Clinical approach to the diagnosis of acid-base disorders.

An ability to rapidly and effectively diagnose and treat acid-base disorders is essential to the management of seriously ill patients. In this paper an approach to the diagnosis of pure and mixed acid-base disorders is presented that is based upon an understanding of the bicarbonate buffer system and a knowledge of the well defined and predictable compensatory responses that occur in association with each of the primary acid-base disorders. With this approach a number of acid-base problems are presented and solved.     

Eating disorders. A review and update.

Anorexia nervosa and bulimia nervosa are prevalent illnesses affecting between 1% and 10% of adolescent and college age women. Developmental, family dynamic, and biologic factors are all important in the cause of this disorder. Anorexia nervosa is diagnosed when a person refuses to maintain his or her body weight over a minimal normal weight for age and height, such as 15% below that expected, has an intense fear of gaining weight, has a disturbed body image, and, in women, has primary or secondary amenorrhea. A diagnosis of bulimia nervosa is made when a person has recurrent episodes of binge eating, a feeling of lack of control over behavior during binges, regular use of self-induced vomiting, laxatives, diuretics, strict dieting, or vigorous exercise to prevent weight gain, a minimum of 2 binge episodes a week for at least 3 months, and persistent overconcern with body shape and weight. Patients with eating disorders are usually secretive and often come to the attention of physicians only at the insistence of others. Practitioners also should be alert for medical complications including hypothermia, edema, hypotension, bradycardia, infertility, and osteoporosis in patients with anorexia nervosa and fluid or electrolyte imbalance, hyperamylasemia, gastritis, esophagitis, gastric dilation, edema, dental erosion, swollen parotid glands, and gingivitis in patients with bulimia nervosa. Treatment involves combining individual, behavioral, group, and family therapy with, possibly, psychopharmaceuticals. Primary care professionals are frequently the first to evaluate these patients, and their encouragement and support may help patients accept treatment. The treatment proceeds most smoothly if the primary care physician and psychiatrist work collaboratively with clear and frequent communication.     

A practical approach to crosslinking

The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH azidobenzoyl hydrazide - ANB- NOS N-5-azido-2-nitrobenzoyloxysuccinimide - ASIB 1-(p-azidosalicylamido)-4-(iodoacetamido)butane - ASBA 4-(p-azidosalicylamido)butylamine - APDP N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide - APG p-azidophenyl glyoxal monohydrate - BASED bis-[-(4-azidosalicylamido)ethyl] disulfide - BMH bismaleimidohexane - BS³ bis(sulfosuccinimidyl) suberate - BSOCOES bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone - DCC N,N-dicyclohexylcarbodiimide - DFDNB 1,5-difluoro-2,4-dinitrobenzene - DMA dimethyl adipimidate·2HCl - DMP dimethyl pimelimidate·2HCl - DMS dimethyl suberimidate·2HCl - DPDPB 1,4-di-(3,2-pyridyldithio)propionamido butane - DMF dimethylformamide - DMSO dimethylsulfoxide - DSG disuccinimidyl glutarate - DSP dithiobis(succinimidylpropionate) - DSS disuccinimidyl suberate - DST disuccinimidyl tartarate - DTSSP 3,3-dithiobis (sulfosuccinimidylpropionate) - DTBP dimethyl 3,3-dithiobispropionimidate·2HCl - EDC or EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride - EDTA ethylenediaminetetraacetic acid disodium salt, dihydrate - EGS ethylene glycolbis(succinimidylsuccinate) - GMBS N--maleimidobutyryloxysuccinimide ester - HSAB N-hydroxysuccinimidyl-4-azidobenzoate - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MES 4-morpholineethanesulfonic acid - NHS N-hydroxysuccinimide - NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid - PMFS phenylmethylsulfonyl fluoride - PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate - SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate - SADP N-succinimdyl (4-azidophenyl)1,3-dithiopropionate - SAND sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate - SANPAH N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate - SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate - SATA N-succinimidyl-S-acetylthioacetate - SDBP N-hydroxysuccinimidyl-2,3-dibromopropionate - SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate - SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SMPB succinimidyl 4-(p-maleimidophenyl) butyrate - SMPT 4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene - sulfo-BSOCOES bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone - sulfo-DST disulfosuccinimidyl tartarate - sulfo-EGS ethylene glycolbis(sulfosuccinimidylsuccinate) - sulfo-GMBS N--maleimidobutyryloxysulfosuccinimide ester - sulfo-MBS m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester - sulfo-SADP sulfosuccinimidyl(4-azidophenyldithio)propionate - sulfo-SAMCA sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate - sulfo-SANPAH sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate - sulfo-SIAB sulfosuccinimidyl(4-iodoacetyl)aminobenzoate - sulfo-SMPB sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate - sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate     

Bioinformatics methods to predict protein structure and function. A practical approach

Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.     

