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.

[Quantitative assessment of improvement in perfusion after coronary bypass grafting: assessed by thallium-201 bull's eye subtraction polar map method].

To assess redistribution (RD) and improvement in regional perfusion after coronary bypass grafting (CABG) on thallium-201 SPECT images quantitatively, the Bull's eye subtraction polar (BS) map was created. BS map was created after subtraction of a normalized bull's eye polar map from another normalized polar map. The quantitative assessment on this map was compared to the visual qualitative analysis in 23 patients (115 segments) who received CABG. All of the improved segments after CABG showed > or = 15% on the BS map. On the other hand, 60% of the non improved segments showed < 10% on the BS map. Furthermore, 67% of the RD segments showing > or = 15% on the BS map before CABG improved in perfusion by > or = 15% on the BS map after CABG. On the BS map, an excellent correlation was observed between the extent of redistribution before CABG and the extent of improvement after CABG. Thus, BS map permits quantitative assessment of improvement in perfusion on T1 SPECT imaging, and this technique seems to be valuable for predicting improvement in perfusion after CABG.

Assessment of myocardial fatty acid metabolism with positron emission tomography at rest and during dobutamine infusion in patients with coronary artery disease.

The myocardial clearance rate of C-11 palmitate as an index of fatty acid oxidation was assessed by means of positron emission tomography (PET) at rest and during dobutamine infusion in seven normal subjects and 10 patients with coronary artery disease. In the normal subjects the clearance half time was homogeneous in the left ventricle at rest and uniformly shortened during dobutamine infusion. In the myocardium at risk, clearance half time tends to be longer in the segments with an abnormal Q wave on ECG, exhibiting regional wall motion abnormality, and supplied by severely stenosed coronary arteries, particularly during dobutamine infusion. These data indicate that fatty acid oxidation may be decreased in infarcted myocardium and associated with regional asynergy. Such an abnormality was most striking in those with severe coronary stenosis during dobutamine infusion. We conclude that PET with the use of C-11 palmitate at rest and during dobutamine is a useful means of identifying impaired fatty acid oxidation and decreased metabolic reserve in patients with coronary artery disease.

[Assessment of myocardial metabolic reserve with positron emission tomography and C-11 acetate].

Positron emission tomography (PET) with C-11 acetate allows noninvasive quantification of myocardial oxidative metabolism. To assess the myocardial metabolic reserve, clearance of C-11 acetate from the myocardium was measured with PET in 8 normal subjects at rest and during the infusion of dobutamine (inotropic agent) or dipyridamole (vasodilator). During dobutamine infusion, the clearance rate constant of C-11 acetate increased significantly (0.063 +/- 0.014 vs 0.109 +/- 0.016, p < 0.002) in good correlation with the rate-pressure product (r = 0.83). During dipyridamole infusion, myocardial acetate clearance also significantly increased (0.088 +/- 0.024, p < 0.05) in correlation with the rate-pressure product (r = 0.54). In normal myocardium, myocardial oxidative metabolism increased in response to increased work load with pharmacological stress. These findings suggest that PET with C-11 acetate and pharmacological stress may be a promising approach for the evaluation of metabolic reserve in patients with ischemic heart disease.

Regional metabolic abnormality in relation to perfusion and wall motion in patients with myocardial infarction: assessment with emission tomography using an iodinated branched fatty acid analog.

The clinical usefulness of single-photon tomography using both a beta-methyl-branched fatty acid analog, 123I-15-(p-iodophenyl)-3-methyl pentadecanoic acid (BMIPP) and 201TI was assessed in 4 normal subjects and 28 patients with myocardial infarction. A homogeneous distribution of the tracer in the left ventricular myocardium was observed in each normal subject. BMIPP uptake was decreased compared to 201TI (discordant) in 17/28 patients (61%) and in 49/196 myocardial segments (25%). Such discordant BMIPP uptake was observed more often in areas of acute myocardial infarction (59% at less than or equal to 4 wk versus 31% at greater than 4 wk after onset) (p less than 0.01) and areas supplied with revascularized arteries (74% for revascularized versus 28% for nonrevascularized areas) (p less than 0.01). In addition, the discordant BMIPP uptake was seen more often in the segments exhibiting a wall motion score lower than the perfusion score (46%) in comparison to segments showing a similar decrease in both the wall motion and perfusion scores (12%) (p less than 0.01). Thus, BMIPP imaging may play a major role in increasing our understanding of the relationship between perfusion and wall motion, particularly in patients with acute myocardial infarction and those who received revascularization therapy.

[Assessment of resting thallium-201 reinjection after stress-delayed Tl imaging (I): Comparison with 24-hour scan and regional wall motion].

