Further improving the cognitive effect profile of electroconvulsive therapy (ECT): the case for studying carbamylated erythropoietin.

Electroconvulsive therapy (ECT) remains the most effective acute treatment for severe depression and several other psychiatric illnesses. However, its use has been limited by concerns about cognitive adverse effects. ECT may cause temporary cognitive impairment in some patients, typically anterograde amnesia for 1-2 weeks after a course of treatment, and circumscribed retrograde amnesia. These cognitive effects largely disappear within days to weeks after treatment. Efforts to find a pharmacological agent to reduce the cognitive effects of ECT have largely been unsuccessful, with the possible exception of thyroid hormone. We review the literature on pharmacological attempts to attenuate ECT's cognitive effects, and propose a novel neuroprotective and neurotrophic agent, carbamylated erythropoietin (CEPO), for this indication.

The barrier method: a technique for calculating very long transition times.

In many dynamical systems, there is a large separation of time scales between typical events and "rare" events which can be the cases of interest. Rare-event rates are quite difficult to compute numerically, but they are of considerable practical importance in many fields, for example, transition times in chemical physics and extinction times in epidemiology can be very long, but are quite important. We present a very fast numerical technique that can be used to find long transition times (very small rates) in low-dimensional systems, even if they lack detailed balance. We illustrate the method for a bistable nonequilibrium system introduced by Maier and Stein and a two-dimensional (in parameter space) epidemiology model.

Harmonic measure for critical Potts clusters.

We present a technique, which we call "etching," which we use to study the harmonic measure of Fortuin-Kasteleyn clusters in the Q-state Potts model for Q=1-4 . The harmonic measure is the probability distribution of random walkers diffusing onto the perimeter of a cluster. We use etching to study regions of clusters which are extremely unlikely to be hit by random walkers, having hitting probabilities down to 10-4600. We find good agreement between the theoretical predictions of Duplantier and our numerical results for the generalized dimension D(q) including regions of small and negative q .

Power spectra of the total occupancy in the totally asymmetric simple exclusion process.

As a solvable and broadly applicable model system, the totally asymmetric exclusion process enjoys iconic status in the theory of nonequilibrium phase transitions. Here, we focus on the time dependence of the total number of particles on a 1-dimensional open lattice and its power spectrum. Using both Monte Carlo simulations and analytic methods, we explore its behavior in different characteristic regimes. In the maximal current phase and on the coexistence line (between high and low density phases), the power spectrum displays algebraic decay, with exponents -1.62 and -2.00, respectively. Deep within the high and low density phases, we find pronounced oscillations, which damp into power laws. This behavior can be understood in terms of driven biased diffusion with conserved noise in the bulk.

Coarsening of "clouds" and dynamic scaling in a far-from-equilibrium model system.

A two-dimensional lattice gas of two species, driven in opposite directions by an external force, undergoes a jamming transition if the filling fraction is sufficiently high. Using Monte Carlo simulations, we investigate the growth of these jams (''clouds''), as the system approaches a nonequilibrium steady state from a disordered initial state. We monitor the dynamic structure factor S(k{x},k{y};t) and find that the k{x}=0 component exhibits dynamic scaling, of the form S(0,k{y};t)=t;{beta}S[over](k{y}t;{alpha}) . Over a significant range of times, we observe excellent data collapse with alpha=12 and beta=1 . The effects of varying filling fraction and driving force are discussed.

A survey of tractors and rollover protective structures in Washington State.

A survey of farms in Washington State was conducted to determine tractor characteristics and the presence of rollover protective structures (ROPS) in a state with more inclusive rules on tractor retrofitting than federal regulations. A total of 544 valid surveys were completed from a proportional random sample across different types of farms. Responders indicated that 58% of tractors overall were equipped with ROPS, and 42% of the tractors without ROPS were exempt from the state rules. Seatbelts on tractors equipped with ROPS were reportedly used "sometimes" or more 30% of the time, and 17% of these tractors had no seatbelt installed. Tractors used for row crop farming were significantly more likely to be equipped with ROPS than those used for tree, vine, or hops farming. Older tractors were used for fewer hours, were less likely to be ROPS-equipped, and were less likely to be operated while wearing a seatbelt. The results were consistent with a positive effect of the Washington State ROPS requirements, demonstrated by the increased percentage of ROPS-equipped pre-1976 tractors, as compared to other states, and by the difference between ROPS-equipped tractors in exempt and non-exempt types of farming. The results point to the need for prevention activities to increase seatbelt use on ROPS-equipped tractors, and for further development of practical protection for tractors operating under overhead obstacles.

