Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants.

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and ß-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l-1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants.

Flunarizine for treatment of partial seizures: results of a concentration-controlled trial.

The National Institutes of Health sponsored a randomized, double-blind, multicenter, placebo-controlled trial of flunarizine (FNR) in epileptic patients receiving concomitant phenytoin (PHT) or carbamazepine (CBZ). Because of FNR's long half-life (up to 7 weeks), a parallel rather than crossover design was used. Each patient received an individualized loading dose and maintenance dosage targeted at a 60-ng/ml plasma FNR concentration. Of 93 patients randomized, 92 provided seizure data for the full 25-week treatment period; one placebo-treated patient dropped out for personal reasons. Fifty-four patients received CBZ only, nine received PHT only, and 30 received both CBZ and PHT. Eighty-seven patients had a history of complex partial seizures, and 60 had secondarily generalized seizures. Eight patients discontinued FNR prematurely, all because of adverse neurologic or psychiatric signs or symptoms; depression was the specific cause in three cases. Calculated maintenance dosages, based on single-dose pharmacokinetic profiles, ranged from 7 to 138 mg/day (mean, 40 mg/day). Plasma FNR concentrations generally exceeded the target, with the highest concentrations observed immediately after loading; excluding the first three treatment weeks and all concentrations after a FNR dosage change, the median plasma FNR concentration was 71.7 ng/ml. The percent reduction from baseline seizure rate was statistically greater (p = 0.002) in the FNR-treated group (mean, 24.4%) than in the placebo-treated group (mean, 5.7%).

Increasing plasma concentration tolerability study of flunarizine in comedicated epileptic patients.

Twelve patients with intractable partial seizures [4 receiving carbamazepine (CBZ), 4 phenytoin (PHT), and 4 both] entered a study of the tolerability of flunarizine (FNR) at specified plasma concentrations. After an 8-week baseline period, a single-dose pharmacokinetic study was performed for each patient to calculate a loading dose and maintenance dosage necessary to achieve a target plasma FNR concentration of 30 ng/ml. The first 8 patients received the loading dose (as divided doses) during a 1-week hospitalization and the maintenance dosage for the ensuing 8 weeks. These patients proceeded to treatment periods with target concentrations of 60 and then 120 ng/ml, using doses based on an assumed linear relation between dose and plasma concentration. The last 4 patients were studied only at the 120- ng/ml target level. Results indicated that this procedure successfully approximated target levels of 30 and 60 ng/ml, but observed concentrations in the last period exceeded the 120-ng/ml target level and continued to increase with time, often necessitating a dosage reduction owing to intolerability. Calculated doses for a given target concentration varied by a factor of 12. The most frequently reported adverse experiences were sedation and increased fatigue; reports of dizziness, headache, and lethargy were also common. Based on this study, a target concentration of at least 60 but < 120 ng/ml is recommended for a controlled clinical trial of the antiepileptic efficacy of FNR.

Felbamate for partial seizures: results of a controlled clinical trial.

Felbamate (2-phenyl-1,3-propanediol dicarbamate) has a favorable preclinical profile in animal models of epilepsy. We present the results of a double-blind, randomized, placebo-controlled clinical trial in patients with partial seizures. Criteria for entry included a requirement for four or more partial seizures per month despite concomitant therapeutic blood levels of phenytoin and carbamazepine. Fifty-six patients (mean age, 31.4 years; 32 men, 24 women) completed the trial. The mean seizure frequencies for the 8-week periods analyzed were felbamate = 34.9, placebo = 40.2. Felbamate was statistically superior to placebo in seizure reduction, percent seizure reduction, and truncated percent seizure reduction. The mean felbamate dosage was 2,300 mg/d. Plasma felbamate concentrations ranged from 18.4 to 51.9 mg/l, mean = 32.5 mg/l. Adverse experiences during felbamate therapy were minor and consisted primarily of nausea and CNS effects. This trial indicates that felbamate is safe and effective in the treatment of comedicated patients with severely refractory epilepsy.

Phenytoin: the pseudosteady-state phenomenon.

After reaching an apparent steady state, plasma phenytoin (PHT) levels may then undergo inexplicable changes, a phenomenon called " pseudosteady state". We evaluated 13 pseudosteady -state periods in 10 inpatients with complex partial seizures. Eleven of the periods occurred after a change in PHT dosage and two after drug withdrawal. The pseudosteady -state period began 2 to 12 days (means = 5.7 days) after dosage change and lasted 5 to 10 days (means = 6.3 days), during which plasma PHT levels were stable (+/- 5%). Plasma PHT levels thereafter fluctuated spontaneously by greater than 25% for 5 to 22 days (means = 10.8 days). A final steady-state level was reached 13 to 31 days (means = 21.4 days) after the first dosage change. Falling plasma PHT levels increased seizure frequency in two patients, and a level of 52 micrograms/ml led to medication toxicity in another.

