Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.     

A PCR-based method for high stringency screening of DNA libraries.

A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described. A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria. Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled. The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers. A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe. This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol. A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR. Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques. PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert. This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal. Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.     

Development of an equation for potentiometric titration of polyelectrolytes using a periodic lattice model. A numerical analysis of interactions among ionizable groups

A numerical method is presented for analysing the potentiometric titration behavior of linear polyelectrolytes. A polyelectrolyte molecule is treated as a one-dimensional lattice containing a large number of lattice points, each of which has an identical ionizable group. In this method, the polyelectrolyte model lattice is divided into identical repeating unit cell systems with a finite number of ionizable groups to calculate the thermodynamic partition function of the system of polyelectrolyte solution. The electrostatic interaction between ionized groups adopted in the present study is given by the Debye-Hückel type screened Coulombic potential. The titration behavior as well as several thermodynamic quantities is derived by making a canonical ensemble summation of all states in a unit cell system under an appropriate periodic boundary condition. This method serves as a model of the two-step ionization often appearing in polyions with strong neighboring interactions such as homo- and copolymers of maleic acid. Several characteristics of the titration behavior of these polyelectrolytes are well reproduced by using a lattice model with pairwise intervals, including the effects of hydrogen bond formation and change in dielectric constant of the medium around an ionizable group. In addition, this method is valid for a more detailed analysis of the titration behavior of polyelectrolytes with various kinds of arrangements of charged groups.     

Behaviour studies of the fungicide cymoxanil in two strains of the fungus Botrytis cinerea and in haemolymph of locust and lobster: I. In situ monitoring by internal surface reversed-phase high-performance liquid chromatography

A method for following the metabolism of the fungicide cymoxanil in various biological media is described. By using a recently developed high-performance liquid chromatographic method, with an internal surface reversed-phase column, it is unnecessary to clean up the sample before analysis. Thus this technique makes monitoring in fungi as well as in arthropod haemolymph easier and faster.     

Evoked potential measurement and analysis on a small laboratory computer

A program is described for the collection and subsequent analysis of somatosensory evoked potentials using a LINC-8 computer. The program allows simple evoked-potentials analysis in centers where a small laboratory computer may be available but sophisticated instrumentation such as a computer of average transients is not available. This program provides an efficient method of easily obtaining information concerning the conduction pathways of the nervous system as well as the cerebral function; the program can be implemented on small laboratory computers which most hospitals currently own, without the associated cost or complexity of additional hardware in the laboratory. Combining utilization of a small laboratory computer with an easily programmable method provides an approach for evoked potential analysis which is well within the financial and technical scope of most neurophysiology laboratories.     

Biotechnological production of pyruvic acid

Pyruvic acid is an important organic acid widely used in the chemical and drug, as well as agrochemical, industries. Compared with the chemical method, biotechnological production of pyruvic acid is an alternative approach because of the low cost. An overview of biotechnological production of pyruvate, including direct fermentative production employing eukaryotic and prokaryotic microorganisms, production by a resting cell method and an enzymatic method as well as the recovery of pyruvate, is discussed. A multi-vitamin auxotrophic yeast strain, Torulopsis glabrata. has been used in the commercial production of pyruvate; emphasis is therefore placed on the mechanism and characteristics of pyruvate production by this strain.     

Détection de l’activation cérébrale : de la spectroscopie à l’imagerie proche infrarouge à fenêtre temporelle

Near infrared spectroscopy is commonly used both in clinical practice and in the biomedical research field in order to monitor tissue perfusion and oxygenation, especially during cerebral activation. A new method is proposed to select detected photons according to their time of flight within tissues. Processing only late photons improves detection sensitivity as well as discrimination between cerebral activation and skin fluctuations. Processing of temporal point spread functions as well as an instrumentation for non contact-volume acquisitions are still in progress and could be the basis of a spectroscopy imaging method.     

Model studies of nonsteady flow using magnetic resonance imaging

A bolus-tracking magnetic resonance imaging (MRI) method has been employed to measure velocity profiles for oscillatory flow with and without a steady flow component as well as pulsatile flow in an axisymmetric tube model. A range of flow conditions within normal physiological limits was tested. The imaged velocity profiles were observed to be generally in accord with theoretical predictions. Instantaneous flow rates calculated from the MR images agreed well with those assessed using an ultrasonic flowmeter. Because MRI is noninvasive and poses few risks to subjects, this technique is potentially useful for studying vascular hemodynamics in vivo.     