Two new classes of conopeptides inhibit the alpha1-adrenoceptor and noradrenaline transporter.

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha1-adrenoceptors (rho-TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi-MrIA and chi-MrIB from the mollusk-hunting C. marmoreus). rho-TIA and chi-MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Novel omega-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes.

omega-Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega-conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega-conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega-conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha(1B-d)) and peripheral (alpha(1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta(3) in Xenopus oocytes. However, the potency of CVID and MVIIA increased when alpha(1B-d) and alpha(1B-b) were expressed in the absence of rat beta(3), an effect most pronounced for CVID at alpha(1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha(1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. (1)H NMR studies reveal that CVID possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 1. Isolation, characterization and chemical synthesis.

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Effects of chirality at Tyr13 on the structure-activity relationships of omega-conotoxins from Conus magus.

The effects of chirality inversions of Tyr13 on the structure-activity relationships of omega-conotoxins MVIIA and MVIIC were examined using a combination of 2D 1H NMR spectroscopy and radioligand binding studies specific for N-type ([125I]GVIA) and P/Q-type ([125I]MVIIC) voltage-sensitive calcium channels (VSCCs). A comparison of the Halpha secondary shifts suggests that the structural scaffolds of MVIIA and MVIIC are little altered by the L- to D- inversion of Tyr13; however, the conformations of several residues in loop 2 (residues 9-14) are significantly altered. The experimentally determined 3D structure of [D-Y13]MVIIA indicates that the positions of key residues in this loop which are involved in the binding of MVIIA to the N-type VSCC (Tyr13, Arg10, and Leu11) are so changed as to render the peptide unrecognizable by its cognate ion channel. The large reduction in potency observed for MVIIA and MVIIC at both N-type and P/Q-type VSCCs is likely to stem from the change in conformation and orientation of loop 2.

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators.

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.

Development of a comprehensive panel of antibodies against the major xenobiotic metabolising forms of cytochrome P450 in humans.

Mono-specific antibodies against the human cytochrome P450 (P450) enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP4A11 and an antibody that binds to CYP2C8, CYP2C9 and CYP2C19 have been produced by immunising rabbits with synthetic peptides representing small regions of each of these P450 enzymes. The specificity of the antibodies was confirmed by immunoblotting using recombinant P450 enzymes and samples of human hepatic microsomal fraction. Each of the antibodies bound only to their respective target P450 enzyme(s). The relative intensity of immunoreactive bands was compared with a variety of P450 activities and correlations were found between CYP1A2 and phenacetin O-deethylase activity, CYP2A6 and coumarin 7-hydroxylase activity, CYP2C9 and tolbutamide 4-hydroxylase activity, CYP2C19 and S-mephenytoin 4-hydroxylase activity, CYP2D6 and debrisoquine 4-hydroxylase activity, CYP2E1 and chlorzoxazone 6-hydroxylase activity, CYP3A4 and midazolam 1'-hydroxylase activity, and CYP4A11 and lauric acid 12-hydroxylase activity. A proportion of the 30 liver samples examined lacked CYP2A6 (7%), CYP2C19 (10%) or CYP2D6 (13%), consistent with the polymorphic expression of these P450 enzymes in human liver. Although CYP3A5 was detected in most individuals (97%), expression was polymorphic with 20% containing substantially higher levels. CYP2B6 was expressed in 20% of the human liver samples, with one sample containing a particularly high level. No immunodetectable CYP1A1 or CYP1B1 was found, consistent with the low level of expression of these P450 enzymes in human liver. The results demonstrate the utility of the antipeptide approach for producing specific antibodies against human P450 enzymes, enabling a comprehensive panel of antibodies against human P450 enzymes to be produced.

Interlaboratory comparison of the assessment of P450 activities in human hepatic microsomal samples.

