SPECTROFLUORIMETRIC ASSAY OF VITAMIN B6 ANALOGUES IN BRAIN TISSUE

Abstract— —A method for the simultaneous assay of the predominant forms of vitamin B₆ occurring in brain tissue was developed. Quantitative release of the vitamers was effected by partial removal of lipids with an organic solvent followed by extraction with aqueous metaphosphoric acid. The compounds were separated by ion exchange column chromatography and estimated fluorimetrically.     

A20 (Tnfaip3) Deficiency in Myeloid Cells Protects against Influenza A Virus Infection

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.     

Inhibition of lecithin:Cholesterol acyltransferase activity in human blood plasma by cigarette smoke extract and reactive aldehydes

Cigarette smoking is a risk factor for atherosclerosis. It is conceivable that reactive chemical components in cigarette smoke may adversely affect reverse cholesterol transport at the level of lecithin:cholesterol acyltransferase (LCAT) and promote atherogenesis. Hence, the effect of cigarette smoke extract (CSE) on the activity of LCAT in human plasma was studied. When incubated with plasma, CSE caused both concentration- and time-dependent losses of LCAT activity. Addition of glutathione, but not ascorbate, to plasma prevented loss of LCAT activity caused by CSE. Incubation of plasma with some reactive aldehydes known to be present in cigarette smoke also inhibited LCAT activity. Among five aldehydes tested, acrolein was the strongest inhibitor of LCAT, with complete enzyme inhibition occurring at 1 mM. Acetaldehyde was the weakest inhibitor of LCAT, with 85% enzyme inhibition at 50 mM. Hexanal, formaldehyde, and malondialdehyde completely inhibited LCAT activity at 10, 50, and 50 mM, respectively. When plasma was incubated with 1 mM acrolein in the presence of 2.5 mM glutathione or dihydrolipoic acid, 100 and 57% of LCAT activity, respectively, remained after incubation. This finding suggests that reactive aldehydes may form adducts with certain free sulfhydryl groups functioning in the active site of LCAT to inhibit enzyme activity. It is concluded that reactive aldehydes are at least partially responsible for the reduction in LCAT activity in plasma treated with CSE.     

Revealing biases inherent in recombination protocols

Background  

The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.     

Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB

Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K _m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe²⁺ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Received: 15 September 1995 / Accepted: 13 November 1995