Development of a competency based training programme to support multidisciplinary working in a combined biochemistry/haematology laboratory.
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The aim of this study was to develop a competency based training programme to support multidisciplinary working in a combined biochemistry and haematology laboratory. The training programme was developed to document that staff were trained in the full range of laboratory tests that they were expected to perform. This programme subsequently formed the basis for the annual performance review of all staff. All staff successfully completed the first phase of the programme. This allowed laboratory staff to work unsupervised at night as part of a partial shift system. All staff are now working towards achieving a level of competence equivalent to the training level required for state registration by the Council for Professions Supplementary to Medicine. External evaluation of the training programme has included accreditation by the Council for Professions Supplementary to Medicine and reinspection by Clinical Pathology Accreditation (UK) Ltd. The development of a competency based training system has facilitated the introduction of multidisciplinary working in the laboratory. In addition, it enables the documentation of all staff to ensure that they are fully trained and are keeping up to date, because the continuing professional development programme in use in our laboratory has been linked to this training scheme. This approach to documentation of training facilitated a recent reinspection by Clinical Pathology Accreditation (UK) Ltd. (+info)
Influence of the extent of haemoglobin hydrolysis on the digestive absorption of haem iron in the rat. An in vitro study.
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This study was designed to assess the haem-peptide interactions which occur during progressive haemoglobin hydrolysis by digestive enzymes and their relationship with haem iron digestive absorption. The behaviour of different haemoglobin hydrolysates was studied using the Ussing chamber model. Hydrolysates were produced from enzyme digestion of bovine haemoglobin at pH 3 by pepsin and at pH 10 by subtilisin. Samples with increasing degrees of hydrolysis (0-15 %) were studied. Biochemical assays (pyridine haemochromogen method and UV absorption spectra) were used to follow haem solubility and haem-peptide interactions in samples. Increasing the hydrolysis level of haemoglobin was associated with an enhanced iron uptake; the highest uptake rate was reached between 8 and 11 % of globin hydrolysis, whichever enzyme was used. The mechanisms rendering iron soluble and available differ between the two enzymes. The comparison between biochemical and absorption data suggests that the formation of soluble peptide-haem complexes was not sufficient to enhance haem iron absorption, since globin-bound iron is poorly absorbed; an efficient absorption occurred only when haem was loosely bound to low molecular weight peptides. (+info)
Biased Brownian dynamics for rate constant calculation.
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An enhanced sampling method-biased Brownian dynamics-is developed for the calculation of diffusion-limited biomolecular association reaction rates with high energy or entropy barriers. Biased Brownian dynamics introduces a biasing force in addition to the electrostatic force between the reactants, and it associates a probability weight with each trajectory. A simulation loses weight when movement is along the biasing force and gains weight when movement is against the biasing force. The sampling of trajectories is then biased, but the sampling is unbiased when the trajectory outcomes are multiplied by their weights. With a suitable choice of the biasing force, more reacted trajectories are sampled. As a consequence, the variance of the estimate is reduced. In our test case, biased Brownian dynamics gives a sevenfold improvement in central processing unit (CPU) time with the choice of a simple centripetal biasing force. (+info)
Bimolecular reaction simulation using Weighted Ensemble Brownian dynamics and the University of Houston Brownian Dynamics program.
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We discuss here the implementation of the Weighted Ensemble Brownian (WEB) dynamics algorithm of Huber and Kim in the University of Houston Brownian Dynamics (UHBD) suite of programs and its application to bimolecular association problems. WEB dynamics is a biased Brownian dynamics (BD) algorithm that is more efficient than the standard Northrup-Allison-McCammon (NAM) method in cases where reaction events are infrequent because of intervening free energy barriers. Test cases reported here include the Smoluchowski rate for association of spheres, the association of the enzyme copper-zinc superoxide dismutase with superoxide anion, and the binding of the superpotent sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidinium acetic acid to a monoclonal antibody fragment, NC6.8. Our results show that the WEB dynamics algorithm is a superior simulation method for enzyme-substrate reaction encounters with large free energy barriers. (+info)
Printing proteins as microarrays for high-throughput function determination.
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Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules. (+info)
The Fas-induced apoptosis analyzed by high throughput proteome analysis.
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The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo. (+info)
Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics.
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The primary structure of the Streptococcus mutans lantibiotic mutacin 1140 was elucidated by NMR spectroscopy, mass spectrometry, and chemical sequencing. The structure is in agreement with other closely related lantibiotics, such as epidermin. A novel method was developed in which mutacin 1140 was chemically modified with sodium borohydride followed by ethanethiol, allowing the differentiation of the thioether-containing residues from the dehydrated residues. This double-labeling strategy provides a simple method to reliably identify all modified lantibiotic residues with a minimal amount of material. While NMR spectroscopy is still required to obtain thioether bridging patterns and thus the complete covalent structure, the double-labeling technique, along with mass spectrometry, provides most of the information in a fraction of the time required for a complete NMR analysis. Thus, with these new techniques lantibiotics can be rapidly characterized. (+info)
The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin.
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The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA. (+info)