A novel technique to determine an 'apparent ke0 ' value for use with the Marsh pharmacokinetic model for propofol.

Debate continues over the most appropriate blood-brain equilibration rate constant (ke0) for use with the Marsh pharmacokinetic model for propofol. We aimed to define the optimal ke0 value. Sixty-four patients were sedated with incremental increases in effect-site target concentration of propofol while using six different ke0 values within the range 0.2-1.2 min(-1). Depth of sedation was assessed by measuring visual reaction time. A median 'apparent ke0' value of 0.61 min(-1) (95% CI 0.37-0.78 min(-1)) led to the greatest probability of achieving a stable clinical effect when the effect-site target was fixed at the effect-site concentration displayed by the target-controlled infusion system, at the time when a desired depth of sedation had been reached. By utilising a clinically relevant endpoint to derive this value, inter-individual pharmacokinetic and pharmacodynamic variability may be accounted for.

Induction of general anaesthesia by effect-site target-controlled infusion of propofol: influence of pharmacokinetic model and ke0 value.

We studied the use of a new ke0 value (0.6 min(-1)) for the Marsh pharmacokinetic model for propofol. Speed of induction and side-effects produced were compared with three other target-controlled infusion systems. Eighty patients of ASA physical status 1-2 were studied in four groups in a prospective, randomised study. Median (IQR [range]) induction times were shorter with the Marsh model in effect-site control mode with a ke0 of either 0.6 min(-1) (81 (61-101 [49-302])s, p < 0.01), or 1.2 min(-1) (78 (68-208 [51-325])s, p < 0.05), than with the Marsh model in blood concentration control (132 (90-246 [57-435])). The Schnider model in effect-site control produced induction times that were longer (298 (282-398 [58-513])s) than those observed with the Marsh model in blood control (p < 0.05), or either effect-site control mode (p < 0.001). There were no differences in the magnitude of blood pressure changes or frequency of apnoea between groups.

Expression of neutrophil gelatinase-associated lipocalin in colorectal neoplastic progression: a marker of malignant potential?

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) has a diverse functional repertoire, involved in the innate immune response as well as cell growth and differentiation. Expression has been linked to malignant disease development and progression. METHODS: Neutrophil gelatinase-associated lipocalin expression was assessed immunohistochemically in 98 colorectal neoplastic lesions (52 cancer polyps (CaPs) and 46 sporadic adenoma/adjacent normal mucosa paired specimens) to investigate association with adenoma progression and early colorectal carcinogenesis. RESULTS: Within CaPs, all adenomatous and carcinomatous epithelium expressed NGAL, with 92% (43 out of 47) and 58% (19 out of 33) epithelial positivity, respectively, as well as positive stromal cell expression. This was significantly increased compared with normal mucosal epithelium (P=0.0001). Neutrophil gelatinase-associated lipocalin positivity was also identified in sporadic low-grade adenomas, in both the epithelial and stromal compartments as compared with adjacent normal mucosa (P=0.0001 and 0.0002), and this increased along with adenoma size >1 cm (P=0.03). CONCLUSION: Neutrophil gelatinase-associated lipocalin is expressed by the majority of human neoplastic colorectal lesions. This phenotypic switch occurs at an early stage in neoplastic progression with clear differential expression between normal mucosa and adenomatous polyps, rather than further downstream in disease progression at the adenoma-carcinoma transformation. Thus, NGAL expression is not a useful biomarker for determining disease progression from adenomatous to malignant colorectal neoplasia.

Specific dermatoses of pregnancy: a review.