1. Although the importance of in vitro technology in supporting drug development is widely accepted, there is no real consensus about which approaches should be taken, which substrates should be used, or on the reliability and application of in vitro data. Consequently, as part of a collaborative project to characterize human liver with respect to the major forms of cytochrome P450, an interlaboratory comparison of the analysis of samples for form-specific activities was undertaken. 2. Microsomal fractions were isolated from five different human liver samples taken from the liver bank maintained at the Royal Postgraduate Medical School (RPMS). Aliquots from the five samples were sent to the 11 collaborating laboratories for characterization using their in-house, form-specific assays for cytochrome P450 activities. Although each laboratory assayed protein concentration, total cytochrome P450 content and enzyme activities were calculated using the protein estimation generated by RPMS to eliminate this possible source of variability. 3. With the exception of one laboratory, all estimates of protein concentration were similar (coefficient of variation, CoV, 9-13%) and the rank-order of the five samples was consistent across the laboratories. There was greater variability in the estimates of total cytochrome P450 content (CoV 28-43%), although again rank order of the samples across laboratories was fairly consistent. 4. The various laboratories used a number of different probe substrates, together with a range of conditions (substrate concentration, time of incubation, amount of protein), to assay for activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. However, apart from the occasional outlier, the five samples were ranked for activity of all these forms of cytochrome P450 with a high degree of consistency by the various laboratories and the choice of substrate had no appreciable effect on the ranking of the samples. 5. While this interlaboratory comparison has shown that greater consistency in the approach to in vivo determination of drug-metabolizing activity is desirable, there was little indication that any particular approach or substrate was superior to the others.

Specific inhibition of human CYP1A2 using a targeted antibody.

The structural similarity of related forms of P450 makes selective immunoinhibition of individual forms notoriously difficult to achieve. We report the use of a targeted antibody to overcome this problem. An antibody was raised against the synthetic peptide, Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile, corresponding to residues 291-302 of human CYP1A2. This sequence of human CYP1A2 is located in a similar position to a proinhibitory region previously identified in rat CYP1A1 and CYP1A2. The antibody bound strongly and specifically to CYP1A2 in human hepatic microsomal fraction. Binding was unaffected by denaturation of the protein. The specificity of the antibody was demonstrated by immunoblotting of human hepatic microsomal fraction where a single immunoreactive band was identified at Mr 54,000. The intensity of this band correlated strongly with high-affinity phenacetin O-deethylase activity of the microsomal fractions. In addition, the antibody bound to a single protein at Mr 54,000 in the microsomal fraction of lymphoblastoid cells expressing human CYP1A2, but not to any other recombinant P450 enzyme. CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of human hepatic microsomal fraction was inhibited >90% by whole antiserum or purified immunoglobulin. This decrease in activity represents complete inhibition of CYP1A2 activity, residual phenacetin O-deethylase activity being due to low-affinity enzymes. In contrast, the antibody, which does not bind to rat CYP1A2, had no effect on CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of rat hepatic microsomal fraction. The antiserum also had no effect on human hepatic microsomal debrisoquine 4-hydroxylase (CYP2D6) or coumarin 7-hydroxylase (CYP2A6) activities, indicating that inhibition was specific to human CYP1A2. These results demonstrate the importance of the region comprising residues 291-302 of human CYP1A2 in the catalytic activity of this enzyme.

Analysis of terfenadine in human plasma using microbore high-performace liquid chromatography-electrospray ionisation mass spectrometry.

An assay based on combined microbore high-performance liquid chromatography-positive ion electrospray ionisation mass spectrometry with selected ion recording has been developed for the measurement of the antihistamine drug terfenadine in human plasma. A deuterated analogue of terfenadine was synthesised for use as an internal standard and extraction of terfenadine was carried out on C18 solid phase extraction columns. The limit of detection of terfenadine in plasma is 0.1 ng/ml and the intra-assay coefficient of variation at 1 ng/ml is 10.1%. Plasma concentrations of terfenadine measured in six normal subjects following a 120 mg oral dose are reported.

The cardiac effects of terfenadine after inhibition of its metabolism by grapefruit juice.

