Flanking sequence specificity determines coding microsatellite heteroduplex and mutation rates with defective DNA mismatch repair (MMR).

The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing -1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A(7)/T(8)) in hMLH1(-/-) cells, but only exon 10 microsatellites fully mutated (A(7)/T(7)) in both hMLH1(-/-) and hMSH6(-/-) backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1(-/-) cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.

The measurement of nitrite in adulterated urine samples by high-performance ion chromatography.

With the increased availability of nitrite-containing commercial products that are used for the adulteration of urine samples in the workplace, it is necessary for laboratories to be able to detect and confirm the presence of nitrite in these samples. We have developed a method to confirm the presence of nitrite in urine samples. The method uses the IonPac AS 14 analytical column with the Dionex series 45001 Bio LC system equipped with an anion self-generating suppressor and conductivity detector. Using a single-point calibration, the method is linear and accurately quantitates nitrite to 12,000 microg/mL. The limit of detection is 30 microg/mL, and the day-to-day precision of the assay has a coefficient of variation (CV) of 4.3% at 1200 microg/mL and 3.8% at 2700 microg/mL of nitrite.

Urinary excretion of 3-methylhistidine as an index of muscle protein catabolism in postoperative trauma: the effect of parenteral nutrition.

The effect of different intravenous nutritional regimens on nitrogen balance and 3-methylhistidine (3-MeHIS) excretion were studied during a 6-day period following major elective surgery in 28 patients. All patients were kept on a synthetic diet 4 days prior to surgery and were given 0.1 g amino acid N and 120 kJ/kg . day. Postoperatively, all patients received parenteral nutrition with approximately 170 kJ/kg . day. Postoperatively, all patients received parenteral nutrition with approximately 170 kJ/kg . day. Three groups of patients were given varying amounts and proportions of amino acids while in one group no amino acids were administered. Preoperatively, urinary 3-MeHIS excretion (determined by a newly developed automatic analyzer) was 240.3 mumole/day +/- 9.2, nitrogen balance was -1.8 g N +/- 0.19. Postoperatively, nitrogen balance was less negative when amino acids were given. The degree of improvement depended on the amount, but not on the composition of nitrogen administered. In all four groups, 3-MeHIS outputs were elevated when compared with preoperative excretion. The 3-MeHIS excretion (mumole/day) was increased more in patients on high amino acid supply than in patients with low or no nitrogen supply. In each of the groups the 3-MeHIS excretion was negatively correlated to the nitrogen balance. Regression analyses suggest that postoperative muscle protein breakdown occurs in relation to the body protein loss. Amino acid administration seems not to decrease muscle protein breakdown, but rather, appears to stimulate protein synthesis, resulting in less net protein loss. The mean rate of muscle protein breakdown in the postoperative state was estimated to be 80 g/day, assuming 4.2 mumole 3-MeHIS per g mixed human muscle protein. This exceeded the mean preoperative breakdown by about 23 g muscle protein per day. This increase might be due to the metabolic response to the trauma and also in part to tissue damage by the surgical procedure.