Expression of natural and synthetic genes encoding herpes simplex virus 1 protease in Escherichia coli and purification of the protein.

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. Studies with HSV-1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation. The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611. The N-terminal processing product (amino acids 1-247) contains the protease domain. To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV-1 protease are required. However, expression of the natural HSV-1 protease gene in Escherichia coli using a T7-promoter-regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene. The HSV-1 protease was purified to homogeneity in three steps using mixed-bed ion-exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.

Inhibitory effects of cimetidine ketoconazole and miconazole on the metabolism of praziquantel.

In order to find ways to increase the usually very low bioavailability of praziquantel, the effect of cytochrome P-450 inhibitors on the metabolism of praziquantel was investigated in rats. Cimetidine, ketoconazole, and miconazole yielded a 90% inhibition of the metabolism of praziquantel in liver microsome preparations from phenobarbital-pretreated rats at concentrations of 2.0, 0.03, and 0.01 mM, respectively. In rats in vivo ketoconazole and miconazole increased the bioavailability of praziquantel by a factor of 2 and 4, respectively in doses of 25 mg/kg. In phenytoin-pretreated rats ketoconazole increased the bioavailability of praziquantel by a factor of 1.4, whereas miconazole yielded a 5-fold increase of the bioavailability. Cimetidine was an effective inhibitor at a dose of 200 mg/kg. These results suggest that the inhibitors tested may suppress the metabolism of praziquantel in humans and consequently increase the bioavailability and blood levels at doses common in human therapy.

Measurement of myocardial fatty acid metabolism: kinetics of iodine-123 heptadecanoic acid in normal dog hearts.

To define the potential of iodine-123 heptadecanoic acid (IHA) for the noninvasive assessment of myocardial fatty acid metabolism with gamma camera imaging, the influence of myocardial oxygen consumption (MVO2) and blood flow (MBF) on extraction and half-times of IHA were investigated in dogs. Following IHA injection into the left circumflex coronary artery, extraction fraction and half-times were derived from the peak and slope of the IHA time activity curve, which consisted of a vascular, early, and late phase. Single-pass extraction fraction of IHA averaged 0.53 +/- 0.11 s.d. at control and was not influenced by MVO2 and MBF. The half-time of the early phase (T = 9.3 min +/- 2.8 s.d. in controls) as well as the ratio between the size of the early and late phase increased with MVO2 (r = 0.82, r = 0.87, respectively). Thus, early phase intracellular turnover of IHA increased, yet clearance of 123I activity was slowed by augmented cardiac work. Preliminary data of HPLC and electrophoretic analysis of myocardial arterial and venous blood samples over time indicate that the early phase is characterized by a decreasing washout of IHA and a relative increase of radioiodine washout. The half-time of the late phase (T = 245 min +/- 156 s.d. at control) was not related to MVO2 and MBF. In conclusion, myocardial fatty acid metabolism cannot be measured from the half-time of the early phase but might be analyzed from the ratio between the size of the early and late phase when using IHA.

Comparative distribution on niobium-95 in maternal and fetal rats and rabbits.

Pregnant rats and rabbits were injected with Nb95 towards the end of gestation. In rats, all maternal tissues showed higher concentrations compared to the fetal organs; the highest ratio was 0.6 in bone. In rabbits a different distribution was found. The fetal bone exhibited a 3.5 times higher concentration of Nb95 than the maternal one.

Study of fetal organ growth in Wistar rats from day 17 to 21.

A total of 1633 Wistar rat fetuses was used to determine weights of the fetus and several fetal organs on days 17 to 21 of gestation. Heart, lung, liver, kidney, stomach, intestine, brain, femur, thyroid and adrenal weights were recorded. Growth curves of the whole body and organs were calculated. A linear semi-log relationship between organ weight and day of gestation was shown. The doubling weight times were 1.5 days for whole bodies and for organs they ranged between 0.9 (spleen) and 3.4 (adrenals) days. A correlation between the rate of organ growth and the start of the organ function was observed.

Transfer and distribution of niobium-95 in adult, fetal, and newborn rats after injection during pregnancy.

Following i.v. injection of Nb-95 into pregnant rats, fetuses and newborns were dissected and measured for radioactivity after several time intervals. At any time only a small quantity of the administered radioactivity was transferred to fetus and newborn and the fetal tissue concentrations were always lower than the maternal ones. The highest ratio (0.6) between fetal and maternal tissue concentrations was found in bone.

Evaluation of 99mTc-dextran as a lymphoscintigraphic agent in rabbits.

Dextran (clinical grade, average mol. wt. 82,200) was labelled with 99mTc and the labelling efficiency was checked by paper and thin-layer chromatography and electrophoresis. The amount of free 99mTcO-4 was always less than 1%. The radiopharmaceutical was injected ID into the web space in hind legs of ten rabbits (200-600 microCi/0.05 ml). Scintigrams were taken at 10-min intervals up to 3 h in three rabbits. The injection site and the hind legs were massaged after injection in the other seven rabbits and scintigrams were taken at 10-min intervals up to 2 h. Blood samples were obtained at 5, 15, 30, 90 and 120 min in both groups. In addition a 180-min sample was also taken in the first group. At the end of the study the rabbits were killed and the popliteal lymph nodes and the organs were removed to be weighed and counted. Our results indicated a high concentration of radioactivity in the popliteal lymph nodes and massage at the injection site increased the average uptake of the popliteal lymph node from 1.12% +/- 0.77% to 4.28% +/- 1.57% at 3 and 2 h, respectively (P less than 0.001). In scintigrams the lymph channels and the nodes were very well visualised. The blood radioactivity levels were too low to present a background problem. With massage 30% of the injected dose was removed from the injection site in 2 h. We have shown that 99mTc-dextran is a good radiopharmaceutical for the visualisation of the lymph system and deserves further experimental and clinical studies.