Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD⁺, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V _max , K _cat , and K _m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.     

Modulation of total ceramide and constituent ceramide species in the acutely and chronically hypoxic mouse heart at different ages

Ceramide has been implicated in regulatory processes vital for cell survival under different stressors, most notably hypoxia. Little has been done to investigate the contributions of the different ceramide species to the regulation of cell survival. This study aims to highlight the patterns of variation in total ceramide and its species in the growing and hypoxic mouse heart. Mus musculus mice were placed in a hypoxic environment at birth. Control animals remained in room air. The hearts were extracted at different time points: 1 day, 1 week, 4 weeks, and 8 weeks. The total ceramide content and the amounts of component species were assayed by a modified diacylglycerol kinase assay and high-performance liquid chromatography-tandem mass spectroscopy, respectively. Data was collected from both ventricles in hypoxic and control conditions. There was significant polycythemia in the hypoxic versus control animals with a nearly twofold increase in hematocrit levels. Hypoxic right ventricle (RV) mass significantly increased over that of controls at different age groups. When ceramide content was compared in the hypoxic versus control animals, there was a significant increase at day 1 and a significant decrease at week 4 in the left ventricle, whereas a significant decrease was found in the RV at 1 week, 4 weeks, and 8 weeks. There was also a differential involvement of the RV with regard to levels of N-palmitoyl-D-erythro-sphingosine (C16-Cer) and its synthetic precursor dihydro-N-palmitoyl-D-erythro-sphinganine (DHC-16-Cer). The decrease in C16-Cer observed in both hypoxic and control RV's over time was paralleled by a significant increase in DHC-16-Cer in hypoxic (142.1+/-15.0 pmol; p<0.05) but not control (52.8+/-4.0 pmol) RV's suggesting a role for DHC-16-Cer in the RV adaptive response to hypoxia. Another species, N-arachidoyl-D-erythro-sphingosine (C20-Cer), was specifically and significantly decreased in the hypoxic RV. These studies support the presence of distinct roles for different ceramide species and their precursors. A better assessment of cyanotic congenital heart disease in light of the mechanism and timing of cardiomyocyte death, will lead to punctual interventions and even novel cardioprotective strategies.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

Sinus aspergilloma in rheumatoid arthritis before or during tumor necrosis factor-alpha antagonist therapy

Introduction  

In 2008, the Food and Drugs Administration required manufacturers of TNFα antagonists to strengthen their warnings about the risk of serious fungal infections in patients with rheumatoid arthritis (RA). Sinus aspergilloma occurs occasionally in RA patients and can progress to invasive Aspergillus disease. The purpose of this study was to describe symptomatic sinus aspergilloma in RA patients treated with TNFα antagonists.     

Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy

The objective of this study was to evaluate the anti-biofilm efficacy of photodynamic therapy by conjugating a photosensitizer (TBO) with silver nanoparticles (AgNP). Streptococcus mutans was exposed to laser light (630 nm) for 70 s (9.1 J cm^?2) in the presence of a toluidine blue O–silver nanoparticle conjugate (TBO–AgNP). The results showed a reduction in the viability of bacterial cells by 4 log₁₀. The crystal violet assay, confocal laser scanning microscopy and scanning electron microscopy revealed that the TBO–AgNP conjugates inhibited biofilm formation, increased the uptake of propidium iodide and leakage of the cellular constituents, respectively. Fluorescence spectroscopic studies confirmed the generation of OH^? as a major reactive oxygen species, indicating type I phototoxicity. Both the conjugates down-regulated the expression of biofilm related genes compared to TBO alone. Hence TBO–AgNP conjugates were found to be more phototoxic against S. mutans biofilm than TBO alone.