High oxygen level in a soaking treatment improves early root and shoot development of black willow cuttings.

Black willow (Salix nigra) stem cuttings are commonly used to stabilize eroded streambanks with survival dependent on rapid development of adventitious roots to maintain plant water balance, absorb nutrients, and provide anchorage and support especially during flood and drought events. Soaking cuttings in water prior to planting increases survival and growth rates, but it is not known whether oxygen content in the soaking water affects the rate of early root and shoot initiation and growth. A laboratory experiment tested the hypothesis that cuttings treated with high oxygen (>95% saturation, 8.62 mg O2 l(-1)) soaking exhibit more rapid initiation and growth of roots and shoots than cuttings treated with low oxygen (<15% saturation, 1.24 mg O2 l(-1)) soaking and control (unsoaked). Root initiation was enhanced in both high and low O2 soaking treatments compared to control (100, 93, and 41%, respectively, n = 27). High O2 soaking led to greater root length than low O2 soaking during the fourth week after planting (26.5 and 12.3 cm on day 22; 27.7 and 19.1 cm on day 27, respectively). Shoot growth was greater in high O2 compared to low O2 soaking on days 36 and 56 after planting (9.3 and 6.3 cm on day 36, 10.7 and 7.2 cm on day 56, respectively). Shoot and root biomass production was stimulated in both soaking treatments, with 200% more biomass production by day 59 compared to control. Results of this study demonstrated that a high oxygen soaking treatment has potential for improving early root and shoot growth, and survival in willow cuttings planted at riparian restoration sites.

Antiviral activity and intracellular metabolism of bis(tButylSATE) phosphotriester of beta-L-2',3'dideoxyadenosine, a potent inhibitor of HIV and HBV replication.

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

In vitro and in vivo metabolism and pharmacokinetics of bis [(t-butyl)-S-acyl-2-thioethyl]-beta-L-2',3'-dideoxy-5-fluorocytidine monophosphate.

Exposure to 10 &M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1(+/-) +/- 46.2 and 242.1 +/- 13.2 pmol/10(6) cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 +/- 29, 23.9 +/- 15.6, and 20.6 +/- 10.5 pmol/10(6) cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 +/- 1.00 and 0.39 +/- 0.15 &M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.

Intracellular metabolism of beta-L-ddAMP-bis(tbutylSATE), a potent inhibitor of hepatitis B virus replication.

beta-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC50 = 0.1 microM. Following a 0- to 72-hrs exposure of human hepatocytes to a 10 microM [2',3'-3H] beta-L-ddAMP-bis(tbutylSATE), the pharmacologically active beta-L-ddATP was the predominant metabolite attaining a concentration of 268.53 +/- 107.97 pmoles/10(6) cells at 2 hrs. In Hep-G2 cell, beta-L-ddATP accounted for 146.8 +/- 29.8 pmoles/10(6) cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of beta-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.

Pharmacokinetics of beta-L-2',3'-dideoxy-5-fluorocytidine in rhesus monkeys.

beta-L-2',3'-Dideoxy-5-fluorocytidine (beta-L-FddC), a novel cytidine analog with an unnatural beta-L sugar configuration, has been demonstrated by our group and others to exhibit highly selective in vitro activity against human immunodeficiency virus types 1 and 2 and hepatitis B virus. This encouraging in vitro antiviral activity prompted us to assess its pharmacokinetics in rhesus monkeys. Three monkeys were administered an intravenous dose of [3H] beta-L-FddC at 5 mg/kg of body weight. Following a 3-month washout period, an equivalent oral dose was administered. Plasma and urine samples were collected at various times for up to 24 h after dosing, and drug levels were quantitated by high-pressure liquid chromatography. Pharmacokinetic parameters were obtained on the basis of a two-compartment open model with a first-order elimination from the central compartment. After intravenous administration, the mean peak concentration in plasma (Cmax) was 29.8 +/- 10.5 microM. Total clearance, steady-state volume of distribution, terminal-phase plasma half-life (t1/2 beta), and mean residence time were 0.7 +/- 0.1 liters/h/kg, 1.3 +/- 0.1 liters/kg, 1.8 +/- 0.2 h, and 1.9 +/- 0.2 h, respectively. Approximately 47% +/- 16% of the intravenously administered radioactivity was recovered in the urine as the unchanged drug with no apparent metabolites. beta-L-FddC exhibited a Cmax of 3.2 microM after oral administration, with a time to peak drug concentration of approximately 1.5 h and a t1/2 of 2.2 h. One monkey in the oral administration arm of the study had a significant delay in the absorption of the aqueous administered dose. The absolute bioavailability of orally administered beta-L-FddC ranged from 56 to 66%.