The effects of protective equipment on styrene exposure in workers in the reinforced plastics industry.

This study was designed to determine whether styrene absorption through the skin results in measurable changes in biologic indicators of styrene exposure using a group of workers engaged in hand lay-up operations during which extensive styrene skin contact occurs. Serial measurements of expired breath and blood styrene and urinary excretion of mandelic and phenylglyoxylic acid were made in eight female workers during 4 consecutive days using different experimental conditions including either (1) gloves/protective clothing alone, (2) respirator alone, (3) both gloves/protective clothing and respirator, or (4) neither respirator nor gloves/protective clothing. A two-by-two factorial design imbedded in a Latin square design allowed estimation of the effects of the two types of protection (i.e., gloves/clothing and respiratory) and any interaction. Levels of styrene in venous blood and expired breath and urine mandelic and phenylglyoxylic acid excretion were no different when gloves/clothes were used as protection compared to no protection at all. Significant reduction in all biologic indices occurred when respiratory protection was used. The results of the study suggest that percutaneous absorption of styrene is not a significant exposure source and does not significantly contribute to the body burden of styrene of workers in the reinforced plastic industry engaged in hand lay-up operations. Respiratory protective devices were the most effective means for reducing styrene absorption. Even though skin absorption of styrene is limited, skin protection is necessary because of the risk of dermatitis.

Responses of normal children and young adults to controlled bicycle exercise.

Exercise responses were obtained from 149 children and young adults (average age 14.5 years) and divided by sex and body surface area (BSA): children with BSA less than 1 m2; children with BSA 1--1.19 m2; males with BSA greater than or equal to 1.2 m2; and females with BSA greater than or equal to 1.2 m2. Total work, mean and maximal power outputs were more affected by body size (height) than age in children with BSA less than 1 m2 and in males and females with BSA greater than or equal to 1.2 m2. Mean systolic pressure increased up to 64% above the preexercise supine value at peak effort, with the level of mean maximal systolic pressure having a positive relationship with body size (height), power output and preexercise sitting systolic pressure in all subgroups except children with BSA 1--1.19 m2. Mean diastolic pressure increased up to 24% above the preexercise supine value at peak effort. ST-segment depression of 1--2 mm was recorded in 12.1% (18 of 149) of the population at peak exercise. These changes occurred in 8.9% of all males and in 16.9% of all females (p greater than 0.1). The data from this study reveal the importance of sex and body size in the clinical interpretation of exercise responses in growing subjects, provide a reference for objective evaluation of subjects with or without cardiac abnormalities and provide a guide for careful monitoring of subjects during an exercise study.

Nonspecific binding as a source of error in thyrotropin radioimmunoassay with polyethylene glycol as separating agent.

We investigated the effects of nonspecific binding on thyrotropin values obtained by radioimmunoassay in which polyethylene glycol is used as precipitant. Differences in nonspecific binding among individual samples were significant (F-test, p less than 0.001, range 5.5 to 14.1%). Non-specific binding and total serum protein were directly correlated (r = 0.472, n = 59; p less than 0.001). Nonspecific binding increased with increasing concentrations of globulins but showed no relation to albumin concentration. If globulin concentration was less than 15 g/L, precipitation of the antigen--antibody complex by polyethylene glycol was incomplete. The mean value for thyrotropin in sera from 67 healthy subjects was 2.7 (SD 0.3) milli-international units per liter (milli-int. unit/L) without individual serum nonspecific binding correction, significantly (p less than 0.005) higher than that with nonspecific binding correction (1.6, SD 0.1, milli-int. unit/L). Evidently, inter-sample variations in nonspecific binding may cause significant errors under these conditions, which can be minimized by taking into account the individual nonspecific binding of each serum sample.

A statistical method for determining normal ranges from laboratory data including values below the minimum detectable value.

Determination of normal ranges from laboratory data containing undectable values is a frequently encountered problem in the radioimmunoassay of peptide hormones. In the past, such determinations usually have been based on the mid-point method or the one-end Winsorized method. A graphic method involving the use of probability paper has also been reported. We propose that the maximum-likelihood estimation is a more appropriate statistical method for the determination of normal range from this type of data (Type I censored data). With this method, the mean and standard deviation, and hence the tolerance limits, can be estimated. We used the maximum-likelihood estimation method to determine the normal range of serum thyrotropin values obtained from 93 healthy subjects, based on a log normal distribution. Although the serum thyrotropin content was undetectable in 14% of the subjects, a normal range could be calculated. Using tolerance limits for 95% coverage of the population with 90% confidence, we calculated the normal range of thyrotropin to be 0.51-5.75 milliunits/L, with a mean value of 1.71 milliunits/L, and predicted that 91.4% of undetectable serum thyrotropin values will fall within the normal range.