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R J Cook

Publications and source records attributed to R J Cook.

At least 73 records · Page 4Linked to original sources
PubMed1995

Enzymatic activities of rat liver cytosol 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase (10-FTH-FDH: EC 1.5.1.6) catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate (H4PteGlu) and CO2 and the NADP(+)-independent hydrolytic cleavage of 10-HCO-H4PteGlu to H4PteGlu and formate. 10-FTHFDH has a 485 amino acid domain at the C-terminus which is 46% identical to aldehyde dehydrogenase (ALDH: EC 1.2.1.3) and contains a conserved active site cysteine (Cys-707). 10-FTHFDH catalyzed NADP(+)-dependent oxidation of propanal and the hydrolysis of p-nitrophenyl acetate (pNPA) in a similar fashion to ALDH. Initial rate studies gave Km values of 46 and 636 microM, respectively, for NADP+ and propanal, while pNPA had a Km of 220 microM. Propanal was able to compete with 10-HCO-H4PteGlu for NADP(+)-dependent oxidation but had no effect on the NADP(+)-independent hydrolase reaction. N-Ethylmaleimide inhibited NADP(+)-dependent 10-HCO-H4PteGlu oxidation but only partially inhibited (65%) hydrolase activity. Disulfiram, a potent inhibitor of cytosolic ALDH, inhibited NADP(+)-dependent propanal oxidation by 10-FTHFDH. We propose that the dehydrogenase reaction of 10-FTHFDH has a mechanism which proceeds through thiohemiacetal and thioester intermediates, similar to that described for aldehyde dehydrogenase. 10-FTHFDH hydrolase activity was dependent on 2-mercaptoethanol and is probably an artifact of the assay system. The N-terminal domain of 10-FTHFDH shows identity to glycinamide ribonucleotide transformylase (EC 2.1.2.2) and contains a putative 10-HCO-H4PteGlu binding site but shows no GAR-TF activity. NADP(+)-dependent oxidation of 10-HCO-H4PteGlu by 10-FTHFDH was inhibited by the folate anti-metabolite, 5,10-dideazatetrahydrofolate, a known GAR-TF inhibitor.

Aldehydes↗
PubMed1995

Recombinant 10-formyltetrahydrofolate dehydrogenase catalyses both dehydrogenase and hydrolase reactions utilizing the synthetic substrate 10-formyl-5,8-dideazafolate.

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) is a bifunctional enzyme, displaying both NADP(+)-dependent dehydrogenase activity for the formation of tetrahydrofolate and CO2, and NADP(+)-independent hydrolase activity for the formation of tetrahydrofolate and formate. A previous report [Case, Kaisaki and Steele (1988) J. Biol. Chem. 263, 1024-1027] claimed that dehydrogenase and hydrolase activities were products of separate cytosolic and mitochondrial forms of this enzyme. Here we report that recombinant 10-formyltetrahydrofolate dehydrogenase carries out both enzymic reactions, proving that a product of a single gene, i.e. one protein, not two, has both activities. The stable synthetic analogue 10-formyl-5,8-dideazafolate can substitute for the labile natural substrate, 10-formyltetrahydrofolate, in both reactions. This was shown with both native and recombinant rat liver enzyme. The Km values for 10-formyl-5,8-dideazafolate were half of those for 10-formyltetrahydrofolate in both the dehydrogenase and hydrolytic reactions. The Vmax, values were similar for both substrates. Both dehydrogenase and hydrolase reactions were dependent on the presence of 2-mercaptoethanol. The pH optima were 7.8 and 5.6 for the dehydrogenase and hydrolase reactions respectively, consistent with the presence of two active sites in the enzyme.

Enzyme Activation↗
PubMed1995

The number needed to treat: a clinically useful measure of treatment effect.

The relative benefit of an active treatment over a control is usually expressed as the relative risk, the relative risk reduction, or the odds ratio. These measures are used extensively in both clinical and epidemiological investigations. For clinical decision making, however, it is more meaningful to use the measure "number needed to treat." This measure is calculated on the inverse of the absolute risk reduction. It has the advantage that it conveys both statistical and clinical significance to the doctor. Furthermore, it can be used to extrapolate published findings to a patient at an arbitrary specified baseline risk when the relative risk reduction associated with treatment is constant for all levels of risk.

