Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

AIMS: To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. METHODS AND RESULTS: The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E. coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E. coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E. coli soluble protein compares favourably on all structural levels. CONCLUSIONS: SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E. coli, which was previously unachievable. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous literature reports have shown that CRM197 can be expressed in E. coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E. coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 .

Increased extravascular forces limit endothelium-dependent and -independent coronary vasodilation in congestive heart failure.

OBJECTIVE: The increase in coronary blood flow (CBF) in response to endothelium-dependent vasodilators is reduced in congestive heart failure (CHF) suggesting endothelial dysfunction. However, increases in extravascular compressive forces secondary to elevated left ventricular diastolic pressure (LVEDP) in CHF might contribute to this abnormality. METHODS: We measured CBF responses to intracoronary doses of the endothelium-dependent vasodilators acetylcholine (ACH) and bradykinin (BK) and the endothelium-independent vasodilator sodium nitroprusside (SNP) in the same eight dogs before (control) and after CHF was produced by 23+/-3 days of rapid ventricular pacing. In five of the dogs with CHF the zero-flow pressure (P(zf)), which reflects extravascular compressive forces in the maximally vasodilated coronary circulation (adenosine) was measured and found to strongly correlate with LVEDP (r=0.91). Coronary vascular resistance (CVR) at each concentration of vasodilator before and after the development of CHF was corrected for estimated coronary back pressure: CVR=(P(Ao)-LVEDP)/CBF, where P(Ao) is mean aortic pressure. RESULTS: CHF resulted in a significant decrease in CBF and increase in heart rate and LVEDP compared to control (P<0.05). The CBF responses to ACH, BK and SNP were all significantly reduced in the failing hearts (P<0.01). However, after correction for the elevated LVEDP in CHF, the response of CVR to the endothelium-dependent vasodilators was not different from normal. CONCLUSION: These findings suggest that endothelium mediated vasodilation is preserved in CHF, but that increased extravascular compressive forces act to limit the increase in CBF.

Regulation of myocardial blood flow by oxygen consumption is maintained in the failing heart during exercise.

The hemodynamic abnormalities and neurohumoral activation that accompany congestive heart failure (CHF) might be expected to impair the increase in coronary blood flow that occurs during exercise. This study was performed to determine the effects of CHF on myocardial oxygen consumption and coronary blood flow during exercise. Coronary blood flow was measured in chronically instrumented dogs at rest, during 2 stages of graded treadmill exercise under control conditions (n=10), and after the development of CHF produced by 3 weeks of rapid ventricular pacing (n=9). In the normal dogs, coronary blood flow increased during exercise in proportion to the increase in the heart rate x the left ventricular systolic blood pressure product (RPP). After the development of CHF, resting myocardial blood flow was 25% lower than normal (P<0.05). Myocardial blood flow increased during the first stage of exercise, but then failed to increase further during the second stage of exercise despite an additional increase in the RPP. Myocardial oxygen consumption during exercise was significantly lower in animals with CHF and paralleled coronary flow. Despite the lower values for coronary blood flow in animals with CHF, there was no evidence for myocardial ischemia. Thus, even during the second level of exercise when coronary flow failed to increase, myocardial lactate consumption continued and coronary venous pH did not fall. In addition, the failure of coronary flow to increase as the exercise level was increased from stage 1 to stage 2 was not associated with a further increase in myocardial oxygen extraction. Thus, cardiac failure was associated with decreased myocardial oxygen consumption and failure of oxygen consumption to increase with an increase in the level of exercise. This abnormality did not appear to result from inadequate oxygen availability, but more likely represented a reduction of myocardial oxygen usage with a secondary decrease in metabolic coronary vasodilation.

Effect of exercise on coronary pressure-flow relationship in hypertrophied left ventricle.

