Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum.

BACKGROUND: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete. RESULTS: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea. CONCLUSIONS: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete-myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.

The abuse potential of zolpidem administered alone and with alcohol.

The abuse potential of zolpidem, alone and in combination with alcohol, was examined in healthy volunteers with a history of social use of alcohol and drugs. Zolpidem, a short-acting imidazopyridine hypnotic with selectivity for a benzodiazepine receptor subtype (BZ1 or omega1), was administered double blind at 0, 10, or 15 mg with alcohol (0.75 g ethanol/kg b.wt.) or with placebo beverage in a randomized, six-way crossover design. Outcome measures included the Drug Effect Questionnaire (DEQ), the Addiction Research Center Inventory (ARCI-40), and the Profile of Mood States (POMS). Blood alcohol concentrations (BACs) were not significantly modified by zolpidem. Relative to placebo, zolpidem and alcohol significantly (p < 0.05) increased drug strength perception, drug-liking, and drug-disliking scores on the DEQ. On the ARCI-40, zolpidem and alcohol significantly increased sedation/intoxication and dysphoria/fear scores, but did not significantly change euphoria/well-being scores. Zolpidem and alcohol were rated more unfavorably than placebo on the POMS. Alcohol did not have additive effects on the subjective ratings for zolpidem. It is concluded that, for this population and at the doses tested, the abuse potential of zolpidem appears to be modest and not increased by alcohol.

The acute effects of zolpidem, administered alone and with alcohol, on cognitive and psychomotor function.

BACKGROUND: Skills performance impairment after acute doses of zolpidem (a short-acting, nonbenzodiazepine hypnotic), alone and with alcohol, was evaluated in 24 subjects. The study was designed to test whether the effects of zolpidem and alcohol are simply additive or reflect potentiation. METHOD: Healthy male volunteers participated in a randomized, six-way crossover study of placebo, zolpidem 10 mg, or zolpidem 15 mg in combination with a placebo beverage or an alcohol dose selected to attain a peak blood alcohol concentration of 0.08% (drug administered double-blind; beverages administered single-blind). A laboratory test battery of four tasks measured concurrent information processing ability (divided attention task), information processing rate (visual backward masking task), immediate memory (Sternberg task), and sustained attention (vigilance task). The battery was repeated three times to measure peak (+45 minutes), postpeak (+130 minutes), and residual (+230 minutes) treatment effects after each dosing. RESULTS: Performance on each test-battery task was significantly impaired (p < .05) by both alcohol and zolpidem (combined and each given alone) during the peak-effect assessment. Residual effects were not observed, with the exception of significant alcohol and drug effects on divided attention performance (p < .05). Analysis of variance tests revealed significant main effects of alcohol and zolpidem, but no significant alcohol-by-drug interactions were found for any measure of skills performance. In general, additive effects of alcohol were detected with zolpidem 10 mg but not with zolpidem 15 mg. CONCLUSION: Although some additive effects of alcohol on performance skills were seen with the lower 10-mg dose of zolpidem, no nonadditive effects were found. That is, alcohol does not appear to potentiate the effects of zolpidem on the various performance skills tested in this population and at the doses and times evaluated. With the exception of persisting deficits (at 4 hours postdose) on the more demanding divided attention task, all other findings were consistent with evidence that zolpidem is a short-acting hypnotic drug.

Alzheimer and vascular dementias and driving. A prospective road and laboratory study.

