The application of a sequential procedure with elimination as a method for the screening for metabolic diseases

A sequential classification procedure with early elimination, for the screening for metabolic diseases, is presented. Asymptotic properties of the procedure are derived in the Appendix and it is shown that the procedure is asymptotically distribution-free under certain assumptions, and asymptotically at least as efficient as a comparable fixed-sample procedure. With the use of data obtained from 36 mentally retarded patients, the procedure was evaluated by means of a bootstrap simulation. The procedure was then applied to this set of data, with satisfactory results and a considerable economy in observations.     

Isolation of glycophorin with deoxycholate

In a previous communication we reported that human erythrocyte glycophorin prepared by the lithium diiodosalicylate phenol procedure contains approximately 10 mol of lithium diiodosalicylate per mol of glycophorin, and further we showed that this bound lithium diiodosalicylate is difficult to remove by detergents or organic solvents (Romans, A.Y. and Segrest, J.P. (1978) Biochim. Biophys. Acta 511, 297–301). In the present communication we report an alternative purification procedure for glycophorin in which sodium deoxycholate is substituted for lithium diiodosalicylate; the sodium deoxycholate is subsequently removed by gel filtration. Utilizing this procedure, 25–30 mg glycophorin are obtained per gram of lyophilized erythrocyte ghosts. The glycophorin prepared by the sodium deoxycholate procedure, after a single gel filtration step, contains less than 1 mol of sodium deoxycholate per mol glycophorin and is colorless compared with glycophorin prepared by the lithium diiodosalicylate procedure, which has a distinct reddish-brown cast.     

Isolation of glycophorin with deoxycholate.

In a previous communication we reported that human erythrocyte glycophorin prepared by the lithium diiodosalicylate phenol procedure contains approximately 10 mol of lithium diiodosalicylate per mol of glycophorin, and further we showed that this bound lithium diiodosalicylate is difficult to remove by detergents or organic solvents (Romans, A.Y. and Segrest, J.P. (1978) Biochim. Biophys. Acta 511, 297-301). In the present communication we report an alternative purification procedure for glycophorin in which sodium deoxycholate is substituted for lithium diiodosalicylate; the sodium deoxycholate is subsequently removed by gel filtration. Utilizing this procedure, 25-30 mg glycophorin are obtained per gram of lyophilized erythrocyte ghosts. The glycophorin prepared by the sodium deoxycholate procedure, after a single gel filtration step, contains less than 1 mol of sodium deoxycholate per mol glycophorin and is colorless compared with glycophorin prepared by the lithium diiodosalicylate procedure, which has a distint reddish-brown cast.     

Analysis of methylthiohydantoins of amino acids by gas-liquid chromatography of their trimethylsilyl derivatives

A procedure is described for the analysis of methylthiohydantoins of amino acids from the Edman degradation of proteins and peptides by gas-liquid chromatography of their trimethylsilyl derivatives. The procedure is applicable to the methylthiohydantoins of all the amino acids commonly found in proteins with the exception of arginine, which did not yield a volatile derivative, and hydroxyproline and hydroxylysine, which were not investigated. Chromatographic separation is achieved in a single run with only one unresolved pair, which can be separated by a supplementary procedure requiring only 4.5 min.     

Radiation diagnosis of venous angiodysplasias

The paper proposes an original procedure for diagnosing venous dysplasias of different sites. The procedure is based on the analysis of the capacities of currently used invasive and noninvasive techniques of radiation visualization. The practical aspects of conduct of routine studies, their potentialities and shortcomings are outlined. The principle of the proposed procedure is a result of a complex use of the capacities of ultrasound visualization and interventional X-ray contrast studies of the vascular system. The procedure has been successfully used in 8 patients. It is concluded that it is promising to examine venous angiodysplasias by means of direct puncture made under ultrasound guidance, followed by mass contrasting and angiography which may be supplemented by sclerosing therapy.     

Frequencies of Twelve Ascus-Types and Arrangement of Three Genes from Tetrad Data

Equations expressing the theoretical frequencies of twelve ascus-types in the tetrad analysis of a triply heterozygous diploid are described. Using these equations, a mapping procedure for a gene X, is proposed. The procedure requires that two genes, X and Y, of the same phenotype be heterozygous and that the map position of Y be known, and that another standard gene, Z, show an independent phenotype from X and Y. This procedure does not require the laborious allelism test of the segregants to determine the allelic 2:2 segregation in tetrads for the X and Y genes, which is indispensable for mapping by the conventional procedure. The exact placement of the X gene on a chromosome is possible by the chi2 minimization procedure in comparison with the expected frequencies of the six ascus-types or four spore-types deduced from the twelve expected ascus-types to give the optimal fit with the observed data.     

A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images

This paper presents a robust two-step segmentation procedure for the study of biofilm structure. Without user intervention, the procedure segments volumetric biofilm images generated by a confocal laser scanning microscopy (CLSM). This automated procedure implements an anisotropic diffusion filter as a preprocessing step and a 3D extension of the Otsu method for thresholding. Applying the anisotropic diffusion filter to even low-contrast CLSM images significantly improves the segmentation obtained with the 3D Otsu method. A comparison of the results for several CLSM data sets demonstrated that the accuracy of this procedure, unlike that of the objective threshold selection algorithm (OTS), is not affected by biofilm coverage levels and thus fills an important gap in developing a robust and objective segmenting procedure. The effectiveness of the present segmentation procedure is shown for CLSM images containing different bacterial strains. The image saturation handling capability of this procedure relaxes the constraints on user-selected gain and intensity settings of a CLSM. Therefore, this two-step procedure provides an automatic and accurate segmentation of biofilms that is independent of biofilm coverage levels and, in turn, lays a solid foundation for achieving objective analysis of biofilm structural parameters.     