Left ventricular (LV) hypertrophy (LVH) secondary to chronic pressure overload is associated with increased susceptibility to myocardial hypoperfusion and ischemia during exercise. The present study was performed to determine whether exercise causes alterations in minimum coronary resistance or effective back pressure [coronary pressure at zero flow (Pzf)] that limit maximum myocardial perfusion in the hypertrophied heart. Ascending aortic banding in 7 dogs increased the LV weight-to-body weight ratio to 7.7 +/- 0.3 g/kg compared with 4.6 +/- 0.2 g/kg in 11 normal dogs (P < 0.01). Maximum coronary vasodilation was produced by intracoronary infusion of adenosine. Under resting conditions, the slope of the pressure-flow relationship (conductance) was significantly lower in the LVH animals than in the normal dogs (7.2 +/- 0.8 vs. 11.9 +/- 0.8 x 10(-2) ml.min-1.g-1.mmHg-1; P < 0.01); the slope correlated with the degree of hypertrophy r = 0.74; P < 0.001). The Pzf measured during total coronary artery occlusion (Pzf,measured) was significantly elevated in LVH compared with normal dogs (25.6 +/- 2.2 vs. 13.0 +/- 1.2 mmHg; P < 0.01); Pzf,measured was positively correlated (r = 0.78, P < 0.0005) with LV end-diastolic pressure measured during total coronary artery occlusion (9.0 +/- 1.1 mmHg in normal dogs and 22.2 +/- 3.2 mmHg in LVH dogs; P < 0.01). Graded treadmill exercise to maximum heart rates of 210 +/- 9 and 201 +/- 8 beats/min in normal and LVH animals, respectively, caused similar decreases in the slope of the pressure-flow relationship in LVH (from 7.7 +/- 0.9 to 6.1 +/- 0.8 x 10(-2) ml.min-1.g-1.mmHg-1; P < 0.01) and normal dogs (from 11.9 +/- 0.8 to 10.0 +/- 0.7 x 10(-2) ml.min-1.g-1.mmHg-1; P < 0.01). However, exercise-induced increases in Pzf,measured were significantly greater in the LVH animals (from 25.6 +/- 2.2 to 40.8 +/- 2.1 mmHg; P < 0.01) than in normal animals (from 13.0 +/- 1.2 to 24 +/- 2.1 mmHg; P < 0.01) (P < 0.01 LVH vs. normal). The greater increase in Pzf paralleled a more pronounced increase in LV end-diastolic pressure in the LVH dogs from 22.2 +/- 3.2 to 39.1 +/- 2.7 mmHg) than in normal dogs from 9.0 +/- 1.1 to 14.2 +/- 2.0 mmHg). The results suggest that exaggerated increases in filling pressure during exercise in the hypertrophied left ventricles contributed to impairment of myocardial perfusion during exercise by augmenting the back pressure, which opposes coronary flow.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha 1-adrenergic tone does not influence the transmural distribution of myocardial blood flow during exercise in dogs with pressure overload left ventricular hypertrophy.

This study was carried out to test the hypothesis that alpha 1-adrenergic activation during exercise causes preferential vasoconstriction of subepicardial coronary resistance vessels, thereby augmenting blood flow to the subendocardium. Studies were performed in 7 dogs in which left ventricular hypertrophy was produced by banding the ascending aorta at 6-9 weeks of age. Animals were studied at approximately 1 year of age when the left ventricular/body weight ratio was 7.7 +/- 0.3 g/kg (mean +/- SE). Left anterior descending (LAD) coronary artery flow was measured with a Doppler velocity flow probe at rest and during a three-stage graded treadmill exercise protocol. The transmural distribution of myocardial blood flow was assessed with radioactive microspheres. Coronary blood flow increased progressively as a function of heart rate and rate-pressure product in response to exercise. In contrast to normal dogs which maintain preferential blood flow to the subendocardium (ENDO) relative to the subepicardium (EPI) during exercise, the ENDO/EPI flow ratio in the hypertrophied left ventricles was 0.88 +/- 0.10 during exercise. Selective alpha 1-adrenergic blockade by infusion of prazosin (10 micrograms/kg) into the LAD decreased mean aortic pressure during exercise from 86 +/- 6 to 76 +/- 4 mmHg (p < 0.05), but did not change coronary pressure, heart rate, left ventricular systolic or enddiastolic pressures, or LVdP/dtmax. Coronary blood flow was not significantly altered by prazosin at rest, but was progressively increased during increasing levels of exercise levels. During the heaviest level of exercise prazosin caused a 22 +/- 3% increase in mean myocardial blood flow which was similar in all transmural layers, with no change in the transmural distribution of perfusion (ENDO/EPI = 0.85 +/- 0.09). These findings demonstrate that alpha 1-adrenergic vasoconstrictor tone limits blood flow during exercise in the hypertrophied left ventricle, but do not support the concept that alpha 1-adrenergic activation augments perfusion of the subendocardium during exercise.

Coronary pressure-flow relationship and exercise: contributions of heart rate, contractility, and alpha 1-adrenergic tone.