Cerebrovascular Disorders↗
PubMed1995

Cysteine 707 is involved in the dehydrogenase activity site of rat 10-formyltetrahydrofolate dehydrogenase.

The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD(+)-dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP+ were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity.

Animals↗
PubMed1995

10-Formyltetrahydrofolate dehydrogenase: identification of the natural folate ligand, covalent labeling, and partial tryptic digestion.

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) was previously identified as a folate-binding protein in rat liver cytosol (R.J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982) by virtue of the tetrahydrofolate polyglutamate tightly bound to the partially purified enzyme. In this current study we provide evidence to show that when liver cytosol was rapidly processed to identify the protein bound folate, large amounts of both 10-formyl- and 5-formyltetrahydrofolate were present. After overnight storage of the cytosol at 5 degrees C before processing, almost no formylfolates were present and the major protein-bound form was tetrahydrofolate. This suggests that 10-formyltetrahydrofolate polyglutamates are tightly bound to the enzyme in vivo and are converted to tetrahydrofolate forms during isolation by the hydrolase activity associated with the enzyme. Covalent binding of the stable folate analogue, 5-formyltetrahydrofolate, to the purified enzyme resulted in 2 mol bound per mole of enzyme subunit. This is consistent with earlier reports suggesting the enzyme is capable of carrying out both oxidative and hydrolytic conversion of 10-formyltetrahydrofolate to tetrahydrofolate at the same time. Partial tryptic digestion of the purified enzyme selectively inhibited dehydrogenase activity of the enzyme but did not affect the hydrolase or aldehyde dehydrogenase activities.

Animals↗
PubMed1995

Baculovirus expression and purification of rat 10-formyltetrahydrofolate dehydrogenase.

The liver cytosolic enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes two reactions: the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. It exists as a tetramer of 99-kDa subunits in rat liver. We expressed rat liver cDNA encoding 10-FTHFDH in insect cells using the pVL 1393 expression vector and MaxBac expression kit. Despite the absence of a leader peptide the recombinant 10-FTHFDH was released from the cells to the culture medium during production in both Sf9 and High five cell lines. The enzyme released into the medium was no less than 80% of the total recombinant 10-FTHFDH. Both enzyme pools, from the medium and from cell extracts, displayed high activity. The maximum expression of 10-FTHFDH was observed 72 h postinfection in High five cells and 96 h postinfection in Sf9 cells. High five cells revealed four times higher expression of the recombinant enzyme per milligram of total cell protein than Sf9 cells. Passage of the cell-free culture medium over an affinity column of 5-formyltetrahydrofolate-Sepharose provided 10-FTHFDH that was more than 95% pure. Additional purification on a Mono Q column resulted in an homogenous preparation of the enzyme. Purified recombinant 10-FTHFDH displayed both dehydrogenase and hydrolase activities, similar to those of the rat liver enzyme, and the recombinant enzyme remained active at least 12 months when stored appropriately. These results show that 10-FTHFDH can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified in two steps by simple chromatographic procedures.

Animals↗
PubMed1995

The influence of bur blade concentricity on high-speed tooth-cutting interactions: a video-rate confocal microscopic study.

