The choroid plexus pseudocyst: sonographic identification and characterization.

Amniocentesis has been advocated by some authors when fetal choroid plexus cysts are discovered, because of their reported association with karyotype abnormalities. We have noted an oval hypoechoic structure projecting into the adjacent choroid plexus, which simulates a cyst when imaged across its short axis, in 39 of 50 consecutive fetuses. This presumably normal structure or pseudocyst may represent the corpus striatum. Knowledge of its existence and benign nature will help avoid diagnostic errors and unnecessary amniocenteses.


Sonographic diagnosis of infantile hypertrophic pyloric stenosis.

Twenty-five consecutive sonographic examinations performed at Flinders Medical Centre for possible infantile hypertrophic pyloric stenosis (IHPS) were analyzed retrospectively. The results and a pyloric muscle index calculated by a formula using length, diameter, muscle thickness, and body weight were compared with the clinical outcome (surgery or conservative management). In the children without IHPS, the calculated pyloric muscle index was less than 0.2, whereas in infants with proven pyloric stenosis, the index was greater than 0.2 (P < 0.001). This result suggested that the published index upper limit of > 0.4 to 0.46 was not valid in our institution. Pyloric length to muscle thickness ratio was also found to predict IHPS. A simplified index, including only length and muscle thickness, is proposed, whereby length (mm) plus 3.64 times thickness (mm), when greater than 25, implies IHPS.


Morantel tartrate release from a long-acting intraruminal device in cattle: pharmacokinetics and gastrointestinal distribution.

The pharmacokinetics and gastrointestinal distribution of morantel tartrate release from a sustained release trilaminate bolus in cattle were investigated over a 98-day period post-treatment. Six Holstein calves (125-150 kg) had permanent indwelling fistulae surgically inserted into the rumen, abomasum and terminal ileum. Samples of jugular blood, feces and ruminal, abomasal and ileal fluids were taken on days -3, 1, 4, 7, 10, 14 and weekly up to 98 days post-bolus administration. Morantel tartrate concentrations were measured by HPLC after extraction and clean-up. Morantel was not detected in plasma at any time after bolus administration. High concentrations of morantel tartrate were found in ruminal, abomasal and ileal fluids and feces over 98 days post-treatment. The morantel peak concentration (Cmax) was achieved at Day 1 post-administration in each of these compartments. The steady-state morantel concentration (Css) was achieved at approximately 10 days post-treatment and maintained for 91-98 days post-treatment in these gastrointestinal compartments. The morantel Cmax, Css, area under the zero (AUC) and first moment (AUMC) of the concentration-time curve were significantly higher (P less than 0.01) in feces than in other compartments. The in vivo drug release profile of this device has been determined. Steady-state concentrations for from 91 to 98 days have been confirmed.


Rifampin disposition in the horse: effects of age and method of oral administration.

The effects of time and method of administration of rifampin with respect to feeding were evaluated in five mature horses. There was a significant (P less than or equal to 0.05) delay in time of maximum serum concentration and an apparent but not significant decrease in oral absorption when rifampin was given as a top dressing on grain as compared with administration in corn syrup 2 h before or 2 h after feeding. Although there were no differences between administration before or after feeding, administration 2 h prior to feeding was selected as the method of choice for future experiments. The effects of age on rifampin disposition were subsequently examined using this method of administration in six, 1-week old foals. Rifampin (10 mg/kg) was given at increasing age from 1 through 10 weeks and the pharmacokinetic disposition parameters compared. There were significant differences in the slope of the elimination phase (beta) and area under the curve (AUC) at 1 week through 6 weeks compared with 10 weeks or with values in the five mature horses.


Pharmacokinetics and intramuscular bioavailability of amikacin in chickens following single and multiple dosing.

