A tale of two unconventional adult diaphragmatic hernias.

Diaphragmatic hernias can be congenital or acquired and are a protrusion of intra-abdominal contents through an abnormal opening in the diaphragm. Acquired defects are rare and occur secondary to direct penetrating injury or blunt abdominal trauma. This case review demonstrates two unconventional cases of large diaphragmatic hernias with viscero-abdominal disproportion in adults. Case 1 is a 27-year-old man with no prior medical or surgical history. He presented following a 24-h history of increasing shortness of breath and left-sided pleuritic chest pain, and no history of trauma. Chest X-ray demonstrated loops of bowel within the left hemithorax with displacement of the mediastinum to the right. Computed tomography (CT) scan confirmed a large diaphragmatic defect causing herniation of most of his abdominal contents into the left hemithorax. He underwent emergency surgery, which confirmed the viscero-abdominal disproportion. He required an extended right hemicolectomy to reduce the volume of the abdominal comtents and laparostomy to reduce the risk of abdominal compartment syndrome and recurrence of the hernia. Case 2 is a 76-year-old man with significant medical comorbidities who presented with acute onset of abdominal pain. He had a history of traumatic right-sided chest injury as a child resulting in right-sided diaphragmatic paralysis. Chest X-ray demonstrated a large right-sided diaphragmatic hernia with abdominal viscera in the right thoracic cavity. CT scan of the chest, abdomen and pelvis demonstrated both small and large bowel loops within the right hemithorax, compression of the right lung and displacement of the mediastinum to the left. The CT scan also demonstarted viscero-abdominal disproportion. Operative management was considered initially but following improvement with basic medical management and no further deterioration, a non-operative approach was adopted. Both cases illustrate atypical presentations of adults with diaphragmatic hernias. In an ideal scenario, these are repaired surgically. When the presumed diagnosis shows characteristics of a viscero-abdominal disproportion and surgery is pursued, the surgeon must consider that primary abdominal closure may not be possible and multiple operations may be necessary to correct the defect and achieve closure. Sacrifice of abdominal viscera may also be necessary to reduce the volume of abdominal contents.

All-Terrain Vehicle(ATV) Injuries - An Institutional Review Over 6 Years.

OBJECTIVE: Quad bike or all-terrain vehicle (ATV) related injuries are a significant cause of trauma and may present with severe or fatal injuries. Most of the literature describing ATV related injuries come from North America and Australasia and data from the United Kingdom is scarce despite a high prevalence of ATV use. The aim of this study was to describe our single centre experience with ATV injuries over a 6-year period from 2010 to 2015. MATERIALS AND METHODS: This is a cohort analysis of 65 patients who presented with ATV related injuries in South West Acute Hospital, UK between 2010 and 2015. RESULTS: 65 patients had ATV injuries. 34 (52%) patients were children between 0 - 17 years of age. 88% (n=57) patients were ejected from the ATV, six got trapped underneath and two had collisions. "Ejection" as a mechanism of injury was significantly more common than the other mechanisms (p<0.0001). Compliance with helmet use was low at 16% (n=10). Extremity (48%) and head and face trauma (43%) were the most common injuries. One (1.5%) patient died while 3 (4.6%) patients had major morbidity. CONCLUSION: ATV injuries are an important cause of trauma admissions and carry a significant risk of morbidity and mortality. Extremity and head trauma are the most common injuries resulting from ATV accidents. More than 50% of the injured are children. Compliance with helmet use is low and calls for legislation and public awareness strategies to reduce the impact of ATV accidents on health care.

Short communication: A printed image quality test phantom for mammography.

This communication describes a novel design for a mammographic image quality test phantom, the final design of which was produced as a radiographer weekly quality assurance phantom for breast screening and symptomatic mammography. The phantom is based on low contrast test features which are built up by superimposing sheets of Mylar overhead projector transparency, on which the test features are printed using a standard LaserJet printer. The required radiation contrast at mammographic energies is produced by the approximately 50% by weight component of iron oxide (Fe(3)O(4)) present in the toner. An easily replicated design of mammographic image quality phantom based on LaserJet printed test features is described. Approximately 40 of these phantoms were constructed, and these have been used successfully for 5 years in both breast screening and symptomatic mammography. The phantom design offers a performance similar to much more expensive mammographic contrast-detail phantoms, but suffers from the disadvantage that high contrast resolution bar patterns cannot be produced using the standard printing process.