Effects of photoisomers of cyclodiene insecticides on liver and microsomal cytochrome P450 in rats.

Threshold dosages of the photoisomers of cyclodiene insecticides, namely photochlordane, photodieldrin, and photoheptachlor, for the induction of hepatic microsomal cytochrome P450 (P450) and liver hypertrophy in male rats were at least one-quarter of those reported for corresponding parent cyclodienes. Maximum increase in total P450 concentration (30%) and demethylases activities (100%) was always respectively one-third or one-tenth of that reported for parent cyclodienes. The P450 isozymic form induced by photoheptachlor resembled that induced by pentobarbital (P4502B1) in its substrate specificity, spectral characteristics, and electrophoretic mobility. The induction of P450 was initially followed by hepatic hypertrophy. However, higher dosages of photoisomers caused wasting and lowered both the liver weight and the activity of aniline hydroxylase while those of mirex and endrin, which also caused wasting and lowered aniline hydroxylase activity, continued causing further hepatic hypertrophy.

Effect of stereoisomerism on the cellular pharmacology of beta-enantiomers of cytidine analogs in Hep-G2 cells.

The beta-L enantiomers of 2',3'-dideoxycytidine (beta-L-ddC) and its 5-fuoro derivative, 2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), were demonstrated to be active against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication in vitro. In the present study, we investigated the cellular pharmacology of beta-L-ddC and beta-L-FddC and compared it with that of beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC). Beta-L-FddC (10 microM) was found to be phosphorylated rapidly in Hep-G2 cells to its 5'-mono-, di-, and triphosphate derivatives with intracellular triphosphate levels achieving 26.6 +/- 10.9 pmol/10(6) cells after 72 hr. In contrast, the active 5'-phosphorylated derivative of beta-D-FddC achieved lower levels with triphosphate levels of only 2.3 +/- 0.5 pmol/ (10(6) cells under the same conditions. Beta-L-ddC was also phosphorylated rapidly. A 5'-diphosphocholine (18 +/- 5.8 pmol/10(6) cells) and a 5'-diphosphoethanolamine (13.6 +/- 0.9 pmol/10(6) cells) derivative were detected in beta-D-FddC-treated cells after 72 hr, whereas in beta-L-FddC- and beta-L-ddC-treated cells, only the 5'-diphosphocholine derivative (10.9 +/- 2.8 and 60.4 +/- 5.7 pmol/10(6) cells, respectively) was detected. Beta-L-FddC-5'-triphosphate (beta-L-FddCTP), beta-D-FddC-5'-triphosphate (beta-D-FddCTP), and beta-L-ddC-5'-triphosphate (beta-L-ddCTP) followed a single phase elimination process with an intracellular half-life (T1/2) of 10.5, 5.7, and 12.3 hr, respectively. Furth ermore, beta-L-FddCTP, beta-D-FddCTP, and beta-L-ddCTP levels of 6.7 +/- 2.3, 0.3 +/- 0.1, and 12.0 pmol/10(6) cells, respectively, were still detectable 24 hr following drug removal. The higher intracellular 5'-triphosphate levels of beta-L-FddC and the extended T1/2 of its 5'-triphosphate are consistent with the more potent in vitro antiviral activity of beta-L-FddC in Hep-G2 cells when compared with its beta-D enantiomer, beta-D-FddC.

Human bites. Guidelines for prompt evaluation and treatment.

Human bites are a relatively common injury. They often involve the hand and may have serious sequelae because of the structures involved, the bacteriologic spectrum, or poor patient compliance with the treatment regimen. A careful history and physical examination are important, and treatment involves good local wound care, appropriate antibiotic coverage (including both a penicillin and a penicillinase-resistant penicillin), immobilization and elevation of the injured area, and close follow-up, as well as early rehabilitation.

