Post-craniotomy analgesia: current practices in British neurosurgical centres--a survey of post-craniotomy analgesic practices.

BACKGROUND AND OBJECTIVE: An evaluation of post-craniotomy analgesia within the University Hospital of Wales Neurosurgical Unit, Cardiff, found that many patients were experiencing moderate to severe pain post-craniotomy. It was therefore decided to undertake a nationwide survey of analgesic practices in order to establish best practice guidelines and benchmark with other units. METHOD: A postal questionnaire was sent to the senior nurse of every Neurosurgical Directorate within the UK inquiring about the current, standard analgesic practices for post-craniotomy patients in their unit. RESULTS: Completed replies were received from 23 of the 33 centres (70%). Intramuscular (i.m.) codeine phosphate was found to be the principal first-line analgesic used post-craniotomy. Only three centres used morphine as the first-line analgesic and only one centre used patient controlled analgesia routinely. The majority of centres (82%) used balanced analgesia. Pain assessments were only carried out in 57% of centres and no centre used a validated pain assessment tool specifically for dysphasic patients. CONCLUSIONS: Codeine phosphate continues to be the mainstay of post-craniotomy analgesia, however, it is proposed that patient controlled analgesia with morphine is an efficacious and safe alternative.

Dynamics of mononuclear cadmium beta-lactamase revealed by the combination of NMR and PAC spectroscopy.

The two metal sites in cadmium substituted beta-lactamase from Bacillus cereus 569/H/9 have been studied by NMR spectroscopy ((1)H, (15)N, and (113)Cd) and PAC spectroscopy ((111m)Cd). Distinct NMR signals from the backbone amides are identified for the apoenzyme and the mononuclear and binuclear cadmium enzymes. For the binuclear cadmium enzyme, two (113)Cd NMR signals (142 and 262 ppm) and two (111m)Cd PAC nuclear quadrupole interactions are observed. Two nuclear quadrupole interactions are also observed, with approximately equal occupancy, in the PAC spectra at cadmium/enzyme ratios < 1; these are different from those derived for the binuclear cadmium enzyme, demonstrating interaction between the two metal ion binding sites. In contrast to the observation from PAC spectroscopy, only one (113)Cd NMR signal (176 ppm) is observed at cadmium/enzyme ratios < 1. The titration of the metal site imidazole (N)H proton signals as a function of cadmium ion-to-enzyme ratio shows that signals characteristic for the binuclear cadmium enzyme appear when the cadmium ion-to-enzyme ratio is between 1 and 2, whereas no signals are observed at stoichiometries less than 1. The simplest explanation consistent with all data is that, at cadmium/enzyme ratios < 1, the single Cd(II) is undergoing exchange between the two metal sites on the enzyme. This exchange must be fast on the (113)Cd NMR time scale and slow on the (111m)Cd PAC time scale and must thus occur in a time regime between 0.1 and 10 micros.

The solution structure of the complex of Lactobacillus casei dihydrofolate reductase with methotrexate.

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase (18.3 kDa, 162 amino acid residues) formed with the anticancer drug methotrexate using 2531 distance, 361 dihedral angle and 48 hydrogen bond restraints obtained from analysis of multidimensional NMR spectra. Simulated annealing calculations produced a family of 21 structures fully consistent with the constraints. The structure has four alpha-helices and eight beta-strands with two other regions, comprising residues 11 to 14 and 126 to 127, also interacting with each other in a beta-sheet manner. The methotrexate binding site is very well defined and the structure around its glutamate moiety was improved by including restraints reflecting the previously determined specific interactions between the glutamate alpha-carboxylate group with Arg57 and the gamma-carboxylate group with His28. The overall fold of the binary complex in solution is very similar to that observed in the X-ray studies of the ternary complex of L. casei dihydrofolate reductase formed with methotrexate and NADPH (the structures of the binary and ternary complexes have a root-mean-square difference over the backbone atoms of 0.97 A). Thus no major conformational change takes place when NADPH binds to the binary complex. In the binary complex, the loop comprising residues 9 to 23 which forms part of the active site has been shown to be in the "closed" conformation as defined by M. R. Sawaya & J. Kraut, who considered the corresponding loops in crystal structures of complexes of dihydrofolate reductases from several organisms. Thus the absence of the NADPH does not result in the "occluded" form of the loop as seen in crystal studies of some other dihydrofolate reductases in the absence of coenzyme. Some regions of the structure in the binary complex which form interaction sites for NADPH are less well defined than other regions. However, in general terms, the NADPH binding site appears to be essentially pre-formed in the binary complex. This may contribute to the tighter binding of coenzyme in the presence of methotrexate.

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.

Domain motions in dihydrofolate reductase: a molecular dynamics study.

