Quantitative determination of free intracellular alpha-keto acids in neutrophils

For the first time, a procedure is described for the quantitative analysis of free alpha-keto acid content in human neutrophils (PMNs) relative to single cell number by reversed-phase fluorescence high-performance liquid chromatography. The procedure is minimally invasive and is unsurpassed in the quality of PMN separation, ease of sample preparation as well as sample stability. This method can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular alpha-keto acid analysis in particularly for the surveillance of severe diseases as well as cellular or organ dysfunction.     

A method of determining the antioxidative activity of biological matter

A procedure is described for determining antioxidation activity of the water-soluble biological material. The procedure is based on the study of kinetics of the reduced 2.6-dichlorophenolindophenol oxidation by air oxygen both with and without the biological material as well as on calculation of the value for the constant of the biological material inhibition of 2.6-dichlorophenolindophenol oxidation as an index of the biological material antioxidation activity.     

A multiple-tubes approach for accurate genotyping of very small DNA samples by using PCR: statistical considerations.

A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. The procedure involves dividing the sample among several tubes, then amplifying and typing the contents of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded fragments in the sample is as small as 30. The procedure remains effective even in the presence of small amounts of laboratory contamination. We find that the multiple-tubes procedure is superior to the standard one-tube procedure, either when the sample is small or when laboratory contamination is a potential problem; and we recommend its use in these situations. Because the procedure is statistical, it allows the degree of certainty in the result to be quantified and may be useful in other PCR applications as well.     

Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer.

A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.     

Automatic procedures for determining amino acid sequences of peptides.

An automatic procedure is described for determining the amino acid sequences of peptides with various lengths and hydrophobicities in a protein sequenator of the Edman-Begg type. A film consisting of Quadrol salts is left in the cup as a hydrated solid phase on which the peptide partitions during solvent extraction. The partitioning of the peptide is facilitated by using benzene and 1-chlorobutane/acetic acid as the sole extractants after coupling. The reproducibility and efficacy of the procedure is illustrated by the sequences obtained with peptides of from 3–29 residues, including several with a series of hydrophobic residues at the C terminus. The procedure is well suited to the completion of the sequence determination on a large peptide following the normal Edman-Begg procedure for proteins.     

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.     

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures

The SOS Chromotest is a quantitative bacterial colorimetric assay for genotoxins. Substantial validation is now available (Quillardet et al., 1985). We describe here in detail the tester strain as well as the effects of the variation of some parameters on the assay. We report a simple spot-test procedure as well as a new standard procedure which incorporate recent technical improvements aimed at simplifying the assay further.     

Largest contour segmentation: a tool for the localization of spots in confocal images

An accurate determination of the 3-D positions of multiple spots in images obtained by confocal microscopy is essential for the investigation of the spatial distribution of specific components or processes in biological specimens. The position of the centroid, as an estimator for the position of a spot, can be calculated on the basis of all voxels that belong to the domain of the spot. For this calculation a domain that defines which voxels belong to the spot must be delimited. To create a boundary for a domain we developed a 3-D image segmentation procedure: the largest contour segmentation (LCS). This procedure is based on an iterative region-growing procedure around each local maximum of intensity. By means of this procedure the position of each spot was determined accurately and automatically. Qualities of the procedure were evaluated by means of simulated test-images as well as 3-D images of real biological specimens.     

Chemometric modelling based on 2D-fluorescence spectra without a calibration measurement

MOTIVATION: 2D fluorescence spectra provide information from intracellular compounds. Fluorophores like trytophan, tyrosine and phenylalanin as well as NADH and flavins make the corresponding measurement systems very important for bioprocess supervision and control. The evaluation is usually based on chemometric modelling using for their calibration procedure off-line measurements of the desired process variables. Due to the data driven approach lots of off-line measurements are required. Here a methodology is presented, which enables to perform a calibration procedure of chemometric models without any further measurement. RESULTS: The necessary information for the calibration procedure is provided by means of the a priori knowledge about the process, i.e. a mathematical model, whose model parameters are estimated during the calibration procedure, as well as the fact that the substrate should be consumed at the end of the process run. The new methodology for chemometric calibration is applied for a batch cultivation of aerobically grown S. cerevisiae on the glucose Schatzmann medium. As will be presented the chemometric models, which are determined by this method, can be used for prediction during new process runs. AVAILABILITY: The MATHLAB routine is free available on request from the authors.     

Double staining of immunoblot using enzyme histochemistry and India ink.

Immunoblotting is a commonly used technique for the immunodetection of specific proteins which have been fractionated by polyacrylamide gel electrophoresis. We describe here a simple procedure for the double staining of immunoblots, first to detect the immunoreactive component(s) by histochemistry using enzyme-conjugated secondary antibodies, and second to visualize the general protein electrophoretogram using India ink. This procedure permits the direct comparison of electrophoretic mobilities between the immunoreactive protein(s) and the total protein population as well as protein standards of known Mr. The experimental advantage of the procedure is that no additional manipulation of the protein samples or the standards is necessary prior to electrophoretic fractionation. In this report, detection of the vitamin D-dependent calcium-binding protein, calbindin-D28K, is used to illustrate the application of the procedure.     

A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).

A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.     