OBJECTIVE: To determine whether the pharmacokinetics and electrocardiographic pharmacodynamics of terfenadine are affected by the concomitant administration of grapefruit juice. METHODS: Six healthy volunteers were recruited for a balanced cross-over study. Each volunteer received 120 mg terfenadine 30 min after drinking 300 ml of either water or freshly squeezed grapefruit juice. The alternative treatment was administered on the second study day 2 weeks later. Measurements of the area under the terfenadine plasma concentration-time curve (AUC), maximum terfenadine concentration (Cmax) and the time to maximum concentration (tmax) were made, and the corrected QT (QTc) interval was measured from the surface electrocardiogram. RESULTS: Terfenadine was quantifiable in plasma in all 6 subjects on both study days for up to 24 h post-dosing. The AUC of terfenadine was significantly increased by concomitant grapefruit administration (median values 40.6 vs 16.3 ng.ml-1.h), as was the Cmax (median values 7.2 vs 2.1 ng.ml-1). The tmax was not significantly increased and there was no significant change in the median QTc interval despite the increased terfenadine levels. The 95% confidence interval for the difference in the change in QTc interval at Cmax was -13 to +38 ms. CONCLUSION: Administration of grapefruit juice concomitantly with terfenadine may lead to an increase in terfenadine bioavailability, but the increase observed in this study did not lead to significant cardiotoxicity in normal subjects. However, this does not exclude the risk of cardiotoxicity in high-risk subjects given greater doses of grapefruit juice over longer periods of time.

Perforation rates after ventilation tube insertion: does the positioning of the tube matter?

A prospective study was performed of children undergoing bilateral ventilation tube insertion. One hundred and twenty-one children aged between 9 months and 10 years 3 months were admitted for surgery for secretory otitis media (glue ear). Each child had a ventilation tube inserted anteriorly in the tympanic membrane of one ear and posteriorly in the tympanic membrane of the other. They underwent regular clinical and audiological assessment until extrusion of the ventilation tubes occurred. Perforations were noted in 2.75% of tympanic membranes (4.6% of the children). The rate with posteriorly placed ventilation tubes was higher than with the anteriorly placed ventilation tubes (3.7% compared with 1.8%) though this is not statistically significant.

Auditory brainstem response screening for hearing loss in high risk neonates.

The present paper reports the findings of a 7 year study evaluating the use of the auditory brainstem response (ABR) as the basis of a hearing screening procedure in a group of newborns at increased risk of hearing impairment. A Special Care Baby Unit (SCBU) population of 417 infants with diverse clinical backgrounds and treatment histories was tested for hearing impairment at birth using ABR audiometry. Some 332 passed the original screen at 30 dBnHL test level in both ears. Of the failure group, 18 did not survive and 32 had some degree of hearing impairment confirmed, nine of which were sensorineural in origin. An increased incidence of persistent middle ear disease was also noted in the failure group. A detailed operational analysis demonstrates that provided appropriate pass/fail criteria are adopted, the ABR technique offers excellent sensitivity and specificity for the detection of significant hearing loss in the test population. Furthermore, the study establishes that implementation of an ABR-based screening programme could reduce the average age at detection of permanent hearing loss by 7 months. A cost assessment shows that the introduction of such a targetted screening procedure could be done at a reasonable outlay.

Dissecting the function of cytochrome P450.

1. The role that P450 plays in the metabolism of drugs and other chemicals is often pivotal in determining the duration and magnitude of their biological effects, so that it is important to identify the specific forms of the enzyme involved. 2. The similarity between different forms of P450 is such that polyclonal antibodies raised by conventional means, even against a homogeneous preparation of the enzyme will often react with several different P450s. 3. Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use. 4. The use of short sequences of amino acids (4-7 residues) predicted to occur in surface loop regions of the protein as immunogens, affords a means of targeting antibodies to specific forms of P450. 5. The terminal amino acid of a peptide, here the C-terminal residue, is very immunodominant in determining the specificity of the resultant antibody. Hence, antibodies against the C-terminal residues of P450 have a high affinity and, in most cases, will be specific. 6. Application of anti-peptide antibodies to the major forms of P450 in human liver revealed the anticipated interindividual variation. However, evidence was found that the polymorphism in CYP3A5 expression is quantitative rather than qualitative. 7. A putative pro-inhibitory region on the surface of mammalian P450, possibly involved in intramolecular electron transport, has been identified, using a combination of directed anti-peptide antibodies and sequence alignment based on secondary structure. 8. Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.