A method for the deductive and unique determination of the values of three parameters involved in fractional functions applicable to relaxation kinetics.

A novel method is proposed to determine deductively and uniquely the values of three parameters, a, b, and c in a fractional function of the form, y=a+bx/(c+x) where x and y are experimentally obtainable variables. This type of equation is frequently encountered in chemistry and biochemistry involving relaxation kinetics. The method of least squares with the Taylor expansion is employed for direct curve fitting of observed data to the fractional function. Approximate values of the parameters, which are always necessary prior to commending the above procedure, can be obtained by the method of rearrangement after canceling the denominator of fractional functions. This procedure is very simple, but very effective for estimating provisional values of the parameters. Deductive and unique determination of the parameters involved in the fractional function shown above can be accomplished for the first time by the combination of these two procedures. This method is extended to include the analysis of relaxation kinetic data such as those of temperature-jump method where the determination of equilibrium concentrations of reactants in addition to the three parameters is also necessary.     

Immunoenzyme determination of total serum ceruloplasmin. Application to Wilson disease

Enzyme immunoassay for ceruloplasmin (CP)*, employing monospecific CP antibodies labeled with horse radish peroxidase was developed. This method permits to determine total content of CP, which is present in Wilson disease patients' blood in enzymatically active and enzymatically inactive forms. The evidence is presented that the method can be used for a direct determination of CP in blood serum. The minimal CP concentration which may be determined by enzyme immunoassay (IEA) is 5.10(-9) g/ml. The method was used for determination of CP concentrations in Wilson disease patients' blood with different disease severity. Analysis of blood samples taken from 6 Wilson disease patients with the use of IEA method revealed similar total CP concentrations. At the same time, the oxidase activities of CP in the blood of different patients varied more than sevenfold.     

Using neural networks as an aid in the determination of disease status: comparison of clinical diagnosis to neural-network predictions in a pedigree with autosomal dominant limb-girdle muscular dystrophy.

Studies of the genetics of certain inherited diseases require expertise in the determination of disease status even for single-locus traits. For example, in the diagnosis of autosomal dominant limb-girdle muscular dystrophy (LGMD1A), it is not always possible to make a clear-cut determination of disease, because of variability in the diagnostic criteria, age at onset, and differential presentation of disease. Mapping such diseases is greatly simplified if the data present a homogeneous genetic trait and if disease status can be reliably determined. Here, we present an approach to determination of disease status, using methods of artificial neural-network analysis. The method entails "training" an artificial neural network, with input facts (based on diagnostic criteria) and related results (based on disease diagnosis). The network contains weight factors connecting input "neurons" to output "neurons," and these connections are adjusted until the network can reliably produce the appropriate outputs for the given input facts. The trained network can be "tested" with a second set of facts, in which the outcomes are known but not provided to the network, to see how well the training has worked. The method was applied to members of a pedigree with LGMD1A, now mapped to chromosome 5q. We used diagnostic criteria and disease status to train a neural network to classify individuals as "affected" or "not affected." The trained network reproduced the disease diagnosis of all individuals of known phenotype, with 98% reliability. This approach defined an appropriate choice of clinical factors for determination of disease status. Additionally, it provided insight into disease classification of those considered to have an "unknown" phenotype on the basis of standard clinical diagnostic methods.     

Contribution of chiral HPLC in tandem with polarimetric detection in the determination of absolute configuration by chemical interconversion method: Example in 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomer series

We report the determination of the absolute configuration of eight stereoisomers in the series of chiral 1-(thi)oxothiazolinyl-3-(thi)oxothiazolinyl toluene atropisomers 1-3, from the known absolute configuration of one stereoisomer, determined by X-ray crystallography. The method uses the affiliation between signs of rotation of polarised light during chemical transformations which preserve the absolute configuration and also during rotation around a single pivot bond producing a compound of known configuration. The use of chiral HPLC in tandem with a chirality detector gives a decisive advantage since such correlation can be performed on a mixture of a very limited quantity of compounds, without tedious purification steps. The method shown as an example in this article, which uses chiral HPLC with chirality detection, may prove useful in many other cases where the determination of the absolute configuration is necessary and where a chemical interconversion method can be used on a microscale.     

Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.     

Radiochemical methods for the assay of phosphorylase in the direction of glycogenolysis.

Two radiochemical procedures were explored for the determination of phosphorylase activity in the glycogenolytic direction. In the "32P assay method' the formation of labelled glucose 1-phosphate from glycogen and [32P]Pi is measured by the radio-activity that remains soluble after the precipitation of phosphomolybdate with triethylamine. In the "14C assay method' the formation of labelled glucose 1-phosphate from peripherally 14C-labelled glycogen and P1 is determined from the radioactivity that remains soluble after the precipitation of glycogen with ethanol. The 14C assay method requires more preparative work but less circumspection than does the 32P assay method. Both radiochemical methods can be applied where the classical spectrophotometric assay fails. They have the same accuracy and reproducibility, and allow more samples to be handled in parallel. They are not intended for use with crude tissue extracts.     

