Rapid detection of CEA mRNA in peritoneal washes using One-Step Nucleic acid Amplification (OSNA) for gastric cancer patients.

BACKGROUND: Carcinoembryonic antigen (CEA) mRNA expression in peritoneal washes from gastric cancer patients has been reported as an indicator for survival or peritoneal recurrence. The whole process of CEA mRNA detection is time- and labor-intensive. We report the potential of One-Step Nucleic acid Amplification (OSNA) as a rapid and simple system for CEA mRNA detection in peritoneal washes. METHODS: A total of 128 peritoneal washes were analyzed by cytological examination including immunocytochemistry. After the cytological examination, the CEA mRNA concentration in the residual cells was measured using the OSNA system. The CEA mRNA concentration in peritoneal washes was compared with the results of the cytological examination. RESULTS: CEA mRNA at concentrations from 10 to 10(7)copies/µL was detected by the OSNA system within 10 min, and an excellent correlation was observed between the logarithmic CEA mRNA concentration and the detection time (r=0.998). The CEA mRNA cutoff value for distinguishing positive and negative cases through cytological examination was identified as 25 copies/µL. At this cutoff value, the concordance rate with the cytological examination was 93.8%. The overall survival in CEA mRNA-positive versus -negative cases identified using the OSNA system was statistically significant. CONCLUSION: This CEA mRNA detection system shows potential for cancer cell detection and for routine use in the clinical laboratory because of its simplicity and rapidity.

Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b(5) reductase for the binding of beta-NADH.

Well-conserved three consecutive Pro residues (Pro247-249) in the NADH-binding subdomain of NADH-cytochrome b(5) reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coli. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6xHis-tag sequence at the NH(2)-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH:ferricyanide reductase and NADH:cytochrome b(5) reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease k(cat) with slight increase in K(m) for the physiological electron donor NADH. However, K(m) values for the electron acceptors (both cytochrome b(5) and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b(5).