Profile analysis of 24-hours measurements of blood pressure

A method is proposed for classifying subjects according to their convex, flat, or concave change patterns of 24-hours blood pressure measurements. To obtain such a classification is useful for detecting subjects who show abnormal change patterns and giving them appropriate medical treatments. Therefore, an appropriate statistic is proposed for detecting a systematic change along the time axis, as well as a statistic with its inverse characteristic appropriate for evaluating the noise variation. The method is based on the ratio of those two types of statistics; it is verified to work well on real data, giving a classification of subjects into four types of subgroups: extreme dipper, dipper, nondipper, and inverted dipper. It also suggests that there might be an ultra-extreme dipper subgroup.     

An Alcian blue (pH 2.5)-PAS-keratin immunoperoxidase method for the simultaneous demonstration of keratin and neutral and acidic mucosubstances

A method is proposed for the simultaneous staining of neutral and acidic, periodate reactive and nonperiodate reactive mucosubstances, glycogen and keratin in paraffin sections. Briefly, sections are stained by the Alcian blue (pH 2.5)-PAS method, followed by a peroxidase-antiperoxidase immunohistochemical stain for keratin. The proposed method modifies an existing method, and expands the range of polysaccharides and mucosubstances which may be demonstrated. The proposed method is easily performed within a single working day and promises to be of value in surgical pathology as well as in studies of bronchial carcinogenesis.     

An Alcian Blue (pH 2.5)-Pas-Keratin Immunoperoxidase Method for the Simultaneous Demonstration of Keratin and Neutral and Acidic Mucosubstances

A method is proposed for the simultaneous staining of neutral and acidic, periodate reactive and nonperiodate reactive mucosubstances, glycogen and keratin in paraffin sections. Briefly, sections are stained by the Alcian blue (pH 2.5)-PAS method, followed by a peroxidase-antiperoxidase imiminohistochemical stain for keratin. The proposed method modifies an existing method, and expands the range of polysaccharides and mucosubstances which may be demonstrated. The proposed method is easily performed within a single working day and promises to be of value in surgical pathology as well as in studies of bronchial carcinogenesis.     

Non-denaturing polyacrylamide gradient gel electrophoresis for the diagnosis of dysbetalipoproteinemia

Dysbetalipoproteinemia, an uncommon but highly atherogenic mixed hyperlipidemia due to the accumulation of remnants of triglyceride-rich lipoproteins, is characterized by cholesterol-enriched VLDL that migrates in the beta-position on agarose gels. The demonstration of a broad beta-band on agarose gel electrophoresis of plasma is an insensitive method and ultracentrifugation is an impractical method of diagnosing this condition. Non-denaturing polyacrylamide gradient gel electrophoresis (PGGE) was investigated as a screening method for the diagnosis of dysbetalipoproteinemia. A minigel procedure separating the Sudan Black prestained apolipoprotein B (apoB)-containing lipoproteins on a 2-8% polyacrylamide gel at 4 degrees C overnight was analyzed for ultracentrifugally and genetically proven dysbetalipoproteinemic subjects as well as matched controls for mixed hyperlipidemia. Visual inspection revealed that the presence of only small VLDL- and IDL-like particles in untreated patients was highly sensitive (72%) and specific (95%) for dysbetalipoproteinemia. Videodensitometric analysis of area under the curve for large and small VLDL, as well as IDL and LDL, permitted even better discrimination in subjects whose profiles included some staining in the LDL-like region. A ratio of area under the curve of more than 0.5 for IDL-LDL allowed for a specificity of 100% and a sensitivity of 89% for the diagnosis of dysbetalipoproteinemia. This modified PGGE system may be useful in screening for dysbetalipoproteinemia.     

A FORTRAN program for testing trend and homogeneity in proportions

A FORTRAN program is provided for testing linear trend and homogeneity in proportions. Trend is evaluated by the Cochran-Armitage method and homogeneity is tested by an overall X2 test as well by multiple pairwise comparisons by the Fisher-Irwin exact method. The program should be easy to implement on any size of computer with a FORTRAN compiler.     

