Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels.

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

An examination of critical care nurses' knowledge and attitudes regarding pain management in hospitalized patients.

The purpose of this study was to determine the effectiveness of an educational intervention focused on pain management. The sample consisted of 30 medical/surgical intensive care nurses (age range, 23 to 62 years) employed in a large metropolitan hospital in the southeastern United States. McCaffery and Ferrell's Nurses' Knowledge and Attitude Survey Regarding Pain was administered at 2 points in time: before and after the educational program. The survey simultaneously measures knowledge and attitude levels regarding pain control. The paired t test was used to test for differences between pre- and posttest scores. The Point-biserial and Spearman's correlation coefficients were computed to examine relationships between selected sociodemographic variables and scores (baseline and change) on the survey. Results revealed a significant increase in scores after the educational intervention (t = 9.60. p = .0005). Additionally, a statistically significant correlation was found between change scores (posttest minus pretest scores) and years of nursing experience (rs = .37, p = .047). However, no significant relationships were found between type of nursing degree and baseline or change scores (rpb = .11, p = .575; rpb = .01, p = .955). These results support previous research findings related to the problem of inadequate pain management in the hospital setting. Further, the findings indicate that education regarding pain control is crucial for current nursing students as well as practicing nurses. The challenge for nurses is to be responsive to and integrate current pain management techniques in an effort to decrease the discomfort of hospitalized patients.

Effects of repeated oral postnatal exposure to chlorpyrifos on open-field behavior in juvenile rats.

Organophosphorus (OP) insecticides have the potential to cause behavioral effects in children. This study was designed to determine if repeated oral exposure of preweanling rats to chlorpyrifos would produce behavioral changes at both pre- and postweanling ages. Treatment occurred every second day beginning on post-natal day (PND) 1, and continued through PND 21. The rats received one of the following regimens: a low-dosage (3 mg/kg) from PND 1-21; a medium dosage (mg/kg from PND 1-5, and then 6 mg/kg from PND 7-21; or a high-dosage schedule of 3 mg/kg on PND 1-5, then 6 mg/kg from PND 7-13, and 12 mg/kg from PND 15-21. There were no differences in body weights among the control-, low-, and medium-dosage groups but the high-dosage group had significantly lower body weights on PND 13-21. An open field was used to measure locomotor activity on PND 10, 12, 14, 16, 18, 20, 25, and 30. There were no differences in locomotor activity levels or treatment effects between males and females. On PND 10, 12, 14, 16, 18, and 20 there was no effect on locomotor activity with any dosage. On days 25 and 30, locomotor activity was significantly decreased with the medium- and high-dosage groups. Brain cholinesterase (ChE) inhibition was about 25-38% on PND 25 and 14-34% on PND 30. On PND 25 but not 30, lung and diaphragm ChE and serum butyrylcholinesterase (BChE), with the high-dosage animals, and heart ChE with the medium- and high-dosage groups were significantly inhibited. There was no significant inhibition of skeletal muscle ChE or serum acetylcholinesterase (AChE) on PND 25 and 30. These data suggest that early postnatal chlorpyrifos exposures will depress locomotor activity in juvenile rats, with the effects most pronounced after brain ChE activity has substantially recovered.

A new chromosome race of Calycadenia pauciflora (Asteraceae: Heliantheae-Madiinae) from Butte County, California.

We initiated a biosystematic study of a recently discovered population of Calycadenia pauciflora in order to evaluate its cytogenetic relationship to previously characterized chromosome races of that species. Cytogenetic analyses of six or more artificially produced individuals of each of the five possible interracial hybrid combinations indicated that the new race (designated Wurlitzer) is differentiated from the other races (Elegans, Healdsburg, Pauciflora, Ramulosa, and Tehama) by the equivalent of 2-4 reciprocal chromosome translocations and in one instance apparently a pericentric inversion. Mean pollen stainability in the hybrids ranged from 13 to 26%. The floral and vegetative features of the new race are very similar to those of races Pauciflora, Ramulosa, and Tehama of C. pauciflora. We ascribe the apparent lack of single-step cytogenetic events in the evolution of the races of C. pauciflora to one or more of the following: (1) (in some cases) the occurrence of saltational chromosome reorganization; (2) extinction of or failure to detect intermediate populations in C. pauciflora; and (3) an insufficient consideration of the possibility of the existence of intermediate races in the closely related species, C. fremontii. We conclude that the C. fremontii-C. pauciflora alliance is one of the most complex and potentially instructive examples of diploid chromosome evolution in plants.

