Biomedical subjects
Publications and source records attributed to D J Walker.
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Artemisinin and its derivatives are important new antimalarials which are now used widely in Southeast Asia. Clinically relevant artemisinin resistance has not yet been reported but is likely to occur. In order to understand how the malaria parasite might become resistant to this drug, we studied artemisinin resistance in the murine malaria parasite Plasmodium yoelii. The artemisinin-resistant strain (ART), which is approximately fourfold less sensitive to artemisinin than the sensitive NS strain, accumulated 43% less radiolabeled drug in vitro (P < 0.01). Within the parasite, the drug appeared to react with the same parasite proteins in both strains. The translationally controlled tumor protein, one of the artemisinin target proteins, did not differ between the strains. No DNA sequence difference was found, but the resistant strain was found to express 2.5-fold-more protein than the sensitive strain (P < 0.01). Thus, the phenotype of artemisinin resistance in P. yoelii appears to be multifactorial.
Glial cell line-derived neurotrophic factor (GDNF) can attenuate the dopamine (DA)-depleting effects of neurotoxic doses of methamphetamine (METH) when given 1 day prior to the METH. The neurotoxic effects of METH may be due, in part, to sustained increases in extracellular levels of DA. It is therefore possible that GDNF may be altering the effects of METH by influencing extracellular levels of DA during the METH treatment. The purpose of the present study was to determine if GDNF has effects on extracellular levels of DA in the striatum by 24-h post-administration. GDNF (10 microgram in 2 microliter vehicle) or vehicle was injected into the right striatum or substantia nigra of anesthetized male rats. The next day the animals were anesthetized again and dialysis probes were positioned in both the right and left striata and perfused with artificial cerebrospinal fluid. Following the collection of baseline samples the rats were administered METH (5 mg/kg, s.c.). The METH injections dramatically increased extracellular DA levels on both sides of the brain. However, levels on the GDNF injected side were significantly greater than levels on the contralateral side. Basal levels of DA were not significantly different between the two sides, but levels of DA metabolites were elevated on the GDNF side. Post-mortem tissue levels of DA metabolites, but not DA, were also elevated in the striatum and substantia nigra. These results indicate that GDNF has significant effects on DA neuron functioning within 24 h of administration and that GDNF can augment DA overflow while inhibiting the neurotoxic effects of METH.
Repeated administration of cocaine often results in tolerance to its effects on operant behavior. The tolerance is often associated with an initial drug effect that results in loss of reinforcement. Cocaine can also produce effects that result in a gain of reinforcement, and it is not known if tolerance will be observed in such a circumstance. The present experiments investigated whether tolerance would develop when cocaine was administered repeatedly to subjects who experienced an increase in the frequency of reinforcement when cocaine was administered acutely. Pigeons were trained to peck a response key under fixed-ratio schedules of food presentation. The ratio value for each pigeon was chosen such that performance indicated that the ratio was relatively large, and produced "ratio strain.". Cocaine was administered acutely (once per week), and then subsequently a dose was chosen and administered before each session. Once performance under the daily drug regimen was stable, other doses occasionally were substituted for the usual daily dose. Acute administration of cocaine (0.3-10.0 mg/kg) revealed substantial increases of 100% or more in response rate, and therefore equivalent increases in rate of food presentation, at some doses. That finding permitted examination of the role of drug-induced increases in rate of reinforcement during repeated administration of a response-rate-increasing dose. Repeated, daily administration of a rate-increasing dose resulted in attenuation of the effects of that dose, and subsequent administration of other doses that previously had increased response rates revealed that these doses, too, had lost their ability to increase rates. That is, "tolerance" developed to the rate-increasing effects, even though the rate increases were associated with more frequent access to food. These findings suggest that "reinforcement gain" may not be sufficient to prevent tolerance from developing to effects of cocaine on operant behavior.
