High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.     

Potential benefit from implantable cardioverter-defibrillator therapy in patients with and without heart failure

BACKGROUND: Whether patients with heart failure derive a benefit from therapy with implantable cardioverter-defibrillators (ICDs) has been questioned. The purpose of this study was to investigate whether New York Heart Association (NYHA) functional class had an impact on the potential benefit from ICD therapy as assessed from data stored in the memory of ICDs. METHODS AND RESULTS: Between 1989 and 1996, 603 patients (77% men; 59% with coronary artery disease and 16% with dilated cardiomyopathy; age, 57+/-13 years; ejection fraction, 44+/-18%) were treated with an ICD with extended memory function (storage of electrograms and/or RR intervals from treated episodes) in combination with endocardial lead systems. The stages of heart failure (NYHA functional class I through III) at implantation were correlated with overall mortality and the recurrence of fast ventricular tachyarrhythmias (>240 bpm) during follow-up. The potential benefit of the device was estimated as the difference between overall mortality and the hypothetical death rate had the device not been implanted. The latter was based on the recurrence of fast and, without termination by the devices, presumably fatal ventricular tachyarrhythmias. In the overall group, a significant difference between hypothetical death rate and overall mortality was observed (13.9%, 23.5%, and 26.6% at 1, 3, and 5 years, respectively) that suggested a benefit from ICD implantation. In patients in NYHA class I, the estimated benefit, which increased over time, was 15.2%, 29.2%, and 35.6% after 1, 3, and 5 years, respectively. In patients in NYHA class II or III, the estimated benefit increased until the third year (21.8% and 21.9%, respectively) and then remained constant until the fifth year (22.9% and 23.8%, respectively). Even those patients in NYHA class III with a history of decompensated heart failure benefited from ICD implantation. CONCLUSIONS: Analysis of stored ECG data suggests that in patients with a history of ventricular tachycardia or ventricular fibrillation, ICD therapy may lead to a prolongation of life in NYHA classes I through III. The initial benefit is greatest in patients in NYHA class II and class III, but the estimated benefit might persist longest for patients in NYHA class I.     

Cooperative mechanisms between leg joints of Carausius morosus II. Motor neuron activity and influence of conditional bursting interneuron

The activity of the motor neuron pools of the protractor coxae muscle and of the thoracic part of the depressor trochanteris muscle during forward walking in the stick insect was investigated, and a spiking local interneuron, able to produce "endogenous bursting" and innervating both motor neuron pools, was identified. Extracellular recordings of the motor neurons innervating the protractor and the thoracic depressor of front, middle, and rear legs, respectively, were made with oil-hook electrodes from the peripheral nerves nl2c and nl4a while the animals were walking on a styrofoam treadwheel. The corresponding leg movements were registered and phase histograms were created with the software Spike2. Intracellular recordings were made in the neuropile of the metathoracic ganglion with glass electrodes filled with the dye Lucifer yellow. In all three legs measured (front, middle, and rear), both motor neuron pools increased their activity during the swing movement. The increase in the activity of the protractor motor neurons started at the end of the stance approximately 100 ms before reaching the posterior extreme position (PEP), and the activity of the large-sized depressor motor neurons increased as soon as the tarsus was lifted at the PEP. A local spiking interneuron was identified that excited both motor neuron pools. In 4 of 23 recordings the interneuron started to burst in synchrony with protractor and thoracic depressor motor neurons. During bursting a depolarizing stimulus reinforced and a hyperpolarizing stimulus inhibited the activity of both motor neuron pools. Thus we conclude that the thoracic part of the depressor trochanteris muscle might be a component of the neuromuscular system that shapes the swing movement. The two proximal joints, subcoxal and coxa-trochanter, connected mechanically via the thoracic part of the depressor trochanteris muscle, are also connected neurally by segmental and intersegmental spiking interneurons (this paper) and by nonspiking local interneurons (see companion paper).     

MiR-205 Is Progressively Down-Regulated in Lymph Node Metastasis but Fails as a Prognostic Biomarker in High-Risk Prostate Cancer

The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for uro-oncologists. The identification of predictive moleculobiological markers allowing risk assessment of lymph node metastasis and systemic progression is essential in establishing effective treatment. In the current study, we investigate the prognostic potential of miR-205 in HRPCa study and validation cohorts, setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly down-regulated in over 70% of the HRPCa samples analysed and that reconstitution of miR-205 causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally, miR-205 is increasingly down-regulated in lymph node metastases compared to the primary tumour indicating that miR-205 plays a role in migration of PCa cells from the original location into extraprostatic tissue. Nevertheless, down-regulation of miR-205 in primary PCa was not correlated to the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients. Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall, we showed that miR-205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic or therapeutic target in HRPCa.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Hypadapter: An adaptive hypertext system for exploratory learning and programming

We have developed an adaptive hypertext system designed to individually support exploratory learning and programming activities in the domain of Common Lisp. Endowed with domain-specific knowledge represented in a hyperspace of topics, the system builds up a detailed model of the user's expertise which it utilizes to provide personalized assistance. Unlike other work emerging in the field of adaptive hypertext systems, our approach exploits domain and user modelling techniques to support individuals in different ways. The system not only generates individualized presentations of topic nodes, but also provides adaptive navigational assistance for link-based browsing. By identifying and suggesting useful hyperlinks according to the user's knowledge state and preferences, the system encourages and guides exploration. While browsing through the hyperspace of topics, the system analyses the user's navigational behaviour to infer the user's learning progress and to dynamically adapt presentations of topics and links accordingly.     

A rapid multi-residue method for the determination of pesticide residues in choi sum, yardlong beans and aubergines

A rapid multi-residue modified QuEChERS method has been developed to analyse 20 pesticide residues in choi sum, yardlong beans and aubergines by gas chromatography with flame photometric and electron capture detectors. Primary secondary amine in dispersive form was omitted in the extraction step but instead packed in a Pasteur glass pipette for column cleanup. The recoveries for all 20 pesticides except chlorothalonil at 10-500 μg kg⁻¹ fortification levels ranged from 74.0% to 117%. Similar recoveries of 81.7-117% were obtained for all organochlorine and pyrethroid pesticides including chlorothalonil using silica gel cleanup. The relative standard deviation obtained was below 14.9%. The limit of quantification was in the range 1-10 μg kg⁻¹. The method has high selectivity for pesticides in vegetables containing high amount of chlorophyll, pigment and coloured compounds such as in choi sum and aubergines. The method was used to analyse 132 vegetable samples from residue survey.     

Zur Anwendung von Lanthanoid-Verschiebungsreagenzien bei der NMR-spektroskopischen Untersuchung von Gemischen monofunktioneller Kohlenwasserstoffe

Application of Lanthanide Shift Reagents in ¹H-N.M.R.-Spectroscopic Investigations of Mixtures of Monofunctional Hydrocarbons The influence of n.m.r. shift reagents on the ¹H-n.m.r.-spectra of mixtures has been investigated. Hydrocarbons of the type CH₃(CH₂)_nX (X =  OH,  CHO,  COR,  COOCH₃,  COOH,  OR with R = Methyl-, Ethyl-, n-Propyl-, n-Butyl- and n = 1,2) served as model substances. The components of the mixtures compete with the shift reagent. Relative equilibrium constants have been calculated to describe this behavior in mixtures. Several equations have been deduced and tested to describe the induced shifts of the protons in mixtures. These equations are applicable to qualitative and quantitative analysis of unknown mixtures.