Family responses to disclosure of self-as-lesbian.

Disclosure or nondisclosure of self-as-lesbian to family is a difficult task. Eleven lesbians aged 25-47 years old described their experiences of disclosure or nondisclosure to family. The primary source of data collection was focus groups. Family disclosure was preceded by recognition of self-as-lesbian. Five story patterns of lesbians and their families related to disclosure or nondisclosure were derived from text analysis. The participants also described missing sociocultural landmarks and symbols for lesbian lives. Implications are included for lesbians who are contemplating disclosure and their health care providers.

Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development.

The effects of repeated oral exposures to the organophosphorus insecticide chlorpyrifos (CPS) on brain muscarinic receptor densities, together with cholinesterase (ChE) activity, were studied in early postnatal rats. Initially, the effects on esterases from lactational exposure to CPS were investigated in young rats by administering CPS (100, 150, or 200 mg/kg subcutaneously in corn oil) to dams 1 day postpartum, yielding a significant body burden of CPS in the dams for possible excretion in the milk. Brain ChE inhibition in pups was less severe than in dams, whereas liver carboxylesterase (CbxE) inhibition in pups was at the same level as in dams. Because of the limited brain ChE inhibition obtained following lactation, pups were exposed to CPS directly by gavage, using 3 dosing regimens to yield a dose response. The rats were gavaged with CPS in corn oil on alternate days from postnatal day (PND) 1 through PND 21. Rats in the low-dosage group received 11 treatments at 3 mg/kg, those in the medium-dosage group received 3 treatments at 3 mg/kg and 8 at 6 mg/kg, and those in the high dosage group received 3 treatments at 3 mg/kg, 4 at 6 mg/kg, and 4 at 12 mg/kg. ChE activity in brain homogenates were inhibited significantly by 29% and 63% in the low- and high-dosage groups, respectively, on PND 22 and by 17% in the high dosage group on PND 40. Muscarinic receptor densities in brain synaptosomes were reduced using 3H-N-methylscopolamine (NMS) and 3H-quinuclidinyl benzilate (QNB) as ligands, with the effects more prominent from 3H-NMS. Densities of both ligands recovered to the control level several days after terminating treatment. The results indicate that pups are apparently exposed to only limited amounts of chlorpyrifos and/or its oxon through the milk when dams are exposed to extremely high chlorpyrifos levels. In addition, repeated direct oral exposures of early postnatal rats to CPS will result in persistent brain ChE inhibition and will transiently reduce muscarinic receptor density.

The interaction of chlorinated alicyclic insecticides with brain GABA(A) receptors in channel catfish (Ictalurus punctatus).

Chlorinated alicyclic insecticides are believed to antagonize the action of the neurotransmitter gamma-aminobutyric acid (GABA) at its receptor in vertebrates. Binding of the specific GABA(A) receptor ligand [35S]-t-butylbicyclophosphorothionate (TBPS) to channel catfish brain P2 membranes suggested a single population of receptors with a Kd (56.6+/-2.6 nM) and Bmax (2435+/-276 fmol/mg protein) that are similar to published values for other fish species. The competition of several chlorinated compounds for TBPS binding was investigated. The most potent inhibitors of TBPS binding were 12-ketoendrin, photoheptachlor epoxide, photoheptachlor, telodrin, and endrin, respectively, with IC50s of 20-90 nM. Photooxychlordane, photo alpha-chlordane, and oxychlordane were intermediate in potency (122-219 nM), as were isodrin, dihydroisodrin, heptachlor epoxide, and alpha-chlordane, which were similar in potency (311-397 nM). Dieldrin, lindane, and dihydroaldrin were much less potent (592-1103 nM). Heptachlor, aldrin, and gamma-chlordane were weak inhibitors of TBPS binding (2073-2738 nM). Chlordene and chlordecone had the lowest potency of all compounds studied (10,201-21,178 nM) with the exception of mirex, which did not inhibit binding at a concentration of 50 microM. There is a good correlation between binding potency and the available toxicity data for several of these compounds in channel catfish. There is also a good correlation between the inhibitory potency in channel catfish by these types of compounds with that in rats.

