Imaging Findings in Children Presenting with CNS Nelarabine Toxicity.

Nelarabine is a nucleoside analog critical for the treatment of patients with T-cell acute lymphoblastic leukemia/lymphoma. However, clinical peripheral and central neurologic adverse events associated with nelarabine administration have been reported. Neuroimaging of brain neurotoxicity has only been described in very few reports in pediatric patients so far. Six children with diagnosed T-cell acute lymphoblastic leukemia who clinically experienced possible, probable, or definite nelarabine-induced toxicity and underwent spine and/or brain MR imaging were reviewed. Neuroimaging findings showed a mixture of patterns including features of acute toxic leukoencephalopathy (seen in 6 cases), posterior reversible encephalopathy syndrome (2 cases), involvement of deep gray structures (1 case) and brainstem (2 cases), cranial and spinal neuropathy (2 cases each), and myelopathy (2 cases). Even though neuroimaging findings are nonspecific, the goal of this article was to alert the pediatric neuroradiologists, radiologists, and clinicians about the possibility of nelarabine-induced neurotoxicity and its broad neuroimaging spectrum.

Cerebellar Watershed Injury in Children.

BACKGROUND AND PURPOSE: Focal signal abnormalities at the depth of the cerebellar fissures in children have recently been reported to represent a novel pattern of bottom-of-fissure dysplasia. We describe a series of patients with a similar distribution and appearance of cerebellar signal abnormality attributable to watershed injury. MATERIALS AND METHODS: Twenty-three children with MR imaging findings of focal T2 prolongation in the cerebellar gray matter and immediate subjacent white matter at the depth of the fissures were included. MR imaging examinations were qualitatively analyzed for the characteristics and distribution of signal abnormality within posterior fossa structures, the presence and distribution of volume loss, the presence of abnormal contrast enhancement, and the presence and pattern of supratentorial injury. RESULTS: T2 prolongation was observed at the depths of the cerebellar fissures bilaterally in all 23 patients, centered at the expected location of the deep cerebellar vascular borderzone. Diffusion restriction was associated with MR imaging performed during acute injury in 13/16 patients. Five of 23 patients had prior imaging, all demonstrating a normal cerebellum. The etiology of injury was hypoxic-ischemic injury in 17/23 patients, posterior reversible encephalopathy syndrome in 3/23 patients, and indeterminate in 3/23 patients. Twenty of 23 patients demonstrated an associated classic parasagittal watershed pattern of supratentorial cortical injury. Injury in the chronic phase was associated with relatively preserved gray matter volume in 8/15 patients, closely matching the published appearance of bottom-of-fissure dysplasia. CONCLUSIONS: In a series of patients with findings similar in appearance to the recently described bottom-of-fissure dysplasia, we have demonstrated a stereotyped pattern of injury attributable to cerebellar watershed injury.

Cerebellar Heterotopias: Expanding the Phenotype of Cerebellar Dysgenesis in CHARGE Syndrome.

BACKGROUND AND PURPOSE: Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness (CHARGE) syndrome is a multisystem developmental disorder associated with a number of well-described clinical and imaging findings, including cerebellar hypoplasia. We observed cerebellar heterotopias on MR imaging in 2 patients with CHARGE, confirmed by postmortem examination. We sought to determine the prevalence and define the characteristics of similar findings on MR imaging for a cohort of patients with CHARGE syndrome. MATERIALS AND METHODS: We performed a retrospective, observational, cross-sectional study to assess the prevalence and characteristic features of cerebellar heterotopias in 35 patients with CHARGE syndrome with available brain MR imaging studies, as well as to evaluate additional features of cerebellar dysgenesis. RESULTS: Cerebellar heterotopias were identified in 27/35 (77%) patients with CHARGE, characteristic in both location and appearance. Additional features of cerebellar dysgenesis were present in 31/34 evaluable patients (91%), including inferior vermian hypoplasia (90%), anteromedial rotation of the inferior tonsils (90%), and disorganized foliation of the cerebellar hemispheres (74%) or superior vermis (16%). CONCLUSIONS: Patients with CHARGE syndrome have a high prevalence of characteristic cerebellar heterotopias and disorganized foliation and abnormal cerebellar morphology, thereby expanding the phenotype of cerebellar dysgenesis in this syndrome.

ß-Hydroxybutyrate Detection with Proton MR Spectroscopy in Children with Drug-Resistant Epilepsy on the Ketogenic Diet.