A practical approach to significance assessment in alignment with gaps.

Current numerical methods for assessing the statistical significance of local alignments with gaps are time consuming. Analytical solutions thus far have been limited to specific cases. Here, we present a new line of attack to the problem of statistical significance assessment.We combine this new approach with known properties of the dynamics of the global alignment algorithm and high performance numerical techniques and present a novel method for assessing significance of gaps within practical time scales. The results and performance of these new methods test very well against tried methods with drastically less effort.     

A practical approach for intracellular protein delivery

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.     

A practical approach to T-cell receptor cloning and expression

Although cloning and expression of T-cell Receptors (TcRs) has been performed for almost two decades, these procedures are still challenging. For example, the use of T-cell clones that have undergone limited expansion as starting material to limit the loss of interesting TcRs, must be weighed against the introduction of mutations by excess PCR cycles. The recent interest in using specific TcRs for cancer immunotherapy has, however, increased the demand for practical and robust methods to rapidly clone and express TcRs. Two main technologies for TcR cloning have emerged; the use of a set of primers specifically annealing to all known TcR variable domains, and 5'-RACE amplification. We here present an improved 5'-RACE protocol that represents a fast and reliable way to identify a TcR from 10(5) cells only, making TcR cloning feasible without a priori knowledge of the variable domain sequence. We further present a detailed procedure for the subcloning of TcRα and β chains into an expression system. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TcR transgene into different expression systems. The presented comprehensive method can be performed in any laboratory with standard equipment and with a limited amount of starting material. We finally exemplify the straightforwardness and reliability of our procedure by cloning and expressing several MART-1-specific TcRs and demonstrating their functionality.     

A review of UVA-mediated photosensitivity disorders

A number of skin conditions are characterised by photosensitivity to UVA. Some of these are exclusively UVA-mediated conditions, while others include UVA in the action spectrum which also include UVB and/or visible light. This review aims to describe this diverse range of conditions for non-dermatologist scientists with an interest in this topic. As such, clinical details, including treatments, are brief and succinct. Recent advances in understanding the pathogenesis of these conditions is highlighted.     

A practical approach to quantitate hepatic excretory function

A statistical comparison of different BSP tests was carried out in normal subjects and in patients with various degrees of chronic liver damage. Only the logarithm of the BSP retention correlated linearly with the physiologically more meaningful determination of the maximal excretory capacity of the liver (BSP Tm, Wheele''s method). A double logarithmic transformation was required to correlate the second exponential component of the BSP plasma disappearance curve (k₂) with the BSP Tm. When the limitations of these methods are kept in mind, the observed statistical relationships can be used to express hepatic functional deterioration in more physiological terms.     

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

Production activity control: A practical approach to scheduling

There is a widely perceived gap within the domain of scheduling for manufacturing systems, namely, many of the methods employed by production supervisors are quite different from those developed by researchers. In a sense, this inconsistency highlights the important fact that much scheduling research has failed to win approval where it matters most, namely, within the manufacturing system. In this article, we argue for a practical approach to scheduling for manufacturing systems, one that we believe can narrow, and possibly bridge, the gap between theory and practice. This approach is based upon a well-defined and modular architecture for scheduling, termedproduction activity control. This architecture is the foundation of our proposed solution to scheduling, since it provides a coherent blueprint for the synthesis of information technology and scheduling strategies. The result of this synthesis is a design tool for production activity control, which allows for detailed and disciplined experimentation with a range of scheduling strategies in a controlled and simulated environment. Due to the unique modular property of the design tool, these strategies may then be implemented live in a flexible manufacturing facility, hence narrowing the gap between scheduling theory and manufacturing practice. Our overall approach is tested through an appropriate implementation in a modern electronics assembly plant.