Results of testing a new method of using antibiotics in nutrient media for the isolation of Shigella: the 2-streak method]

A new variant of media with antibiotics for isolation of Shigella, i.e. a method of 2 streaks each containing different antibiotics was tested in analysis of excrements from patients with acute dysentery. It was found that the new method is more effective than the well known method of gradient plates (isolation of Shigella in one series of the experiments amounted to 85.2 and 64.7 per cent respectively, and in the other series of the experiments the respective figures were 95.4 and 89.3 per cent). Its efficiency was lower as compared to the procedure of inoculation onto 2 plates, i.e. onto the media with and without an antibiotic (isolation of Shigella was 67.5 and 77.4 per cent respectively). The new method provided a higher frequency of Shigella isolation as compared to inoculation onto the media without an antibiotic, as well as onto any of the media used with one antibiotic. The method of 2 streaks offers wider possibilities for choosing the antibiotics for adding to the nutrient medium, as well as estimation of the antibioticograms and species structures of Shigella distributed in a concrete area.     

“Virtues” of apartheid

The traditional clinical method, which has served medicine well for over 100 years, had its origins in the integration of physical examination with morbid anatomy in early 19th-century France. Now this method is showing signs of failing to meet some contemporary needs. In particular, there is no means for understanding the inner experience of patients. Previous models of a transformed method have not grappled with the problem of validation. Data on the inner experience of patients are not open to empiric validation in the same way as clinical data. The process of understanding the meaning of an illness is not, therefore, scientific in the conventional sense. There are, none the less, rigorous methods for validating the results of this form of inquiry, notably those of phenomenology. A transformed method should aim to understand the meaning of an illness for the patient as well as provide a clinical diagnosis. The transformation will require a change in the epistemology of medicine and an educational process that encourages reflection and growth of self-knowledge.     

On the determination of optimal levels in phytosociological classification

A method is introduced to compare results of a clustering technique at different levels of abstraction, or of different clustering techniques. The method emphasizes within cluster homogeneity as well as discontinuities between clusters. It has been derived from Hogeweg's method with some important changes. First each cluster is handled separately to determine the ratio between homogeneity and similarity to the nearest neighbour cluster. For a given clustering a weighted average value is computed over all clusters. This average value is standardized using an expected average value for a cluster configuration with the same number of clusters having the same sizes. A low level of the ratio between expected and observed values is supposed to indicate an optimal clustering. A derivation of the criterion is given and results from three sets of data with different properties are evaluated.     

Model based calculation of substrate/inducer feed-rate profiles in fed-batch processes for recombinant protein production

A model-based feed-rate profile optimization problem is discussed for the fed-batch recombinant protein production. Two optimization procedures, an evolutionary programming technique and a simplified method using the dynamic programming concept, are discussed and compared. Modeling as well as experimental results are presented.     

On the theory of Cherenkov emission in a plasma

The Cherenkov emission of transverse-longitudinal waves in an anisotropic plasma is considered by applying a Hamiltonian method and by drawing an analogy between the equations for the Cherenkov emission of purely transverse and purely longitudinal waves in isotropic media and the equations for the emission of transverse-longitudinal electromagnetic waves in a highly anisotropic medium (a magnetized plasma). A formula for the emitted power is derived, as well as an expression for the directional pattern of the emitted waves in an anisotropic plasma.     

Detection of breaking points in helices linking separate domains

A novel method is proposed to predict whether two domains connected by a helical link can mutually reorient themselves, as well as where the helix can be distorted to allow the domain-domain movements. The method, based on analysis of the variation of the a.d.p. values along the helix link, is applied to three proteins--calmodulin, lysozyme, and hemagglutinin--for which both the domain-domain flexibility and the helix fragment responsible for it are well documented. The helix regions that are variously distorted to permit domain-domain reorientation are well predicted. The method is also applied to colicin Ia and shows that an inter-domain rearrangement can take place as previously postulated. The prediction of the helix breaking point should prove useful in interpreting structural data and in defining the domain borders automatically for proteins built by domains connected by helical links.