Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe.

BACKGROUND: Pancreatic fistula (PF) remains the most serious complication after digestive surgery. It is difficult to prevent because of the inability to visualize the leakage of pancreatic juice during surgery or to evaluate the protease activity of leaked fluid, which is responsible for PF formation. METHODS: The fluorescence intensities of a chymotrypsin probe (glutaryl-phenylalanine [corrected] hydroxymethyl rhodamine green with added trypsin) in pancreatic juice and in intestinal or abdominal fluids drained after pancreatic resection were evaluated. The chymotrypsin probe was sprayed on to filter papers that had been placed on the resected pancreatic stump in patients undergoing pancreaticoduodenectomy or central pancreatectomy. The ability of this technique to visualize the leakage of pancreatic juice and predict postoperative PF formation was assessed. RESULTS: The fluorescence intensity of the chymotrypsin probe in 76 fluid samples correlated positively with amylase levels (r(s) = 0.678, P < 0.001). The fluorescence patterns of the pancreatic stump were classified grossly into the three types: duct (fluorescence signal visualized only on the stump of the main pancreatic duct, 16 patients), diffuse (ductal stump and surrounding pancreatic parenchyma, 7) and negative (no fluorescence signal, 7). Symptomatic PFs developed in 13 of 23 patients with duct- or diffuse-type fluorescence, but in none of the seven patients with negative-type fluorescence (P = 0.008). CONCLUSION: The chymotrypsin probe enabled determination of the protease activity in drained pancreatic fluid samples and allowed real-time visualization of pancreatic juice leakage during surgery.

New quantitative methods for evaluation of dynamic changes in QT interval on 24 hour Holter ECG recordings: QT interval in idiopathic ventricular fibrillation and long QT syndrome.

OBJECTIVES: To introduce a nomogram of the normal QT interval at various heart rates measured from 24 hour Holter ECG recordings in healthy subjects with respect to age and sex and to use the nomogram to characterise dynamic changes in QT interval in patients with idiopathic ventricular fibrillation (IVF) and the long QT syndrome (LQT). METHODS: The study group consisted of 422 subjects: 249 healthy men ranging in age from 21-88 years (mean (SD) 47 (20) years) and 173 healthy women ranging in age from 21-85 years (47 (19) years). In addition, seven men with IVF ranging in age from 33-53 years (43 (9) years) and five women with LQT ranging in age from 20-55 years (37 (14) years) were studied. For each subject, QT interval and heart rate were determined automatically from 24 hour Holter ECG digital data-namely, QT interval was measured from signal averaged ECG waves obtained by averaging consecutive sinus beats during each 15 second period over 24 hours. Data were grouped and averaged at an interval of 5 beats/min for heart rates ranging from 46-120 beats/min. RESULTS: In healthy subjects aged < 50 years and > or = 50 years QT intervals were longer in women than in men. QT intervals were longer in both men and women aged > or = 50 years than in ages < 50 years. From these findings a nomogram of QT interval at varying heart rates adjusted for age (younger group aged < 50 years or older group aged > or = 50 years) and sex was determined. In patients with IVF, QT intervals were significantly shorter at slower heart rates than normal values obtained from the nomogram. In patients with LQT, QT intervals were significantly longer at both faster and slower heart rates than normal values. CONCLUSIONS: The nomogram of QT interval at varying heart rates adjusted for sex and age could be used to assess dynamic changes of QT interval of various pathological conditions. For example, patients with IVF had shorter QT interval at slower heart rates, a finding suggestive of arrhythmogenicity of this specific syndrome at night. Patients with LQT had prolonged QT interval at specific heart rate ranges depending on their genotype.

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers.

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.

Spectral characteristics of human atrial fibrillation waves of the right atrial free wall with respect to the duration of atrial fibrillation and effect of class I antiarrhythmic drugs.

The aim of this study was to use fast Fourier transform analysis to clarify the characteristics of human atrial fibrillation (AF) waves with respect to the duration of AF and the effect of class I antiarrhythmic drugs. Twenty-two patients (10 paroxysmal AF, 12 persistent AF) without organic heart disease were studied by conventional electrophysiological methods. Electrograms were recorded from the right atrial free wall during AF and spectral analysis was performed for 35s (16 consecutive 4096-ms epochs with 50% overlap) and the fibrillation cycle length (FCL) was calculated from the peak frequency. Mean FCL and SD were determined from 16-epoch data, and the temporal variability of FCL was defined as the SD of FCL. Paroxysmal AF had a longer mean FCL than persistent AF (178+/-26ms vs 139+/-16 ms, p<0.001) and AF duration had a significant inverse correlation with mean FCL (r=-0.79, p<0.001). The temporal variability of FCL was significantly greater in paroxysmal AF than in persistent AF (p<0.05) and there was a significant positive correlation between the mean FCL and the temporal variability of FCL (r=0.66, p<0.001). In 8 of 18 patients given a class I antiarrhythmic drug (cibenzoline or procainamide), AF was terminated and in those patients the mean FCLs before administration of class I drugs were significantly greater than in patients without AF termination. With respect to mean FCL before drug administration, conversion occurred in 100% of patients with FCL > or =168 ms and in 17% of those with FCL <168 ms. A longer duration of AF shortens the mean FCL, which is consistent with atrial electrical remodeling. Class I drugs prolong the mean FCL above a critical level and will terminate AF, which can be estimated from the mean FCL before drug administration.

Cell-cycle-regulated transcription of A- and B-type plant cyclin genes in synchronous cultures.

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A-type cyclins, and CYM belongs to the group of B-type cyclins. In a fashion similar to the pattern of expression seen for A-type and B-type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell-cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A-type cyclins, and hence CYS along with the soybean cyc1GM can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell-cycle-dependent expression to the beta-glucuronidase (GUS) reporter gene: the CYS promoter directed S-phase-specific expression, whereas the CYM promoter drove M-phase-specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.

A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis.

Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products are post-labeled with two different fluorescent dyes in one tube, and analyzed by an automated capillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE-SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged. Thirty-three of 34 known mutations in fragments of three unrelated sequence contexts up to 741 bp were detected using one electrophoresis condition at the confidence level of <0.3% false positive. All the mutations were detected by analyzing at two temperatures. The described system has the advantage of little human intervention, short analysis time, high sensitivity, and objectivity of data interpretation.