Clinical aspects of a phase I trial of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent.

The antitumour action of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is mediated through tumour-selective antivascular effects and cytokine induction. This clinical phase I trial was conducted to examine its toxicity, maximum tolerated dose, pharmacokinetics (PK) and pharmacodynamics (PD). A secondary objective was to assess its antitumour efficacy. DMXAA was administered every 3 weeks as a 20-min i.v. infusion. Dose escalation initially followed a modified Fibonacci schema but was also guided by PK and toxicity. A total of 63 patients received 161 courses of DMXAA over 19 dose levels ranging from 6 to 4900 mg m(-2). DMXAA was well tolerated at lower doses and no drug-related myelosuppression was seen. Rapidly reversible dose-limiting toxicities were observed at 4900 mg m(-2), including confusion, tremor, slurred speech, visual disturbance, anxiety, urinary incontinence and possible left ventricular failure. Transient prolongation of the corrected cardiac QT interval was seen in 13 patients evaluated at doses of 2000 mg m(-2) and above. A patient with metastatic cervical carcinoma achieved an unconfirmed partial response at 1100 mg m(-2), progressing after eight courses. The results of PK and PD studies are reported separately. DMXAA has antitumour activity at well-tolerated doses.

The Legs For Life Screening for Peripheral Vascular Disease: results of a prospective study designed to improve patient compliance with physician recommendations.

PURPOSE: To determine how compliance with recommendations made by physicians during the 2000 Legs For Life National Screening for Peripheral Vascular Disease (PVD) and Leg Pain is affected through the use of (i) simple and concise patient information and recommendation cards and (ii) a "targeted" postscreening follow-up plan. MATERIALS AND METHODS: Patients were initially screened for PVD by completion of the Legs For Life Risk Factor Assessment form and determination of bilateral ankle/brachial indexes (ABIs). Each patient then met with an interventional radiologist or vascular surgeon. Patients with normal ABIs (>1.0 bilaterally) or mildly abnormal ABIs (<1.0 but >0.90) were classified as having no risk and low risk, respectively. Patients with ABIs of 0.70-0.89 were classified as having moderate risk for PVD and patients with ABIs <0.69 were classified as having high risk for PVD. Physicians reviewed the Risk Factor Assessment form with each patient and made specific lifestyle improvement recommendations. For the year 2000 screening, patients classified at moderate and high risk for PVD received special instructions and a card containing clearly printed information on the purpose of the Legs For Life screening, their level of risk for PVD, specific recommendations for follow-up, and phone numbers to call to help arrange for that follow-up. Two weeks after the screening, a second copy of this card was mailed to each moderate- and high-risk assessed patient. Four months later, each of these patients was contacted by telephone to determine if they had pursued additional care or testing. RESULTS: A total of 185 patients were screened, 42 (23%) of whom were determined to be at moderate or high risk for PVD. Four months after the screening, 39 (93%) of these patients were available for follow-up. Twenty (51%) patients had received no further medical advice or treatment. Nineteen (49%) patients had pursued further medical care which included physician consultation (n = 19; 100%), noninvasive Doppler evaluation (n = 10; 26%), diagnostic arteriography (n = 2; 5%), initiation of pharmacologic therapy for claudication (n = 1; 3%), percutaneous intervention (n = 1; 3%), or vascular surgery (n = 1; 3%). Seventeen of 39 patients (44%) reported that claudication-type leg pain was still a concern and/or lifestyle-limiting problem. CONCLUSION: Patients can be provided with problem-focused information and succinct physician recommendations at and soon after a screening for PVD, which can contribute to enhanced patient compliance. However, a host of personal, social, health, and physician-related issues still prevent a large percentage of patients from achieving relief of PVD-associated leg pain.

Effectors of HIV-1 protease peptidolytic activity.

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.

Treatment of hemodialysis catheter-associated fibrin sheaths by rt-PA infusion: critical analysis of 124 procedures.