To examine the genetic contribution of HLA and non-HLA genes in the etiopathogenesis of rheumatoid arthritis (RA), 60 Caucasian multiplex families were identified and DNA analyzed for over 52 markers including DRB1, DQA1 and DQB1 alleles. Many of the markers were chosen because of close proximity to candidate genes suggested by previous studies or models of pathogenesis. Sibling pair analysis (SIBPAL), relative pair analysis (RELPAL) and linkage studies using two different models of inheritance suggested linkage for the MHC and two additional chromosomal regions: chromosome 2 (D2S443 near CD8 and IGk; 2p13-2p11.1), and chromosome 15 (CYP19-estrogen synthase; 15q15). No support was found for two chromosomal regions, 1p36 and 3q13, recently suggested by other studies. We used transmission disequilibrium testing (TDT), conditional logistic regression, and segregation analysis to study the contributions that the shared epitope and TNF-c have in contributing to risk for RA. These studies provide additional evidence that the association of HLA alleles in RA patients from multiplex families is similar to that observed in sporadic disease, suggest candidate regions for further analysis and find additional support for an association of TNF-c alleles with RA susceptibility.
OBJECTIVE: To validate the objective monitoring of ambulatory activity as an outcome measure for rheumatoid arthritis (RA). METHODS: We have compared ambulatory activity to a range of currently favoured outcome measures, ranging from subjective opinions to X-ray damage, in a population of 93 RA sufferers. RESULTS: Correlations were stronger with measures of joint damage and disability, and less strong with measures of disease activity. Sensitivity to change was good. Three different interventions were compared for the quantity of the response, and the results agree with clinical experience, with steroid injection of the knee and use of non-steroidal anti-inflammatory drugs (NSAIDs) having a similar response and the provision of surgical shoes producing a more modest increase in ambulation. CONCLUSION: The measurement of ambulatory activity has validity for RA assessment. It provides different but related data to the currently used measures. It is objective, relevant, quantifiable and of unlimited scale. It could be used to quantify interventions aimed at increasing ambulation, in carefully constructed studies.
The subjective, psychomotor, and physiological effects of three opioid mu-receptor agonists were studied in healthy volunteers using a cumulative-dosing procedure. Sixteen volunteers with no history of drug abuse received i.v. injections of saline (SAL), morphine (MOR), hydromorphone (HM), or meperidine (MEP) in a randomized double-blind crossover design. Subjects received 1 injection/h for the first 4 h, and a 3-h recovery period followed. SAL was injected first during each session, then SAL or increasing doses of each drug were administered every hour for the next 3 h. The absolute doses per injection were MOR: 2.5, 5, and 10 mg/70 kg; HM: 0.33, 0.65, and 1.3 mg/70 kg; and MEP: 17.5, 35, and 70 mg/70 kg. These injections resulted in cumulative doses of MOR: 2.5, 7.5, and 17.5; HM: 0.33, 0.98, and 2.28; and MEP: 17.5, 52.5, and 122.5 mg/70 kg. Subjects completed mood forms and psychomotor tests, and physiological measures were recorded at various times after each injection and during recovery. MEP tended to produce the most intense effects immediately after drug injection, which dissipated rapidly. MOR produced the mildest effects but was associated with unpleasant side effects during recovery and after the session. HM's effects were stronger than MOR's, and the recovery from HM was slower than with MEP. None of the opioids produced consistent effects that are typically associated with abuse liability. Orderly dose-response functions suggested that our cumulative-dosing procedure is an efficient way of determining dose-response functions for multiple opioids within the same subjects within the same study.
Artemisinin and its derivatives are important new antimalarial drugs. When Plasmodium falciparum-infected erythrocytes are incubated with [10-3H]dihydroartemisinin, several malaria-specific proteins become labeled. One of these proteins is the P. falciparum translationally controlled tumor protein (TCTP) homolog. In vitro, dihydroartemisinin reacts covalently with recombinant TCTP in the presence of hemin. The association between drug and protein increases with increasing drug concentration, plateauing at approximately 1 drug/TCTP molecule. By Scatchard analysis, there appear to be 2 hemin binding sites on TCTP with dissociation constants of approximately 18 microM. When the single cysteine moiety is blocked by pretreatment with iodoacetamide, hemin binding is not affected, whereas drug binding is reduced by two-thirds. Thus, TCTP reacts with artemisinin in situ and in vitro in the presence of hemin and appears to bind to hemin. The function of the malarial TCTP and the role of this reaction in the mechanism of action of artemisinin await elucidation.
