A polarographic method for measuring dissolved nitric oxide.

A polarographic method for measuring the concentration of authentic nitric oxide (NO) in aqueous solutions is described. When solutions of NO were injected into aqueous solutions containing dissolved oxygen, NO reacted with oxygen to give nitrite. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). We observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range (20-100 nmol NO-2) (R2 = 0.89). These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic NO solutions. There are several advantages to measuring consumed oxygen by polarographic methods: The method is simple and can be performed immediately prior to utilization of the NO solutions, it does not require any NO calibration standards and it fully discriminates between NO and oxidation products of NO. An additional advantage is, therefore, that stock solutions of NO can be stored for an indefinite period of time prior to measurement. Both the preparation of authentic NO solutions and the measurement of their NO content are therefore simplified. We also demonstrate that a slow infusion of authentic NO solution to cell suspensions in vitro makes it possible to study the rapid, reversible biological effects of NO.

Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953.

The major outer membrane protein, FomA, of the Gram-negative human oral pathogen Fusobacterium nucleatum functions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such as Streptococcus sanguis and Porphyromonas gingivalis. We describe here the cloning of fomA from F. nucleatum in E. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of the fomA gene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the whole fomA gene. The ATCC 10953 fomA gene was cloned under the phosphate limitation-inducible phoE promoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.

Analysis of RNA by capillary electrophoresis.

Analytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2-8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (< 0.3%) were suited for the separation of high molecular mass RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60-300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.

Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.

The monomeric fluorescent dye, SYBR Green I, was investigated and compared with the dyes YO-PRO-1 and thiazole orange (TO) for their application in capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection of double-stranded DNA (dsDNA). DNA fragments were injected by hydrodynamic pressure and separated in a replaceable matrix of hydroxypropyl methylcellulose. For all 3 dyes, optimal concentrations were established and efficient separations of DNA fragments ranging in size from 75 to 12216 bp were obtained. The most promising results in terms of linear detection range were achieved with SYBR Green I. At the optimal dye concentration, fluorescence intensity versus DNA concentration was linear over more than three orders of magnitude (4 pg/microliters to 30 ng/microliters). Limit of detection (LOD) with SYBR Green I was approximately 80 fg of dsDNA (240 zmol of a 200-bp fragment). Similar LOD was obtained with YO-PRO-1, whereas TO resulted in lower sensitivity. Precision in both fluorescence intensity and migration time was high (relative standard deviation, RSD < 3.6%; n = 10) for dsDNA fragments complexed with SYBR Green I. In conclusion, SYBR Green I is fluorescent dye well suited for efficient separation and quantitative, sensitive, and precise determination of dsDNA by CE-LIF.