PURPOSE: To prospectively evaluate the efficacy of a low-dose, 3-hour infusion of recombinant tissue plasminogen activator (rt-PA) for the treatment of hemodialysis catheter (HDC)-associated fibrin sheaths. This report expands the authors' experience with this technique over that previously reported. MATERIALS AND METHODS: Fifty-five patients with end-stage renal disease (38 women, 17 men) undergoing catheter-directed hemodialysis treatment were evaluated for 124 episodes of HDC dysfunction. This patient group had a mean age of 57 years and an age range of 23-92 years. Radiographic contrast studies and/or clinical evaluation were consistent with the presence of a fibrin sheath on the arterial and/or venous port in all cases. Each patient underwent a thrombolytic infusion consisting of 2.5 mg rt-PA in 50 mL normal saline at 17 mL/h (3-hour infusion) per port. All infusions were performed in the interventional radiology recovery room on an outpatient basis. Patients were followed prospectively for technical success, complications, catheter patency, and long-term outcome. RESULTS: The technical success rate, defined as return of effortless manual aspiration and infusion capability from both ports followed by at least one successful dialysis session, was 91%. No patient was excluded from rt-PA therapy because of contraindications, and the procedure-related complication rate was zero percent. A Kaplan-Meier survival analysis yielded primary patency rates at 30, 60, 90, and 120 days of 0.55, 0.36, 0.25, and 0.15 (SE <.10), respectively; secondary patency rates at 60, 120, 180, and 240 days were 0.70, 0.46, 0.30, and 0.27 (SE <.10), respectively (P < 001). At the end of the study period, all 52 surviving patients continued to undergo catheter-directed hemodialysis and 34 (65%) were using the same catheter present at the time of entrance into the study. Of the 18 patients (35%) requiring catheter exchange, 16 (89%) did for persistent malfunction after rt-PA therapy, one (5.5%) for infection, and one (5.5%) for a fractured hub. CONCLUSION: Thrombolytic therapy with use of a 2.5-mg rt-PA infusion through each port over a 3-hour period would appear to be a safe method for treating HDC-associated fibrin sheaths. Immediate return of catheter function is achieved in most patients, obviating more invasive techniques. Primary patency rates are relatively short, but catheters that fail can be retreated, resulting in secondary patency rates that are substantial and significantly improved.

The Legs for Life screening for peripheral vascular disease: compliance with physician recommendations in moderate- and high-risk assessed patients.

PURPOSE: To determine compliance within a community with recommendations made by physicians during the 1999 Legs for Life National Screening for Peripheral Vascular Disease (PVD) and Leg Pain. MATERIALS AND METHODS: Patients were initially screened for PVD by completion of the Legs for Life risk factor questionnaire and determination of bilateral ankle/brachial indexes (ABIs). Each patient subsequently met with an interventional radiologist or vascular surgeon. Patients with normal ABIs (>1.0 bilaterally) or mildly abnormal ABI(s) (<1.0 but >0.90) were classified at no and low risk for PVD, respectively; patients with ABI(s) of 0.70-0.89 were classified at moderate risk for PVD; and patients with ABI(s) <0.69 were classified at high risk for PVD. Risk factors for PVD were assessed by the consulting physician and discussed with all patients. Recommendations were made for additional evaluation and/or follow-up care, if necessary. Seven months after screening, patients who were assessed at moderate and high risk for PVD were contacted by telephone to determine if they had pursued additional care or testing. RESULTS: A total of 205 patients were screened for PVD, 48 (23%) of whom were determined to be at moderate to high risk. Forty-four (92%) patients were available for follow-up. At 7 months after screening, 31 (70%) patients had received no further medical advice or treatment. Thirteen (30%) of these patients had completed a follow-up appointment, but only three with a physician specializing in peripheral vascular disease. None of the patients had clinical follow-up with an interventional radiologist. Five (11%) patients had undergone noninvasive Doppler evaluation and one (2%) had undergone diagnostic arteriography. No patient had undergone any form of percutaneous or surgical intervention. CONCLUSION: Patient compliance with physician recommendations after outpatient screening for PVD is low. The Legs for Life screening program could be considered successful in that it provides for patient education and the identification of moderate to high-risk patients. Physicians participating in this program may have to modify their approach to patient screening and follow-up if a concomitant goal is to deliver specialty care.

Hemodialysis catheter-associated fibrin sheaths: treatment with a low-dose rt-PA infusion.