BACKGROUND: Some dermatoses worsen during pregnancy, some improve, yet others have unpredictable course. OBJECTIVE: To conduct evidence-based search, and review of current management of specific dermatoses of pregnancy. METHODS: Comprehensive literature search was conducted, with Medline and Cochrane Database regarding skin diseases in pregnancy from 1990- 2005. International pharmaceutical abstracts science search (1997- 2003) was used for search references found in the articles. All articles selected for inclusion in this review were evaluated critically with regards to their impact factor, source, and evidence based contribution on this topic as measured by their citation index and the journals they were published in. This review was limited to specific dermatoses of pregnancy generally and some of the skin disorders modified by pregnancy. RESULTS: Intrahepatic cholestasis of pregnancy should be managed as high risk pregnancies as several investigations have shown foetal complications. Recent randomised trials have demonstrated beneficial effects of ursodeoxycholic acid (UDCA) in intrahepatic cholestasis of pregnancy (ICP). Pruritice ruption of pregnancy is associated with multiple pregnancies. It has variable clinical features and has to be differentiated from pemphigoid gestationis, which is associated with an increased incidence of both prematurity and small for date's babies. Prurigo of pregnancy is extremely itchy with papules appearing on the extensor surfaces of the limbs and trunk. It has no maternal risk. The eruptions in pruritic folliculitis of pregnancy clear spontaneously in the postpartum period, with no associated morbidity either in the mother or baby. CONCLUSION: Some skin diseases like obstetric cholestasis may have adverse foetal outcome, while other disorders like pruritic folliculitis of pregnancy have no significant effect on either the mother or baby.

The effect of anaesthesia and aortic clamping on cardiac output measurement using arterial pulse power analysis during aortic aneurysm repair.

The LiDCO plus monitor (LiDCO Ltd, Cambridge, UK) uses pulse contour analysis of the arterial pressure waveform to indicate changes in stroke volume and cardiac output. Calibration against a lithium indicator dilution method is required to permit display of absolute values in addition to trends. The effect of haemodynamic changes during anaesthesia and surgery on this calibration factor has not previously been studied. Therefore, we investigated whether it remained constant during elective abdominal aortic aneurysm surgery in 15 patients. Comparison between the calibration factor values at different time points was made by repeated recalibration throughout the peri-operative period. Calibration factor increased by a mean of 53% after anaesthesia (epidural plus general) (p = 0.03) and decreased by a mean of 40% after aortic clamping (p = 0.0001). Recalibration should be undertaken after induction of anaesthesia and after aortic clamping if absolute values of cardiac output and stroke volume are required.

Climate indices, rainfall onset and retreat, and malaria in Nigeria.

BACKGROUND & OBJECTIVES: Rainfall in western sub-Saharan Africa is related to seasonal shifts of the Inter-tropical Convergence Zone, which moves northward early in the year, retreating in the second half of the year. The objective of the present study was to determine significant relationships between onset and retreat timing and climate indices. The relationship between timing and malaria case reporting was then evaluated. METHODS: Relationships between published rainfall onset and retreat dates for Nigeria from 1971- 2000 were evaluated in relation to pairs of climate indices using response surface analysis. Graphical representation of the response surface in relation to the underlying data was used to identify instances of overfitting. Association of onset and retreat timing with published case reporting records was evaluated using graphical and correlation analysis. RESULTS: Onset timing and rate of advance were related to ENSO (El Niño-Southern Oscillation), in combination with the Northern Annular Mode (NAM), while retreat timing was related to NAO (North Atlantic Oscillation), in combination with the East Pacific (EP) or West Pacific (WP) index, depending on location. Later onset was associated with faster northward progression of onset. Retreat date at Kano, the most northerly of the study locations, increased over the period 1990-2000, with higher case reporting for Nigeria as a whole being associated with the last three years of that period. INTERPRETATION AND CONCLUSION: Rainfall retreat occurs much faster than onset, with onset and retreat timing and rate of onset advance being related to combinations of climate indices rather than to a single index. Threshold for determining a "rainy" day would influence results. The increase in national case reporting with delayed retreat at Kano may be related to the extension of the short transmission period in the north.

How useful are the BSUG database outcome criteria: validation using the MESA questionnaire.

Urinary stress incontinence is common, but there is a wide range of prevalence which might account for variations in definition of incontinence and variations in study methodology. Our study assessed the validity and reliability of the British Society of Urogynaecology's (BSUG) database subjective outcome scores after the tension-free vaginal tape (TVT), by correlating these with the changes in the Medical Epidemiologic and Social Aspects of Ageing (MESA) questionnaire score. A total of 100 women with urodynamic stress incontinence underwent TVT, completed a MESA questionnaire preoperatively and at 6 months postoperatively. We also collected information about three outcome measures of the BSUG database, patients' global impression of outcome and stress and urge symptom analyses. Our study showed that the postoperative patients' global impression of outcome improved significantly in 85% of cases and had 73.89% reduction in mean MESA scores (p < 0.001). The outcome measures of the BSUG database relates well to symptom improvement, based on MESA scores and these subjective assessments currently used by the BSUG's database are a valid assessment of TVT outcome.

