A novel AKT3 mutation in melanoma tumours and cell lines.

Recently, a rare activating mutation of AKT1 (E17K) has been reported in breast, ovarian, and colorectal cancers. However, analogous activating mutations in AKT2 or AKT3 have not been identified in any cancer lineage. To determine the prevalence of AKT E17K mutations in melanoma, the most aggressive form of skin cancer, we analysed 137 human melanoma specimens and 65 human melanoma cell lines for the previously described activating mutation of AKT1, and for analogous mutations in AKT2 and AKT3. We identified a single AKT1 E17K mutation. Remarkably, a previously unidentified AKT3 E17K mutation was detected in two melanomas (from one patient) as well as two cell lines. The AKT3 E17K mutation results in activation of AKT when expressed in human melanoma cells. This represents the first report of AKT mutations in melanoma, and the initial identification of an AKT3 mutation in any human cancer lineage. We have also identified the first known human cell lines with naturally occurring AKT E17K mutations.

Audit of oxygen use in emergency ambulances and in a hospital emergency department.

BACKGROUND: Oxygen is widely used but poorly studied in emergency medicine, with a limited evidence base for its use in specific conditions. There are safety concerns about the underuse of oxygen in patients with critical illness and its overuse in conditions such as chronic obstructive pulmonary disease (COPD). A baseline audit was required to assess current practice prior to the introduction of new national emergency oxygen guidelines in late 2008. METHODS: The use of pulse oximetry and oxygen therapy was audited in patients brought by ambulance to the "majors" section of the emergency department (ED) in a university hospital. Oxygen therapy in the ambulance and the ED was subsequently documented. Oxygen use in ambulances was compared with Joint Royal Colleges Ambulance Liaison Committee (JRCALC) guidance and with subsequent patient management. RESULTS: The ambulance and ED records of 1022 patients were audited manually. Oxygen saturation (SpO(2)) was recorded for 90% of patients, 17% of whom had SpO(2) <94% at some time and 7% had SpO(2) <90%, including 33% of patients with COPD and 5.5% of patients without COPD. 34% of patients received oxygen in the ambulance and almost half of these had oxygen discontinued in the ED. Only 62% of ambulance oxygen use was in accordance with JRCALC guidance, but most "undertreated" patients were stable normoxaemic patients for whom guidance recommends high-flow oxygen. Only 58% of patients with COPD were correctly identified in the ambulance and 73% of these patients were treated with flow rates >4 l/min (equivalent to >35% oxygen). CONCLUSIONS: Oxygen use in ambulances is very common, equivalent to 2.2 million episodes annually in the UK. The quality of oxygen use is suboptimal, especially for patients with COPD. Emergency oxygen therapy will become simpler when new evidence-based UK emergency oxygen guidelines are published, and it is hoped that future audits will show better protocol adherence.

Phase I trial of weekly scheduling and pharmacokinetics of titanocene dichloride in patients with advanced cancer.

PURPOSE: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of a weekly schedule of titanocene dichloride (TD) and to define the pharmacokinetics of titanium in plasma and urine. PATIENTS AND METHODS: Twenty patients with a median age of 58 years received 83 courses of TD. TD was given as 1-hour infusion at escalating doses from 70 to 185 mg/m2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). RESULTS: At the fifth dose level (185 mg/m2/wk), a variety of DLTs were seen in five patients: fatigue in three, bilirubinemia in one, and hypokalemia in two. A further six patients were treated at 140 mg/m2; only one had dose-limiting creatinine elevation and this dose was therefore defined as the MTD. No myelosuppression or alopecia were observed. One patient with adenocarcinoma of unknown primary had a minor response. Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (t1/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a one-compartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% +/- 2.5% those of TPTi. CONCLUSION: The MTD of TD given on a weekly schedule is 140 mg/m2, with cumulative, but reversible creatinine and bilirubin elevation being the DLTs.

Two crystal forms of the extracellular domain of type I tumor necrosis factor receptor.

