Localization of biotransformational enzymes along the crypt-villus axis of the rat intestine. Evaluation of two cell isolation procedures

Rat intestinal epithelial cells were isolated by EDTA-chelation, combined with gentle shaking (modified Weiser procedure) or with strong longitudinal vibration (Harrison/Webster procedure). Both methods yield large numbers of viable cells and are relatively easy to use. Electronmicroscopical and biochemical data indicate that cell fractions from different levels of the villous region can be obtained only by the modified Weiser procedure. When strong mechanical forces are involved (Harrison-Webster procedure) the villus epithelium is released according to an all-or-nothing process. The biotransformational capacity of cell fractions, obtained from different levels of the villi by the modified Weiser procedure, was investigated. It was shown that the rate of metabolism of 7-ethoxycoumarin and 1-naphthol was substantially higher in lower villous cells than in cells isolated from the upper villous region. O-Deethylation of 7-ethoxycoumarin decreases from 145 +/- 13 pmole/min mg cell protein (72 +/- 4% conjugated) in lower villous cells to 62 +/- 12 pmole/min mg cell protein (37 +/- 6% conjugated) in tip cells. Glucuronidation of 1-naphthol decreased from 495 +/- 23 pmole/min mg cell protein (lower villous cells) to 137 +/- 13 pmole/min mg cell protein (tip cells).     

Evaluation of different DNA sampling techniques for the application of the real-time PCR method for the quantification of cyanobacteria in water

AIMS: To evaluate different types of sample storage and DNA extraction techniques for the real-time PCR quantification of cyanobacteria in water. METHODS AND RESULTS: Two different filter types for the cell harvest of Microcystis sp. and Planktothrix spp. that were either freeze-dried or stored frozen, and two different methods for DNA extraction were compared. DNA extraction was achieved by standard phenol-chloroform extraction or by a faster commercially available purification kit (DNeasy, QIAGEN). In general there was good agreement between the cell number equivalents of phycocyanin (PC) genotypes that were estimated using the Taq nuclease assay (TNA) between both filter types and the storing of samples. The standard DNA extraction procedure gave higher numbers of PC genotypes when compared with the DNeasy procedure. TNA results obtained from Planktothrix from natural samples extracted with the standard procedure revealed a significant correlation with the cell numbers estimated via the microscope. CONCLUSIONS: Freeze-drying of samples gives quantifiable data. The standard DNA extraction is considered to be the most reliable and accurate, although the DNeasy procedure is useful for early warning monitoring. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of quantitative genotype analysis in cyanobacteria from freeze-dried samples collected during recent and past sampling programmes.     

Parametric variance function estimation for nonnormal repeated measurement data.

Zeger and Liang (1986, Biometrics 42, 121-130) proposed a procedure for analyzing nonnormal longitudinal data in the context of the generalized linear model. This procedure is extended to model variance heterogeneity, allowing the observations to come from distributions with different scale parameters. Loss of efficiency is evaluated when heterogeneity of scale factor is ignored.     

Comparison of Methods for Coccidioidomycosis Complement Fixation

A Laboratory Branch Task Force of the National Communicable Disease Center has proposed a standardized complement fixation procedure (LBCF) and an adaptation of this to microtitration techniques (MT) as uniform methods for performing complement fixation (CF) tests. A common procedure should make CF results from one laboratory more comparable to another. In addition, it would be preferable if the common procedure reproduced the titer levels of a testing procedure which is to be replaced, particularly when valid clinical interpretations have been derived from the latter. Replicated sets of sera were tested by the LBCF, MT, and the standard Smith CF procedure for coccidioidomycosis. Results with all three procedures were highly reproducible within an acceptable one-tube variation of a twofold dilution series, but the frequency of one-tube variations was greater with the MT method than with the other two. There was no statistical difference in the titers obtained with the Smith and LBCF procedures, but there was a significant difference when the MT results were compared to those with the Smith method. The LBCF method should be acceptable as a standardized and uniform CF procedure for coccidioidomycosis, subject to comparative testing between different laboratories.     

Normalizing upper trapezius EMG amplitude: Comparison of ramp and constant force procedures

The present study compared three procedures for normalization of upper trapezius surface electromyographic (EMG) amplitudes: (a) a ramp procedure (providing data in per cent of maximal voluntary contraction, MVC); (b) a constant force procedure based on two reference contractions (two-force procedure) (%MVC) and (c) a procedure expressing muscle activation in per cent of a reference voluntary electrical activity (%RVE). The study also evaluated the repeatability of the ramp and the RVE procedures and estimated the force exertion (%MVC) corresponding to the RVE. To illustrate the ergonomic effect of different normalization procedures, trapezius EMG during two work tasks was compared after normalization by the two-force and the RVE procedures. Fifteen subjects participated in the whole study. We found that force estimates obtained by the ramp procedure equation could be translated to force estimates obtained by the two-force procedure by the equation: %MVC_2force = − 0.6 + 0.9*%MVC_ramp, although with a considerable imprecision due to large inter-individual differences. In the ramp procedure, the intra-individual test-retest coefficient of variation (CV) depended on the force level; it was 45% at 5% MVC and 10% at 30% MVC. The CV of the RVE was 15%. The reference contraction used in the RVE procedure corresponded from 13–79% MVC (median 33%MVC). The load reducing effect of an ergonomic intervention was less obvious with the RVE procedure than with the two-force procedure due to a larger inter-individual variation. The advantages and disadvantages of the different procedures are discussed.     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

Efficient transfection and expression of heterologous genes in PC12 cells

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.     