This study aimed to determine the degree of eccentricity between different tungsten carbide bur manufacturing techniques and to study the effect of bur inaccuracy on dental enamel. Error in bur concentricity may arise from malalignment of the steel shaft and carbide head in a two-piece construction bur. Cutting blades rotate at multiple radii from the shaft axis, potentially producing vibrations and damage to the cut substrate. Techniques now allow for the manufacture of one-piece tungsten carbide burs with strength adequate to withstand lateral loading. A comparison of tungsten carbide dental cutting tools revealed the true extent of concentricity errors. Variation in alignment of the cutting head and shaft in the two-part constructions incurred between 20 and 50 microns of additional axial error. High-speed cutting interactions with dental enamel between carbide burs were studied by means of a video-rate confocal microscope. A cutting stage fitted to a Tandem Scanning Microscope (TSM) allowed for real-time dynamic image acquisition. Images were captured and retrieved by means of a low-light-level camera recording directly to S-VHS videotape. Videotape showing the interactions of high-speed rotary cutting instruments (at 120,000 rpm) were taken under simulated normal wet-cutting environments, and the consequent damage to the tooth tissue was observed as it occurred. Concentrically engineered bur types produced a superior quality cut surface at the entry, exit, and advancing front aspects of a cavity, as well as less subsurface cracking. Imaging of the coolant water film local to recent cutting operations showed regular spherical cutting debris of 6 to 18 microns diameter from the concentric tools, whereas the less-well-engineered burs produced ragged, irregular chips, with 25-40 microns diameter debris, indicating far more aggressive cutting actions. This study has shown that there is reduced substrate damage with high-concentricity carbide burs.

Dental Cavity Preparation↗
PubMed1995

Interim analyses in 2 x 2 crossover trials.

A method is presented for performing interim analyses in long term 2 x 2 crossover trials with serial patient entry. The analyses are based on a linear statistic that combines data from individuals observed for one treatment period with data from individuals observed for both periods. The coefficients in this linear combination can be chosen quite arbitrarily, but we focus on variance-based weights to maximize power for tests regarding direct treatment effects. The type I error rate of this procedure is controlled by utilizing the joint distribution of the linear statistics over analysis stages. Methods for performing power and sample size calculations are indicated. A two-stage sequential design involving simultaneous patient entry and a single between-period interim analysis is considered in detail. The power and average number of measurements required for this design are compared to those of the usual crossover trial. The results indicate that, while there is minimal loss in power relative to the usual crossover design in the absence of differential carry-over effects, the proposed design can have substantially greater power when differential carry-over effects are present. The two-stage crossover design can also lead to more economical studies in terms of the expected number of measurements required, due to the potential for early stopping. Attention is directed toward normally distributed responses.

Antiemetics↗
PubMed1994

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6). NH2-terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted.

Animals↗
PubMed1994

Interim monitoring of bivariate responses using repeated confidence intervals.

A method for interim monitoring is described for bivariate response data arising in clinical trials. This procedure centers on a sequence of rectangular joint confidence regions obtained by inverting the hypothesis testing-confidence interval relationship when the testing procedure is governed by a bivariate group sequential design described by Cook and Farewell [4]. The advantage of this type of design over other possible multivariate sequential designs is that the marginal analyses are maintained throughout the trial. This approach proves to be particularly advantageous for active control trials in which a region of therapeutic equivalence is specified for two responses related to efficacy. Two examples illustrate the design and analysis aspects of this approach. The procedure is presented in the context of normally distributed responses; however, adaptations to handle bivariate binary and bivariate failure time data are indicated. Generalizations of this procedure to handle multivariate response data are straightforward.

Administration, Inhalation↗
PubMed1994

Prolactinomas in men masquerading as invasive skull base tumours.

Four giant prolactin-secreting tumours invading the skull base are described. All of them occurred in men. The presenting features were sufficiently diverse to be clinically misleading. We advocate the estimation of prolactin levels before embarking on complicated skull base surgery in men where doubt remains regarding the diagnosis after clinical and radiological study.

Adolescent↗
PubMed1994

Guidelines for monitoring efficacy and toxicity responses in clinical trials.

There is currently a need for clinical trial methodology that allows formal consideration of toxicity responses. Since a complete evaluation of an experimental therapy addresses both relative efficacy and relative toxicity, general methods for handling bivariate response data are of interest. A procedure for sequentially analysing both efficacy and toxicity data is presented. The procedure is designed to allow early termination due to efficacy results, toxicity results, or both. The method is based on modified marginal sequential analyses, accounting for bivariate correlated responses and multiple analyses over time. The theory is presented in the context of normally distributed responses. Extensions to bivariate failure time data are indicated and an example from a kidney transplant study demonstrates the procedure.