The soluble extracellular domain of human type I tumor necrosis factor receptor (sTNFrI) is a 161 residue polypeptide found in serum and urine. This domain tightly binds tumor necrosis factors (TNF) alpha and beta and, as part of the whole receptor, initiates the powerful biological effects of TNF. The extracellular domain, typical of other TNF receptor superfamily members, comprises four cysteine-rich motifs. We have obtained two crystal forms of the sTNFrI. One crystal form is grown at pH 3.7 with MgSO4 as the precipitant. These crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 78.5 A, b = 85.5 A and c = 67.5 A. A data set to 2.0 resolution has been collected for these crystals. Tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions a = 69.0 A and c = 185.5 A are obtained using methylpentanediol as precipitant at pH 8.5. Data to 2.8 A have been measured from these crystals. It appears that both unit cells may contain two molecules in the asymmetric unit. These crystal structures of sTNFrI may reveal possible conformational differences between receptor localized on the cell surface (high pH), the receptor in the endosomal compartments (low pH) and the receptor in a complex with tumor necrosis factor beta. An accurate structure of the receptor and an understanding of its mechanism will provide a basis for rational drug design.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein.

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(1-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/IGF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Treatment of end-stage chronic obstructive pulmonary disease with double lung transplantation.

Six patients with end-stage emphysema (age 44 +/- 2 years) underwent double lung transplantation (Tx) from June 1988 through May 1990. All suffered from severe inanition and required oxygen therapy. The ischemic time was 193 +/- 28 minutes. Post-Tx immune suppression was OKT3 (14 days), cyclosporine (trough levels of 150 +/- 25 ng/ml), azathioprine to keep WBC at 3,000 to 5,000/cu mm (1 to 3.0 mg/kg/day) and following OKT3, a tapering prednisone regimen. Two rejection episodes that occurred in two patients on post-Tx day 5 and 10 were treated with bolus doses of methylprednisolone. The mean hospital stay was 32 +/- 7 days (range, 20 to 69 days). Four patients required treatment of cytomegalovirus (CMV) infection: gastritis (+donor, +recipient) in one and CMV pneumonia in two (+donor, -recipient). A fourth (+donor, -recipient) had right-sided Candida empyema six weeks post-Tx, developed CMV and staphylococcal sepsis, and died 64 days post-Tx. One patient required pyloroplasty eight weeks post-Tx and one patient underwent tracheal suture line repair at eight weeks. During a follow-up of 81 patients months (range, 8 to 24 months), one patient had developed Epstein-Barr viral (EBV) induced lymphoproliferative disease in the lung and one patient had developed EBV lymphoma. Three patients are at work, one is continuing rehabilitation, and one is at home. Double lung Tx offers a definitive benefit to patients with emphysema; however, a prolonged postoperative course can be expected. Viral infections remain serious but treatable problems.

A short synthetic pathway to a fully-functionalized southern hemisphere of the antitumor macrolide bryostatin 1.

[reaction--see text] An 18-step asymmetric synthesis of the bryostatin 1 "southern hemisphere" fragment (1) has been developed. Key steps include an aldol reaction between 6 and 7 and a dehydration to establish the (E)-exocyclic alkene in 2 and a stereoselective Luche reduction and protection with TESOTf to access 1.

A synthetic strategy for the cyclodepsipeptide core of the antitumor antibiotic verucopeptin.

[reaction: see text]. An efficient [2 + 2 + 2]-fragment condensation strategy is described for obtaining the cyclodepsipeptide core of verucopeptin. The 19-membered macrocycle was established through a Carpino HATU mediated macrolactamization, which proceeded in good yield under high-dilution conditions.

Control of olefin geometry in the bryostatin B-ring through exploitation of a C(2)-symmetry breaking tactic and a Smith-Tietze coupling reaction.

A completely stereocontrolled asymmetric synthesis of an advanced B-ring synthon for the bryostatin family of antitumor agents is reported. Noteworthy features of our synthesis include the Smith-Tietze bis-alkylation reaction between 12 and 13 en route to C(2)-symmetrical ketone 10 and the totally stereoselective conversion of 10 into triol 18 via a Grignard addition tactic. Triol 18 was converted to epoxide 3 in nine steps, and an acid-catalyzed intramolecular Williamson etherification reaction completed the synthesis of 2.

Estimating antemortem drug concentrations from postmortem blood samples: the influence of postmortem redistribution.