Implementation of a patient management system in a children's hospital.

Implementing systems to the clinical areas within the hospital setting, is increasing as rapidly as technology is changing. This paper describes the requirements and problems identified during an implementation of a patient management system to a major Paediatric Hospital. The system implemented was developed within the I.S. Department of the hospital and provides a high degree of functionality and ease of use for clinical and clerical staff. It also offers case-based interfaces to clinical and business management systems outside the patient care setting.

Design & implementation of an automated questionnaire.

This paper describes a system which allows the patient to answer a structured questionnaire in private, at their own speed and without pressure using a touch-screen personal computer. A clinical report is generated immediately and used to help guide a clinician in a face to face consultation with the patient. Trials in a clinical setting have shown a positive response by both patients and clinicians.

Kinetic analysis of the in vitro inhibition, aging, and reactivation of brain acetylcholinesterase from rat and channel catfish by paraoxon and chlorpyrifos-oxon.

In rats, the phosphorothionate insecticide parathion exhibits greater toxicity than chlorpyrifos, while in catfish the toxicities are reversed. The in vitro inhibition of brain acetylcholinesterase (AChE) by the active metabolites of the insecticides and the rates at which these inhibitor-enzyme complexes undergo reactivation/ aging were investigated in both species. Rat AChE was more sensitive to inhibition than catfish AChE as demonstrated by greater bimolecular rate constants (ki) in rats than in catfish. In both species, chlorpyrifos-oxon yielded higher ki's than paraoxon. The higher association constant (KA) of chlorpyrifos-oxon than paraoxon in both species and the lack of significant differences in the phosphorylation constants (kp) suggest that association of the inhibitor with AChE is the principal factor in the different potencies between these two inhibitors. In catfish, the ki of chlorpyrifos-oxon was 22-fold greater than that of paraoxon, while in rats it was 9-fold greater, suggesting that target site sensitivity is an important factor in the higher toxicity of chlorpyrifos to catfish but not in the higher toxicity of parathion to rats. No spontaneous reactivation of phosphorylated catfish AChE occurred and there were no differences in the first oder aging constants (ka) between compounds. For phosphorylated rat AChE, there were no differences in the first order reactivation constants (kr) but the ka for chlorpyrifos-oxon was significantly greater than that for paraoxon. This difference suggests that the steric positioning of the diethyl phosphate in the esteratic site is not the same between the two compounds, leading to differences in aging.

Biochemical mechanisms contributing to species differences in insecticidal toxicity.

Comparison of published LD50 or LC50 levels for a variety of insecticides in several vertebrate species indicate that a wide range of toxicity levels exist, and these cannot be easily predicted within either a chemical group or within a species. There is a relatively limited data base documenting interactions between insecticides and other chemicals, either agricultural or non-agricultural; however, the fact that all major insecticide groups perturb nervous system function as their primary mechanism of acute toxicity suggests the potential for interactions. Studies in our laboratories on a select group of phosphorothionate insecticides in rats indicated that brain acetylcholinesterase sensitivity to inhibition by the oxons, the active metabolites of the phosphorothionates, does not correlate with acute toxicity levels. The activities and properties of hepatic cytochrome P450-mediated activation (desulfuration) and detoxication (dearylation) of the phosphorothionates as well as of A-esterase-mediated hydrolysis of oxons contribute substantially to understanding the acute toxicity levels in rats, as does the sensitivity of the protective aliesterases to phosphorylation. However, in the channel catfish, the acetylcholinesterase sensitivity to oxon inhibition reflects the acute toxicity level of these same insecticides, and may be largely responsible for determining the acute toxicity level in this species. Thus, metabolism of insecticides appears to be far more influential in some species than others in determining the toxicity elicited.

Inhibition and aging of channel catfish brain acetylcholinesterase following exposure to two phosphorothionate insecticides and their active metabolites.