The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.
The subjective, psychomotor, and physiological effects of analgesic doses of oral codeine and morphine were examined in 12 healthy volunteers. Subjects ingested placebo, morphine 20 or 40 mg, or codeine 60 or 120 mg in a randomized, double-blind, crossover design. The smaller and larger doses of each drug were putatively equianalgesic, and the cold-pressor test was included to test this assumption. Codeine and morphine increased ratings of "feel drug effect" but had little effect on other subjective measures, including the Addiction Research Center Inventory, visual analog scales, and adjective checklists. The few subjective effects that were observed were modest and were dose-related for morphine but not for codeine. The drugs did not affect performance on Maddox-Wing, digit-symbol substitution, coordination, auditory reaction, reasoning, and memory tests. Dose-related decreases in pupil size (miosis) were observed following codeine and morphine. Ratings of pain intensity decreased in a dose-related manner for morphine but not for codeine. Plasma codeine and morphine levels varied as an orderly function of dose. These results suggest that oral codeine and morphine are appropriate drugs for outpatient pain relief because they are effective analgesics at doses that have only modest effects on mood, produce few side effects, and do not impair performance. The results also suggest a possible ceiling effect of codeine on analgesia and subjective effects.
Cysticercoids as well as 6-, 10-, and 14-day Hymenolepis diminuta were evaluated in terms of enzymatic activities related to phosphoenolpyruvate (PEP) utilization and mitochondrial succinate accumulation. The data obtained support a transition toward anaerobic electron-transport-dependent succinate accumulation, characteristic of adult H. diminuta, with development from cysticercoid to adult. This transition was reflected most prominently in the increasing activities of PEP carboxykinase (PEPCK), malate dehydrogenase, NADPH-->NAD+ transhydrogenase, and fumarate reductase. Developmental increases in PEPCK/pyruvate kinase (PK), fumarate reductase (FR)/NADH oxidase (NO), and FR/succinate dehydrogenase (SDH) activity ratios were also apparent. Evaluations of "egg-free" immature, mature, and pregravid-gravid segments of adult H. diminuta revealed that in general the greater levels of activity were associated with the immature and mature segments. Whereas FR/NO and FR/SDH ratios remained relatively constant in segment comparisons, the greatest PEPCK/PK ratio was associated with the pregravid-gravid segment.
Pneumocystis carinii pneumonia (PCP) is a frequent opportunistic infection in AIDS patients. Large numbers of HIV-infected individuals take prophylactic medications to prevent this illness. The development of drug resistance, while expected, cannot be monitored by classical means, since the organism cannot be cultivated in vitro. Two drug target genes, dihydropteroate synthase (DHPS) and cytochrome b, have been cloned and sequenced from human-derived P. carinii. Mutations leading to amino acid substitutions in the active sites of both proteins have been detected in patients receiving prophylaxis with sulfonamides and sulfones (DHPS inhibitors) and with atovaquone (cytochrome b inhibitor), suggesting that drug resistance may indeed be developing.
Atovaquone (Mepron, 566c80) is an effective agent against Pneumocystis carinii, which probably acts by binding to cytochrome b and inhibiting electron transport. To assess the possibility that atovaquone resistance might be developing, the genes for the cytochrome b from P. carinii sp. f. carinii and P. carinii sp. f. hominis were partially sequenced. Eight of 10 patient isolates had cytochrome b genes with the same amino acid sequence. The P. carinii cytochrome b genes from 2 of 4 patients who had atovaquone prophylaxis failure contained mutations resulting in amino acid changes in one of the ubiquinone (coenzyme Q) binding sites (Qo). These mutations are homologous to mutations in other microorganisms that confer resistance to similar inhibitors. Variations in the sequence of the P. carinii cytochrome b gene suggest but do not prove the development of drug resistance.