The pharmacokinetics of amikacin were studied in healthy mature female chickens (n = 6). Single doses of amikacin were injected as an i.v. bolus (10 mg/kg) and i.m. (20 mg/kg) into the same birds with a 30-day rest period between treatments. Amikacin was determined by the fluorescence polarization immunoassay method. The i.v. pharmacokinetics could be described by a two-compartment model with a t1/2 alpha of 0.150 +/- 0.064 h and a t1/2 beta of 1.44 +/- 0.34 h. The total body clearance was 0.109 +/- 0.017 1/h/kg and the volume of distribution at steady-state was 0.193 +/- 0.060 l/kg. Following a single i.m. injection, the peak plasma concentration (Cmax) was 50.79 +/- 4.05 micrograms/ml and occurred at 0.50 +/- 0.26 h. The i.m. extent of absorption was 91.2 +/- 17.6%. Simultaneous modeling of i.v. and i.m. results provided estimates of an absorption half-life of 0.480 +/- 0.158 h. The i.m. pharmacokinetics after repeated administration were studied following the tenth dose (20 mg/kg, every 8 h). The Cssmax was 38.58 +/- 6.96 micrograms/ml and occurred at 0.79 +/- 0.37 h, and the biological half-life of amikacin was 1.86 +/- 0.47 h. The multiple dosing yielded peak concentrations of 39 micrograms/ml and trough concentrations of 3.26 micrograms/ml. Based on these data, the recommended amikacin dosage in chickens is 20 mg/kg body weight every 8 h.


Formulation and (bio)availability problems of drug formulations in birds.

Most drugs used for the treatment of birds are not formulated for birds. Therefore the availability of drugs for birds and their administration routes largely depend on formulations available for man, mammals and to some extent poultry. The problems with the application of existing formulations, the drug concentrations and the many different avian species are discussed. The desire of the avian veterinarian for the combination of several active compounds presents special problems. This again requires extra data on the interactions in galenic, pharmaceutic, pharmacokinetic and dynamic phases, which are generally not readily available.


Bioavailability and bioinequivalence of drug formulations in small animals.

Differences in bioavailability of many drugs from their various dosage forms have been shown to be relatively common in human medicine. Although comparable bioavailability ('bioequivalence') is though to ensure comparable clinical effectiveness and safety ('therapeutic equivalence'), the relationship between bioinequivalence and therapeutic inequivalence is less clear. Thus the prevalence of clinically important differences in bioavailability is unknown. While similar concerns have arisen about drug products used in small animal practice, there have been few investigations and some earlier reports are incomplete. However, there are indications of bioinequivalence with enteral formulations of ampicillin, aspirin, chloramphenicol, digoxin, mitotane, oxytetracycline, penicillin V and theophylline. Other studies have suggested bioequivalence with enteral formulations of chloramphenicol, digoxin, phenytoin, oxytetracycline and thyroxine. Limited data for injectable preparations showed bioinequivalence with chloramphenicol and possibly oxytetracycline. There is no reason to expect formulation-related bioinequivalence to be less prevalent in veterinary than in human medicine. Indeed, it may be more common in veterinary practice because other potential influences on bioavailability (food, diseases, other drugs, etc.) are frequently ignored, and cheaper generic products are often favoured for economic reasons.


Bioavailability and bioequivalence of veterinary drug dosage forms, with particular reference to horses: an overview.

The route of administration and formulation of the dosage form affect the bioavailability (rate and extent of absorption) of a drug and may thereby influence the intensity and duration of the pharmacological effect. Location of injection site may affect the plasma concentration profile of drugs administered as aqueous suspensions or sustained release parenteral preparations (procaine penicillin G). When absorption influences the rate of elimination ('flip-flop' phenomenon), the apparent half-life of the drug will be increased (cefazolin sodium, i.m.; meclofenamic acid, p.o.). Absorption generally approximates a first-order process and either the absorption half-life or the mean absorption time (statistical moment term) will provide an estimate of the rate of absorption. The method of corresponding areas is the usual technique employed in estimating the extent of absorption (systemic availability). Inherent in this technique is the assumption that clearance of the drug remains unchanged. In horses, the time of feeding relative to oral dosing has been shown to affect systemic availability (rifampin, trimethoprim) and pattern of absorption (phenylbutazone). Oral paste formulations (trimethoprim-sulphadiazine, ivermectin) are convenient to administer, allow precision in dosage compared with powders or granules added to feed, and could provide sustained release. Assessment of bioequivalence is based on relative bioavailability, using a reference dosage form, together with a measure of the uncertainty (variance) of the estimate. Bioequivalence relies on the concept that preparations of a drug which provide essentially equivalent plasma concentration profiles should produce the same therapeutic effect.


Effect of inhalation anaesthetics on total respiratory resistance in conscious ponies.

