Engineering a waste management enzyme to overcome cancer resistance to apoptosis: adding DNase1 to the anti-cancer toolbox.

Cancer treatment is often complicated by resistance to conventional anti-cancer treatment and to more recently developed immunotherapy and gene therapy. These therapeutic modalities aim at activating death pathways within cancer cells. Attempts to activate the apoptotic death pathway, by overexpressing proapoptotic signals, are compromised by cancer defense mechanisms, which disrupt the apoptotic-signaling cascade downstream of the overexpressed component. Here, we describe a therapeutic option of triggering apoptosis without activating the apoptotic-signaling cascade or using the native apoptosis executioner nuclease. We have engineered Deoxyribonuclease-1 (DNase1), a waste-management enzyme, by deleting its signal peptide, adding a nuclear localization signal, and mutating its actin-binding site. Apoptosis studies and colony-forming assay for assessing cell viability were conducted in apoptosis-resistant Mel-Juso human melanoma cells. The modified DNase1 reduced cell viability by 77% relative to controls. It also induced typical microscopic features of cellular apoptosis, such as Terminal Transferase dUTP Nick-End Labeling-positive cells and DNA fragmentation. Quantification of apoptosis by Laser scanning cytometry demonstrated high-killing efficiency of 70-100%. The results suggest that this modified DNase1 can efficiently eliminate apoptosis-resistant cancer cells through apoptosis. Coupled to different tissue-specific gene expression elements, this recombinant DNase1 may serve as a platform for eliminating a variety of cancer types.

Visualizing antibody-catalyzed retro-aldol-retro-Michael reactions.

We developed a visible detection system for antibody-catalyzed retro-aldol-retro-Michael reactions. Aldolase antibody 38C2 catalyzed the reaction of substrate 1 to provide 6-bromo-2-napthol that forms a visible colored azo dye with diazonium salts. This system has potential for the screening of novel catalysts.

A randomised, double-blind comparison of the angiotensin II receptor antagonist, irbesartan, with the full dose range of enalapril for the treatment of mild-to-moderate hypertension.

OBJECTIVE: To compare the anti-hypertensive efficacy, safety, and tolerability of irbesartan with those of the full dose range of enalapril in patients with mild-to-moderate hypertension. DESIGN AND METHODS: A total of 200 patients were randomised to irbesartan 75 mg or enalapril 10 mg (once daily). Doses were doubled at Weeks 4 and/or 8 if seated diastolic blood pressure (DBP) was > or = 90 mm Hg. Trough blood pressure was measured after completion of a 4- to 5-week placebo lead-in period and again after 2, 4, 8, and 12 weeks of treatment. MAIN OUTCOME MEASURES: Efficacy was evaluated by determining the change from baseline in trough seated blood pressure and the proportion of patients normalised (seated DBP <90 mm Hg) at Week 12. Safety and tolerability were assessed by adverse events reported by physicians, by patients in response to a specific-symptoms questionnaire, by open-ended questioning of patients by physicians, and by clinical laboratory evaluations. RESULTS: Both treatments significantly lowered blood pressure with no significant difference in efficacy between treatment groups. At Week 12, the percentage of patients titrated to either enalapril 40 mg or irbesartan 300 mg was 24% and 28%, respectively. The frequency of overall adverse events was similar in both groups. The incidence of cough in the enalapril and irbesartan groups was 17% and 10%, respectively. In contrast to other AII receptor antagonists, there was no change in uric acid concentrations with irbesartan. CONCLUSIONS: Irbesartan was as effective as the full dose range of enalapril. Irbesartan also demonstrated an excellent tolerability profile.

Comparison of the angiotensin II receptor antagonist irbesartan with atenolol for treatment of hypertension.

In this multicenter, double-blind study, the antihypertensive efficacy and safety of irbesartan were compared with those of atenolol in patients with mild-to-moderate hypertension. Following a 4- to 5-week placebo lead-in period, 231 patients with seated diastolic blood pressure (SeDBP) 95-110 mmHg were randomized to irbesartan 75 mg or atenolol 50 mg once daily for 24 weeks. Doses were doubled at Week 6 for SeDBP > or = 90 mmHg. At Week 12, or anytime thereafter, doses were doubled for SeDBP > or = 90 mmHg if not done at Week 6, and hydrochlorothiazide and then nifedipine were added. Efficacy was determined by change from baseline in blood pressure and by therapeutic response rates. Safety was assessed by monitoring adverse events (AEs). Both treatments significantly lowered blood pressure from baseline. There were no significant differences between treatment groups with respect to blood pressure changes or therapeutic response. Atenolol significantly reduced seated heart rate compared with irbesartan at Week 12. The incidences of serious AEs and discontinuations due to AEs were approximately twice as high in the atenolol group compared with the irbesartan group. Thus, in comparison to atenolol, irbesartan < or = 150 mg provided at least equivalent blood pressure control while demonstrating an excellent safety and tolerability profile.

