Synthesis of nucleoside 5'-O-alpha,beta-methylene-beta-triphosphates and evaluation of their potency towards inhibition of HIV-1 reverse transcriptase.

A polymer-bound alpha,beta-methylene-beta-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5'-O-alpha,beta-methylene-beta-triphosphates. Among all the compounds, cytidine 5'-O-alpha,beta-methylene-beta-triphosphate inhibited RNase H activity of HIV-1 reverse transcriptase with a K(i) value of 225 microM.

Grapefruit juice, lyophilized grapefruit juice, and powdered whole grapefruit inhibit cytochrome P450-mediated triazolam hydroxylation by beagle dog liver microsomes.

Coadministration of grapefruit juice (GFJ) has been proposed to enhance the systemic availability and decrease the required dose of drugs such as cyclosporine that are extensively metabolized in the intestine and liver. Although GFJ inhibits human cytochrome P450 (CYP) 3A, effects on dog CYP have not yet been reported. Consequently, we determined whether GFJ inhibits triazolam hydroxylation by Beagle dog liver microsomes (DLM) using human liver microsomes (HLM) as positive control. Results were compared with the effects of lyophilized GFJ and commercially-available powdered grapefruit capsules, which may be more convenient dosage forms. GFJ inhibited alpha-hydroxytriazolam formation in both DLM and HLM with similar IC(50) (inhibitor concentration producing a 50% decrease in reaction velocity) values of 0.56% and 0.52% (v/v), respectively. Lyophilized GFJ and powdered grapefruit also inhibited DLM alpha-hydroxytriazolam formation with IC(50) values of 0.76 and 1.2 mg/mL, respectively. Consistent with mechanism-based enzyme inhibition, preincubation of DLM with any of the grapefruit products for 20 min resulted in significant enhancement of inhibition of triazolam alpha-hydroxylation by 8-20%. The results indicate that 16 g of lyophilized GFJ or 23 g of powdered grapefruit would be equivalent to dosing 100 mL of GFJ. In vivo pharmacokinetic interaction studies are needed to confirm these in vitro findings.

Isolated nephron segments from rabbit models of obstructive nephropathy.

Micropuncture and microcatheterization studies have been used extensively to investigate the pathophysiologic alterations in renal function induced by urinary tract obstruction. The present isolated tubule microperfusion studies were designed to examine the intrinsic alterations in segmental nephron function induced by 24 h of bilateral (BUO) and unilateral (UUO) urinary tract obstruction. Following UUO superficial proximal convoluted tubule reabsorption rate (J(v)) was not different from contralateral control (0.75+/-0.08 vs. 0.73+/-0.11 nl/mm per min, NS). Following UUO J(v) in juxtamedullary proximal convoluted tubules (JMPCT) was reduced 32% (0.69+/-0.06 vs. 0.47+/-0.04 nl/mm per min, P < 0.02). Following UUO J(v) in proximal straight tubules (PST) was reduced 52% (0.25+/-0.02 vs. 0.12+/-0.03, P < 0.01). Thick ascending limb (T-ALH) function was assessed by measurement of ability to lower perfusate chloride ion concentration (DeltaCl). Following UUO DeltaCl was reduced 76% (-39+/-9 vs. -9+/-1 meq/liter, P < 0.001). Cortical collecting tubule (CCT) function was assessed by measurement of antiduiretic hormone (ADH)-dependent osmotic water flow. Following UUO osmotic water flow was reduced 76% (0.90+/-0.08 vs. 0.22+/-0.04 nl/mm per min, P < 0.01) and this ADH resistance could not be overcome by cAMP. Nephron segments were then examined following relief of BUO. There were no differences in intrinsic function following relief of BUO when compared with UUO. We conclude that in UUO and BUO (a) the intrinsic tubular defects are identical, (b) the natriuresis noted is due, in part, to disordered JMPCT, PST, and T-ALH NaCl reabsorption, (c) the impaired concentrating ability is due, in part, to depressed function in T-ALH and ADH resistance of the CCT, and (d) the ADH resistance occurs at a site distal to the intracellular generation of cAMP.

Study of chloride transport across the rabbit cortical collecting tubule.