We examined the impeding effects of exercise on coronary blood flow by analyzing exercise-induced changes in the pressure-flow relationship during maximal coronary vasodilation with adenosine in chronically instrumented dogs and assessed the individual contributions produced by heart rate, contractility, and alpha 1-adrenergic vasoconstriction. Treadmill exercise that increased heart rate from 118 +/- 6 beats/min at rest to 213 +/- 8 beats/min (P < 0.01) decreased maximum coronary blood flows by decreasing the slope of the linear part of the pressure-flow relationship for coronary pressures > or = 30 mmHg (slopeP > or = 30) from 12.3 +/- 0.9 to 10.9 +/- 0.9 ml.min-1 x g-1 x mmHg-1 (P < 0.01) and increasing the measured coronary pressure at zero flow (P zf,measured) from 12.6 +/- 1.2 to 23.3 +/- 2.0 mmHg (P < 0.01). Atrial pacing at 200 beats/min caused an increase of P zf,measured from 15.0 +/- 1.6 to 18.3 +/- 2.1 mmHg (P < 0.05) with no change in slopeP > or = 30. While pacing continued, infusion of dobutamine (20 micrograms.kg-1 x min-1 i.v.) increased contractility to levels similar to those during exercise but caused no significant change in coronary blood flow, as a decrease of the slopeP > or = 30 was compensated for by a slight decrease in P zf,measured. alpha 1-Adrenergic blockade with intracoronary prazosin (10 micrograms/kg) did not prevent the exercise-induced increase of P zf,measured but abolished the decrease of the slopeP > or = 30. When the increases in heart rate, contractility, and alpha 1-adrenergic vasoconstriction were prevented, exercise still increased P zf,measured from 15.8 +/- 2.1 to 21.8 +/- 2.6 mmHg (P < 0.05) but had no effect on the slopeP > or = 30. This residual increase in P zf,measured correlated with the concomitant increase in left ventricular filling pressure. In conclusion, exercise-induced decreases of maximum coronary blood flow were explained by increases in heart rate, alpha 1-adrenergic vasoconstriction, and left ventricular filling pressure, with a minimal contribution of contractility.

Effect of superoxide dismutase and catalase on regional dysfunction after exercise-induced ischemia.

This study was designed to test the hypothesis that the oxygen free radical scavengers superoxide dismutase (SOD) and catalase may reduce myocardial "stunning" after exercise-induced ischemia. To test this hypothesis, 8 mongrel dogs performed treadmill exercise for 10 min in the presence of a flow-limiting coronary artery stenosis. Regional left ventricular function was measured with ultrasonic microcrystals implanted to measure regional wall thickening. Regional myocardial perfusion was measured with radioactive microspheres. The combination of SOD (5 mg/kg iv) and catalase (5 mg/kg iv) did not affect heart rate, blood pressure, coronary artery flow, or regional myocardial blood flow at rest, during exercise, or in the postexercise period. SOD and catalase had no effect on regional wall thickening at rest before exercise. During exercise in the absence of a coronary artery stenosis, thickening was slightly lower during SOD and catalase infusion (27 +/- 11.0 vs. 30.8 +/- 11.5%, SOD vs. control P = 0.05). During exercise in the presence of a coronary artery stenosis, there was no difference in thickening. Infusion of SOD and catalase affected neither the transient rebound function occurring early after exercise nor the prolonged period of stunning. These results indicate that the myocardial stunning that follows exercise-induced ischemia is unlikely to be mediated by oxygen free radicals.

Effect of exercise intensity and duration on regional function during and after exercise-induced ischemia.