OBJECTIVE: Quantifiable outcome measures for disabling diseases such as osteoarthritis (OA) of the knee are necessary in order to compare the impact of different interventions competing for financial resources. Current subjective and questionnaire data are not satisfactory for such study. In this study, we examine the potential of the direct measurement of ambulatory activity as such a measure. POPULATION: Patients with X-ray evidence of OA of the knee recruited to studies of anti-inflammatory agents (n = 29). Patients with OA of the knee awaiting knee replacement surgery (n = 28). METHODS: Comparison of various standard measures with total energy output data from an activity monitor. RESULTS: Spearman rho correlations of ambulatory energy output (number of steps x average amplitude of steps) correlated with other measures. Correlation with physician's opinion was greater than with patient's opinion (r = 0.4 and 0.2, respectively). There was no correlation with visual analogue pain scale or OA severity index. Correlation with scales of the Nottingham Health Profile questionnaire were not significant either for mobility (r = - 0.15) or for pain (r = - 0.13). There was, however, a significant correlation between poor sleep and increased activity (r = 0.34, P < 0.05). Correlation with Kellgren X-ray grade was significant (r = - 0.45, P = 0.01). Patients recruited to anti-inflammatory studies were 69% more active than those awaiting replacement surgery. CONCLUSION: The monitoring of ambulatory activity shows some construct and discriminant validity, and is worthy of further study.
In Experiment 1 pigeons pecked a key that was illuminated with a 501-nm light and obtained food by doing so according to a variable-interval (VI) schedule of reinforcement, the mean value of which differed across groups: either 30 s, 120 s, or 240 s. The pigeons in all three groups were trained for 10 50-min sessions. Generalization testing was conducted in extinction with different wavelengths of light. Absolute and relative generalization gradients were similar in shape for the three groups. Experiment 2 was a systematic replication of Experiment 1 using line orientation as the stimulus dimension and a mean VI value of either 30 s or 240 s. Again, gradients of generalization were similar for the two groups. In Experiment 3 pigeons pecked a key that was illuminated with a 501-nm light and obtained food reinforcers according to either a VI 30-s or a 240-s schedule. Training continued until response rates stabilized (> 30 sessions). For subjects trained with the 30-s schedule, generalization gradients were virtually identical regardless of whether training was for 10 sessions (Experiment 1) or until response rates stabilized. For subjects trained with the VI 240-s schedule, absolute generalization gradients for subjects trained to stability were displaced upward relative to gradients for subjects trained for only 10 sessions (Experiment 1), and relative generalization gradients were slightly flatter. These results indicate that the shape of a generalization gradient does not necessarily depend on the rate of reinforcement during 10-session single-stimulus training but that the effects of prolonged training on stimulus generalization may be schedule dependent.
OBJECTIVE: To quantify any increase in short term spontaneous ambulatory activity resulting from the use of nonsteroidal antiinflammatory drugs (NSAID) in patients with rheumatoid arthritis (RA). METHODS: Double blind placebo controlled crossover study of 8 women with RA using conventional assessments and the Numact activity monitor. RESULTS: Patients' ambulatory activity was 50% greater during NSAID treatment compared to placebo treatment. Effect size calculated at 0.62, suggesting good sensitivity to change. The increased activity occurred late morning. CONCLUSION: Spontaneous ambulatory activity is an objective and relevant measure of disability.
Key pecking by pigeons was maintained by a fixed-ratio 15 or a fixed-interval 15 s schedule of food presentation. Sessions consisted of six blocks that included a 4 min blackout then 2 min of schedule time or six food presentations. Cumulative dose-response curves were assessed by injecting saline at the start of the second block and increasing cocaine doses at the onset of subsequent blocks. Repeated cumulative dosing often shifted dose-response curves to the left, and these effects appeared to be due to the reliable correlation between smaller and larger doses administered early and later in the session. When saline was substituted for the larger doses later in the session, dose-response curves initially remained shifted to the left, and continued substitution eventually resulted in curves shifting to the right. Cumulative dosing was compared with two noncumulative dosing procedures: (a) pre-session dosing and (b) one cocaine injection at the block in which the same cumulative dose would be tested, with saline injections at the other blocks (except the first). Noncumulative dosing tended to produce more reliable (repeatable) results than did cumulative dosing, and at least one interpretation is the possibility of conditioning factors that may be present in cumulative-dosing but not in noncumulative-dosing procedures.
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