Factors affecting the termination of cardiovascular actions of 10,10-difluoro,13-dehydroprostacyclin.

Experiments were conducted to determine why 10,10-difluoro,13-dehydroprostacyclin (DF2-PGI2) has a long vascular relaxant activity in vitro but like PGI2 has a short duration of effect in vivo. DF2-PGI2 produced depressor responses in anesthetized dogs which were not affected by nephrectomy suggesting that the kidney was not responsible for the termination of action. DF2-PGI2 given intravenously or into the ascending aorta produced depressor responses of a similar magnitude but injection of the same doses into the hepatic portal circulation resulted in a large attenuation of responses. The data suggest hepatic, but not pulmonary, metabolism of DF2-PGI2. Injection or infusion of PGI2 and DF2-PGI2 into the hindlimb circulation caused vasodilatation of a similar duration suggesting diffusion from tissue sites as another mechanism of termination of action.

On the effects and mechanism of action of the antihypertensive agent TR 3369 (5-methoxytryptamine beta-methylcarboxylate) in spontaneously hypertensive rats.

The effects of the serotonin (5-HT) analog TR 3369 (5-methoxytryptamine beta-methylcarboxylate) on blood pressure and heart rate of spontaneously hypertensive rats (SHR) were examined. In conscious SHR, TR 3369 caused reductions in blood pressure without importantly changing heart rate in doses ranging from 1 to 30 mg/kg p.o. TR 3369 was found to have no significant antagonistic effects on alpha-, beta-, or 5-HT receptors, nor did the drug inhibit adrenergic neuronal or ganglionic function. A slight but unimportant effect on angiotensin II pressor responses was noted. Therefore, the data are in agreement with the suggestion that TR 3369 acts through a central mechanism of action. The 5-HT antagonist cinanserin had little effect on blood pressure of SHR when administered alone, whereas it markedly reduced the duration, but not the magnitude, of the TR 3369 antihypertensive action in SHR. It is suggested that at least a portion of the antihypertensive effect of TR 3369 involves activation of central 5-HT receptors.

International science--an overview.

Scientific projects that succeed as international cooperative efforts are those related to subjects that transcend national frontiers, are costly, have long-range objectives rather than short-term commercial aims, and correspond with the political objectives of the countries involved. Yet the best context for the all science is the global community, which is also the best hope for humanity. The global community is still generations away and scientists must continue to work for it, by seeking the international dimensions of science as individuals and participating in governmental as well as nongovernmental international scientific organizations.

Pre- and postjunctional inhibition of vascular sympathetic function by captopril in SHR. Implication of vascular angiotensin II in hypertension and antihypertensive actions of captopril.

The present study was designed to examine the effects of treatment of SHR with captopril, teprotide, and saralasin on vascular and cardiac responses to sympathetic nerve stimulation and angiotensin I and II (AI, AII) and norepinephrine (NE). A single dose of captopril (10 mg/kg i.v. as well as 10 and 100 mg/kg p.o.) caused significant and marked inhibition of pressor responses to sympathetic nerve stimulation in pithed SHR but cardiac responses were unaffected. Pressor responses to AI were abolished but those to AII and NE were not significantly altered. Neither teprotide nor saralasin caused consistent inhibition of sympathetic responses despite total blockade of AI and AII response respectively. Selective inhibition of pressor but not cardiac responses to sympathetic nerve stimulation was obtained after 2 weeks, 3 and 6 months of daily oral doses of captopril. In addition, postjunctional pressor responses to AI, AII, and NE were also significantly inhibited by chronic captopril treatment. Infusion of AII, bilateral nephrectomy, or pretreatment with indomethacin alone in pithed SHR receiving captopril had no effect on the inhibition of pressor responses to sympathetic stimulation. However, the combination of pretreatment of indomethacin and infusion of AII completely restored sympathetic function in SHR receiving captopril. These studies suggest that captopril has a selective inhibitory effect on vascular responses to sympathetic nerve stimulation but not on cardiac responses. Moreover, this effect may have a prejunctional component since, after acute treatment, there is no inhibitory effect on responses to AII or NE. Since, under appropriate conditions, the inhibition can be reversed by AII infusion but not nephrectomy, it is suggested that this inhibition occurs at the vascular level by inhibition of local AII formation by captopril, a site not accessible to teprotide or saralasin.