BACKGROUND: Transient reversible myocardial dysfunction has been documented after episodes of exercise-induced ischemia. This study was undertaken to determine whether the duration or intensity of exercise affects the severity of postischemic dysfunction in this setting. METHODS AND RESULTS: Ten dogs were instrumented with ultrasonic microcrystals for measurement of wall thickening, with circumflex coronary artery flow probes, and with hydraulic occluders. Dogs performed low-intensity exercise, which was sufficient to increase coronary perfusion 50% above control, and high-intensity exercise, which was sufficient to double coronary blood flow. To investigate the effects of exercise intensity on postischemic dysfunction, we had dogs perform high-intensity exercise for 5 minutes in the presence of a stenosis. On the alternate day, dogs performed low-intensity exercise for 10 minutes in the presence of a stenosis. These two protocols provide equivalent coronary flow debts. Mean transmural blood flow during high-intensity exercise without stenosis (2.61 +/- 0.54 ml/min/g) was significantly higher than that during low-intensity exercise (1.74 +/- 0.61 ml/min/g, p less than 0.002). During high-intensity exercise with coronary artery stenosis, subendocardial blood flow was significantly lower than that during low-intensity exercise with stenosis (0.64 +/- 0.40 versus 1.08 +/- 0.28 ml/min/g, p less than 0.02). This difference in subendocardial perfusion was associated with greater degrees of regional dysfunction during exercise (circumflex wall thickening was 44 +/- 23% of control for high-intensity exercise versus 60 +/- 18% of control for low-intensity exercise, p less than 0.01). In addition, from 10 to 30 minutes after exercise, wall thickening in myocardium perfused by the circumflex coronary artery remained significantly lower after high-intensity exercise than that after low-intensity exercise. To assess the effects of exercise duration on the severity of postischemic dysfunction, we had dogs perform low-intensity exercise in the presence of a coronary stenosis for 10 minutes and low-intensity exercise for only 5 minutes on alternate days. Systolic wall thickening was significantly lower after low-intensity exercise for 10 minutes than after low-intensity exercise for 5 minutes. CONCLUSIONS: High-intensity exercise results in greater degrees of subendocardial hypoperfusion and greater degrees of regional dysfunction both during and after exercise-induced ischemia than does low-intensity exercise. Second, exercise duration also exerts an effect on the severity of postischemic dysfunction, although the magnitude of this effect is less important than the effect of exercise intensity.

Kinetics of the DNA binding of 2-substituted anthraquinones.

The DNA-binding properties of the 2-substituted-1,4-dihydroxyanthraquinones 5-12 were examined. Compounds 5 and 6 were synthesized in this study, 7-12 were already available. Spectral studies were consistent with intercalation of 5, 6 and 8 into DNA. Affinity constants were in the range of 3 x 10(4)M-1. Compounds 7 and 10-12 showed no DNA binding, presumably being sterically excluded from binding. The kinetics of the DNA interaction of 5, 6 and 8 were studied. There is a biphasic process. This may reflect an initial reaction, with drug from this first mode of binding moving into the second binding mode (a sequential process) or independent binding of drug in two sets of sites (a parallel process). The methods used in this study do not allow us to discriminate between these models. However, if the parallel binding model is correct, the compounds show sequence selectivity of binding, and provide a lead for further development of neutral DNA-binding ligands with sequence selectivity.

Dissociation kinetics of DNA-anthracycline and DNA-anthraquinone complexes determined by stopped-flow spectrophotometry.

The first order rate constants for the dissociation of daunorubicin, doxorubicin, and 1-; 1,4-; 1,5-; and 1,8-; N,N-diethylaminoethylamino-substituted anthraquinones from calf thymus DNA were determined using stopped-flow spectrophotometry. Sodium dodecyl sulphate was used to disrupt the equilibrium. In all cases there was an increase in the rate constant with temperature. The dissociation rate constants at 20 degrees, 25 degrees and 37 degrees, were in the order 1-; much greater than 1,8-; greater than 1,4-; greater than daunorubicin and doxorubicin greater than 1,5-disubstituted anthraquinone. The 1,5-disubstituted anthraquinone (VII) thus shows the slowest rate of dissociation from DNA; the DNA complex dissociating more slowly than the DNA complexes of the anthracyclines, daunorubicin and doxorubicin. The result is consistent with the data from computer graphics modelling studies [39] which show that DNA-breathing (transient base pair unstacking) has to occur to allow the docking of the 1,5-disubstituted anthraquinone (VII) into the receptor site. Hence once the 1,5-disubstituted anthraquinone molecule has intercalated into DNA, DNA-breathing is required before dissociation can take place. This is not necessary with the other compounds (though the 1,4-disubstituted anthraquinone (V) can bind in this manner as well). So the very slow dissociation of the DNA/1,5-disubstituted anthraquinone complex relative to that of the DNA complexes of the other compounds examined here, supports the proposed mode of binding [39].

Low doses of 2,4,5-trichlorophenoxyacetic acid are behaviorally teratogenic to rats.

Pre-natal exposure of rats to 2,4,5-T has long-term effects on behavior. A test for novelty responding detects abnormalities after exposure to a single dose as low as 6 mg/kg on day 8 of gestation. This is well below doses reported to be morphologically teratogenic, and raises concern for human exposure.