Analysis of Variance↗
PubMed1993

Neurologic changes during carotid endarterectomy under cervical block predict a high risk of postoperative stroke.

BACKGROUND: This study was undertaken to confirm a previous report that patients having neurologic changes with carotid artery clamping were at greater risk of developing permanent postoperative neurologic complications after carotid endarterectomy. METHODS: Superficial and deep cervical plexus blocks were performed in 389 patients undergoing carotid endarterectomy. The patients were premedicated and sedated to a level that allowed awake neurologic assessment. Intraoperative neurologic changes were recorded and all patients were examined postoperatively by an independent anesthesiologist to record postoperative neurologic outcome. RESULTS: Trial carotid artery cross clamping resulted in 24% of patients having neurologic changes that usually responded to declamping and shunt insertion. Postoperative permanent neurologic complications occurred in 2.6% of patients, but were more common in patients who had neurologic changes associated with carotid artery cross clamping (6.6% compared to 1.1%, P < 0.01). Thrombosis of the carotid artery was the most common finding in patients who underwent reexploration of the carotid artery after developing postoperative neurologic changes. CONCLUSIONS: This study confirms that patients undergoing carotid endarterectomy under cervical plexus block who have intraoperative neurologic changes have a sixfold increase in the chance of developing a postoperative stroke. This high-risk group may benefit from antithrombotic therapies to improve their outcome.

Aged↗
PubMed1993

The Westmead Head Injury Project. Physical and social outcomes following severe head injury.

This study reports the physical outcomes of 181 survivors of severe head injury [Glasgow Coma Score (GCS) 8 or less] following aggressive head injury management which included early triage, evacuation and resuscitation, the use of ventilation in the Intensive Care Unit and intracranial pressure (ICP) measurement and control. At the conclusion of the study period of 2 years after the head injury, 71% had achieved a Glasgow Outcome Score (GOS) of 1, 17% GOS 2, 10% GOS 3 and 2% GOS 4. Physical recovery continued during the 2 years. Locomotor independence was achieved in 93% despite a motor disorder in 59%. About 90% were considered independent for basic life functions, e.g. feeding, bathing, dressing or toileting. Speech disorders were present in 19%. A return to an occupation, either a job or studies, at or below the pre-morbid level was achieved in 68%. Most patients were cared for at home by family or relatives. The majority of patients make a reasonable physical recovery following severe head injury with independence in locomotion and basic life skills. It was our impression that the neuropsychological sequelae of cognitive and behavioural disorders were a major impediment to re-assimilation into society.

Activities of Daily Living↗
PubMed1993

The Westmead Head Injury Project outcome in severe head injury. A comparative analysis of pre-hospital, clinical and CT variables.

A prospective study of 315 consecutive patients with a severe head injury was undertaken to study factors contributing to mortality and morbidity, both in the pre-hospital and hospital phases. Entry criteria were a Glasgow Coma Scale (GCS) score of 8 or less after non-surgical resuscitation within 6 h of the injury, or a deterioration to that level within 48 h. Patients with gunshot wounds or who were dead on arrival were excluded. End points of the study were either death or at 6 months after the injury. Predictors of mortality were increasing age, the presence of hypotension, a low GCS, abnormal motor responses and pupillary non-reactivity. In the 167 patients in whom intracranial pressure (ICP) was measured, raised ICP and failure to respond to treatment for raised ICP also predicted mortality. Three CT predictors of mortality were the presence of cerebral oedema, intraventricular blood and the degree of midline shift. When analysed using logistic regression, the most accurate model (accuracy 84.4%) included increasing age, abnormal motor responses and the three CT indicators. Analysis of the data for 'good' (Glasgow Outcome Score (GOS) 1 and 2) vs 'poor' (GOS 3 and 4) survival at 6 months was also performed using logistic regression. The model which provided the most accurate prediction of poor outcome included age, hypotension and three different CT characteristics, subarachnoid blood, intracerebral haematoma or intracerebral contusion (accuracy 72.5%).

Adolescent↗