Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.     

Isolation and characterization of folded fragments released by Staphylococcal aureus proteinase from the non-histone chromosomal protein HMG-1

HMG-1 was isolated from newborn calf thymus without exposure to overt denaturing conditions. The purified protein was digested under several solvent conditions with the proteinase (endoproteinase GluC) from Staphylococcus aureus strain V8. We found that the preferred site of attack by the enzyme on HMG-1 was influenced markedly by ionic strength and temperature. In 0.35 M NaCl/50 mM Tris-phosphate (pH 7.8) at 37 degrees C, cleavage near the junction between the A and B domains is predominant, as previously reported by Carballo et al. (EMBO J. 2 (1983) 1759-1764). However, in 50 mM Tris-phosphate (pH 7.8) lacking NaCl and at 0 degrees C, cleavage between the B and C domains strongly predominates. Three major products of the digestions were purified and characterized. The fragment consisting of domains B and C was found by circular dichroism to contain a substantial amount of helix. This re-emphasizes the importance of avoiding overt denaturing conditions when working with members of the HMG-1 family.     

DoesAgapetus fuscipes cultivate algae in its case?

The presence of algae within the cases ofAgapetus fuscipes was investigated. Cases recognised as dirty or clean with the naked eye had more and less algal growth, respectively. Larvae in the former survived significantly longer when starved in the laboratory. It is suggested that the presence of algae within the cases would be of ecological advantage during periods of flood.     

Identification of psychrotrophic pseudomonads from goats' milk by computer-assisted analysis of carbon source assimilation data

The results of carbon source assimilation tests on a group of psychrotrophic pseudomonas were compared with published data for established Pseudomonas taxa, using computer-assisted numerical taxonomic analysis and a modified diagnostic computer program. Several phenons were not grouped at the biovar level by numerical taxonomic analysis. Identification of strains by the diagnostic program revealed heterogeneity among those in the unassigned phenons, and supported a continuum concept among the fluorescent pseudomonads.     

A numerical taxonomic study of proteolytic and lipolytic psychrotrophs isolated from caprine milk

Lipolytic and proteolytic psychrotrophs were isolated from raw and pasteurized goats' milk, which had been stored at 5 degrees C for 7 d. The 241 strains isolated and 20 reference strains were examined by 149 biochemical, physiological, and morphological tests. The results yielded 195 characters suitable for taxonomic analysis. Computer-assisted complete linkage analysis, using the Jaccard coefficient, produced 22 phenons at 75% S. The results showed that Pseudomonas fluorescens was the predominant psychrotrophic bacterium, but that Pseudomonas fragi was dominant in some milk samples. Strains of Serratia liquefaciens and Flavobacterium balustinum were also identified.     

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.     

Kinetic analysis of the cerebral creatine kinase reaction under hypoxic and hypoglycaemic conditions in vitro. A 31P-n.m.r. study.

1. The tissue concentration of phosphocreatine (PCr) and the pseudo-first-order rate constant of creatine kinase (kf) were monitored in superfused guinea-pig brain slices in vitro by using 31P-n.m.r. techniques. 2. Superfusion of slices in low oxygen partial pressure (pO2 approx. 16 kPa) decreased tissue PCr concentrations by 48% but ATP concentrations were unchanged. Regression analysis revealed a significant negative correlation between the PCr concentration in hypoxic tissue and the increase in the rate constant, kf. Nevertheless the forward flux through the enzyme (Jf = kf.[PCr]) declined under these conditions. 3. Lowering the glucose concentration to 0.2 or 0.1 mM decreased PCr concentrations by 29% and 48% respectively; here ATP concentrations as well as PCr concentrations also decreased. Only in the presence of the lower glucose concentration (0.1 mM) was kf increased. However, unlike the situation in hypoxic tissue, Jf was maintained at control rates. 4. In spectra obtained in the presence of low oxygen or low glucose concentrations, a resonance attributable to tissue inorganic phosphate became dectectable. This observation is discussed in terms of known changes in tissue phosphate concentrations and possible alterations in cytoplasmic pH.