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.     

A simple method for the correction of the inverted nipple

A procedure is described in which a direct approach to the underlying pathology of the inverted nipple is undertaken. The inverted nipple is raised as desired by freeing it from the surrounding tissue by vertical and horizontal undermining and then is stabilized by purse-string suture. It can be performed as an office procedure under local anesthesia. This procedure is simple, reliable, not time-consuming, leaves no visible scars, and requires no special or bulky dressing.     

A batchwise purification procedure of neurofilament proteins

A rapid batchwise purification procedure for neurofilament proteins from bovine spinal cord is described. A crude filament fraction can be obtained by treating the tissue with Triton X-100, followed by centrifugation through sucrose. From this crude filament fraction the protein is processed through a batch purification procedure using hydroxyapatite in 8 M urea. With this procedure, approximately 0.5 g of purified neurofilament protein is obtained from a single bovine spinal cord in less than 3 days.     

An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Purification and some properties of rat intestinal ornithine decarboxylase

Ornithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr = 53,000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr = 52,000). Values for the Km for L-ornithine (0.1 mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5 x 10(5) nmol CO2/mg protein/30 min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr = 52,000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.     

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.     

Efficiency of a Two-Stage Procedure for Survival Studies

A two-stage procedure using occurrences of a surrogate endpoint regarding a true clinical endpoint has been proposed (FLANDRE, O'QUIGLEY and BROOKMEYER, 1994). The procedure allows estimation of the survival function S(t) reliably and comparison of treatment groups. The aim of this paper is to investigate the efficiency of such a two-stage procedure compared to a one stage procedure, i.e. a classical study. The efficiency is estimated by the ratio of the standard derivation of S(t) for the classical procedure to the standard derivation of S(t) for the two-stage procedure and represents the precision on the estimation of S(t). Standard deviation of S(t) is approximate by the delta-method. Using the exponential model. some numerical results are presented.     

Comment on analyzing flow cytometric data for comparison of mean values of the coefficient of variation of the G1 peak.

BACKGROUND: The coefficient of variation (CV) is often used to characterize and summarize the flow cytometry analysis of nuclear DNA of the Go/G1 peak in a cell population within an individual organism. CV values are frequently used in subsequent statistical analysis to compare experimental groups of individuals. METHODS: We explain why the conventional analysis of variance, linear comparisons and regressions that employ the F and t-tests are not appropriate for analyzing CV data sets. The weighted least squares procedure which utilizes the chi-square test is presented as an adequate method. We further explain why this type of data needs to be analyzed by this procedure. RESULTS: To illustrate the application of the weighted least squares procedure, we analyzed a real data set that had been previously analyzed by conventional methods. We found that a non-significant result (p = 1) using the latter was significant when re-analyzed with the weighted least squares procedure (p = 0.032). CONCLUSIONS: Significant differences between treatments established by the weighted least squares often go unidentified by the conventional analysis. Use of the weighted least squares procedure is recommended for analyzing CV data sets.     

A Procedure for Clustering Means of Unequal-Sized Samples in ANOVA

The proposed procedure “SECOLICO” is based on the sequential construction of linear contrasts. After an analysis of variance the procedure is able to classify the treatments represented by mean values of unequal-sized samples made at random into distinguishable groups. For the purpose of illustrating the procedure “SECOLICO” a simplified example is given.     

A simple procedure for separating the subunits of ovine and bovine pituitary interstitial cell stimulating hormone

A simple, effective procedure for separating ovine or bovine interstitial cell stimulating hormone into their subunits has been described with a yield of 70–80%. The β subunit isolated by the new procedure behaves differently from that obtained by the countercurrent distribution method, and is in a more native state. In addition, full biological activity is consistently restored upon recombination of the subunits isolated by the new procedure.     

The determination of folylpolyglutamate synthetase.

A relatively rapid radiochemical procedure for the determination of folylpolyglutamate synthetase activity is presented in this communication. The procedure is based on measurement of the incorporation of radioactive l-glutamate into tetrahydropteroylpolyglutamate on incubation with a tetrahydrofolate. After deproteinating the incubation mixtures with trichloroacetic acid, folate is separated from radioactive glutamate by an adaptation of a procedure generally employed in the isolation of folate from natural materials, i.e., adsorption on columns of charcoal from which it is subsequently eluted with aqueous-alcoholic ammonia containing mercaptoethanol and counted. The procedure is applicable to monitoring purification of the enzymes and to the study of their properties.The technique for separating a radioactive product of enzyme action from a radioactive precursor with a column of charcoal, that has been developed for this procedure is applicable also to other radiochemical enzyme determinations requiring the separation of an aromatic from an aliphatic metabolite.     

On the validity of using semilogarithmic plots to determine initial velocities of enzyme-catalyzed reactions

It has been proposed (Johnston & Diven, 1969a) that it is valid to use semilogarithmic (first-order) plots of the extent of reaction versus time for graphic determination of initial velocities of enzyme-catalyzed reactions. This proposition suggests the assumption that the initial velocity error expected to be introduced by the proposed procedure is smaller than or comparable to the error introduced by the customary graphic procedure. The latter is based on the assumption that the progress curves of enzyme-catalyzed reactions have an initial linear segment of sufficient duration to permit accurate determination of slope. The validity of the procedure proposed by Johnston and Diven is examined in this report. It is concluded that the procedure is applicable to a very small class of enzyme-catalyzed reactions and only under certain experimental conditions.