Evaluation of a new method of assessing depth of sedation using two-choice visual reaction time testing on a mobile phone.

The utility of two-choice visual reaction time testing using a specially programmed mobile telephone as a measure of sedation level was investigated in 20 healthy patients sedated with target controlled infusions of propofol. At gradually increasing target concentrations visual reaction time was compared with patient-assessed visual analogue scale sedation scores and an observer-rated scale. Propofol sedation caused dose-dependent increases in visual reaction time and visual analogue scale scores that were statistically significant when the calculated effect-site concentration reached 0.9 microg.ml(-1) (p < 0.05) and 0.5 microg.ml(-1) (p < 0.01) respectively. While visual analogue scale scores were more sensitive at lower levels of sedation than visual reaction time, the latter demonstrated marked increase in values at higher levels of sedation. Visual reaction time may be useful for identifying impending over-sedation.

Antenatal dexamethasone treatment transiently alters diastolic function in the mouse fetal heart.

Endogenous glucocorticoid action is important in the structural and functional maturation of the fetal heart. In fetal mice, although glucocorticoid concentrations are extremely low before E14.5, glucocorticoid receptor (GR) is expressed in the heart from E10.5. To investigate whether activation of cardiac GR prior to E14.5 induces precocious fetal heart maturation, we administered dexamethasone in the drinking water of pregnant dams from E12.5 to E15.5. To test the direct effects of glucocorticoids upon the cardiovascular system we used SMGRKO mice, with Sm22-Cre-mediated disruption of GR in cardiomyocytes and vascular smooth muscle. Contrary to expectations, echocardiography showed no advancement of functional maturation of the fetal heart. Moreover, litter size was decreased 2 days following cessation of antenatal glucocorticoid exposure, irrespective of fetal genotype. The myocardial performance index and E/A wave ratio, markers of fetal heart maturation, were not significantly affected by dexamethasone treatment in either genotype. Dexamethasone treatment transiently decreased the myocardial deceleration index (MDI; a marker of diastolic function), in control fetuses at E15.5, with recovery by E17.5, 2 days after cessation of treatment. MDI was lower in SMGRKO than in control fetuses and was unaffected by dexamethasone. The transient decrease in MDI was associated with repression of cardiac GR in control fetuses following dexamethasone treatment. Measurement of glucocorticoid levels in fetal tissue and hypothalamic corticotropin-releasing hormone (Crh) mRNA levels suggest complex and differential effects of dexamethasone treatment upon the hypothalamic-pituitary-adrenal axis between genotypes. These data suggest potentially detrimental and direct effects of antenatal glucocorticoid treatment upon fetal heart function.

Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy.

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

Fluorescence-activated single cell sorting of human embryonic stem cells.

Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.

Low temperature magnetic circular dichroism spectra of met- and myoglobin derivatives.

1. Low temperature magnetic circular dichroism spectra of high and low spin derivatives of metmyoglobin and myoglobin have been measured in the Soret and high wavelength regions. 2. The large difference in intensity of the Soret magnetic circular dichroism bands suggest that a correlation exists between the signal intensity and spin state of the heme-iron. 3. From a comparison of the high and low spin sepctra of the myoglobin derivatives it is concluded that oxymyoglobin contains between 10 and 20% of a ferrous high spin component below 100 degrees K.

Assignment of the axial ligands of the haem in milk lactoperoxidase using magnetic circular dichroism spectroscopy.

The absorption and MCD spectra of ferric lactoperoxidase from milk and its cyanide and fluoride derivatives have been measured in the near infrared and visible wavelength regions both at room temperature and at 4.2 K. By comparison with the MCD spectra of haemoproteins of known axial ligation, which also contain protohaem IX, it has been possible to arrive at suggestions for the axial ligation in lactoperoxidase. At room temperature oxidized lactoperoxidase has the haem iron in the high-spin state, and the results indicate that the proximal ligand of the haem iron is a histidine imidazole and that the sixth ligand is probably a carboxylate ion. At 4.2 K oxidized lactoperoxidase converts almost totally to a low-spin form, changing the sixth ligand to a histidine imidazole, which is in the imidazolate form.