The inhibition and aging of acetylcholinesterase (AChE) in fingerling channel catfish (lctalurus punctatus) brain tissue was studied after single in vivo exposures to high levels of chlorpyrifos (0.25 mg/L), chlorpyrifos-oxon (7 micrograms/L), parathion (2.5 mg/L), or paraoxon (30 micrograms/L). Exposure to both parent compounds produced identical initial inhibition (95%), but in the later sampling times there was significantly more inhibited AChE in the chlorpyrifos-treated fish than in the parathion-treated fish (47% and 28%, respectively, on d 16). There were higher levels of aged AChE following chlorpyrifos exposure than following parathion exposure, but differences were not significant. Exposure to both oxons produced initial inhibition greater than 90%, and patterns of recovery and aging were statistically similar between both compounds; no significant inhibition was observed after d 11. The similar patterns of inhibition, recovery, and aging between the two oxon treatments, which have similar lipophilicities, suggest that the greater amount of AChE inhibition and aging observed in the chlorpyrifos-treated fish compared with the parathion-treated fish probably results from the higher lipophilicity of chlorpyrifos than of parathion. Overall, the prolonged brain AChE inhibition exhibited in catfish exposed to phosphorothionates is not the result of aging of the inhibited enzyme but is the result of either a slow rate or a lack of spontaneous reactivation.

Inhibition patterns of brain acetylcholinesterase and hepatic and plasma aliesterases following exposures to three phosphorothionate insecticides and their oxons in rats.

Rats were administered high sublethal intraperitoneal dosages of the phosphorothionate insecticides parathion, methyl parathion, and chlorpyrifos, and their oxons. Acetylcholinesterase activities in cerebral cortex and medulla oblongata and aliesterase activities in liver and plasma were monitored at 2 hr and 1, 2, and 4 days after exposure. The maximal inhibition of brain acetylcholinesterase activity was not immediate with parathion and chlorpyrifos, reflecting the time required for bioactivation of the phosphorothionates as well as the effectiveness of the aliesterases to inactivate much of the hepatically generated oxons. In contrast, brain acetylcholinesterase activities were more quickly inhibited following administration of paraoxon and chlorpyrifos-oxon, which do not require bioactivation. Brain acetylcholinesterase was also rapidly inhibited following administration of methyl parathion and methyl paraoxon, reflecting the low sensitivity of the aliesterases to methyl paraoxon. Aliesterases were inhibited to a greater extent than acetylcholinesterase at each sampling time with parathion and chlorpyrifos and their oxons, whereas the reverse was true with methyl parathion and methyl paraoxon. All of the above patterns correlate with the in vitro sensitivities of acetylcholinesterase and aliesterases to the oxons. The very prolonged inhibition of esterase activities following chlorpyrifos treatment probably results from its substantially greater lipophilicity compared to the other compounds, which would allow it to be stored and released for gradual bioactivation. The data reported indicate that the disposition and effects of different phosphorothionate insecticides will be influenced by the sensitivities of target and nontarget esterases for their oxons and by their lipophilicity, and that predictions of in vivo responses can be made from in vitro data.

Acute effects of the organophosphate paraoxon on schedule-controlled behavior and esterase activity in rats: dose-response relationships.

The effects of acute intraperitoneal administration of paraoxon on behavioral and biochemical parameters were studied in male rats. Rats were trained to press a lever under an FR10 schedule of reinforcement. Rats were injected with 3 sublethal doses of paraoxon (0.5, 0.75, and 1.0 mg/kg) and performance was monitored for four days after exposure. Response rates were depressed significantly for days 1 and 2 with 0.75 and 1.0 mg/kg, but not 0.5 mg/kg, even though there was inhibition of brain and plasma cholinesterases at all doses. Performance recovered prior to brain AChE recovery. There was no clear-cut threshold of brain AChE inhibition required to yield performance deficits, nor was there a direct correlation between significant inhibition in peripheral enzymes which could serve as markers (plasma aliesterases, butyrylcholinesterase, non-iso-OMPA-sensitive cholinesterase, and hepatic aliesterases) and performance deficits, suggesting that other noncholinergic targets may play a role in OP-induced behavioral deficits.