Search PubMed⌕ Search

Biomedical subjects

H Kambara

Publications and source records attributed to H Kambara.

At least 37 records · Page 2Linked to original sources
PubMed1996

Paternity testing by the detection of D1S80 VNTR using fluorescence image analyzer (Dualcolour system).

An improved method for DNA polymorphism typing of D1S80 VNTR locus and its application to paternity testing are described. For accurate estimation of the length of polymorphic DNA fragments, the size marker was labeled with fluorescence different from that of PCR primers, and co-electrophoresed as an internal standard. The dualcolour system of fluorescence image analyzer was used to detect the fragments and determine their size. This internal marker method could successfully overcome the problems of band pattern distortion and tailing, besides it allows easy and accurate interpretation of the DNA types. Our results indicate that the internal marker method is much more accurate than the method of using size marker in gel, even with the presence of distortion or tailing of the band patterns. Family studies applying this method showed complete agreement between the observed and predicted types.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Fragment walking for long DNA sequencing by using a library as small as 16 primers.

A DNA sequence can be rapidly and efficiently determined by digesting it into segments small enough to be sequenced at one time, then assembling them into contigs by searching for overlaps. Fragment walking achieves this without subcloning or preparing many kinds of primers; fragments obtained by digesting a template DNA are sequenced in parallel directly from the fragment mixture by using a set of 16 primers. Since the sequence adjacent to each cutting site is determined together with the fragment sequence, the contiguous fragment can be easily determined. The complete template DNA sequence can thus efficiently determined. The sequencing of pUC19 (2.7 kb) from both sides was done in one step by using only 15 fragments. The redundancy was only about 1.3, greatly reducing base-reading redundancy. Automating this strategy would increase the speed and efficiency of large-scale DNA sequencing.

Base Sequence↗
PubMed1996

Temperature profile of buffer-filled electrophoresis capillaries using air convection cooling.

Temperature profiles of buffer-filled capillaries (capillary temperature), using both natural and forced air convection for cooling, were obtained both theoretically and experimentally in order to achieve stable capillary electrophoresis for accurate DNA sequencing analysis. The capillary temperature was calculated using the temperature dependency of the buffer resistivity, and the threshold values of the electric field and the capillary temperature for temperature instability were characterized. The capillary temperature became unstable at about 420 V/cm and at about 62 degrees C regardless of the outer diameter of the capillary; these values approximately coincide with the measured values obtained using a very thin thermocouple. The sets of values of electric field, air temperature, and air flow velocity in forced air cooling were obtained to keep the capillary temperature at the 40-60 degrees C needed for accurate DNA sequencing in a Tris-boric acid-EDTA buffer.

Buffers↗
PubMed1996

Characteristics of single-stranded DNA separation by capillary gel electrophoresis.

The optimization of electrophoretic conditions for fast separation of single stranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4% T, 5% C gel at 100-300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4% T, 5% C gel were analyzed by the reptation theory and compared with those in a 9% T, 0% C matrix. The reptation plots indicate that the onset of reptation in a 4% T, 5% C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9% T, 0% C matrix. The 4% T, 5% C gel proved superior to the 9% T, 0% C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4% T, 5% C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.

Acrylic Resins↗
PubMed1996

Selecting and amplifying one fragment from a DNA fragment mixture by polymerase chain reaction with a pair of selective primers.

A new method for selecting and amplifying a single DNA fragment from a mixture is proposed. This method is applicable for the rapid classification of DNA fragments from a mixture and for preparation of sequencing templates. DNAs of several to tens of kilobases (kb) are digested with a four-base recognition restriction enzyme to produce smaller fragments. The complementary strand extension reactions are then carried out to produce fluorophore-labeled DNA fragments from the digestion products. These fragments can be rapidly classified according to their terminal-base sequences and their sizes are analyzed by capillary-array gel electrophoresis (CAGE). Electropherograms are used to characterize the fragments and to select polymerase chain reaction (PCR) primers. Any fragment in a digestion mixture can be amplified by PCR with a pair of primers selected from a primer pool by referring to the electropherograms of the fragments. This method was successfully used to compare the electropherograms of two different DNA strands and to sequence a several-kb DNA fragment without subcloning. Combined with CAGE, this method could be used to dramatically simplify DNA fragment analysis.