Low temperature EPR and MCD studies on cytochrome b-558 of the Bacillus subtilis succinate: quinone oxidoreductase indicate bis-histidine coordination of the heme iron.

Bacillus subtilis cytochrome b-558 was expressed in high amounts in Escherichia coli, solubilized from membranes with detergent and purified free from other hemoproteins. The cytochrome possibly contains two heme groups. To determine the axial ligands to the low-spin heme and the heme rhombicity, the cytochrome was analyzed using low-temperature electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopy. The combined results exclude bis-methionine, bis-lysine and histidine-methionine coordination. Bis-histidine coordination of the heme(s) with a near perpendicular orientation of the imidazole planes is strongly suggested by the highly axial low-spin EPR signals and the intense near infrared MCD spectrum (delta epsilon = 380 M-1.cm-1 at 4.2 K and 5 T) of the charge-transfer band at 1600 nm.

Low-temperature magnetic circular dichroism spectra and magnetisation curves of 4Fe clusters in iron-sulphur proteins from Chromatium and Clostridium pasteurianum.

The magnetic circular dichroism (MCD) spectra of the 4Fe clusters in the iron-sulphur proteins high-potential iron protein from Chromatium and the 8Fe ferredoxin from Clostridium pasteurianum have been measured over the wavelength range 300-800 nm at temperatures between approx. 1.5 and 50 K and at magnetic fields up to 5 tesla. In both cases the proteins have been studied in the oxidized and reduced states. The reduced state of high-potential iron protein gives a temperature-independent MCD spectrum up to 20 K, confirming the diamagetism of this state at low temperature. The MCD spectrum of samples of oxidized ferredoxin invariably show the presence of a low concentration of a paramagnetic species, in agreement with the observation that the EPR spectrum always shows a signal at g = 2.01. The paramagnetic MCD spectrum runs across the whole of the wavelength range studied and therefore most probably originates from an iron-sulphur centre. The diamagnetic component of the MCD spectrum of oxidized ferredoxin is very similar to that of reduced high-potential iron protein. The low-temperature MCD spectra of oxidized high-potential iron protein and reduced ferredoxin reveal intense, temperature-dependent bands. The spectra are highly structured with that of high-potential iron protein showing a large number of electronic transitions across the visible region. The MCD spectra of the two different oxidation levels are quite distinctive and should provide a means of establishing the identity of these state of 4Fe clusters in more complex proteins. MCD magnetisation curves have been constructed from detailed studies of the field and temperature dependence of the MCD spectra of the two paramagnetic oxidation states. These plots can be satisfactorily fitted to the theoretically computed curves for an S = 1/2 ground state with the g factors experimentally determined by EPR spectroscopy. The low-temperature MCD spectra of the reduced 2Fe-2S ferredoxin from Spirulina maxima are also presented and MCD magnetisation curves plotted and fitted to the experimentally determined g factors.

A spectroscopic investigation of the structure and redox properties of Escherichia coli cytochrome b-562.

The six-coordinate monohaem ferricytochrome b-562 from Escherichia coli exhibits two haem-linked pH-dependent transitions detected by NMR and optical spectroscopy. Only one of these transitions, that of the Fe(III)-coordinated His-102, is detected by EPR and MCD; the ionisation of a haem propionate is not. Both ionisations are redox-state-dependent and the midpoint redox potential of the protein is markedly pH-dependent. Over the pH range 5.0 to 8.5 the potential drops from 260 mV to 110 mV and at least five single proton ionisations are responsible for this. In addition to the two spectroscopically identified ferricytochrome ionisations, there are at least three unidentified ionisations, two of which occur in the ferrous protein. From a consideration of the X-ray structure, together with NMR data, it seems probable that at least one of these ionisations involves an amino acid carboxylate. The X-ray structure also suggests that the relatively low pKa of His-102 is a result of its proximity to Arg-98. However, an appreciable interaction between these groups requires that the solution conformation differs slightly from the X-ray structure. The fast rate of electron self-exchange, over 4 X 10(6) M-1 X s-1 at 315 K and pH* 7, may be a reflection of the fact that, as shown by the X-ray structure, a large amount of the haem and axial histidine ligand are exposed at the molecular surface with an asymmetric distribution of charged groups surrounding them.