OBJECTIVE: To characterize on-the-road, behind-the-wheel driving abilities and related laboratory performances of subjects with mild Alzheimer's disease (AD) and vascular dementia. DESIGN: Prospective, experimental study involving two mild dementia and three age and health control groups. Road test reliability and validity were assessed. SETTING: Greater western Los Angeles. Subjects were enrolled from the community by referral and from the Veterans Affairs dementia and diabetes clinics. PARTICIPANTS: Eighty-seven driving subjects were enrolled; 83 completed the study. A sample of eligible dementia clinic subjects consisting of 15 mild AD patients met National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association probable AD criteria, while 12 met Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition and Hachinski diagnostic criteria for multi-infarct dementia (vascular dementia). Clinic control subjects consisted of 15 age-matched patients with diabetes and without a history of stroke or dementia. Community controls consisted of 26 healthy, age-matched, older subjects (> 60 years) and 16 young subjects (20 to 35 years). MAIN OUTCOME MEASURES: Drive score from the Sepulveda (Calif) road test and laboratory measures of attention, perception, and memory. RESULTS: The drive scores in the mild AD group (mean, 22.1; SD, 3.8) and in the vascular dementia group (mean, 24.0; SD, 7.8) differed significantly (P < .001 studentized range test) from the drive scores in the diabetic control group (mean, 31.5; SD, 3.9), the older control group (mean, 32.6; SD, 2.8), and the young control group (mean, 33.6; SD, 3.2). Drive score among the three control groups did not vary significantly. Short-term memory (Sternberg), visual tracking, and Folstein Mini-Mental State Examination scores correlated best with drive score, with a cumulative R2 of 0.68. Drive score and number of collisions and moving violations per 1000 miles driven were negatively correlated (r = -0.38; P < .02). CONCLUSIONS: Based on this study, type and degree of cognitive impairment are better predictors of driving skills than age or medical diagnosis per se. Specific testing protocols for drivers with potential cognitive impairment may detect unsafe drivers more effectively than using age or medical diagnosis alone as criteria for license restriction or revocation.

Laboratory study of drug-related performance changes.

Performance decrements associated with therapeutic drugs may increase the risk of many common activities that require intact skills. However, accident data that define such risks are difficult to obtain, and the assessment of skill decrements often must be based on data obtained from the laboratory study of drug effects. The validity and reliability of conclusions drawn from laboratory data are a function of experiment design and measurement methods. Critical issues include the assumptions, rationale, and hypotheses underlying laboratory measurement of skills performance. Drug doses and dosing regimens, subject characteristics, and response measures are key variables that limit data interpretation. The design of reported experiments, including the selection of a battery of laboratory tests, reveals the underlying issues, and data from the experiments demonstrate both valid conclusions and constraints on interpretation.

Metocurine kinetics in patients undergoing operations requiring cardiopulmonary bypass.

Metocurine kinetics were determined in 10 patients undergoing operations requiring hypothermic cardiopulmonary bypass (CPB) and nine patients of similar age undergoing operations of similar duration but not requiring CPB. The metocurine dosage regimen was a bolus of 0.3 mg/kg given concomitantly with the commencement of an infusion at a rate of 0.04 mg/kg/hr; this regimen was designed to produce and maintain a plasma metocurine concentration associated with 95% depression of the twitch response. Metocurine kinetics were affected minimally by hypothermic CPB. The kinetic parameters including volumes of distribution at steady state of 0.35 L/kg and 0.34 L/kg and elimination clearances of 1.3 ml/min/kg and 1.1 ml/min/kg in the control and CPB groups, respectively, are in agreement with the results of others. Changes in neuromuscular blockade with the onset of CPB and the return to original blockade intensities with rewarming suggest a decreased sensitivity to the effects of metocurine at lower temperatures.

Cardiopulmonary bypass in patients with malignant renal neoplasms.

A case report of a patient with malignant renal neoplasm who underwent emergency pulmonary embolectomy during operation is presented. Knowledge of degree of venous extension, appropriate operative positioning of the patient and consideration of elective cardiopulmonary bypass are suggested ways to reduce morbidity and mortality from massive pulmonary emboli in these patients.

Status of women in academic anesthesiology.

The authors compared anesthesia faculties with the rest of medical school faculties at each of four academic ranks and found a significant difference in proportion of men and women anesthesia faculty members at the assistant professor rank only (P less than 0.001). When the faculty status of women and men academic anesthesiologists was examined a significant difference was found in rank distribution in age groups 40 to 44 (P less than 0.005) and 45 to 49 (P less than 0.001), where there was a deficit of professors and a surfeit of instructors among women. Significant differences in distribution continued at age 50-54 (P less than 0.01), 55-59 (P less than 0.001), and 60-64 (P less than 0.005), primarily at professor and assistant professor ranks. In addition, there was significantly lower prevalence of board certification (P less than 0.001) and level of responsibilities for women (P less than 0.001). There was no significant difference in tenure status.

Effects of diazepam (Valium) and trait anxiety on human physical aggression and emotional state.