Clinical value of resting reinjection of 1 mCi (37 MBq) of Tl after stress-delayed scan was assessed in comparison with 24 hr delayed scan and regional wall motion (RWM) in 37 patients with coronary artery disease. Of 101 segments with initial perfusion abnormality, concordant findings were observed after Tl reinjection in 67 segments (66%). But redistribution (RD) after Tl reinjection was observed in 19 of the 52 persistent defect (PD) segments (37%), and complete RD was observed 15 of the 43 segments (35%) where 3 hr scan showed incomplete RD. On the other hand, concordant findings were observed on 24 hr delayed scan in 11 of the 20 segments with perfusion abnormality (55%). RD on 24 hr delayed scan was observed in 5 of the 13 PD (38%), but it was not apparent in 3 of the 7 segments where 3 hr scan showed RD. Furthermore the segments which showed RD after Tl reinjection in PD segments tend to have less severe RWM abnormality than the sustained PD segments (RWM score: 1.65 +/- 1.17 vs. 2.29 +/- 1.05, p less than 0.05). These data indicate that Tl reinjection which permits assessment of RD with good quality images on the same day is considered as a valuable means for assessing myocardial viability.

[Assessment of myocardial viability by quantitative analysis of stress Tl-SPECT--comparison with FDG-PET].

Stress Tl-201 tomography (SPECT) is widely used for evaluating myocardial viability. To assess its value, redistribution (RD) on SPECT was compared with metabolic imaging using FDG. Thirty patients with coronary artery disease underwent stress-3 hour Tl-201 SPECT and PET using N-13 ammonia and FDG. RD was classified into 4 grading, including complete RD (CR), incomplete RD (IR), persistent defect (PD) and additional minimal RD (MR) defined as no definite RD on visual analysis but faint RD with Bull's eye quantitative analysis (QNT). All but one segment with CR or IR were viable regions (normal or ischemic regions) by PET. Of 74 segments without RD on visual analysis, 31 segments (42%) had RD by QNT (MR). All of them were viable regions by PET. Thus, QNT identified 31 segments (63%) of the metabolically viable segments which the visual Tl-201 analysis did not show RD and classified as myocardial scar. However, even such QNT cannot detect ischemic myocardium in 18 segments (42%) containing metabolic activity on PET. These data indicate that QNT of RD on Tl-201 SPECT is considered as a valuable means for assessing myocardial ischemia.

[Serial assessment of cardiac function during and after exercise by an ambulatory ventricular function monitor (VEST)].

Cardiac function was serially assessed during and after exercise by an ambulatory ventricular function monitor (VEST) in 31 patients who received coronary angiography. Based on the study of fluctuation during the baseline recording, greater than or equal to 6% change in ejection fraction (EF) was considered significant. The serial changes in EF during exercise was divided into 5 types, including continuous increase (type A), initial increase but return to the baseline (type B), no change (type C), initial increase but later decrease below the baseline (type D), and continuous decrease (type E). Among 8 normal subjects, their EF changes during exercise showed type A in 3, type B in 2, type C in 2, and type D in 1. Among 21 patients with coronary artery disease, the EF changes showed type A in 5, type B in 4, type C in 4, type D in 5 and type E in 3. Thus, there was a significant overlap in EF response between normal and coronary patients. However, every patient showing type A and B had single-vessel disease, and 63% of them had persistent thallium defect without redistribution. After the exercise, 29 patients showed rapid increase in EF. The time to the peak EF was significantly longer in coronary patients (1.88 +/- 1.24 min) than that in normal cases (0.88 +/- 0.55 min) (p less than 0.05) particularly in patients with multi-vessel disease (2.22 +/- 1.29 min). In addition, those showing type C, D or E tended to have a longer time to peak EF and more increase in EF after exercise than those showing type A or B. These data suggest that VEST is suitable for continuous measurement of cardiac function during and after exercise which provided valuable indices for assessment of severity of ischemia in coronary artery disease.

Radiocardiographic assessment of dobutamine and isosorbide dinitrate therapy in patients with mitral stenosis and pulmonary congestion.

Simultaneous hemodynamic and radiocardiographic measurements were performed on 10 patients with mitral stenosis and pulmonary congestion for evaluating the acute effects of dobutamine (DB, 5 micrograms/kg/min), isosorbide dinitrate (ISD, 10 mg sublingually) or a combination of the two. DB alone produced a significant increase of the cardiac index (CI) from 2.9 +/- 0.1 to 3.7 +/- 0.2 L/in/m2 (p less than 0.01), but a modest increase in pulmonary artery diastolic pressure (PADP) and in pulmonary blood volume by approximately 15%, respectively. ISD alone caused a decline in PADP from 26 +/- 2 to 18 +/- 1 mmHg (p less than 0.001), in right heart volume from 300 +/- 36 to 215 +/- 18 ml/m2 (p less than 0.05) and in left heart volume from 321 +/- 28 to 248 +/- 20 ml/m2 (p less than 0.05), but no change in the CI. Combined administration of the two agents resulted in favorable alterations in both hemodynamic variables: PADP decreased from 26 +/- 2 to 20 +/- 1 mmHg (p less than 0.01) and the CI increased from 2.9 +/- 0.1 to 3.3 +/- 0.1 L/min/m2 (p less than 0.05). Thus, DB alone had a tendency to aggravate pulmonary venous congestion in our patients, while ISD is effective in reducing the congestive manifestations of heart failure due to its venodilating effects but less beneficial in increasing the CI. The combined therapy of DB and ISD appears to be extremely effective in restoring an adequate cardiac output and in relieving the symptoms of pulmonary vascular congestion in the presence of mitral stenosis.