Recent micropuncture studies have suggested that the collecting tubule may be involved in the regulation of extracellular fluid volume. The present studies were designed to evaluate chloride transport across the in vitro-perfused rabbit cortical collecting tubule inasmuch as chloride ion would ultimately affect extracellular fluid volume. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate. Four groups of studies were conducted. In groups one and two, tubules from rabbits not receiving desoxycorticosterone (DOCA) were compared to tubules from rabbits which had received DOCA (5 mg/day) for 1 wk. In groups three and four, tubules were obtained only from rabbits not receiving DOCA. In group one, sequential bidirectional chloride fluxes were measured. The ratio of chloride efflux to influx was 0.99+/-0.04 in tubules obtained from rabbits not receiving DOCA whereas it was 1.28+/-0.09 in tubules obtained from rabbits receiving DOCA, suggesting stimulation of net chloride flux under these conditions. In group 2, chemical chloride concentration and osmolality of the collected fluid were measured. Neither the chemical chloride concentration nor the osmolality of the collected fluid decreased significantly below their respective perfusion fluid values in tubules from non-DOCA-treated rabbits but there was a significant decrease in the chemical chloride concentration (10-42 meq/liter) and osmolality (10-42 mosmol/kg H(2)O of the collected fluid in tubules from DOCA-treated rabbits. In group three, unidirectional chloride permeabilities from lumen-to-bath were determined during the passage of current down the perfusion pipette. The alterations of the average lumen potential, -35+/-4 and +28+/-2 mV, did not influence unidirectional chloride movement suggesting that the cortical collecting tubule is quite impermeable to chloride. In group four, unidirectional chloride permeability from lumen-to-bath was measured before and after substitution of NaCH(3)SO(4) for sodium chloride in the bath. Replacement of chloride by CH(3)SO(4) reversibly decreased the apparent chloride permeability from 2.41+/-0.50 to 0.69+/-0.08 (x10(-5) cm/s) demonstrating that (36)Cl permeability is dependent on the chemical concentration of chloride.THE CURRENT STUDIES DEMONSTRATE THAT: (a) the cortical collecting tubule is able to reabsorb salt under the modulation of circulating mineralocorticoids and, thus, may participate in overall volume homeostasis; (b) the chloride permeability and the major portion of isotopic chloride flux across the cortical collecting tubule is via exchange diffusion; and (c) under certain circumstances the cortical collecting tubule may act as a diluting segment.

A survey of lightning policy in selected division I colleges.

OBJECTIVE: The purpose of this research was to investigate the hazards of lightning for participants in outdoor athletics and to determine the existence of, and assess the nature of, lightning safety policy at the collegiate level. DESIGN AND SETTING: We used data from the National Severe Storms Laboratory in Norman, Oklahoma, and from a survey of Division I institutions. SUBJECTS: The 48 National Collegiate Athletic Association Division I (football) universities in Florida, Michigan, Pennsylvania, North Carolina, and New York. MEASUREMENTS: Athletic trainers at all of the selected 48 Division I institutions responded to the telephone survey. RESULTS: Florida, Michigan, Pennsylvania, North Carolina, and New York led the country in lightning deaths and injuries from 1959-1994. Only 8% (n = 4) of the institutions surveyed in these states have a written policy regarding lightning safety. CONCLUSIONS: This study demonstrated the lack of lightning safety policy in the surveyed universities and the need for a systematic plan of action to make fields safer for all who are involved in outdoor sport activities.

Pomegranate juice and pomegranate extract do not impair oral clearance of flurbiprofen in human volunteers: divergence from in vitro results.

Nutrient interactions with prescription drugs are a topic of ongoing basic and clinical research. Pomegranate juice and a 1-g capsule containing pomegranate extract were evaluated in vitro and in vivo as inhibitors of cytochrome P450 2C9 (CYP2C9), with flurbiprofen serving as the index substrate. Fluconazole was the positive control inhibitor. The in vitro 50% inhibitory concentration (IC(50)) values for pomegranate juice and extract were below 1% (vol/vol), with no evidence of mechanism-based (irreversible) inhibition. In clinical studies, flurbiprofen pharmacokinetics were unchanged by pomegranate juice or extract as compared to a low-polyphenol placebo control beverage. However, fluconazole significantly reduced the oral clearance of flurbiprofen. Despite inhibition of CYP2C9 in vitro, pomegranate juice and extract had no effect on CYP2C9 activity in human subjects, and can be consumed by patients taking CYP2C9 substrate drugs with negligible risk of a pharmacokinetic interaction.

Isolated nephron segments in a rabbit model of ischemic acute renal failure.

Renal artery occlusion has been extensively used in animal models to cause acute renal failure. The present isolated tubule microperfusion studies were designed to examine the transport characteristics of multiple nephron segments of the rabbit after 60 min of total renal ischemia. Preliminary studies showed that this maneuver produced significant and persistent elevations of serum creatinine. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate and studied at 37 degrees C. Four nephron segments were examined. Ischemia reduced proximal convoluted tubule fluid reabsorption 77% (0.72 +/- 0.11 vs. 0.14 +/- 0.06 nl . mm-1 . min-1, P less than 0.01) and cortical proximal straight tubule fluid reabsorption 88% (0.54 +/- 0.10 vs. 0.06 +/- 0.03 nl . mm-1 . min-1, P less than 0.005). Ischemia reduced the ability of the thick ascending limb of Henle's loop to lower perfusate chloride ion concentration 60% (-47 +/- 9 vs. -19 +/- 3 meq/liter, P less than 0.02) and its diluting ability 49% (-87 +/- 15 vs. -44 +/- 7 mosmol/kg H2O, P less than 0.01). Ischemia reduced the antidiuretic hormone-dependent osmotic water permeability of the cortical collecting tubule 59% (0.0203 +/- vs. 0.0083 +/- 0.0020 cm/s, P less than 0.01). Morphologic alterations were noted in the proximal segments but not in the distal segments of the nephron. The current studies demonstrate that 60 min of renal ischemia impairs the transport capability of all proximal and distal nephron segments studied.

Assessment of microwave-based clinical waste decontamination unit.