BACKGROUND AND PURPOSE: The ketogenic diet, including both classic and modified forms, is an alternative to antiepileptic medications used in the treatment of drug-resistant epilepsy. We sought to evaluate the utility of proton MR spectroscopy for the detection of ß-hydroxybutyrate in a cohort of children with epilepsy treated with the ketogenic diet and to correlate brain parenchymal metabolite ratios obtained from spectroscopy with ß-hydroxybutyrate serum concentrations. MATERIALS AND METHODS: Twenty-three spectroscopic datasets acquired at a TE of 288 ms in children on the ketogenic diet were analyzed with LCModel using a modified basis set that included a simulated ß-hydroxybutyrate resonance. Brain parenchymal metabolite ratios were calculated. Metabolite ratios were compared with serum ß-hydroxybutyrate concentrations, and partial correlation coefficients were calculated using patient age as a covariate. RESULTS: ß-hydroxybutyrate blood levels were highly correlated to brain ß-hydroxybutyrate levels, referenced as either choline, creatine, or N-acetylaspartate. They were inversely but more weakly associated with N-acetylaspartate, regardless of the ratio denominator. No strong concordance with lactate was demonstrated. CONCLUSIONS: Clinical MR spectroscopy in pediatric patients on the ketogenic diet demonstrated measurable ß-hydroxybutyrate, with a strong correlation to ß-hydroxybutyrate blood levels. These findings may serve as an effective tool for noninvasive monitoring of ketosis in this population. An inverse correlation between serum ß-hydroxybutyrate levels and brain tissue N-acetylaspartate suggests that altered amino acid handling contributes to the antiepileptogenic effect of the ketogenic diet.

Asymmetric Meckel Cave Enlargement: A Potential Marker of PHACES Syndrome.

BACKGROUND AND PURPOSE: PHACES syndrome is a complex of morphologic abnormalities of unknown cause and includes posterior fossa abnormalities; head and neck infantile hemangiomas; arterial, cardiac, and eye anomalies; and sternal or abdominal wall defects. Accurate identification of the syndrome is important for optimal treatment. The purpose of this study was to investigate the incidence of asymmetric Meckel cave enlargement, a potential novel imaging marker, in a population of patients referred for evaluation of possible PHACES syndrome. MATERIALS AND METHODS: Eighty-five patients referred for neuroimaging evaluation of possible PHACES syndrome were identified and stratified on the basis of their ultimate clinical PHACES diagnosis categorization into PHACES, possible PHACES, or not PHACES. MR imaging studies were subsequently reviewed for the presence or absence of unilateral Meckel cave enlargement, with the reviewer blinded to the ultimate PHACES syndrome categorization. RESULTS: Twenty-five of 85 patients (29%) were ultimately categorized as having PHACES or possible PHACES according to consensus guidelines. Asymmetric Meckel cave enlargement was present in 76% (19/25) of these patients and in 82% (19/23) of only those patients with definite PHACES. This finding was present in none of the 60 patients determined not to have PHACES syndrome. In 7/19 patients (37%) with this finding, subtle MR imaging abnormalities consistent with PHACES were missed on the initial MR imaging interpretation. CONCLUSIONS: Asymmetric Meckel cave enlargement was a common feature of patients with PHACES in our cohort and may serve as a novel imaging marker. Increased awareness of this imaging feature has the potential to increase the diagnostic accuracy of PHACES.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Šresolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies.

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of alpha-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His(6) tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured alpha-helix as predicted from the sequence.

Crystallization and preliminary X-ray diffraction analysis of L-threonine dehydrogenase (TDH) from the hyperthermophilic archaeon Thermococcus kodakaraensis.

The enzyme L-threonine dehydrogenase catalyses the NAD(+)-dependent conversion of L-threonine to 2-amino-3-ketobutyrate, which is the first reaction of a two-step biochemical pathway involved in the metabolism of threonine to glycine. Here, the crystallization and preliminary crystallographic analysis of L-threonine dehydrogenase (Tk-TDH) from the hyperthermophilic organism Thermococcus kodakaraensis KOD1 is reported. This threonine dehydrogenase consists of 350 amino acids, with a molecular weight of 38 kDa, and was prepared using an Escherichia coli expression system. The purified native protein was crystallized using the hanging-drop vapour-diffusion method and crystals grew in the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 124.5, c = 271.1 A. Diffraction data were collected to 2.6 A resolution and preliminary analysis indicates that there are four molecules in the asymmetric unit of the crystal.

Crystallization and preliminary X-ray diffraction analysis of calexcitin from Loligo pealei: a neuronal protein implicated in learning and memory.

The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 A suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 A resolution and yields data of high quality using synchrotron radiation.

The impact of aromatase mechanism on other P450s.