DNA↗
PubMed1995

DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA.

A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.

Animals↗
PubMed1995

Effects of nisoldipine in silent myocardial ischemia after healing of acute myocardial infarction.

Silent myocardial ischemia occurring after acute myocardial infarction is classified as Cohn type II and has a frequency of 20-30% in all patients with acute myocardial infarction. Follow-up data of patients with either silent or anginal ischemia show a poor prognosis. Thus, all ischemic episodes occurring after myocardial infarction should be treated aggressively. Many multicenter studies have evaluated whether drug treatment can improve prognosis or protect from a nonfatal second attack. Calcium antagonists, especially those that increase heart rate, have not been considered as drugs of choice for this purpose, despite the many beneficial effects shown on myocardial tissue in experimental studies. In the study reported here, the effect of nisoldipine on postinfarction silent myocardial ischemia was evaluated by ambulatory left ventricular function monitoring. Ten patients were selected for study who showed silent myocardial ischemia after their first acute infarction. Blood pressure fell significantly (p < 0.05) after 4 weeks of treatment with nisoldipine (5-10 mg/day), but heart rate showed no change at rest. Exercise time improved (p < 0.05), with increased peak double products. During exercise, there was no significant change in end-diastolic volume but there was a marked improvement in end-systolic volume, and at the submaximal point the ejection fraction was significantly (p < 0.05) increased. Ejection fraction at rest also improved. The deterioration in ejection fraction due to dipyridamole was ameliorated by nisoldipine. Ejection fraction and blood pressure improved during the calculation test, and work performance also improved.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Gender difference in long-term prognosis after myocardial infarction--clinical characteristics in 1000 patients. The Kyoto and Shiga Myocardial Infarction (KYSMI) Study Group.

To examine gender differences in the long-term prognosis of patients with myocardial infarction, 1000 patients with myocardial infarction were studied after coronary arteriography. Over a follow-up period of 3.3 +/- 2.0 years, 65 patients died from cardiac causes and 301 experienced cardiac events (death, reinfarction and revascularization). Overall, the 5-year cardiac mortality was 8%: that in females (12.4%) was significantly higher than that in males (6.6%) (p = 0.0073). The overall 5-year cardiac event-rate was 35%, with no significant difference between females and males (41.1% vs 33.3%). Univariate analysis revealed that differences in age (57.8 +/- 9.8 years in males vs 64.8 +/- 8.9 years in females, p < 0.0001), presence of smoking habit, obesity, hypercholesterolemia, hypertension, heart failure, right coronary artery disease, nicorandil administration, hypolipidemic, diuretic and anti-hypertensive treatment, and warfarin administration were present between men and women. The mortality rate in elderly females tended to be higher than that in their male counterparts. Multivariate analysis demonstrated that number of diseased vessels, post-infarction angina and left main trunk disease were significant predictors for cardiac death in both sexes, while gender was not. Therefore, gender did not appear to affect the long-term prognosis after age-adjustment among patients with myocardial infarction in the Western part of Japan.

Adult↗
PubMed1995

[Sudden death among 1,000 patients with myocardial infarction: incidence and contributory factors. KYSMI Study Group].

Clinical indicators and the incidence of sudden death among 1,000 consecutive patients (816 males and 184 females) with myocardial infarction were investigated between 1983 and 1987 after coronary arteriography. Twenty-four patients died suddenly during a study period of 3.3 +/- 2.0 yrs. The yearly incidence of sudden death calculated by the Kaplan-Meier method was 0.7%, nearly half of the incidence of cardiac death (1.5%/yr). Univariate analysis demonstrated statistically significant differences in the number of cases with coronary thrombolysis in the acute phase, degree of coronary artery stenosis, the frequency and degree of congestive heart failure and angina in the acute and chronic phase, the presence of serious post-infarction arrhythmia, ejection fraction, recurrent myocardial infarction, and administration of digitalis and diuretics. between the sudden-death group and the cardiac and other patient groups. There was also a tendency to higher frequency of history of diabetes mellitus, presence of acute mitral regurgitation, nitrates and nicorandil treatment, and coronary artery bypass graft in the sudden-death group. Multivariate analysis revealed that digitalis and nitrates treatment and left anterior descending artery involvement were major contributing factors. Re-infarction, left ventricular ejection fraction, and nicorandil therapy were not significant and minor contributing factors. The incidence of sudden death increased with digitalis therapy (Odds' ratio of 9.59), and left anterior descending artery disease, and decreased with nitrates (0.34).

