For trial use in the local chemotherapy of cancer by a combination of cytosine deaminase (EC 3.5.4.1) and 5-fluorocytosine (J. Biotechnol., (1985), 2, 13-21), 40 U of partially purified cytosine deaminase was obtained from 500 g of commercial compressed bakers' yeast. The enzyme, which is unstable, was immobilized to stabilize it by the use of commercial epoxy-acrylic beads (Eupergit C). The immobilized enzyme was made into enzyme capsules with cellulose tubing for dialysis to encapsulate it or urethane polymer to entrap it, which materials are biocompatible. The activity of the intact cellulose capsules thus made was 0.4% that of the immobilized enzyme inside. The enzyme capsules also were stable. Ten days after the cellulose capsules were implanted in rats, 25% of the starting activity remained. When the polyurethane capsules were tested in vitro for 9 mo for thermostability at 37 degrees C, the activity decreased rapidly (with a half-life of 28 d) during the first 4 mo, and then slowly (half-life, about 100 d) during the next 5 mo. A calculation to transform the biphasic decline into a sum of the exponential decline of two components of enzymic activities with different strengths and half-lives showed that the larger half-life was 5 mo.

Antineoplastic Agents↗

PubMedDate not supplied

Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates.

Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.

Adenocarcinoma↗

PubMedDate not supplied

Reversal of adrenaline-induced contraction in the rabbit pulmonary artery by clonidine.

Characterization of clonidine which reversed the adrenaline-induced contractions was assessed using the rabbit pulmonary arterial segment. Clonidine in high concentrations (10(-5)-10(-4) mol/l) contracted this vascular tissue. Nevertheless, an adrenaline (3 X 10(-6)mol/l)-induced contraction, which can be markedly depressed by phentolamine, was reversed to relaxation by pre-addition of 3 X 10(-5)mol/l clonidine. This reversal was abolished by 10(-5) mol/l propranolol, a beta-adrenoceptor antagonist. The cumulative concentration-contraction curve for adrenaline was shifted to the right by 10(-5) mol/l clonidine without further alteration at 10(-4) mol/l, suggesting a partial agonistic feature of clonidine for alpha-adrenoceptors. Contractions evoked by 5 X 10(-5) mol/l clonidine alone were effectively reduced by prazosin (IC50 = 1.7 X 10(-7) mol/l) or yohimbine, (IC50 = 3.3 X 10(-5) mol/l) and those by phenylephrine (10(-5) mol/l) were suppressed as well (prazosin, IC50 = 3.4 X 10(-8) mol/l; yohimbine, IC50 = 5.0 X 10(-6) mol/l). However, the depolarization-induced contractions with 30 mmol/l KCl were virtually unaltered by both antagonists. These results suggest that the reversal action of clonidine on the adrenaline-induced contraction is primarily mediated by a partial agonistic property for postsynaptic alpha 1-adrenoceptors.

Animals↗

PubMedDate not supplied

Cholinergic neuromodulation by ATP, adenosine and its N6-substituted analogues in guinea-pig ileum.

The effects of ATP, adenosine and N6-substituted adenosines, adenosine receptor agonists, on the twitch contraction of guinea-pig ileum evoked by transmural stimulation were evaluated. Adenosine and ATP produced an immediate and concentration dependent inhibition of the twitch, IC50 being 1.1 X 10(-5) mol/l and 1.2 X 10(-5) mol/l, respectively. N6-l-phenylisopropyl adenosine (L-PIA), N6-cyclohexyl adenosine (CHA) and N6-allyl adenosine also induced inhibitions which were gradual and persistent, IC50 being 2.6 X 10(-8), 2.7 X 10(-8) and 5.4 X 10(-7) mol/l, respectively. Dipyridamole (10(-7) mol/l), an adenosine uptake inhibitor, markedly augmented the inhibition evoked by adenosine and ATP, but not that by three N6-substituted adenosines, while theophylline (10(-4) mol/l) almost completely antagonized the inhibitory effects of all purine compounds. IC50 value of adenosine in the presence of dipyridamole (5 X 10(-7) mol/l) was shifted to the left about 50 times from the control, whereas that of L-PIA was virtually unchanged. Tissue-medium ratios indicating uptake of [3H]adenosine, [3H]ATP and [3H]CHA into the segment were 3.23 (s.e.m. = 0.59), 3.59 (s.e.m. = 0.78) and 0.41 (s.e.m. = 0.04), respectively. These results suggest that not only adenosine and ATP but also these N6-substituted adenosines are potent agonists for the P1 receptor, implying a similarity between P1 and A1 receptor in a functional role and these purine compounds may thereby modulate cholinergic neurotransmission by altering adenylate cyclase activity.

Adenosine↗

PubMedDate not supplied

Cardiovascular effects of brovincamine and possible mechanisms involved.

Cardiovascular effects of brovincamine (BV) and possible modes of action were studied in dogs, rabbits and guinea-pigs. In pentobarbitone-anaesthetized dogs, intravenous administration of BV (1.6-12.8 mg/kg) induced a dose-dependent decrease of blood pressure and heart rate, a slight and transient stimulation of respiration, and a prolongation of the R-R interval in ECG without changes of wave pattern. In the isolated atria of guinea-pigs, BV showed no effect at 10(-7) and 10(-6) g/ml, but dose-dependently inhibited both myocardial contractile force and heart rate at the higher concentrations of 10(-5) and 10(-4) g/ml. In pentobarbitone-anaesthetized dogs, BV (0.1-1.6 mg/kg) administered to the vertebral or carotid artery dose-dependently increased the blood flow in the respective artery. Intravenous administration of BV (0.8-12.8 mg/kg) also exhibited a dose-dependent increase of the vertebral and carotid blood flow, but the increase was a little reduced at the higher dose of 12.8 mg/kg, probably resulting from marked hypotension and negative chronotropism. Intravenous administration of BV at 6.4 mg/kg induced a marked increase of the coronary flow, a slight increase of the renal flow and a slight decrease of the femoral flow. 11-Bromovincaminic acid (BVA, 0.1, 0.2 mg/kg), a major metabolite of BV, given intravenously or to the internal carotid artery, did not exert actions on the blood pressure, heart rate and carotid blood flow, implying that it is an inactive metabolite. In rabbit pulmonary arterial strips, BV showed no influence on effects of noradrenaline and adenosine but inhibited KCl-induced contracture. This inhibitory effect of BV, but not papaverine, was antagonized by an increase of external Ca2+ concentration. In conclusion, BV rather selectively increases the cranial and coronary blood flow. The vasodilative effects of BV seem to involve an inhibition of depolarization dependent Ca2+ slow channel. A major metabolite of BV, BVA, is inactive.

Animals↗