Separating the effects of salience and disparity on the rate of observing.

Pigeons producing deliveries of grain on a mixed variable-interval, extinction schedule by pecking a center key could also produce discriminative stimuli on concurrent variable-interval schedules by pecking the left or right observing key. The stimuli produced by each observing key were varied independently. In the first experiment, the negative discriminative stimulus was at the far end of the spectrum from the key illumination accompanying the mixed schedule and from the positive discriminative stimulus. When the magnitude of the difference between the latter two stimuli (salience) was varied, more pecks occurred on the observing key producing the larger of the two differences than on the key producing the smaller difference. In the second experiment, the stimulus accompanying the mixed schedule was at the far end of the spectrum, and the magnitude of the difference between the two discriminative stimuli (disparity) was varied. The proportion of pecks occurring on each observing key shifted systematically in the direction of the key producing the larger difference. The salience of the discriminative stimuli and their disparity each has an independent influence on the frequency of observing when the other is controlled, but the effect of the salience appears to be the more substantial.

The acquisition of observing.

Pigeons were exposed to stimuli correlated with the presence or absence of a variable-interval 60-second schedule of reinforcement only while they depressed a crossbar or "perch." In the first experiment, the stimuli were different tilts of a line displayed on the key. When the difference in brightness between the line and the background (salience) was maximal, seven of eight birds acquired the discrimination, but when the difference was reduced by 50%, only one succeeded. In the second experiment, wavelength of chamber illumination served as the relevant dimension. Neither experiment showed a large effect attributable to the magnitude of the difference (disparity) between the positive and the negative stimulus. Individual differences in time spent observing were positively correlated with level of discrimination in the presence of the stimuli. All birds produced the positive stimulus for a greater proportion of the available time than they did the negative stimulus. This may be the mechanism that provides selective reinforcement of observing. Finally, the formation of a discrimination was analyzed in terms of changes in the proportion of time spent in contact with the discriminative stimuli.

Associative learning in neonatal rats revealed by cardiac response patterns.

Infant rat pups (3-4 days of age) that received a single pairing of a novel odor (CS) with illness later responded to the CS with sustained accelerations in heart rate (HR); a different novel odor evoked deceleratory HR responses. Control pups responded to the CS and the second novel odor with cardiac deceleration. In a second experiment, rat pups that received three pairings of a novel odor with a cold (10 degrees C) temperature reinforcement displayed a similar pattern of HR responses, i.e., acceleration to the CS and deceleration to the novel odor. Cardiac response patterns are a useful measure of learning in infant mammals. The directional modulations of HR found in these experiments compare favorably with previous interpretations of "orienting" and "defensive" reactions derived from studies of HR responses in humans.

Taste aversions to mother's milk: the age-related role of nursing in acquisition and expression of a learned association.

The development of taste aversion learning to novel cues contained in mother's milk was examined in rat pups. Pups receiving distinctive mild by experimenter-delivered oral infusions followed by toxicosis formed an aversion to the dam's diet. Robust aversions were learned as early as Day 10 and were retained for at least 11 days. When the same distinctive milk was obtained directly from a foster mother through nursing, however, only weanling-age pups (over 20 days) formed an aversion. X-ray analysis of nipple location in the mouths of suckling pups suggested that pups between the ages of 10 and 21 days receive milk at a similar tongue locus. Flavored milk was then delivered at specific time intervals in controlled quantities through tongue cannulas implanted at loci corresponding to the nipple position shown by the X-rays. Cannulated preweanling pups that were attached to a nipple during milk delivery failed to associate the taste cue with illness, whereas both preweanlings off the nipple and weanlings on the nipple acquired aversions to the taste cue in the milk. The evidence obtained in these experiments suggests that pups of all ages are incapable of expressing a taste aversion in a nursing situation and that preweanling pups in particular are also dificient in acquiring aversions within a suckling context. The inability of preweanling pups to acquire taste aversions in a nursing situation appears to result from a failure to associate taste cues with illness rather than a failure to detect taste cues obtained from a nipple.