Total respiratory resistance was measured rapidly and non-invasively in 6 conscious ponies before and after they inhaled approximately 25% of the minimal anaesthetic concentration (0.25 MAC) of either enflurane, halothane, or isoflurane, over a 10 min period. The forced random noise (FRN) method was used to measure the impedance over the frequency range of 5 to 40 Hz and its real part, the resistance, was extracted from these impedance measurements. At the concentrations used, halothane appeared to have no effect on the total respiratory resistance; enflurane and isoflurane seemed to increase it but the changes were not statistically significant.


Prescribing of drugs for food-producing animals in Norway. Information about withdrawal times.

The prescribing of drugs for food-producing animals in Norway was investigated with special emphasis on written information given about withdrawal times. The study was designed as a cross-sectional prescription survey. Of 1518 prescriptions for food-producing animals, it was concluded that 1224 of the prescriptions were for drugs requiring withdrawal times for meat, milk or eggs. Of these 1224 prescriptions, 82.8% were for veterinary preparations, 6.6% were for human preparations and 10.6% were for other drugs. For 20.8% of the prescriptions, information about withdrawal time(s) was missing. For prescriptions for veterinary preparations this figure was 5.9%, and for prescriptions for human preparations and other drugs 95.1% and 90.8%, respectively. For veterinary preparations approved for the intended species, as many as 99.2% gave information about withdrawal times on the drug container label. Lack of information about withdrawal times might give rise to drug residues in food for human consumption and thus pose a potential hazard to human health.


Effect of erythromycin on L-threonine transport in rabbit jejunum in vitro.

Several antibiotics characterized by different molecular structures are known to affect some intestinal activities. Some of them have been described as inhibitors of the intestinal sugar and amino acid transport with different mechanisms. Erythromycin (EM) is a macrolide antibiotic acting as a motilin agonist and thus stimulating the gastrointestinal motor activity. Since several substances which increase the motor activity of the gastrointestinal tract may produce effects on the intestinal absorption of nutrients, the present study has been carried out to determine whether erythromycin affects the L-threonine intestinal absorption. The results obtained indicate that erythromycin diminishes the L-threonine intestinal transport, probably at the mucosal border level. Two groups of experiments carried out, with Na(+)-deprived medium and ouabain-enriched medium, might indicate that erythromycin action could be due to either a direct or an indirect action on the Na(+)-dependent L-threonine transport located in the brush border.


Mathematical description of the concentration of oxytetracycline and penicillin-G in tissue cages in calves as related to the serum concentration.

A mathematical model based on Fick's laws of diffusion describing the concentration of drug in tissue cage models was elaborated. The model takes into account differences in protein binding, tissue cage geometry and serum pharmacokinetics. The validity of the model was tested against experimental data obtained from a tissue cage model in calves by simultaneous fitting to serum and tissue cage fluid (TCF) data in a non-linear least-squares regression computer program. Concentrations of penicillin-G (pen-G) in serum and TCF following intravenous (i.v.) administration of potassium pen-G were adequately described by the mathematical model. Concentrations in TCF after intramuscular (i.m.) administration of the same drug and of procaine pen-G could be predicted by the mathematical model. Concentrations of oxytetracycline (OTC) in serum and TCF following i.v. administration and continuous i.v. infusions were also adequately described by the model, and TCF concentrations after i.m. administration of the same drug could be roughly predicted. The results indicate that pen-G and OTC have the same permeability coefficient for transport from serum to TCF.


Bioavailability of oral penicillins in the horse: a comparison of pivampicillin and amoxicillin.

The pharmacokinetics of ampicillin and amoxicillin following intravenous administration at a dose rate of 15 and 10 mg/kg respectively were studied in four healthy adult horses. Pharmacokinetics of pivampicillin and amoxicillin were studied after oral administration to four healthy adult horses. Pivampicillin, a prodrug of ampicillin, was administered orally to starved and fed horses at a dose rate of 19.9 mg/kg, which is equivalent on a molecular basis to 15 mg/kg ampicillin. Amoxicillin was administered orally to starved horses only, at a dose rate of 20 mg/kg. Ampicillin and amoxicillin concentrations in plasma, synovial fluid and urine were determined. Mean biological half-life of intravenously administered ampicillin and amoxicillin was 1.72 and 1.43 h respectively, whilst the distribution volume (Vss) appeared to be 0.180 and 0.192 1/kg. Orally administered pivampicillin and amoxicillin were rapidly absorbed. A maximum concentration in plasma of 3.80 micrograms/ml was reached 2 h after administration of pivampicillin to starved horses; in fed horses a maximum concentration of 5.12 micrograms/ml was reached 1 h after administration. After oral administration of amoxicillin a maximum concentration of 2.03 micrograms/ml was reached after 1 h. The (absolute) bioavailability of pivampicillin administered orally was 30.9% in starved horses and 35.9% in fed horses. The bioavailability of amoxicillin administered orally was 5.3% in starved horses.