Adult↗
PubMed1994

Separation of long DNA fragments by capillary gel electrophoresis with laser-induced fluorescence detection.

Long DNA fragments were separated by capillary gel electrophoresis with laser-induced fluorescence detection, and two approaches to obtaining high-resolution separation of DNA fragments were examined. One method achieved high resolution at the expense of detection time, while the other achieved both high resolution and fast detection. The former approach used a 300 cm long capillary with a gel concentration of 4% T and an electric field strength of 70 V/cm. The resolution limit in this electrophoresis was 800 bases with a resolution value of 0.5 for adjacent peaks. Under these conditions, the measurement time was 44 h. The latter approach used a 200 cm long capillary with a gel concentration of 3% T and an electric field strength of 170 V/cm. The resolution limit in this electrophoresis was 680 bases with a resolution value of 0.5 for adjacent peaks, and the measurement time was only 10 h. With both approaches, fragments with less than 600 bases were efficiently separated. The resolution values for adjacent peaks with less than 500 bases are greater than 1.0, and those for peaks with less than 100 bases are greater than 2.7. These approaches thus improve the accuracy of the base sequence determination.

DNA↗
PubMed1994

DNA sequencing directly from a mixture using terminal-base-selective primers.

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3' terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the HindIII digestion products of lambda phage.

Bacteriophage lambda↗
PubMed1994

DNA sequencing using ultra small amounts of reagents and template.

One of the key points in the genome project is finding ways to reduce the running cost in DNA sequencing. One way is to use a highly-sensitive fluorescent DNA sequencer, where only trace amounts of template DNA and reagents are needed. An experimental protocol optimized for the trace amounts of DNA analysis was established by using the hybridization reaction rate coefficient of primers on template DNA, which was estimated to be 7.5 x 10(5) M-1sec-1 at 37 degrees C. One femtomole of template DNA with 0.001 unit of modified T7 DNA polymerase (Sequenase Ver. 2.0) and also 0.45 fmol of M13 template DNA with 0.01 unit of Taq DNA polymerase were enough to sequence DNA of up to 400 bases.

Bacteriophage M13↗
PubMed1994

Detection of exercise-induced silent ischemia and the sequence of ischemic events in coronary artery disease by radionuclide ambulatory ventricular function monitoring.

Twenty-seven patients with stable coronary artery disease were continuously monitored with an ambulatory radionuclide ventricular function monitor (VEST) during exercise to determine the prevalence of silent ischemia and the temporal sequence of events during ischemic episodes. Exercise-induced ejection fraction abnormality was considered a < 6% increase in the control value lasting for more than 60 sec. Patients performed exercise for 424 +/- 111 sec during VEST recording. Seventeen exercise-induced ejection fraction abnormalities were observed in 17 patients, of which eight (47%) were silent electrocardiographically and 12 (71%) were silent symptomatically. In all of the patients, exercise-induced ischemia occurred in a temporal sequence of ejection fraction abnormalities, ST depressions and then symptoms. In nine patients with ejection fraction and ST abnormalities, ejection fraction abnormalities occurred earlier (199 +/- 87 sec) than ST depressions (321 +/- 117 sec; p < 0.01). In five patients with symptoms (399 +/- 151 sec), the sequence was ejection fraction abnormalities (205 +/- 64 sec; P < 0.05) followed by ST depressions (266 +/- 101 sec; P < 0.05) and symptoms (399 +/- 151 sec; P < 0.01). After termination of exercise, ejection fraction (49 +/- 12 sec) recovered sooner than symptoms (102 +/- 27 sec; p < 0.01) or ST abnormalities (191 +/- 96 sec; p < 0.01). Thus, hemodynamic monitoring by VEST may be helpful in detecting ischemic episodes in coronary artery disease which remain electrocardiographically or symptomatically silent. Furthermore, exercise-stress induces a cascade of temporal changes in ischemic events which may be helpful in understanding the pathophysiology of ischemia.