Analysis of a centric shift in the S11 chromosome of Aiolopus strepens (Orthoptera: Acrididae)

A centric shift in the S₁₁ chromosome of Aiolopus strepens, previously described as a pericentric inversion (Cabrero & Camacho, 1982), was analyzed by using the C-banding procedure. The location of breakage points as well as the posibilities of pericentric inversion and three-break transposition are discussed. The relationship between C-bands and NOR location as revealed by both silver staining and a double procedure Ag-NOR C-banding is also discussed.     

Sugar and Alcohol Stabilization of Yeast in Sweet Wine

Secondary fermentation of sweet wine was prevented by the Delle stabilization procedure. For this procedure, advantage is taken of the inhibitory effects of high concentrations of sugar as well as of alcohol. Thus, relatively small amounts of wine spirits were added to fermenting musts to obtain stability, as compared to the conventional procedure in which larger amounts of alcohol are added and the inhibitory effect of alcohol only is considered. The Delle value is a function of the concentrations in the wine, after spirits addition, of alcohol and sugar. Delle values which gave stable wine were dependent on time of alcohol addition, on strain of wine yeast, and on composition of wine spirits. Fractional addition of spirits, concentration of SO(2), and clarity of must had little effect on the Delle value. Sensory comparison of wines especially prepared for tasting by the Delle procedure and by the conventional procedure showed the wines made by the Delle procedure to be superior in quality. Under proper storage conditions, the Delle wines were shown to be microbiologically stable and resistant to wine spoilage organisms.     

Quantitative fitting of atomic models into observed densities derived by electron microscopy

A new methodology for fitting atomic models into density distributions is described. This approach is based on a global density correlation analysis that can be optionally supplemented by biochemical as well as biophysical data. The procedure is completely general and enables an objective evaluation of the resulting docking in the light of available biochemical and biophysical information as well as density correlation alone. In this paper we describe the implementation of the algorithm and its application to two biological systems. In both cases the procedure provided an interface model on the atomic level and located parts of the structure that were missing in the atomic model but present in the electron-microscopic construct. It also detected and quantified conformational changes in actomyosin complexes.     

Subcutaneous mastectomy. The excess skin problem.

Subcantaneous mastectomy through a lateral approach, with preservation of the nipple and areola on a dermal pedicle, removing the excess skin at the time of aubautaneous mastectomy, is a safe procedure which results in esthetically acceptable breasts. The surgical approach greatly facilitates the removal of the entire glandular portion of the breast. The need for a second surgical procedure is eliminated. Lateral biopsy scars can be reinforced by the dermal sling support, thereby decreasing the chances of exposure of the implant. The implant is also successfully and easily held in position by the use of the dermal-fat sling support. The nipple and areola survive quite well on the dermal pedicle, with preservation of contractility and sensation, as well as of blood supply.     

The use of a multi-channel kalman filter algorithm in structural analysis of the epileptic EEG

An extension of the Kalman filter algorithm to the multi-channel case is presented and its application as a segmenting procedure in the analysis of the epileptic EEG is discussed. An analytical example of structural analysis, using the segments extracted by the proposed filter, is presented for a particular set of 4-channel EEG recordings. This analysis is shown to be especially fruitful if the autoregressive coefficients - a by product of the filtering procedure - are used to estimate the information flow between the channels by the calculation of partial as well as directed coherences for the representative segments.     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

A simple, rapid, and sensitive procedure for the assay of endoproteases using Coomassie brilliant blue G-250

A rapid colorimetric method for the assay of proteolytic enzymes based on the binding of Coomassie brilliant blue G-250 to unhydrolyzed protein substrate is described. Considerable assay time is saved since the method does not require the separation of the hydrolyzed products from the undergraded protein substrate. The procedure is applicable to crude as well as purified preparations of various proteolytic enzymes and compares well with the procedure of M. L. Anson.     

Translation of specific vaccinia virus RNAs purified as RNA-DNA hybrids on potassium iodide gradients.

A procedure has been developed for purifying specific mRNAs by hybridization to fragments of DNA and isolation of the hybrids by potassium iodide equilibrium buoyant density centrifugation. The hybrids obtained are essentially free of unhybridized RNA as well as double-stranded RNA. Moreover, the RNA in the hybrids is undamaged and can be translated in vitro. Application of this procedure to mapping vaccinia virus genes is described. A total of 34 polypeptides have been assigned to three regions of the viral genome.     

Optimized selective N-methylation of peptides on solid support.

Peptides containing N(alpha)-methylamino acids exhibit interesting therapeutic profiles and are increasingly recognized as potentially useful therapeutics. Unfortunately, their synthesis is hampered by the high price and nonavailability of many N(alpha)-methylamino acids. An efficient and practical three-step procedure for selective N-methylation of peptides on solid support is described. The procedure was based on the well known solid-phase N-methylation of N(alpha)-arylsulfonyl peptides, which was improved by using dimethylsulfate and the less expensive DBU as base. Every step of the procedure, amine activation by an o-nitrobenzenesulfonyl group, selective N-methylation and removal of the sulfonamide group, was optimized in respect of time and economy. The described optimized three-step procedure is performed in 35 min without solvent changes, instead of 3 h. Tripeptides (Fmoc-Phe-MeXaa-Leu-OH) containing N-methylated common amino acids were also prepared using the optimized procedure to demonstrate its compatibility with these amino acids. The described procedure allows an efficient synthesis of N(alpha)-methylamino acid containing peptides in a very short time using Fmoc solid-phase peptide synthesis.