The pharmacokinetics and pharmacodynamics of furosemide in the anaesthetized dog.

The correlation between pharmacokinetics and dynamics of furosemide was investigated in anaesthetized dogs. After intravenous administration (i.v.) of furosemide (5 mg/kg), the plasma concentration declined rapidly with bioexponential decay. The half-life (t1/2 beta) of the late phase of elimination was 0.931 +/- 0.187 h and the apparent volume of distribution at steady state was 0.25 +/- 0.043 l/Kg. The total clearance (Cltot) was 0.435 +/- 0.031 l/h/kg, in which the renal clearance was 0.260 +/- 0.020 (about 60% of Cltot). The change in rate of urinary excretion of furosemide was similar to the plasma concentration decay curve. The diuretic effect of furosemide was accompanied by an extreme increase in the excretion rate of sodium and chloride, but not potassium. The relationships between the diuretic response and the plasma concentration or the urinary excretion rate of furosemide was depicted by sigmoidal dose-response curves in both cases. The half-maximum effect was obtained at 1.5 micrograms/ml of plasma concentration or at 80 micrograms/min of excretion rate of furosemide.


Synovial and serum levels of triamcinolone following intra-articular administration of triamcinolone acetonide in the horse.

Seven mature thoroughbred horses, weighing between 400 and 541 kg, were each injected intra-articularly into three joints with 6 mg/joint of triamcinolone acetonide (Vetalog). The fourth joint, the control, was injected with saline. Synovial fluid was taken from all four legs of the horses on days 1, 2, 3, 4, 5, 6, 7, 8, 11, and 15 following the injections. Triamcinolone acetonide was assayed by a radioimmunoassay. Blood was collected at 1, 2, 4, 6, 12 h and on days 1, 2, 3, 4, 5, 6, 7, 8, 11, and 15 following injection of either triamcinolone or saline. Both cortisol and triamcinolone were assayed. The results show that the synovial fluid level of triamcinolone was 7.5 micrograms/ml 1 day following treatment and decreased to 10 ng/ml by the 4th day. These low levels were maintained for approximately 14 days. By the 15th day, the triamcinolone was below a detectable level. Serum levels of triamcinolone increased to 3 ng/ml within 1 h and further increased to a peak of 4.3 ng/ml at 4th h. The level then decreased to 2 ng/ml at 24 h and to nearly an undetectable level in 48 h. The mean level of serum cortisol, on the other hand, gradually decreased as the serum level of triamcinolone increased. As the serum level of triamcinolone reached an undetectable level on the 2nd day, the serum cortisol level gradually increased and returned to the pre-administration level by the 5th day. These results showed that the intra-articular administration of triamcinolone maintained triamcinolone in the synovial fluid for 4-14 days and that the triamcinolone reached the blood within 1 h. The serum level of triamcinolone was maintained for 2 days and resulted in the inhibition of adrenal function for 4 days.


Effects of phenothiazine and thiabendazole on bovine dorsal pedal vein contractility induced by ergonovine and serotonin; potential for alleviation of fescue toxicity.

Phenothiazine and thiabendazole were studied for their ability to antagonize venoconstriction induced by ergonovine, and the biogenic amine serotonin, in the isolated dorsal pedal vein of cattle. The two compounds are commercially available, approved for usage in cattle and have been reported to reverse some of the toxic effects associated with the intake of Acremonium coenophialum-infested fescue forage by cattle. Neither compound had any antagonistic activity against venoconstriction induced by ergonovine. However, thiabendazole did have some activity against venoconstriction induced by serotonin. Ergot alkaloids are known to cause venoconstriction through effects on biogenic amine receptors, including serotonergic receptors, and since thiabendazole has anti-serotonin activity, part of the reported beneficial effects of thiabendazole in alleviating fescue toxicity may be due to the anti-serotonin activity of the drug. Further work is needed to determine if phenothiazine and thiabendazole have any effect on other types of alkaloids that are present in A. coenophialum-infested fescue.