AIMS: To compare blood drug concentrations during life with postmortem drug concentrations measured from a peripheral site and a central site. METHODS: Coroner's cases from October 1990 to July 1997 were reviewed. Six cases had data on both antemortem and postmortem blood drug concentrations. The postmortem to antemortem ratio was compared with the postmortem central to peripheral ratio, using cardiac blood as a central site and femoral blood as a peripheral site. RESULTS: Drugs that have a high postmortem central to peripheral ratio; that is, drugs that exhibit considerable postmortem redistribution, also have high postmortem to antemortem ratios. CONCLUSIONS: A large degree of error can arise from attempting to estimate antemortem drug concentrations and the ingested dose from postmortem measurements. The chosen site and technique for postmortem blood sampling can greatly influence the concentration of drug measured.

A microassay for urinary phenol using capillary gas chromatography and optimised enzymic hydrolysis.

An improved capillary gas chromatography method is described for the assay of urinary phenol for the assessment of benzene exposure. Enzymatic hydrolysis with an extract of Helix pomatia is used to liberate phenol from its glucuronide and sulphate conjugates in urine and has the advantage in that complete hydrolysis occurs and clean chromatograms are produced. Furthermore, an internal standard (2,4-xylenol) has been incorporated in the method enabling rapid and single step extraction of phenol. The microassay can be used to measure phenol concentrations as low as 1 mg/l in urine thus making it a suitable method for monitoring benzene exposure.

Variable-ratio schedules of timeout from avoidance: effects of anxiolytic drugs.

Concurrent performances in rats were studied under conditions where responses on one lever postponed shock on an unsignaled avoidance schedule, and responses on another level produced periods of signaled timeout from avoidance on a variable-ratio schedule. This procedure resulted in relatively high rates of responding on the timeout lever, and provided a baseline which permitted simultaneous evaluation of drug effects on two different types of negative reinforcement (shock postponement vs timeout). Chlordiazepoxide and ethanol selectively increased responding on the timeout lever at low doses, while higher doses decreased responding on both levers. Two 5-HT(1A) agonists, buspirone and 8-OH-DPAT, had different effects. Buspirone decreased responding across all effective doses, but 8-OH-DPAT increased responding on both the timeout and avoidance levers, with greater increases noted in responding maintained by timeout. These results replicate and extend previous findings, and support the notion that traditional anxiolytic drugs like chlordiazepoxide and ethanol may increase the reinforcing properties of escape from an avoidance schedule. Differences between the behavioral effects of buspirone and 8-OH-DPAT may reflect differential activity at the 5-HT(1A) receptor or the dopaminergic properties of buspirone.

Academic promotion and tenure in U.S. family medicine units.

The authors interviewed by telephone the heads (or their representatives) of 101 of the 120 family practice units in U.S. medical schools in 1987. Each respondent was asked for his or her personal perceptions of the relative importances of research, teaching, patient care, and administrative activities in the academic promotion process. Respondents were also asked for their views of their units' and institutions' perceptions of the importances of the same four activities in the promotion process, as well as other related questions about promotion and tenure. The findings indicate that there is still a significant incongruence between the value structure of most family practice units and that of their institutions but that many family practice units are beginning to achieve parity of promotion and tenure with other departments in their institutions.

Reduced long-term respiratory morbidity after treatment of respiratory syncytial virus bronchiolitis with ribavirin in previously healthy infants: a preliminary report.

Previously healthy infants less than 6 months of age with severe respiratory syncytial virus bronchiolitis who required hospitalization were identified from hospital records. Infants had been treated either conservatively (control group, n = 19) or with ribavirin added to conservative management (study group, n = 22). All infants underwent a 1-year follow-up after the initial illness. There was a significant reduction in the prevalence of reactive airway disease in the group treated with ribavirin (P < 0.05) compared with the control group, both in terms of the proportion of patients developing airway reactivity (59% vs. 89%) and the number of episodes of reactive airway disease (31 vs. 70). Our data suggest that ribavirin reduces the prevalence of airway reactivity.

High-performance liquid chromatographic assay of plasma thalidomide: stabilization of specimens and determination of a tentative therapeutic range for chronic graft-versus-host disease.

Thalidomide is now widely used to treat chronic graft-versus-host disease, but its use is associated with non-teratogenic side effects such as peripheral neuropathy. To examine the value of monitoring plasma concentrations of the drug in such patients, we have developed a high-performance liquid chromatographic (HPLC) assay. The method uses 0.5 mL plasma, is linear to 10 mg/L and had a detection limit of 0.2 mg/L. Thalidomide in plasma specimens was unstable at physiological pH but could be stabilized for several weeks by simple acidification. We describe a protocol for monitoring patients treated with thalidomide which permits convenient transportation and storage of specimens and report, provisionally, that plasma concentrations in the range 1-7 mg/L are therapeutically effective in chronic graft-versus-host disease without adverse side effects.

