proteins and accumulation of denatured hemoglobin in stored red cells

Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.     

Proteome phenotyping of acid stress-resistant mutants of Lactococcus lactis MG1363

To cope with medium acidity, Lactococcus lactis has evolved a number of inducible mechanisms commonly referred as acid stress response. To better understand the molecular basis of this response, several mutants constitutively tolerant to acidity were previously obtained by insertional random mutagenesis of L. lactis MG1363. Mutants in which the GMP synthase gene (i.e. guaA), the (p)ppGpp synthase gene (i.e. relA*) or the high affinity phosphate transport system (i.e. pstS) are inactivated are further characterized in this study. 2-DE was performed and showed that 42, 26, and 35 protein spots are positively deregulated in the guaA, relA*, and pstS mutants, respectively, as compared to the wild-type strain. Most of these proteins were identified by MS. Proteomes comparison of the mutants guaA, relA*, and pstS as well as the acid adaptation proteome of the wild-type strain revealed (i) the presence of numerous overlaps and (ii) that only five proteins were overexpressed in the four conditions, suggesting that these proteins play a crucial role in the constitutive acid stress tolerance of the mutants and in the acid tolerance response of the wild-type strain.     

Flux change in basophil membrane is not the main pathogenesis for hypersensitivity

The oxidation process is one of the most important natural processes. Oxidative change in hypersensitivity is believed to be an important process in the pathogenesis. However, the clear explanation on the transmembrane flux change ofbasophil and its correlation to hypersensitivity pathogenesis has never been reported. Here, the author determines the transmembrane oxidation flux in basophil. The simulation test to determine the oxidation flux change based on nanomedicine technique is used. Of interest, no change of flux can be detected. Therefore, this work can support the finding that the oxidation flux change is not an important part in the pathogenesis of basophil-related hypersensitivity.