Experimental findings from a number of laboratories have converged to show that the conversion of androgens into oestrogen, catalysed by aromatase, involves three distinct reactions which occur at a single active site. That each one of these reactions belongs to a different generic type was revealed by chemical consideration, together with our (18)O-experiments. In particular, these findings highlighted the fact that the third reaction in the sequence occurs by a novel process for which a number of plausible mechanisms have been considered. The scrutiny of these mechanisms has involved either studies on aromatase itself, or on related enzymes which catalyse the aromatase type of cleavage reaction as generalized in equation 1: [equation: see text]. The acyl-carbon cleavage reaction of equation 1 is catalysed by sterol 14alpha-demethylases, accounts for several side-chain fission products formed by CYP17 (17alpha-hydroxylase-17,20-lyase), and constitutes a weak property of certain drug metabolizing P450s, when given aliphatic aldehydes as substrates. From cumulative studies on these enzymes, consensus is beginning to emerge that the acyl-carbon fission may be promoted by the FeIII-OOH intermediate, formed during the catalytic cycles of P450s. The precedent for the direct involvement of the FeIII-OOH species in the reaction of equation 1 is influencing our thinking regarding the mechanism of the conventional hydroxylation reaction. The status of knowledge surrounding the current debate on these issues will be reviewed.

Interaction of human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5.

Human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1-98, followed by a membrane insertable C-terminal tail, residues 99-133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (core-tail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signal-core) and the latter containing the C-terminal tail of the native rat protein (signal-core-tail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and core-tail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signal-core-tail was 55% as efficient. The core and signal-core constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.

The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14 alpha-demethylase of Candida albicans (other names are: lanosterol 14 alpha-demethylase, P-45014DM, and CYP51).

The Candida albicans sterol 14 alpha-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 mu mol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14 alpha-demethylase, 3 beta-hydroxylanost-7-en-32-al and 3 beta-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3 beta-hydroxylanost-7-en-32-al with the recombinant 14 alpha-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17 alpha-hydroxylase-17,20-lyase (P-45017 alpha,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14 alpha-demethylase is considered.

Mechanistic kinship between hydroxylation and desaturation reactions: acyl-carbon bond cleavage promoted by pig and human CYP17 (P-450(17)alpha; 17 alpha-hydroxylase-17,20-lyase).

Using homogeneous pig and recombinant human CYP17, the mechanism of the acyl-carbon bond fission involved in the direct cleavage of pregnenolone was studied. It was found that the formation of androsta-5,16-dien-3 beta-ol (5,16-diene) and androst-5-ene-3 beta,17 alpha-diol (17 alpha-hydroxyandrogen) from pregnenolone was catalyzed by both the isoforms and that the two conversions were dependent on the presence of cytochrome b5 (cyt b5). 3 beta-Hydroxyandrost-5-ene-17 beta-carbaldehyde (aldehyde), an analogue of the physiological substrate pregnenolone, was handled as a substrate by both isoforms of CYP17. The aldehyde underwent cleavage to produce the 5,16-diene plus the 17 alpha-hydroxyandrogen, at rates approximately 8- and 3-fold higher than any physiological reaction catalyzed, in the absence of cytochrome b5, by the pig and human CYP17 isoforms, respectively. The stereochemistry of the reaction was studied using the aldehyde labeled with 2H at three strategic positions, 16 alpha, 16 beta, and 17 alpha, with incubations performed under both 16O2 and 18O2. The results showed that the formation of the 5,16-diene is attended by the removal of the 16 alpha-hydrogen atom; all three 2H atoms are retained in the formation of 17 alpha-hydroxyandrogen and its 17 alpha-hydroxyl oxygen originates from O2. Irrespective of the nature of the substrate, or the enzymic conditions used, the 5,16-diene and 17 alpha-hydroxyandrogen were produced in similar ratios, suggesting that their genesis is closely linked. Both the compounds may be envisaged to arise from a peroxy adduct that fragments to give a carbon radical that then undergoes either a disproportionation or an oxygen-rebound reaction. The conclusion was supported by isotope-partitioning experiments when the conversion of a mixture of the unlabeled aldehyde and its isotopomer, containing 2H at 16 alpha as well as 16 beta, led to the enrichment of 2H in 17 alpha-hydroxyandrogen. It is suggested that the mechanistic kinship between hydroxylation and olefin formation, revealed by the present study, also applies to conventional hydroxylation and desaturation reactions.

Modulation of the activity of human 17 alpha-hydroxylase-17,20-lyase (CYP17) by cytochrome b5: endocrinological and mechanistic implications.