Aged↗
PubMed1993

Prognostic value of an increase in fluorine-18 deoxyglucose uptake in patients with myocardial infarction: comparison with stress thallium imaging.

OBJECTIVES: This study was undertaken to evaluate the prognostic value of an increase in fluorine (F)-18 deoxyglucose uptake compared with clinical, angiographic and stress thallium findings in patients with myocardial infarction. BACKGROUND: Positron emission tomography (PET) imaging using F-18 deoxyglucose has been applied to assess tissue viability in patients with coronary artery disease. We hypothesized that patients with a myocardial segment with augmented F-18 deoxyglucose uptake are at high risk for a future cardiac event. METHODS: One hundred fifty-eight consecutive patients with myocardial infarction referred for F-18 deoxyglucose PET and stress thallium scans were studied. Follow-up was obtained in 84 patients at a mean interval of 23 months to investigate prognostic implications of radionuclide studies. RESULTS: Seventeen patients had a cardiac event during the follow-up interval. Univariate analysis showed that an increase in F-18 deoxyglucose uptake was the best predictor of a future cardiac event (p = 0.0006), followed by the number of stenosed vessels (p = 0.008). In the multivariate analysis, when an increase in F-18 deoxyglucose uptake was entered into the model, only angiographic variables had an independent prognostic value, whereas no other radionuclide variables showed significant prognostic value. Among patients who did not show redistribution, a future cardiac event was observed more often in patients with than in those without an increase in F-18 deoxyglucose uptake (p < 0.05). CONCLUSIONS: Thus, an increase in F-18 deoxyglucose uptake seemed to be the best predictor of a future cardiac event among all clinical, angiographic and radionuclide variables in this study of stable patients with myocardial infarction. Even when a stress thallium-201 scan does not show redistribution, those patients who have an increase in F-18 deoxyglucose uptake in a PET study may be at risk for a future cardiac event, and these patients may need aggressive treatment to prevent a future cardiac event.

Aged↗
PubMed1993

Long-term prognosis after myocardial infarction: univariate and multivariate analysis of clinical characteristics in 1,000 patients. Kyoto and Shiga Myocardial Infarction (KYSMI) Study Group.

To examine the clinical outcome for patients with myocardial infarction and to analyze clinical predictors for long-term prognosis, 1,000 patients were studied retrospectively. Between January 1983 and December 1987, 1,002 consecutive patients with myocardial infarction, who resided in the Kyoto and Shiga districts, were reviewed after coronary arteriography, but in two patients medical records were not located. During 3.3 +/- 2.0 years, 75 patients died of cardiac causes and 301 experienced cardiac events (death, reinfarction, and revascularization). Overall 5-year cardiac mortality was 8% and cardiac event rate was 35%. Among determinants of age, smoking, hypertension, diabetes mellitus, heart failure, postinfarction angina, serious arrhythmia, mitral regurgitation, digitalis and diuretics administration, ejection fraction (EF), left main trunk disease, and number of diseased coronary arteries selected by univariate analysis, multivariate analysis revealed that heart failure, EF, number of coronary vessel disease, diabetes, and mitral regurgitation were the important predictors of cardiac death. For cardiac events, multivariate analysis demonstrated that the number of diseased coronaries, postinfarction angina, and left main trunk disease were significant predictors. Therefore, impaired left ventricular function and myocardial ischemia appear to be important markers for cardiac death, but impaired left ventricular function does not appear to be a predictor for cardiac events. The data obtained in this study will be useful in the assessment of patients with myocardial infarction and will be of clinical significance in predicting mortality and cardiac events.

Adult↗