Development and characterization of a FIA system for selective assay of l-ascorbic acid in food samples

An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration curve in 25–400 μmol/L range was R ² = 0.99.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

Littoral macrophyte–periphyton complexes in two Hungarian shallow waters

Periphyton developing on the surfaces of emergent and submerged aquatic plants has a significant influence on water quality. The periphyton types that form on various plant species can be characterized by their mass values, the proportion of the present organic and inorganic fractions, as well as their chlorophyll-a contents. Studies on periphyton complexes constituting integrated biomonitoring systems are useful to gain essential long-term information about the performance of shallow water bodies. The filtering and settling effect of Phragmites and other aquatic plants, as well as their periphyton was examined and clearly observable in the water areas and non-flooded aquatic habitats belonging to the second phase of Kis-Balaton Protection System, as it was indicated by the mass values and ash contents. The periphyton forming on the aquatic vegetation that annually develops in Kisköre Reservoir and yields a considerable biomass has a critical part in influencing water quality. The only difference (p<0.05) was found in the ash content of the periphyton, being lower in Kis-Balaton (48.64 ± 2.29 S.E., %) and higher in Kisköre Reservoir (57.42 ± 2.54 S.E., %). This paper presents the dry mass of the periphyton, as well as its ash and chlorophyll-a content, and the results obtained on the composition of the alga species of the periphyton.

     

Left ventricular pressure-volume relationship in a rat model of advanced aging-associated heart failure

Aging is associated with profound changes in the structure and function of the heart. A fundamental understanding of these processes, using relevant animal models, is required for effective prevention and treatment of cardiovascular disease in the elderly. Here, we studied cardiac performance in 4- to 5-mo-old (young) and 24- to 26-mo-old (old) Fischer 344 male rats using the Millar pressure-volume (P-V) conductance catheter system. We evaluated systolic and diastolic function in vivo at different preloads, including preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (+dP/dt), and its relation to end-diastolic volume (+dP/dt-EDV) as well as the time constant of left ventricular pressure decay, as an index of relaxation. The slope of the end-diastolic P-V relation (EDPVR), an index of left ventricular stiffness, was also calculated. Aging was associated with decrease in left ventricular systolic pressure, +dP/dt, maximal slope of the diastolic pressure decrement, +dP/dt-EDV, PRSW, ejection fraction, stroke volume, cardiac and stroke work indexes, and efficiency. In contrast, total peripheral resistance, left ventricular end-diastolic volume, left ventricular end-diastolic pressure, and EDPVR were greater in aging than in young animals. Taken together, these data suggest that advanced aging is characterized by decreased systolic performance accompanied by delayed relaxation and increased diastolic stiffness of the heart in male Fischer 344 rats. P-V analysis is a sensitive method to determine cardiac function in rats.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.