Using NADPH-cytochrome P-450 reductase as electron donor the homogeneous pig 17 alpha-hydroxylase-17,20-lyase (CYP17) was shown to catalyse the conversion of delta 5, as well as delta 4, steroids (pregnenolone and progesterone respectively) predominantly into the corresponding 17 alpha-hydroxylated products. The latter were then cleaved by the lyase (desmolase) activity of the enzyme into androgens. Cytochrome b5 stimulated both these activities, but its most noticeable effect was on the formation of delta 16-steroids, which compulsorily required the presence of cytochrome b5. These results on the pig enzyme confirm the original findings [Nakajin, Takahashi, Shinoda and Hall (1985) Biochem. Biophys. Res. Commun. 132, 708-713]. The human CYP17 expressed in Escherichia coli [Imai, Globerman, Gertner, Kagawa and Waterman (1993) J. Biol. Chem. 268, 19681-19689] was also purified to homogeneity and was found to catalyse the hydroxylation of pregnenolone and progesterone without requiring cytochrome b5. Like the pig CYP17, the human CYP17 also catalysed the cytochrome b5-dependent direct cleavage of pregnenolone into the delta 5,16-steroid, but unlike it the human enzyme did not cleave progesterone at all. 17 alpha-Hydroxypregnenolone was, however, cleaved into the corresponding androgen but only in the presence of cytochrome b5. 17 alpha-Hydroxyprogesterone was a poor substrate for the human CYP17; although it was converted into androstenedione in the presence of cytochrome b5 its K(m) was 5 times higher and Vmax. 2.6 times lower than those for the hydroxylation of progesterone. The endocrinological and mechanistic implications of these results are discussed.

Mechanism of the acyl-carbon cleavage and related reactions catalyzed by multifunctional P-450s: studies on cytochrome P-450(17)alpha.

It is now well-known that conventional cytochrome P-450s catalyze hydroxylation reactions using an iron mono-oxygen species, the structure of which, as inferred from chemical model studies, may be drrepresented by the following canonical forms: FeV==O<-->(.+)FeIV==O<-->FeIV--O(.). Certain multifunctional P-450s, notably those involved in steroid biosynthesis, catalyze, in addition to hydroxylation reactions, an acyl-carbon cleavage process in which the participation of an iron peroxide intermediate, FeIII--OOH, has been suggested. However the possibility still exists that the C--C bond cleavage may also occur using the FeV==O species. We have scrutinized the chemical consequences of involving either an FeV==O or an FeIII--OOH species for five different C--C bond cleavage reactions. With respect to the status as well as the origin of hydrogen and oxygen atoms, in four of the examples the mechanism involving the FeV==O species makes the same prediction as that using the iron peroxide intermediate, that is, the incorporation of an atom of oxygen from O2 into acyl part of the cleaved fragment. The fifth example, however, involving the formation, with pig testes microsomes, of 17 alpha-hydroxyandrogen (androst-5-ene-3 beta,17 alpha-diol) from pregnenolone, presents an interesting contrast--in this case different outcomes are predicted by the two mechanisms. These possibilities have been experimentally evaluated using substrates stereo- and regiospecifically labeled with heavy isotopes and incubated with pig testes microsomes under either 16O2 or 18O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanistic studies on aromatase and related C-C bond cleaving P-450 enzymes.

Some P-450 systems, notably aromatase and 14 alpha-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C-C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17 alpha-hydroxylase-17,20-lyase that participates in hydroxylation as well as C-C bond cleavage process. The C-C bond cleavage reactions catalysed by the above enzymes conform to the general equation: [formula: see text] It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed FeIII-O-O. species is converted into FeIII-O-OH and the heterolysis of the oxygen-oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example FeV = O<==>(+.)FeIV = O<==>FeIV-O.. One of these, FeIV-O. behaves like an alkoxyl radical and participates in hydrogen abstraction from C-H bond to produce FeIV-OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from FeIV-OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C-C bond cleavage reaction occurs through the participation of the FeIII-O-OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl-carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views, "The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a "running" commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive "running" commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners".

The general medical record. Concepts and suggestions for implementation.

Our view of a general medical record consists of a combination of distinct departmental- and specialty-specific medical records and an organizing kernel that contains arguably critical information. Because this system allows each clinical entity to evolve its own system, clinical priorities do not have to be negotiated or compromised. Additionally, subsystem or departmental medical records can be easily revised without disturbing the general medical record because of the modular design. Although the system seems robust with respect to design considerations, only implementation can provide adequate tests.

Human factors in rural road crashes.

An in-depth study of 79 vehicle crashes on rural roads in an area of about 100 km radius around Adelaide examined sociodemographic and psychophysiological characteristics of the drivers and riders involved. In many respects this sample of crashes was similar to a much larger number of police-reported crashes in the same area but included: relatively more crashes with severe or fatal injuries; more crashes on divided roads, on sealed roads and on curves; and more crashes involving trucks. Alcohol and lack of seat belt use were shown to be major problems in these rural crashes. The drivers and riders most strongly associated with these particular problems were males, in blue collar occupations and with limited education; they tended to be aged 30 years or more in the case of alcohol abuse, and were likely to be under 30 years in the case of restraint misuse. The attitudes of these drivers and riders, and other characteristics likely to have contributed to their involvement in a crash, are discussed. There is a need to develop specific and effective countermeasures to reduce drink-driving and increase seat belt wearing in rural areas.