Fluorescence displacement method for the determination of receptor-ligand binding constants.

The equilibrium constant for the binding of a spectroscopically invisible ligand to its protein receptor can be determined in a competition experiment, by using a structural analog that contains a reporter group (fluorophor). A novel mathematical treatment of the multiple equilibria allows the analysis to be performed under tight-binding conditions. The equilibrium equation for mixtures of two mutually competitive tight-binding ligands can be expressed in a recursive form, a form in which the dependent variable appears on both sides and the solution is found iteratively. The algorithm is also applicable to the special case of weak binding, where the concentration of the bound ligand can be neglected in the mass balance. The fluorescence displacement method is demonstrated on the determination cyclophilin binding to cyclosporin A (CsA), in competition with its fluorescent derivative, [D-Lys(Dns)]8-CsA.     

Studying biomolecular complexes with pulsed electron-electron double resonance spectroscopy

The function of biomolecules is intrinsically linked to their structure and the complexes they form during function. Techniques for the determination of structures and dynamics of these nanometre assemblies are therefore important for an understanding on the molecular level. PELDOR (pulsed electron-electron double resonance) is a pulsed EPR method that can be used to reliably and precisely measure distances in the range 1.5-8?nm, to unravel orientations and to determine the number of monomers in complexes. In conjunction with site-directed spin labelling, it can be applied to biomolecules of all sizes in aqueous solutions or membranes. PELDOR is therefore complementary to the methods of X-ray crystallography, NMR and FRET (fluorescence resonance energy transfer) and is becoming a powerful method for structural determination of biomolecules. In the present review, the methods of PELDOR are discussed and examples where PELDOR has been used to obtain structural information on biomolecules are summarized.     

Sonic hedgehog maintains the identity of cortical interneuron progenitors in the ventral telencephalon

Fate determination in the mammalian forebrain, where mature phenotypes are often not achieved until postnatal stages of development, has been an elusive topic of study despite its relevance to neuropsychiatric disease. In the ventral telencephalon, major subgroups of cerebral cortical interneurons originate in the medial ganglionic eminence (MGE), where the signaling molecule sonic hedgehog (Shh) continues to be expressed during the period of neuronogenesis. To examine whether Shh regulates cortical interneuron specification, we studied mice harboring conditional mutations in Shh within the neural tube. At embryonic day 12.5, NestinCre:Shh(Fl/Fl) mutants have a relatively normal index of S-phase cells in the MGE, but many of these cells do not co-express the interneuron fate-determining gene Nkx2.1. This effect is reproduced by inhibiting Shh signaling in slice cultures, and the effect can be rescued in NestinCre:Shh(Fl/Fl) slices by the addition of exogenous Shh. By culturing MGE progenitors on a cortical feeder layer, cell fate analyses suggest that Shh signaling maintains Nkx2.1 expression and cortical interneuron fate determination by MGE progenitors. These results are corroborated by the examination of NestinCre:Shh(Fl/Fl) cortex at postnatal day 12, in which there is a dramatic reduction in cell profiles that express somatostatin or parvalbumin. By contrast, analyses of Dlx5/6Cre:Smoothened(Fl/Fl) mutant mice suggest that cell-autonomous hedgehog signaling is not crucial to the migration or differentiation of most cortical interneurons. These results combine in vitro and ex vivo analyses to link embryonic abnormalities in Shh signaling to postnatal alterations in cortical interneuron composition.     

Accounting for linkage disequilibrium among markers in linkage analysis: impact of haplotype frequency estimation and molecular haplotypes for a gene in a candidate region for Alzheimer's disease

OBJECTIVES: Linkage disequilibrium (LD) between closely spaced SNPs can be accommodated in linkage analysis by specifying the multi-SNP haplotype frequencies, if known. Phased haplotypes in candidate regions can provide gold standard haplotype frequency estimates, and may be of inherent interest as markers. We evaluated the effects of different methods of haplotype frequency estimation, and the use of marker phase information, on linkage analysis of a multi-SNP cluster in a candidate region for Alzheimer's disease (AD). METHODS: We performed parametric linkage analysis of a five-SNP cluster in extended pedigrees to compare the use of: (1) haplotype frequencies estimated by molecular phase determination, maximum likelihood estimation, or by assuming linkage equilibrium (LE); (2) AD families or controls as the frequency source; and (3) unphased or molecularly phased SNP data. RESULTS: There was moderate to strong pairwise LD among the five SNPs. Falsely assuming LE substantially inflated the LOD score, but the method of haplotype frequency estimation and particular sample used made little difference provided that LD was accommodated. Use of phased haplotypes produced a modest increase in the LOD score over unphased SNPs. CONCLUSIONS: Ignoring LD between markers can lead to substantially inflated evidence for linkage in LOD score analysis of extended pedigrees with missing data. Use of marker phase information in linkage analysis may be important in disease studies where the costs of family recruitment and phenotyping greatly exceed the costs of phase determination.     