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.     

Classical molecular interaction potentials: improved setup procedure in molecular dynamics simulations of proteins.

The latest version of the classical molecular interaction potential (CMIP) has the ability to predict the position of crystallographic waters in several proteins with great accuracy. This article analyzes the ability of the CMIP functional to improve the setup procedure of the molecular system in molecular dynamics (MD) simulations of proteins. To this end, the CMIP strategy is used to include both water molecules and counterions in different protein systems. The structural details of the configurations sampled from trajectories obtained using the CMIP setup procedure are compared with those obtained from trajectories derived from a standard equilibration process. The results show that standard MD simulations can lead to artifactual results, which are avoided using the CMIP setup procedure. Because the CMIP is easy to implement at a low computational cost, it can be very useful in obtaining reliable MD trajectories.     

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

Purification and characterization of osteopontin from human milk

Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.     

Construction of integrated genetic linkage maps by means of a new computer package: Join Map

A computerized procedure to contruct integrate genetic maps is presented. The computer program (J oin M ap ) can handle raw data from F₂s, backcrosses and recombinant inbred lines, as well as listed pair-wise recombination frequencies. The procedure is useful for combining linkage data that have been collected in different experiments; the result is a mathematical alignment of the distinct genetic maps. Data from single experiments can be dealt with as well. In view of the fast growing amount of linkage information for molecular markers, which is often being generated by different research groups, integrated maps provide useful information on the map position of genes and DNA markers.
The procedure performs a sequential build-up of the map and, at each step, a numerical search for the best fitting order of markers. Weighted least squares is used for the estimation of map distances.     

Application of short column gel permeation in the study of protein-protein interactions

A simple and rapid procedure based on the gel filtration principle is described together with its applicability to the study of protein-protein interactions including subunit-subunit and enzyme-enzyme interactions. Using this procedure, it is shown that phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) interact with a stoichiometry of one PGK molecule combining with one monomeric subunit of GPDH. This interaction has been observed with both enzymes being from the same, as well as from different, species. The K_d values for rabbit muscle PGK and porcine muscle GPDH complex and that for the rabbit muscle PGK and yeast GPDH complex are found to be (4.5 ± 2.0) × 10⁻⁷ M and (6.5 ± 1.7) × 10⁻⁷ M, respectively. The specificity of bienzyme association is stronger when enzymes are from the same species than when they are from different species.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.     

猪口蹄疫病毒与猪肠道病毒在细胞培养中的基因重组——病毒重组及一个重组株的初步鉴定

利用两株不同理化和生物学特性的不同属小RNA病毒,即口蹄疫病毒(FMDV)与猪肠道病毒(EV),建立了病毒基因重组,克隆筛选和鉴定的方法,初步证实,在实验室条件下它们可以进行基因重组,本文为研究自然界病毒变异机理,不同属病毒的基因重组提供了依据。     

A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains

In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.     

Development of an ELISA for Evaluation of Swab Recovery Efficiencies of Bovine Serum Albumin

After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32–80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0–60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.     

Heterogeneous inbred family (HIF) analysis: a method for developing near-isogenic lines that differ at quantitative trait loci

Abtract  Analysis of near-isogenic lines (NILs) that differ at quantitative trait loci (QTL) can be an effective approach for the detailed mapping and characterization of individual loci. Although NILs are useful for genetic and physiological studies, the time and effort required to develop these lines have limited their use. Here we describe a procedure to identify NILs for any region of the genome that can be analyzed with molecular or other genetic markers. The procedure utilizes molecular markers to identify heterogeneous inbred families (HIFs) that segregate for a genomic region of interest. Each HIF is isogenic at the majority of loci in the genome, but NILs differing for markers linked to QTL of interest can be extracted from segregating families. The application of this procedure is described for two QTL associated with seed weight in sorghum. A population of 98 HIFs was screened with two RAPD markers from different linkage groups that were associated with seed weight. Three segregating families were identified for each marker. The progeny of these HIFs were characterized for the segregation of seed weight and other yield components and for markers flanking each QTL. NILs derived from each HIF had significantly different seed weights confirming the presence of at least two loci that influence seed weight in sorghum. Received: 16 September 1996 / Accepted: 25 April 1997     

clonality V.0.4: a randomization-based program to test for heterozygosity-genet size relationships in clonal organisms

clonality V.0.4 is a program for testing heterozygosity-genet size relationships in clonal organisms using a randomization procedure. The software has been developed under the Borland Delphi developing environment and a Windows-executable version is freely downloadable from http://gemi.mpl.ird.fr/SiteSGASS/Prugnolle/ClonalityPage.html. The program compares the observed F(IS) of the population with the F(IS) expected if genets (multilocus genotypes present in multiple copies within the population) were chosen randomly from the set of different multilocus genotypes. The randomization procedure is performed with the same number of genets and the same number of repetitions per genet as what is observed in the original data set.