PURPOSE: To prospectively evaluate the efficacy of a low-dose, 3-hour recombinant tissue plasminogen activator (rt-PA) infusion for the treatment of hemodialysis catheter (HDC)-associated fibrin sheaths. MATERIALS AND METHODS: Seventeen patients with end-stage renal disease (female, n = 11; male, n = 6), who were undergoing catheter-directed hemodialysis, were evaluated for 28 episodes of HDC dysfunction. This patient group ranged in age from 25 to 92 years (mean, 57 years). Radiographic contrast and/or clinical evaluation were consistent with the presence of a fibrin sheath on either the arterial and/or venous port in all cases. Patients subsequently underwent a thrombolytic infusion consisting of 2.5 mg rt-PA in 50 mL normal saline at a rate of 17 mL/h (3-hour infusion) per port. All infusions were performed in the interventional radiology recovery room, on an outpatient basis. Patients were followed-up prospectively for technical success, complications, catheter patency, and long-term outcome. RESULTS: The immediate technical success rate, defined as return of manual aspiration and infusion capabilities to both ports, was 100%. No potential patients required exclusion from thrombolytic therapy secondary to contraindications, and no procedure-related complications occurred. The arithmetic mean and median catheter patency at the end of the study was 41 and 25 days, respectively (range, 1-116 days). A Kaplan-Meier survival analysis yielded a 30-, 60-, and 90-day probability of patency of 0.67, 0.61, and 0.51, respectively. At the end of the study period, all 17 patients remained on catheter-directed hemodialysis and 13 (76%) were utilizing the same catheter present at the time of entrance into the study. CONCLUSION: Thrombolytic therapy utilizing a 2.5-mg rt-PA infusion through each port during a 3-hour period would appear to be a safe, efficient method for treating HDC-associated fibrin sheaths. Three-month patency rates are comparable to those reported for other methods of restoring function to HDC catheters, including new catheter placement, catheter exchange over a guide wire, thrombolytic infusions with urokinase, and percutaneous fibrin sheath stripping.

Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation.

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] >> [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.

Active site generation of a protonically unstable suicide substrate from a stable precursor: glucose oxidase and dibromonitromethane.

Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH(3)CCl=NO(2)(-) and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH(0)(+), pK(a) approximately 3.5) and substrate (CHBr=NO(2)(-), pK(a) approximately 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO(2)(-) is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr(2)NO(2), thereby generating the desired protonically unstable suicide substrate in situ (E(r) + CHBr(2)NO(2) --> E(o) + CHBr=NO(2)(-) + HBr + H(+)). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO(2)(-) is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO(2)(-) at the active site are protonation and release or oxidation to HCOBr (or HCONO(2)). Strong support for these conclusions comes from (1) the brisk evolution of CH(3)CBr=NO(2)(-) (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH(3)CBr(2)NO(2), (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1, 5-dihydro-FAD.

Strand-separating activity of hepatitis C virus helicase in the absence of ATP.

HCV helicase [E(wt)] catalyzed strand separation of a short DNA duplex (F21:HF31) formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a 3'-fluorescein-tagged 21-mer (F21) complementary to the 5'-end of HF31. Strand separation was monitored by the fluorescence increase associated with the formation of F21 from F21:HF31. In the presence of ATP, the strand-separating activity was catalytic. In the absence of ATP and with E(wt) concentrations greater than that of F21:HF31, a biphasic fluorescence increase was observed at 25 degrees C. The late phase of this reaction was assigned to the separation of F21 from F21:HF31. The ATP-independent strand-separating reaction occurred more rapidly in the absence of Mg(2+) than in its presence. This result correlated with a lower T(m) value of F21:HF31 in the absence of 3.5 mM Mg(2+) than in its presence (45 vs 63 degrees C). The stoichiometry for the strand-separating reaction in the absence of ATP was 8 mol of E(wt) per mole of F21:HF31 separated into single-stranded F21 and HF31. The dissociation constants of HCV helicase for F21, HF31, and F21:HF31 in the absence of Mg(2+) were 0.6 +/- 0.4, 6 +/- 1, and 7.3 +/- 0.9 nM, respectively. Histidinyl-tagged E(wt) [hE(wt)] and a mutant enzyme [hE(V432A)] were prepared. hE(wt) and E(wt) bound F21 and HF31 with similar affinities and had similar ATP-dependent helicase activities, whereas hE(V432A) bound F21 and HF31 with affinities similar to that of E(wt) but had greatly reduced ATP-dependent helicase activities. In contrast to E(wt) and hE(wt), hE(V432A) did not support the ATP-independent strand-separating reaction. Consequently, the ATP-independent strand-separating reaction was not only the result of the high affinity of the enzyme for single-stranded DNA. The enzyme preferentially used duplex DNA with a 3'-tail for the ATP-dependent helicase reaction. In contrast, the enzyme strand-separated blunt-ended, 5'-tailed, and 3'-tailed duplex DNA equally effectively in the ATP-independent strand-separating reaction.