Evidence for prejunctional inhibition of norepinephrine release by captopril in spontaneously hypertensive rats.

Pretreatment of spontaneously hypertensive rats (SHR) with the converting enzyme inhibitor captopril (10 or 100 mg/kg p.o.) had no effect on pressor responses to angiotensin II or norepinephrine whereas the response to angiotensin I was markedly inhibited. In contrast, pressor responses to sympathetic stimulation in pithed SHR were inhibited by captopril whereas the positive chronotropic responses to stimulation were unaltered. These results suggest that captopril causes a prejunctional inhibition of norepinephrine release to sympathetic nerve stimulation which is selective for the vasculature. This is probably due to inhibition of angiotensin II formation in the vasculature.

Cardiovascular effects of alloxan diabetes in normotensive and spontaneously hypertensive rats.

The diabetogenic effects of alloxan (50 mg/kg i.v.) were recorded for 6 weeks in normotensive (WKY) and spontaneously hypertensive (SHR) rats. While systolic blood pressure increased in diabetic WKY, SHR exhibited a reduction in systolic blood pressure over the 6-week period. Heart rate of treated SHR, but not WKY, was reduced significantly during the course of diabetes. 6 weeks postalloxan, the pressor responses to spinal cord stimulation and various vasoactive agents were significantly reduced in both diabetic groups as compared to their age-matched controls. However, in vitro preparations taken from diabetic and control WKY rats showed similar responsiveness to vasoactive agents. The data suggest that circulatory factors contribute to the decreased in vivo responsiveness.

Mediation of enhanced reflex vagal bradycardia by L-dopa via central dopamine formation in dogs.

L-Dopa (5 mg/kg i.v.) decreased blood pressure and heart rate after extracerebral decarboxylase inhibition with MK-486 (25 mg/kg i.v.) in anesthetize MAO-inhibited dogs. In addition, reflex bradycardia caused by injected norepinephrine was significantly enhanced by L-dopa, DL-Threo-dihydroxyphenylserine had no effect on blood pressure, heart rate or reflex responses to norepinephrine. FLA-63, a dopamine-beta-oxidase inhibitor, did not have any effect on the hypotension, bradycardia or reflex-enhancing effect of L-dopa. Pimozide did not affect the actions of L-dopa on blood pressure and heart rate but completely blocked the enhancement of reflexes. Removal of the carotid sinuses caused an elevation blood pressure and heart rate and abolished the negative chronotropic effect of norepinephrine. However, L-dopa restored the bradycardia caused by norepinephrine in addition to decreasing blood pressure and heart rate. 5-HTP (5 mg/kg i.v.) decreased blood pressure and heart rate and decreased the reflex bradycardia to norepinephrine. It is concluded that L-dopa enhances reflex bradycardia through central alpha-receptor stimulation. Furthermore, the effects are mediated through dopamine rather than norepinephrine and do not require the carotid sinus baroreceptors.

Centrally mediated increased reflex vagal bradycardia after L-dopa in monoamine oxidase-inhibited anesthetized dogs.

In monoamine oxidase-inhibited dogs whose peripheral dopa decarboxylase was inhibited with MK-486, L-dopa (K mg/kg i.v.) caused significant hypotension and bradycardia. In addition, reflex bradycardia but not pressor responses to norepinephrine were markedly enhanced by the drug. Similar results were obtained after intracerebroventricular administration of L-dopa (0.5 mg/kg) in monoamine oxidase-inhibited dogs not receiving MK-486. Proparnolol prevented the bradycardia caused by L-dopa but was without effect on the reflex facilitation. Both atropine and phentolamine had no effect on L-dopa bradycardia but prevented the facilitatory effect on reflexes. Inhibition of both peripheral and central dopa decarboxylase with Ro 4-4602 prevented all the effects of L-dopa. Infusions of dopamine which had obvious cardiovascular effects had no effect on reflex responses to norepinephrine. It is concluded that L-dopa, after decarboxylation, causes a centrally mediated enhancement of reflex vagal bradycardia. Pharmacological analysis of this enhancement suggests that this effect appears to be a result of central alpha receptor stimulation.