[Assessment of left ventricular function at rest and during exercise by gated cardiac blood pool scan and pressure measurement of the pulmonary artery by Swan-Ganz catheter].

Gated cardiac blood pool scan and hemodynamic parameters by Swan-Ganz catheter were simultaneously measured in five normal subjects (Group A), five patients with coronary artery disease (CAD) without angina on exercise (Group B) and nine patients with CAD with angina on exercise (Group C) in order to evaluate left ventricular function at rest and during supine bicycle ergometer exercise. Work load was gradually increased every 3 minutes and each data were acquired using a digital computer. There was a fairly good correlation (r = 0.83) between stroke volume by thermodilution method and stroke counts corrected by counts/ml of blood on gated cardiac blood pool scan, which were obtained by subtraction of end-systolic counts (ESC) from end-diastolic counts (EDC) in the region of interest arranged at the left ventricle. Stroke counts obtained by this method are considered to reflect a change in the left ventricular volume during exercise. As to the correlation between pulmonary capillary wedge pressure (PCWP) and stroke work index (SWI) at peak exercise, there was a large increase in SWI (78.9 +/- 17.6 to 112.3 +/- 30.0 g X m/m2) with a small rise in PCWP (10.0 +/- 1.9 to 16.2 +/- 4.0 mmHg) in Group A. In Group B, SWI was increased (57.8 +/- 15.8 to 92.9 +/- 25.2 g X m/m2) with a moderate rise in PCWP (11.8 +/- 3.7 to 20.0 +/- 3.1 mmHg) at peak exercise. In Group C, there was a trivial increase in SWI (74.0 +/- 16.0 to 81.2 +/- 29.3 g X m/m2) despite a marked rise in PCWP (13.1 +/- 3.1 to 33.4 +/- 8.9 mmHg). Similar relationship was present between SWI and EDC. On mild exercise of 25w, however, the rise in PCWP was more prominent rather than the increase in EDC in Group C. This indicates that the left ventricle in Group C is operating on a very steep portion of the diastolic pressure-volume curve.

Noninvasive assessment of T-wave abnormalities on precordial electrocardiograms in middle-aged professional bicyclists.

Six middle-aged, active, professional bicyclists with T-wave abnormalities on precordial ECGs were studied noninvasively. Twenty-five aged-matched bicyclists without T-wave abnormalities served as the control subjects. Increased voltage of SV1 + RV5 was demonstrated in all subjects. A 5-year follow-up study revealed that these abnormalities of T-wave inversion became more pronounced with age, except in one case. VCGs showed enlargement of anterior QRS loop and discordant T loop, in all cases. On echocardiography, thickness of both the interventricular septum and the left ventricular posterior wall, and left ventricular mass were significantly increased compared with the control group. 201Tl myocardial scintigraphy at rest and during exercise revealed no regional perfusion defects of the tracer in either case. We conclude that: (1) T-wave abnormalities of precordial ECGs in six middle-aged athletes were progressive in nature; and (2) these electrocardiographic abnormalities seem to be related to left ventricular hypertrophy induced by steady and strenuous training rather than to coronary artery disease.

A factor analytic assessment of prognosis in ischemic heart disease using thallium-201 myocardial scintigraphy, radiocardiograhy and orthogonal electrocardiography.

One hundred and three subjects with ischemic heart disease (IHD) were followed-up in an attempt to assess the most important factors affecting morbidity and mortality. At the time of initial examination 23 patients had congestive heart failure. During the one year follow-up after the examination 11 patients died. Various parameters including perfusion defects (SCORE) on 201 Tl-myocardial scintigrams, radiocardiographic hemodynamic data and orthogonal electrocardiographic parameters were analysed at the onset of the study, according to the method of "factor analysis". The factor analytic technique enabled a summary of the data obtained from these non-invasive approaches into five major interpretable components termed factors 1 to 5; factor 1 as "the extent of myocardial ischemia", factor 2 "heart size", factor 3 "cardiac pump function", factor 4 "peripheral blood volume" and factor 5 "QRS forces". The relationship between these five factors with a one-year mortality rate was then investigated. Of these factors, factor 1 which included SCORE, spatial G/QRS, T/QRS and QRS-T angle proved to be the most important as related to morbidity and mortality in IHD. Factors 2 and 3 were also shown to be useful prognostic indicators of subsequent mortality. Thus, a combination of these non-invasive parameters is useful not only to diagnose IHD but also serves as one form of evaluation of the prognosis in patients with IHD.