The effects of diazepam (10 mg orally) and trait anxiety on the aggressive behavior of normal male undergraduates were studied in a competitive reaction time task paradigm which entails shock setting with an increasingly provocative "opponent." Diazepam produced an aggression-enhancing effect which was specifically shown only by the low-trait anxious group under low provocation and generally shown by all groups under conditions of higher provocation. Reductions in state anxiety following diazepam ingestion were seen most clearly in the high- and, somewhat, the moderate-trait anxious groups. In contrast, the low-trait anxious group evidenced an increase in depression but little change in anxiety. The results are consistent with reports of the ability of antianxiety drugs to disinhibit suppressed behaviors. Moreover, trait anxiety appears to mediate the effects of diazepam on both mood states and aggressive behavior.

Changes of body temperature and heat in cardiac surgical patients.

Changes in body temperature were assessed in ten adult patients undergoing surgery involving cardiopulmonary bypass (CPB) and induced hypothermia. Intraoperatively, in comparable time intervals before CPB and after rewarming, the patients lost body heat. Between the time of induction of anaesthesia and CPB, the temperature of blood in the pulmonary artery fell 1.46 (SD 0.28 degrees C); between CPB and the end of surgery the fall was 1.55 (SD 0.86 degrees C). The extent of spontaneous hypothermia did not correlate with the amount of subcutaneous fat. Hypothermia was induced to obtain a stable deep body temperature of 27.2 (SD 1.3) degrees C, when mean skin temperature averaged 2 degrees C higher. The CPB machine returned approximately 2000 kJ of heat in the rewarming period, to produce pulmonary artery and mean skin temperatures of 37.1 (SD 0.7) degrees C and 31.4 (SD 2.1) degrees C respectively. Intraoperative deep body temperatures demonstrated the expected exponential relationship with metabolic rate. Postoperatively, increase in metabolic rate was associated with rising deep body and skin temperatures. Low resistance to the flow of heat toward the skin surface was demonstrated by low postoperative values for thermal insulation, which may indicate good peripheral perfusion seen during continuing vasodilator therapy.

Serial assessment of left ventricular performance following coronary artery bypass grafting. Early postoperative results with myocardial protection afforded by multidose hypothermic potassium crystalloid cardioplegia.

Forty patients who recently underwent coronary artery bypass graft (CABG) operations had serial hemodynamic and scintigraphic studies. Multidose hypothermic potassium crystalloid cardioplegia was used for myocardial protection and newer techniques in anesthetic management and perioperative patient care were also employed. The method of equilibrium cardiac gated blood pool (GBP) scintigraphy was used to obtain perioperative changes in global ejection fraction (EF) and regional wall motion (RWM). Ninety percent of patients displayed a decrease in EF 2 hours postoperatively when compared to their preoperative values. This change was also associated with a fall in cardiac index (CI) and left ventricular stroke work index (LVSWI). Twenty-four hours postoperatively, EF and CI recovered to preoperative levels, but LVSWI remained depressed. Seven days postoperatively, global EF had improved to a value greater than the preoperative one (50% +/- 3% versus 57% +/- 4%, p < 0.05). Perioperative changes in RWM followed the same pattern as EF, but recovery in this index of regional contractility was faster than EF, since maximal improvement was observed 24 hours postoperatively. Thus transient left ventricular dysfunction is common immediately after CABG, but recent advances in myocardial protection and perioperative management are associated with short-term increases in regional and global left ventricular function documented by noninvasive GBP imaging.

Lidocaine treatment of experimental cutaneous lesions from potassium chloride injection.

Potassium chloride (KCl) given subcutaneously in high concentrations causes necrosis of skin, possibly from vasoconstriction around the injection site. The authors studied guinea pigs given subcutaneous injections of various volumes and concentrations of KCl and observed the severity of the cutaneous lesions. In further experiments, therapeutic agents were injected subcutaneously 10 minutes after KCl infiltration. The severity of cutaneous lesions was not affected by various volumes of KCl of the same concentration, but was correlated positively with increasing concentrations of the salt when concentration was varied. Dextrose, 5%, and sodium bicarbonate, 1 M, had no effect on the cutaneous lesions caused by KCl, while hyaluronidase, 150 U/ml, lessened them. Lidocaine, 1%, a vasodilator, eliminated cutaneous lesions caused by KCl. Kcl-induced lesions may be due to vasocontriction, which can be relieved by lidocaine.