Acyclovir (Zovirax) pharmacokinetics in Quaker parakeets, Myiopsitta monachus.

The pharmacokinetics of intravenous (i.v.) and intramuscular (i.m.) single-dose administration of acyclovir were determined in Quaker parakeets. After i.v. injection at a dose of 20 mg/kg of acyclovir, elimination half-life was estimated at 0.65 h, volume of distribution at steady state was 627.65 ml/kg, and clearance was 11.22 ml/kg/min. The estimated pharmacokinetic values after i.m. injection at a dose of 40 mg/kg of acyclovir were an elimination half-life of 0.71 h and a bioavailability of 90.1%. The peak plasma acyclovir concentration occurred at 15 min when the drug was administered i.m. Plasma concentrations of acyclovir were undetectable 4-6 h after i.v. administration and 6-8 h after i.m. administration. Oral (capsules) and intravenous (sodium salt) formulations of acyclovir were given by gavage at 80 mg/kg. Peak concentrations with the sodium salt formulation were lower and developed more slowly than with the capsules. In studies designed to detect excessive drug accumulation or adverse side effects, acyclovir was administered i.m. at 40 mg/kg every 8 h for 7 days. Plasma concentrations were determined 15 min after (peak) and just prior to drug administration (trough). In another study acyclovir was gavaged at a dose of 80 mg/kg every 8 h for 4 days. Acyclovir plasma concentrations were determined just prior to and 2 h after drug administration. In both experiments, the birds maintained normal appetite and weight and did not exhibit excessive drug accumulation. Acyclovir plasma concentrations ranging from 2.07 +/- 1.09 micrograms/ml to 3.93 +/- 1.13 micrograms/ml were maintained for 4 days when acyclovir was administered in the feed and water (sole source of food and water).(ABSTRACT TRUNCATED AT 250 WORDS)


Quantitative electroencephalography for measurement of central nervous system responses to diazepam and the benzodiazepine antagonist, flumazenil, in isoflurane-anaesthetized dogs.

Quantitative EEG was assessed in six dogs anaesthetized with 1.8% end-tidal isoflurane concentration and following diazepam (0.2 mg/kg i.v.) administration. Ventilation was controlled to maintain normocapnia. Five dogs were subsequently given the benzodiazepine antagonist, flumazenil (0.04 mg/kg i.v.), and quantitative EEG was recorded. One dog received a saline injection following diazepam (as a control) and quantitative EEG was recorded for an additional 2.5 h. Heart rate, arterial blood pressure, esophageal temperature, arterial pH and blood gas tensions, end-tidal CO2 tension and end-tidal isoflurane concentration were monitored throughout the study. A 21 lead linked-ear montage was used for recording EEG. Quantitative EEG data were stored on an optical disc for analysis at a later date. Values for absolute power of EEG were determined for theta, delta, alpha, and beta frequencies. Cardiovascular parameters remained stable throughout the study. Diazepam administration was associated with decreased absolute power in all frequencies of EEG at all electrode sites. The duration of diazepam-induced decreased absolute power of EEG was at least 3 h in one dog. Administration of flumazenil antagonized diazepam-induced decreased absolute power of EEG in all frequencies at all electrode sites. We conclude that quantitative EEG provides a relatively non-invasive, objective measure of diazepam- and flumazenil-induced changes in cortical activity during isoflurane anaesthesia.


Methimazole-mediated modulation of netobimin biotransformation in sheep: a pharmacokinetic assessment.