Molecular dynamics simulations have been carried out on the enzyme dihydrofolate reductase from Lactobacillus casei complexed with methotrexate, NADPH and 264 crystallographic water molecules. Analysis of correlations in atomic fluctuations reveal the presence of highly correlated motion (correlation coefficient > 0.6) in the region between residues 30 to 35 and 85 to 90 leading to the identification of two domains, an "adenosine-binding domain" and a "large domain", which rotate by 3 to 4 degrees with respect to each other. The strongest correlation (> 0.6) within the large domain involves a coupling between the motions of the "teen-loop", and the spatially contiguous loops linking beta 6-beta 7 and beta 7-beta 8. Moreover, there is a significant correlation (approximately 0.5) between the adenosine fragment of NADPH and the pteridine and p-aminobenzoyl fragments of methotrexate, which are separated by approximately 17 A, and is lost on removal of "rigid-body" motion from the original trajectory. This provides support for the idea that the relative motion of the two domains is a means by which the occupation of the binding site for the adenosine end of the coenzyme can affect methotrexate binding and vice versa. Quasiharmonic vibrational analysis of the trajectory reveals that the overall dynamics of the system are governed by domain motions whose contributions are dominant at low frequencies. In addition, different low-frequency modes are responsible for separately coupling the adenosine-binding site and parts of methotrexate.

The conformation of coenzyme A bound to chloramphenicol acetyltransferase determined by transferred NOE experiments.

The conformation of coenzyme A bound to chloramphenicol acetyltransferase has been studied in solution by NMR methods. Transferred nuclear Overhauser enhancement (NOE) and rotating frame NOE (ROE) experiments were used to determine the conformation of the bound coenzyme. Experiments were carried out at five mixing times and two temperatures, and with normal and perdeuterated enzyme, to ensure (1) that the fast exchange condition was satisfied and (2) that the results were not complicated by spin diffusion involving enzyme protons. The data were analysed using a general approach involving combined exchange and relaxation matrices. For the binary complex of coenzyme A (CoA) and enzyme, the conformation of CoA was calculated by using distance constraints derived from the intensities of 71 NOE and 33 ROE cross-peaks between coenzyme protons. The conformation of the adenosine moiety of CoA in the structure deduced by NMR is very close to that seen in the crystal structure of this complex, while the pantetheine moiety is clearly less extended. Essentially the same conformation was obtained whether or not the calculations included the protein (with appropriate intermolecular energy terms). The difference between the NMR and X-ray structures is interpreted in terms of the existence of two conformations of the CoA-enzyme complex. Support for this model comes from measurements of the coenzyme dissociation rate constant; NMR (lineshape analysis and transferred NOE experiments) gives estimates of koff approximately 3700 s-1 at 298 K and approximately 500 s-1 at 280 K, both significantly greater than estimates by fluorescence stopped-flow measurements. For the ternary complex of CoA, chloramphenicol and enzyme, 71 NOE cross peaks between protons of coenzyme A and a further ten cross-peaks between protons of coenzyme A and chloramphenicol were measured. Starting with a model derived from the crystal structures of the two binary complexes (in the absence of crystallographic data for the ternary complex) the conformations and relative positions of the two ligands were refined using the distance constraints derived from these NOEs. The conformation of the adenosine part of CoA is the same as in the binary complex, while the pantetheine arm is more extended and approaches close to the bound chloramphenicol molecule. The model of the ternary complex is discussed in terms of the information available on the mechanism of the enzyme.

Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin.

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1-12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed.

A novel aldehyde reductase with activity towards a metabolite of aflatoxin B1 is expressed in rat liver during carcinogenesis and following the administration of an anti-oxidant.

In contrast with fractions from control animals, an aldehyde reductase, which catalyses the reduction of aflatoxin B1-dihydrodiol, in the dialdehyde form at physiological pH values, to aflatoxin B1-dialcohol, is expressed in cytosolic fractions prepared from rat livers bearing pre-neoplastic lesions, or following treatment with the anti-oxidant ethoxyquin. This expression parallels the development of resistance to the toxin. Unlike the aflatoxin B1-dihydrodiol, the dialcohol does not undergo binding to protein. This enzyme activity could play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Correlated n.m.r. and m.s. spectra are provided in Supplementary Publication SUP 50171 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A.T.C.C. 6633.

A variant of the peptide antibiotic subtilin has been isolated from Bacillus subtilis A.T.C.C. 6633, and its structure has been shown to be [N alpha-succinyl-Trp1]subtilin. The chemical structure of a fragment derived by tryptic hydrolysis of the variant is shown to be N alpha-succinyl-Trp-Lys by 1H and 13C n.m.r., fast-atom-bombardment m.s. and total chemical synthesis [N alpha-Succinyl-Trp1]-subtilin is produced later in the growth of the bacterium than is subtilin; reverse-phase h.p.l.c. analysis shows that after 24 h growth the ratio subtilin/[N alpha-succinyl-Trp1]subtilin is approx. 1:2. Although [N alpha-succinyl-Trp1]subtilin retains significant antibacterial activity, it is 10-20 times less active than subtilin.