Kinetic analysis of the effects of mutagenesis of W501 and V432 of the hepatitis C virus NS3 helicase domain on ATPase and strand-separating activity.

Two hydrophobic residues, W501 and V432, in the nucleic acid (NA) binding pocket of the HCV helicase domain (E) were mutagenized in an effort to investigate contributions of these residues to substrate affinities and to enzymatic activities. The affinities of wild-type [hE(wt)] and mutant enzymes [hE(W501F), hE(W501A), and hE(V432A)] for NA and ATP were determined by monitoring changes in the intrinsic protein fluorescence, in the fluorescence of fluorescently tagged nucleic acid, and in the enzymatic activity. The steady-state kinetic parameters of the mutant enzymes for ATP hydrolysis (at saturating concentrations of NA) were similar to those of hE(wt). hE(W501F), hE(W501A), and hE(V432A) had strand-separating activities that were 136%, 3.8%, and 3.1% of that of hE(wt). The processivities of hE(W501F), hE(W501A), and hE(V432A) were reduced relative to that of hE(wt). The reduced processivities of hE(W501F) and hE(W501A) were primarily due to an increase in the rate of dissociation of E. ATP from E.ATP.NA. The reduced processivity of hE(V432A) was primarily due to a reduction in the intrinsic forward rate constant for strand separation. This result suggested that V432 may constitute part of the forward "stepping" motor of E. hE(W501A) and hE(V432A) did not display a dominant negative phenotype in a steady-state helicase assay with hE(wt). hE(wt) stored in the presence of beta-mercaptoethanol was covalently modified at three cysteinyl residues. The biological significance of the potential reactivity of these cysteinyl residues on hE(wt) is unknown.

Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn(2+) holoenzyme are similar.

Recombinant Escherichia coli cytosine deaminase is purified as a mixture of Zn(2+) and Fe(2+) forms of the enzyme. Fe(2+) is removed readily by o-phenanthroline to yield apoenzyme (apoCDase) that contains <0.2 mol of Zn(2+)per mol of subunit. ApoCDase was efficiently reconstituted to Zn(2+)CDase by treatment with ZnCl(2). The interaction of cytosine with apoCDase and Zn(2+)CDase was investigated at pH 7.5 and 25 degrees C by monitoring changes in intrinsic protein fluorescence. The values for the kinetic data K(1), k(2), and k(3) for Zn(2+)CDase were 0.25 mM, 80 s(-1), and 38 s(-1), respectively. The value for k(-2) was statistically indistinguishable from zero. The analogous values for K(1), k(2), and k(-2), (k(3)=0) for apoCDase were 0.157 mM, 186 s(-1) and approximately 0.8 s(-1), respectively. The overall dissociation constant of apoCDase for cytosine was 0.00069 mM, whereas the K(m) of Zn(2+)CDase for cytosine was 0.20 mM. The pre-steady state phase of the reaction was associated with an absorbance increase at 280 nm that was attributed to solvent perturbation of the spectrum of cytosine or enzyme. Formation of the Fe(2+)CDase-cytosine complex was too rapid to monitor by these techniques.

Replication-associated activities of purified human papillomavirus type 11 E1 helicase.

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replication and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of difficulties in expressing and purifying the protein. Herein, we report a method for the overexpression of full-length, untagged E1 (73.5 kDa) in baculovirus-infected Trichoplusia ni insect cells and the purification to homogeneity using a two-step procedure. The purified protein is a nonspecific NTPase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations were made in the putative NTPase domain to verify that the activities observed were encoded by E1. Purified mutant D523N had negligible ATPase and helicase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Secondary structure determination by circular dichroism revealed a large percentage of alpha-helical structure consistent with secondary structure predictions. These data define a fundamental set of biochemical and kinetic parameters for HPV E1 which are a critical prerequisite to future mechanistic studies of the enzyme.

alpha-fluoro-beta-alanine: effects on the antitumor activity and toxicity of 5-fluorouracil.