The effects of modulation of liver microsomal sulphoxidation on the disposition kinetics of netobimin (NTB) metabolites were investigated in sheep. A zwitterion suspension of NTB was given orally at 7.5 mg/kg to sheep either alone (control treatment) or co-administered with methimazole (MTZ) orally (NTB + MTZ oral treatment) or intra-muscularly (NTB + MTZ i.m.) at 3 mg/kg. Blood samples were taken serially over a 72 h period and plasma was analysed by HPLC for NTB and its major metabolites, i.e. albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2). Only trace amounts of NTB parent drug and ABZ were detected in the earliest samples after either treatment. There were significant modifications to the disposition kinetics of ABZSO in the presence of MTZ. ABZSO elimination half-life increased from 7.27 h (control treatment) to 14.57 h (NTB + MTZ oral) and to 11.39 h (NTB + MTZ i.m.). ABZSO AUCs were significantly higher (P less than 0.05) for the NTB + MTZ oral treatment (+55%) and for the NTB + MTZ i.m. treatment (+61%), compared with the NTB alone treatment. The mean residence times for ABZSO were 12.66 +/- 0.68 h (control treatment), 18.85 +/- 2.35 h (NTB + MTZ oral) and 17.02 +/- 0.90 h (NTB + MTZ i.m.). There were no major changes in the overall pharmacokinetics of ABZSO2 for the concomitant MTZ treatments. However, delayed appearance of this metabolite in the plasma resulted in longer ABZSO2 lag times and a delayed Tmax for treatments with MTZ.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative pharmacokinetics of the photosensitizer tin-etiopurpurin in dogs and rats.

Photodynamic therapy is a promising new treatment for local eradication of cancer. Little work has been done to define the pharmacokinetics of photodynamic drugs or the variability in drug disposition that may occur between different species and pathophysiological states of tissues. Pharmacokinetic studies of tin-etiopurpurin (SnET2), a lipophilic photosensitizer, were conducted on six Beagle dogs and six Sprague-Dawley rats. Blood was collected up to 24 h following drug administration for measurement of tin-etiopurpurin concentration. Dogs and rats were euthanatized 24 h post-administration and tissues were collected for drug analyses. The plasma drug concentrations were best described by a 2-compartment model (Ct = Ae-alpha t + Be-beta t). Median distribution and elimination half-lives were 0.24 and 0.34 h and 10.21 and 5.25 h for dogs and rats, respectively. The apparent volumes of distribution were 4.26 +/- 1.75 L/kg for dogs and 1.84 +/- 0.36 L/kg for rats. Systemic clearance was 7.56 +/- 2.45 ml/kg/min and 6.63 +/- 0.91 ml/kg/min for dogs and rats, respectively. Drug was detected in all tissues analyzed 24 h after drug administration. Drug was detected only sporadically in skin and muscle and was generally below the limit of detection of the assay. Where comparisons could be made, concentrations of SnET2 were significantly greater in all tissues except jejunum of rats compared to dogs 24 h after drug administration.


Clorsulon pharmacokinetics in sheep and goats following oral and intravenous administration.

Clorsulon was measured in plasma and urine of sheep and goats after administration of a single intravenous (i.v.) and after a single oral dose of 7 mg/kg. A three-compartment model with elimination occurring from the central compartment was determined to best describe the i.v. data, whereas a one-compartment model with a single exponential absorption phase best described the oral plasma data. The bioavailability of orally administered clorsulon was approximately 55% in goats and 60% in sheep. Peak plasma concentrations occurred at 14 h and 15 h after oral administration in goats and sheep, respectively. Absorption from the gastro-intestinal tract effectively prolonged the elimination of clorsulon by increasing the elimination half-life from 17 to 28 h in sheep and from 12 to 23 h in goats for the i.v. and oral routes, respectively. In both goats and sheep, approximately 50% of the i.v. dose was recovered in urine as parent drug at 48 h after administration, whereas 41% and 30% of the dose was recovered after oral administration for goats and sheep, respectively. The elimination rate constant (kel) in goats was nearly twice as large as the value determined in sheep, and the urea under the i.v. plasma curve in goats was only 63% of the value in sheep indicating that goats are more effective in their capacity to eliminate clorsulon than are sheep. These differences in drug disposition between sheep and goats may account for the reduced efficacy of clorsulon reported in goats.


Plasma concentrations of flunixin in the horse: its relationship to thromboxane B2 production.