Solution structures of nisin A and its two major degradation products determined by n.m.r.

The conformations of nisin and two major degradation products, nisin-(1-32)-peptide (nisin1-32) and des-delta Ala5-nisin1-32 (where delta Ala is alpha beta-didehydroalanine), in aqueous solution have been determined from n.m.r. data. Sequential assignments of the peptides using correlation spectroscopy ('COSY'), homonuclear Hartmann-Hahn spectroscopy ('HOHAHA'), nuclear Overhauser enhancement spectroscopy (NOESY), relayed NOESY and rotating-frame nuclear Overhauser spectroscopy (ROESY) experiments are presented, including stereospecific assignments of beta-methylene protons of the lanthionine residues. ROESY experiments are also used to detect flexible regions in the polypeptide chain. A dynamic-stimulated-annealing approach is used for structural determination. It can be concluded that all these peptides are flexible in aqueous solution, with no experimental evidence of preferred overall conformations; the only defined conformational features are imposed by the presence of the lanthionine residues. Low-temperature studies also reveal that des-delta Ala5-nisin1-32 adopts conformations similar to those when the ring is intact, suggesting that the loss of activity of this degradation product is due to the absence of the delta Ala5 residue rather than to the conformational consequences of ring-opening.

Sequence-specific resonance assignment and conformational analysis of subtilin by 2D NMR.

Subtilin, a 32-amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus subtilis ATCC6633. The chemical structure has been confirmed by the unambiguous sequence-specific assignment of its 1H NMR spectrum. Detailed NMR analysis revealed that subtilin is a rather flexible molecule; the only observed conformational contraints were those imposed by the cyclic structures created by the lanthionine and 3-methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformations.

Relationship of pain to the radiological anatomy of the hip joint in adults treated for congenital dislocation of the hip as infants: a long-term follow-up of patients treated by three methods.

Sixty female patients with congenital dislocation of the hip treated by three methods were followed up to a mean age of 27.3 years. Standard radiographic measurements of hip joint morphology, including stereoscopic measurements of acetabular and femoral neck anteversion, were made. The Severin score of the hip joint, but not acetabular or femoral neck anteversion, was related to symptoms. Hips treated by femoral derotation osteotomy had superior results to hips treated by traction reduction alone. Both of these methods gave much better results than treatment by closed manipulative reduction. The "normal" opposite hip was morphologically abnormal, although usually asymptomatic.

13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure.

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165.

The results of early posterior release in resistant club feet. A long-term review.

Seventy resistant club feet in 46 patients were treated within the first six months of life by posterior release as an incident in continuing conservative care and were later assessed at an average age of 15 years. Assessment was made upon strict clinical criteria, including appearance, function and degree of pain during activity; the results were excellent in 22 feet, good in 19, and poor in 29. We also studied the correlation between the radiographs and the final outcome. From these data we propose certain indications for the timing and technique of this type of management, and also discuss the relative merits of this approach compared with more conventional radical release operations.

A 1H-n.m.r. study of casein micelles.

The 1H-n.m.r. spectrum of casein micelles consists of a small number of moderately sharp (linewidth approx. 60 Hz) resonances superimposed on the envelope of very broad lines expected for particles of this size. These sharp lines resemble, in chemical shift and relative intensity, the spectrum of the isolated 'macropeptide' released from the micelles by treatment with chymosin. The sharp lines in the casein micelle spectrum are further sharpened by addition of chymosin and broadened markedly by addition of ethanol. These observations are consistent with the proposal that the 'macropeptide' (the C-terminal 64 residues of K-casein) forms flexible 'hairs' on the surface of the micelles.

Avulsion of the distal pole of the patella in cerebral palsy. A cause of deteriorating gait.

Avulsion of the distal pole of the patella in spastic children with a flexed knee gait may upset the equilibrium of the knee and cause deterioration in walking. We emphasise the clinical features, discuss the mechanism and outline the principles of treatment.

Trochanteric advancement for premature arrest of the femoral capital growth plate.

Premature closure of the growth plate of the femoral capital epiphysis associated with limitation of abduction and gluteal insufficiency is described. The indications for trochanteric advancement and the results obtained in the late treatment of Perthes' disease and of congenital dislocation are discussed. Nine hips treated after Perthes' disease had satisfactory results, in that all were improved; but in eight hips with deformity arising from congenital dislocation the outcome was less favourable.

Talectomy for arthrogryposis multiplex congenita.

Eighteen patients (34 feet) with arthrogryposis multiplex congenita treated by talectomy for rigid equinovarus deformity were reviewed. The average follow-up was 11 years. Twenty-four feet (71%) were considered satisfactory; the remainder were improved. Seven feet required further operations to correct recurrence of the deformity, but finally all could be fitted with boots or shoes and all patients could walk. The history of talectomy is reviewed and the operative details described.