We have shown previously that (R)-5-fluoro-5,6-dihydrouracil (FUraH(2)) attenuates the antitumor activity of 5-fluorouracil (FUra) in rats bearing advanced colorectal carcinoma. Presently, we found that alpha-fluoro-beta-alanine (FBAL), the predominant catabolite of FUra that is formed rapidly via FUraH(2), also decreased the antitumor activity and potentiated the toxicity of FUra. In rats treated with Eniluracil (5-ethynyluracil, GW776), excess FBAL, in a 9:1 ratio to FUra, produced similar effects when administered 1 hr before, simultaneously with, or 2 hr after FUra. FBAL also decreased the antitumor activity of FUra in Eniluracil-treated mice bearing MOPC-315 myeloma at a 9:1 ratio with FUra, but not at a 2:1 ratio. FBAL did not affect the antitumor activity of FUra in mice bearing Colon 38 tumors. We also evaluated the effect of thymidylate synthase (TS) and thymidine kinase (TK) from tumor extracts after FUra +/- Eniluracil +/- FBAL treatment. The activity of TK was similar among the three groups at both 18 and 120 hr. There was also no difference in TS inhibition ( approximately 35%) at 18 hr. However, significantly more TS inhibition was observed in the Eniluracil/FUra group than in the FUra-alone group at 120 hr. FBAL did not alter the effect of Eniluracil/FUra in TS inhibition. Neither FUraH(2) nor FBAL affected the IC(50) of FUra in culture. Thus, the effect of FBAL did not result from direct competition with FUra uptake or immediate anabolism. Either another downstream catabolite that is not formed in cell culture is the active agent, or the effect requires the complexity of a living organism or an established tumor.

Chromium: friend or foe?

Chromium is an essential nutrient with many natural sources, but several investigators have presented evidence that healthy persons often have an inadequate intake of this metal. Advocates of its use as a dietary supplement believe that people with diabetes, lipoprotein abnormalities, and increased risk of cardiovascular disease may benefit from such supplementation. Others suggest a therapeutic role for chromium in obese people and in those who seek to improve their body composition for other reasons, but research provides little support for these uses. For the general public, current data do not warrant routine use of chromium supplements, whose risk-benefit function has not yet been adequately characterized.

Phase I study of the cytotoxic agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA) is a new DNA-intercalating drug with a dual mode of cytotoxic action that is thought to involve topoisomerases I and II. On the basis of novelty of action and promising preclinical activity against solid tumours in mice, DACA was selected for clinical trial under the auspices of the Cancer Research Campaign, United Kingdom. We report the phase I findings of a 3-h infusion regimen, repeated 3-weekly, of escalating doses through 18-1000 mg/m2 given to 31 patients with solid malignancies. A maximum tolerated dose (MTD) of 750 mg/m2 was identified, with 3 of 6 cycles being abandoned at 1000 mg/m2. Dose-limiting toxicity took the form of infusional arm pain, in some cases associated with facial discomfort, that was of rapid onset and subsided quickly on the cessation of infusion. The mechanism is unclear but is modulated to some extent by the rate of drug delivery, and it was unaffected in this study by concurrent anti-inflammatory or opiate medication. No host or tumour anti-proliferative activity was observed at these doses, and only minimal toxicity of any other kind was evident. Animal data suggest that the MTD achieved with this schedule may be sub-therapeutic in humans. It is therefore important that efforts be continued to explore methods of giving higher doses of DACA.

The use of high density metal foils to increase surface dose in low-energy clinical electron beams.

BACKGROUND AND PURPOSE: This paper describes a practical method of elevating the surface dose of clinical electron beams in the energy range 3-12 MeV using thin high density metal foils (tin and lead) as an alternative to tissue equivalent bolus. Because, relative to water, these materials exhibit a high scattering power to stopping power ratio, the desired dose elevation may be achieved with less energy loss than conventional bolus and consequently a gain in therapeutic interval. METHODS: The foil thickness required to raise the surface dose to 90% off peak, for a given electron energy, was calculated using published scattering and stopping power data. An empirical expression is derived to facilitate calculation of foil thickness (tin or lead) to produce a given surface dose. RESULTS AND CONCLUSIONS: Measurements were made to confirm the predictions of the derived expression and were found to be in good agreement.

Expression and characterization of the HCV NS3 helicase domain.

The hepatitis C virus (HCV) NS3 protein has two distinct biochemical domains. The N-terminal 20 kDa has serine protease activity (see Chapter 31 ) and the C-terminal 50 kDa has both nucleoside triphosphatase (NTPase) and helicase activities (1-4).