Analysis of etorphine in postmortem samples by HPLC with UV diode-array detection.

Etorphine is a synthetic narcotic analgesic usually used in veterinary medicine. It possesses an analgesic potency up to 1000 times greater than morphine and is therefore used in low doses, primarily for tranquilising large animals. For veterinary use, etorphine is usually available in its commercial formulation as Immobilon, when in combination with acepromazine or methotrimeprazine. Due to the potency of etorphine, only very low doses are required to produce adverse or fatal effects. This paper describes a method for detecting and quantifying etorphine using HPLC with UV diode array detection (HPLC-DAD) and demonstrates the advantage of the technique for the detection of Immobilon at low doses. In a forensic case involving Immobilon, the etorphine concentrations measured in postmortem femoral vein and heart blood specimens were 14.5 and 23.5 micrograms/l, respectively. No etorphine was detected in the urine. To our knowledge this is the first time postmortem etorphine concentrations have been reported.

Behavioral effects of caffeine and other methylxanthines on children.

Subjective, performance-enhancing, dependence-producing, and adverse effects of methylxanthines are examined, based on computerized searches (i.e., Medline and PsycLIT). High doses (> 3 mg/kg) of caffeine in children who consume little caffeine produce negative subjective effects such as nervousness, jitteriness, stomachaches, and nausea. Whether lower doses produce positive subjective effects has not been adequately tested. Caffeine appears to slightly improve vigilance performance and decrease reaction time in healthy children who habitually consume caffeine but does not consistently improve performance in children with attention deficit-hyperactivity disorder. Early studies suggest caffeine self-administration and withdrawal can occur in some adolescent soda drinkers.

Applications of an HPLC-DAD drug-screening system based on retention indices and UV spectra.

An analytical database, using high-performance liquid chromatography (HPLC) with diode array ultraviolet (UV) detection, of over 250 toxicologically relevant drugs has been extensively applied to both clinical and forensic toxicology. This general drug screening system, based on a mixed-phase (octadecyl/cyanopropyl) column and gradient reversed-phase HPLC, can identify a wide range of basic, acidic, and neutral drugs and metabolites, some of which are not amenable to gas chromatography and thin-layer chromatography. Compounds are identified using both retention index (RI) value (calculated by interpolation between a series of reference drug markers) and UV spectral data. It has been previously shown that this chromatographic system provides long-term reproducibility and has potentially useful selectivity differences compared with those based on octadecylsilane columns. Development has been undertaken to improve the speed and practicality of the system for emergency toxicology screening by increasing the rate of the elution gradient, while maintaining the applicability of the RI database. The database has also been found to be an important tool in determining optimum strategies for the adaptation of the system for quantitative analyses of drugs and metabolites under isocratic conditions.

A previously unidentified acepromazine metabolite in humans: implications for the measurement of acepromazine in blood.

High-performance liquid chromatography-diode-array detection results obtained during the investigation of two cases involving acepromazine prompted us to study the stability of the drug in blood. It was found that acepromazine can undergo in vitro conversion by human red blood cells to 2-(1-hydroxyethyl)promazine, a product that has been reported as a minor urinary metabolite in horse urine but not previously identified in humans. Further, our analytical findings in the two cases examined suggest that 2-(1-hydroxyethyl)promazine may be the major unconjugated metabolite of acepromazine in humans. These findings have important implications for the analytical toxicology of acepromazine.

Applications of, and future challenges for, genetic vaccines.

Genetic vaccines have progressed significantly since the first demonstration of the technology in 1992. When Sanford and Johnston first developed the idea, two applications were envisaged. One was as a new, simple, possibly more effective, method for delivering vaccines. The other was as a new tool to explore the immune system and to discover new vaccines. As there has been relatively little emphasis on the latter, we provide three examples of the potential uses of genetic immunization for discovery/manipulation. One of these technologies may have important implications for the safety of the vaccines. Finally, we propose that the clinical application of genetic vaccines may be limited by inadequate delivery systems and propose the characteristics of an ideal system.