A clinical waste decontamination unit that used microwave-generated heat was assessed for operator safety and efficacy. Tests with loads artificially contaminated with aerosol-forming particles showed that no particles were detected outside the machine provided the seals and covers were correctly seated. Thermometric measurement of a self-generated steam decontamination cycle was used to determine the parameters needed to ensure heat disinfection of the waste reception hopper, prior to entry for maintenance or repair. Bacterial and thermometric test pieces were passed through the machine within a full load of clinical waste. These test pieces, designed to represent a worst case situation, were enclosed in aluminium foil to shield them from direct microwave energy. None of the 100 bacterial test pieces yielded growth on culture and all 100 thermal test pieces achieved temperatures in excess of 99 degrees C during their passage through the decontamination unit. It was concluded that this method may be used to render safe the bulk of of ward-generated clinical waste.

Prior mannitol and furosemide infusion in a model of ischemic acute renal failure.

Tubular transport abnormalities have recently been characterized in a rabbit model of ischemic acute renal failure (ARF). These studies demonstrated severe observable morphologic and functional changes in the proximal nephron together with functional changes in the distal nephron. Tubular debris was often produced by perfusion of proximal nephron segments. In the present study, agents used to prevent ARF were tested in this rabbit model of ARF. Rabbits were infused with either 5% body wt 5% manitol or 20 micrograms . kg-1 . min-1 furosemide in 5% body wt normal saline for the 60 min preceding 60 min of total renal ischemia. Mannitol 1) prevented the development of ARF, 2) maintained fluid reabsorption in the proximal convoluted tubule (PCT) (0.59 +/- 0.03 vs. 0.52 +/- 0.1 nl . mm-1 . min-1) and proximal straight tubule (PST) (0.34 +/- 0.05 vs. 0.39 +/- 0.07 nl . mm-1 . min-1), 3) depressed NaCl reabsorption in the cortical thick ascending limb of Henle's loop (TALH), and 4) did not prevent a decrease in ADH-mediated osmotic water flow in the cortical collecting tubule (CCT). Furosemide 1) partially preserved renal function, 2) partially protected the PCT (0.63 +/- 0.05 vs. 0.38 +/- 0.04 nl . mm-1 . min-1) and PST (0.32 +/- 0.04 vs. 0.22 +/- 0.02 nl . mm-1 . min-1), and 3) did not change the transport capacity of the TALH or the ADH response of the CCT. Preservation of proximal nephron integrity was also reflected by the absence of debris formation. There is a direct relation between an agent's ability to protect the functional integrity of the cells of the proximal nephron and its ability to preserve renal function.

Logographic reading by TMR adults.

Ten moderately retarded adults learned a set of 16 logographic signs. Subjects were asked to demonstrate sentences formed from the signs using dolls and other toys. They were also asked to pick out signs and form a sentence after watching the experimenter demonstrate an event. All subjects were able to demonstrate sentences with only minor errors. Only about half were completely successful in constructing four- and five-word sentences. The method of construction of most subjects revealed a semantic--syntactic disengagement; the signs were chosen in nonsentence order, apparently in order of salience of the referents. After collecting the signs, most subjects were able to put them in correct sentence order. The results support the feasibility of a logographic system for nonreading retarded adults.

A limitation on the law of effect.

Nineteen mildly or moderately retarded subjects were presented 32 oddity-training trials per day for 10 days with all new etimuli presented on each trial. Six subjects learned oddity, approaching an asymptote of 100 percent correct. Six others showed a strong preference (78 percent) for the odd stimulus but failed to improve. Seven remained in the performance range of 50 to 60 percent without improving. On eight trials per day, the two specific cues of an oddity display, chosen randomly, were presented alone as probes for specific-cue learning. Probe trials were above chance for all subjects. These results show that attention to the relevant dimension, demonstrated by oddity preference, plus adequate reinforcing conditions, indicated by specific-cue learning, did not ensure oddity learning. We concluded that subjects do not necessarily learn about the cues that control behavior. The operation of differential forgetting and/or rehearsal of relative vs. specific vs. specific cues was proposed as a likely explanation.

In vitro perfusion of the isolated dog glomerulus.

Recent technological advances allowing direct in vivo measurements of the determinants of glomerular ultrafiltration have greatly expanded our understanding of that process. In addition, these in vivo studies have clarified the dynamics of glomerular ultrafiltration in a number of physiologic and pathophysiologic conditions. Despite this progress, important issues remain unresolved and beyond the scrutiny of in vivo techniques. We have therefore devised a technique for in vitro glomerular perfusion of the isolated dog glomerulus. In eight glomeruli perfused at physiologic rates, the glomerular filtration rate averaged 39 nl/min and the filtration fraction was 0.19. Filtration pressure disequilibrium was observed in all studies and thus allowed calculation of a unique value for the ultrafiltration coefficient. That parameter averaged 2.34 nl/(min X mmHg). Morphologic studies employing transmission electron microscopy indicate that isolated perfused glomeruli remain ultrastructurally intact. The method for glomerular isolation and in vitro perfusion is presented in detail, the results obtained are compared with published in vivo results, and the advantages offered by the technique are discussed.