The effects of the intravenous (i.v.) administration of 1.1 mg/kg of flunixin meglumine on thromboxane B2 (TxB2) concentrations were studied in sedentary and 2-year-old horses in training. The baseline TxB2 serum concentrations generated during clotting were 2.89 +/- 0.81, 2.19 +/- 0.25 and 0.88 +/- 0.12 ng/ml for the 2-year-old Thoroughbreds in training, sedentary horses under 10 and over 10 years old, respectively. There was a significant difference in baseline TxB2 concentrations between older and younger horses (P less than 0.005). Significant reduction in TxB2 production from baseline were noted at 1 (P less than 0.01) and 4 h (P less than 0.01) but not at 8 h after flunixin administration. The percent reduction in serum TxB2 concentration at 1 h after the administration of flunixin was 68.6 +/- 7.3 and 45.2 +/- 6.8 for the training and sedentary horses, respectively; the differences were significant (P less than 0.04). Serum concentrations of TxB2 returned to baseline values by 12-16 h after flunixin administration. The results of this study indicate a difference in the TxB2 concentrations of older vs. younger horses and a difference in the suppression of TxB2 after the administration of flunixin in 2-year-old Thoroughbreds in training compared to sedentary horses. The results of this study suggest that the detection of low concentrations of flunixin in urine 24 h post-administration may not represent pharmacologic effective concentrations of flunixin in plasma.


Identification and cloning of a novel heterogeneous nuclear ribonucleoprotein C-like protein that functions as a transcriptional activator of the hepatitis B virus enhancer II.

Liver specificity of hepatitis B virus (HBV) replication has been attributed to the action of its second enhancer (EII). We report here the characterization of EII and the subsequent isolation of a novel liver-specific DNA-binding protein which binds to and activates EII. The cDNA clone of the protein, designated E2BP, was isolated from a lambda gt11 expression library constructed from the hepatoma cell line HuH-6 which was screened with a binding site probe derived from EII. Sequence analysis of E2BP revealed 86.6% homology with a rat heterogeneous nuclear ribonucleoprotein C protein sequence, while conformational studies suggest a helix-loop-helix motif as a DNA-binding site. Cloned E2BP expressed in human fibroblasts by transient transfection displayed EII binding and activating characteristics similar to those of native E2BP in hepatocytes.


Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.


Enhancement of polyhedrin nuclear localization during baculovirus infection.

Polyhedrin is the major component of the nuclear viral occlusions produced during replication of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). Since viral occlusions are responsible for the horizontal transmission of AcMNPV in nature, the biosynthesis, localization, and assembly of polyhedrin are important events in the viral replication cycle. We recently defined the sequence requirements for nuclear localization and assembly of polyhedrin. In this study, we examined the localization of polyhedrin at different times of infection. The results showed that nuclear localization of polyhedrin becomes more efficient as the occlusion phase of infection progresses. Several different factors were identified that might contribute to this overall effect, including a higher rate of polyhedrin nuclear localization and a higher rate of polyhedrin biosynthesis. We also examined the biosynthesis and processing of polyhedrin in cells infected with an AcMNPV few polyhedra (FP) mutant, which produces smaller numbers of viral occlusions that contain few or no virions. Compared with wild type, the FP mutant produced polyhedrin more slowly and localized it to the nucleus less efficiently at the beginning of the occlusion phase of infection (24 h postinfection). This supported the idea that the efficiency of polyhedrin nuclear localization is tightly coupled to its rate of biosynthesis. It also revealed that expression of the viral 25K gene, which is inactivated in the FP mutant, is directly or indirectly associated with an enhancement of polyhedrin biosynthesis and nuclear localization at the beginning of the occlusion phase of infection. This enhancement effect appears to be necessary to ensure the normal assembly of viral occlusions.


Sulfation of the human immunodeficiency virus envelope glycoprotein.

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.


Down-regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells is accompanied by an increase in factor binding.

In transformed cells, the E1A gene of adenovirus type 12 (Ad12) represses transcription of class I genes of the major histocompatibility complex. The tumorigenic potential of Ad12-transformed cells correlates with this diminished class I expression. In contrast, the E1A gene of the nontumorigenic Ad5 does not affect class I expression. We show here that a transfected reporter chloramphenicol acetyltransferase plasmid driven by an H-2K promoter (-1049 bp) was expressed at much lower levels in Ad12- than in Ad5-transformed mouse cells. Analysis of mutant constructs revealed that only 83 bp of H-2 DNA, consisting of the enhancer juxtaposed to the basal promoter, was sufficient for this differential expression. Whereas the H-2 basal promoter alone was somewhat less active in Ad12-transformed cells, the H-2 TATA box itself did not appear to be important. The H-2 enhancer proved to be the principal element in Ad12 E1A-mediated repression, since (i) substitution of the H-2 enhancer by simian virus 40 enhancers overcame the repression, and (ii) when juxtaposed to either its native or heterologous basal promoters, the H-2 enhancer was functional in Ad5- but not Ad12-transformed cells. Mobility shift assays showed that there is a DNA-binding activity to the 5' site (R2 element) of the enhancer that is significantly higher in Ad12- than in Ad5-transformed cells. These results suggest that decreased class I enhancer activity in Ad12-transformed cells may, at least in part, be due to the higher levels of an enhancer-specific factor, possibly acting as a repressor.


Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.


Negative regulation of the major histocompatibility complex class I enhancer in adenovirus type 12-transformed cells via a retinoic acid response element.

In cells transformed by the highly oncogenic adenovirus type 12 (Ad12), the viral E1A proteins mediate transcriptional repression of the major histocompatibility class I genes. In contrast, class I transcription is not reduced in cells transformed by the nononcogenic Ad5. The decreased rate of class I transcription is, at least in part, the result of a reduced major histocompatibility complex class I enhancer activity in Ad12-transformed cells and correlates with an increase in the levels of a DNA-binding activity to the R2 element of the enhancer (R. Ge, A. Kralli, R. Weinmann, and R. P. Ricciardi, J. Virol. 66:6969-6978, 1992). Employing transient transfection assays, we now provide direct evidence that the R2 element can confer repression in Ad12- but not Ad5-transformed cells. Repression by R2 was observed only in the presence of the positive enhancer element R1 and was dependent on (i) the number of the R2 elements and (ii) the relative arrangement of R2 and R1 elements. The putative R2-binding repressor protein, R2BF, was similar in molecular weight and binding specificity to members of the thyroid hormone/retinoic acid (RA) receptor family. RA treatment abrogated the R2-mediated repression in Ad12-transformed cells and had no effect on the activity of R2/R1-containing promoters in Ad5-transformed cells. These results are consistent with the presence of an R2-binding repressor in Ad12-transformed cells. In the absence of RA, the repressor compromises enhancer activity by interfering with the activity of the positive cis element R1. RA treatment of Ad12-transformed cells may render the repressor inactive.


Localization of an immunodominant domain on baculovirus-produced parvovirus B19 capsids: correlation to a major surface region on the native virus particle.

An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.


Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.


Role of ribosomes in Semliki Forest virus nucleocapsid uncoating.

The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.


Serial backcross analysis of genetic resistance to mousepox, using marker loci for Rmp-2 and Rmp-3.

At least three genes from C57BL/6 mice that mediate dominant resistance to lethal mousepox were isolated and transferred onto a susceptible DBA/2 background. Three [(C57BL/6 x DBA/2)F1 x DBA/2] male mice that survived infection were selected as founders on the basis of different complements of marker loci for two resistance genes, Rmp-2r (Hc1) and Rmp-3r (H-2Db). They were crossed with DBA/2 mice, male progeny were infected with ectromelia virus, and the cycle was repeated with surviving male progeny through seven backcross generations. Two founders carried a marker locus for Rmp-2r or Rmp-3r, and the third carried neither marker locus. Resistance pedigrees were analyzed for passage of marker loci. From the three founders, resistance was passaged through multiple generations, producing backcross lines with intermediate-male-resistance phenotypes (20% resistant). Females of backcross lines with intermediate male resistance had high resistance (> 50%). High-resistance backcross lines (40% male resistance) also developed from the founders that carried marker loci for Rmp-2r and Rmp-3r, and marker loci were passaged through all generations of high resistance but not intermediate-resistance lines. About one-third of all resistant mice in high-resistance lines sired by mice that carried marker loci for Rmp-2r and Rmp-3r did not carry the respective marker locus. In lines that carried Rmp-2r, this was apparently not the result of recombination between Rmp-2r and Hc1, because Rmp-2 was not in the predicted location on chromosome 2 and because mice that did not inherit Hc1 transmitted significantly less male resistance than Hc1-positive mice, although female resistance remained high. These results confirmed that C57BL/6 mice have redundant resistance mechanisms, two of which are controlled at least in part by Rmp-2r and Rmp-3r, and provided evidence for a fourth resistance gene, herein presumptively named Rmp-4, which protects females more than males and which may be epistatic to Rmp-2.