Competitive binding assays for high-affinity binders in the presence of endogenous ligands: application to biotin-binding proteins

Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. We have developed a mathematical model for such systems where radioligand and endogenous ligand are structurally identical. Data which relate radioligand binding at equilibrium as a function of sample volume can be plotted such that the concentrations of endogenous ligand and binder are graphically determined; however, a more precise determination may be done by nonlinear regression with the aid of a microcomputer. The method is demonstrated for the assay of biotin-binding proteins in the presence of a range of endogenous biotin concentrations below and above that required to saturate the binding sites.     

On the three-dimensional reconstruction of icosahedral particles

The problem of reconstructing an isolated particle from its projections, using spherical harmonics to represent the scattering density, is reformulated. A technique that makes explicit and efficient use of the symmetry to describe the angular part of the scattering density is developed in full detail for the particular case of icosahedral symmetry. It provides a very concise method, where the effort is concentrated in the determination of the radial part of the Fourier transform of the particle scattering density. The method allows individual-image model-independent view determination. An illustrative numerical example is given.     

The management of ureteral stone; a guide for physicians in general practice

General practitioners are usually the first to observe a patient with ureteral stone, and often they practice in communities where consultation with a urologist is not immediately available. Study of the records of 423 patients with ureteral stone observed in private practice indicates that by following a definite system for diagnosis, relief of pain, determination of contralateral function, control of infection, and prophylaxis, general practitioners can manage the disease in the great majority of cases, and can determine when treatment by a specialist is imperative.     

In vitro expression and analysis of the 826 human G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in all humanphysiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826humanGPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.     

Defining and solving the essential protein-protein interactions in HIV infection

The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV - host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV.     

A new approach for obtaining sequential assignment of large proteins

A novel NMR experiment for obtaining sequential assignment of large proteins and protein complexes is described. The proposed method takes full advantage of transverse relaxation optimized spectroscopy (TROSY) and utilizes spin-state-selection to distinguish between intraresidual and sequential connectivities in the HNCA-TROSY-type correlation experiment. Thus, the intra- and interresidual cross peaks can be identified without relaying magnetization via carbonyl carbon, which relaxes very rapidly at the high magnetic fields where TROSY is most efficient. In addition, the presented method enables measurement of several scalar and residual dipolar couplings, which can potentially be used for structure determination of large proteins.     

Analyses of sedimentation equilibrium data

A numerical procedure is presented which can quite adequately compute the molecular weight averages as a function of solute concentration from sedimentation equilibrium data for homogeneous systems and for monomer-dimer associating systems with a possible extension to heterogeneous systems where monotonic variation in the weight average molecular weight is observed such as in weakly associating or dissociating systems. The procedure utilizes the method of orthogonal polynomials for curve fitting which allows for a rapid determination of best fit with minimal round off error. The procedure is particularly applicable in cases where the concentration of solute at the meniscus can be considered to be neither appreciable and reasonably well determined as in low speed sedimentation equilibrium experiments, nor essentially zero as in high speed sedimentation equilibrium experiments where the calculations become somewhat more simplified. The use of moderate speed sedimentation equilibrium has the advantage of providing a more broad concentration distribution in the centrifuge cell which yields more extensive information concerning dissociating systems yet still provides results at low solute concentrations where most solutes can be considered to be behaving ideally.     

Testing a method for measuring dissolved oxygen concentrations in microhabitats in fresh water

It was previously known that polyethylene film is permeable to oxygen, and that water-filled polyethylene bags can be used for determination of dissolved oxygen in fresh-water habitats: analysis of oxygen concentrations in the bag water indicates the concentrations in the habitat where the bag has been resting for some time. Field experiments show that the bag method (with minor modifications) is reasonably accurate for ecological studies (a deviation of 0–0.20 ml O₂/l as compared with samples collected with a Ruttner water sampler).The bag method is simple in use. Its main advantage is its usefulness in many microhabitats in fresh-water where other methods may fail. Oxygen deficit in a dense helophyte stand is demonstrated using the bag method.     

Wildlife disease elimination and density dependence

Disease control by managers is a crucial response to emerging wildlife epidemics, yet the means of control may be limited by the method of disease transmission. In particular, it is widely held that population reduction, while effective for controlling diseases that are subject to density-dependent (DD) transmission, is ineffective for controlling diseases that are subject to frequency-dependent (FD) transmission. We investigate control for horizontally transmitted diseases with FD transmission where the control is via culling or harvest that is non-selective with respect to infection and the population can compensate through DD recruitment or survival. Using a mathematical model, we show that culling or harvesting can eradicate the disease, even when transmission dynamics are FD. Eradication can be achieved under FD transmission when DD birth or recruitment induces compensatory growth of new, healthy individuals, which has the net effect of reducing disease prevalence by dilution. We also show that if harvest is used simultaneously with vaccination, and there is high enough transmission coefficient, application of both controls may be less efficient than vaccination alone. We illustrate the effects of these control approaches on disease prevalence for chronic wasting disease in deer where the disease is transmitted directly among deer and through the environment.