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H B Fackrell

Publications and source records attributed to H B Fackrell.

At least 19 recordsLinked to original sources
PubMed1998

Senescent erythrocytes exhibit a single-hit response to staphylococcal alpha toxin.

In vitro aged rabbit erythrocytes are 2- to 3-fold less sensitive than fresh erythrocytes to lysis by the alpha toxin of Staphylococcus aureus. Previously, we correlated the loss in hemolytic sensitivity as cells aged with the proteolytic degradation of Band 3, a putative binding site for staphylococcal alpha toxin. Here we separated young and old erythrocytes by density gradient centrifugation and compared their patterns of lysis by alpha toxin. Derivative plots of lytic-time curves exhibited two lytic peaks for young erythrocytes versus a single peak for the aged erythrocytes. Hit analysis showed that the first peak in the young cells was generated by the monomeric form of the toxin while the second peak involved the polymeric form. Similar analysis of the single peak in the aged erythrocytes showed that only a monomeric form of the toxin is involved in the lysis. Furthermore, analysis of alpha toxin receptor (Band 3) indicated that the receptor is gradually lost as erythrocytes age. This loss in Band 3 is proportional to the decrease in the rate of lysis by alpha toxin. Based on these comparisons we suggest that the intact Band 3 promotes polymeric assembly of the alpha toxin which becomes disrupted by the age-related changes occurring in the receptor.

Animals↗
PubMed1994

Differential action of staphylococcal alpha-toxin on young and old erythrocytes.

Band 3 of rabbit erythrocytes is a binding site for staphylococcal alpha-toxin. Young erythrocytes have a functionally intact band 3 of 100 kD that is hydrolysed as the cells age into a 35-kD fragment. Consequently, aging of erythrocytes renders them more resistant to the action of the toxin. Thus the age of a blood sample will reduce the sensitivity of the hemolytic assay and contribute to the variation of this assay.

Animals↗
PubMed1991

Age-related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ.

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.

Aging↗
PubMed1990

POLAR: a menu driven computerized analysis of fluorescence polarization data.

While fluorescence polarization is a powerful tool for the measurement of ligand-receptor (e.g. antigen-antibody) interactions, its application in routine clinical chemistry and immunochemistry has been limited because collection of data was error prone, and because analysis of data previously required tedious and imprecise manual graphical methods. We present the series of linear and curvilinear regressions required to calculate parameters (p', Q', pf, Qf, Pb, Qb, Fb max, a and Ko) and their corresponding standard errors. The values of a and Ko are comparable to those obtained by the method of finding the minimal the sum of squares. A menu operated computer program that uses these regressions, simplifies the collection, analysis and presentation of fluorescence polarization data. A simple spread sheet system permits the insertion, deletion or modification of data. The results are presented in graphical and tabular form, either on a monitor, a printer or an X-Y plotter. The system has its own graphical routines.

Algorithms↗
PubMed1990

Image analysis of biological systems with microcomputers.

A microcomputer based system was developed to acquire and analyse images of biological specimens for the purpose of identification, classification and quantification. The system uses a video camera to capture images of biological samples either directly or through a microscope. The signal from the camera is digitized by an imaging board for subsequent processing through a PC-XT. From the processed image, simple features such as the cell enclosed area and the cell optical density were extracted for the purpose of cell identification or quantification. Two examples: (a) the quantification of DNA in blood cells and (b) the identification of hybridoma cells are given to illustrate the use of the system. The method is versatile and can easily be modified to adapt to other biological applications.

Algorithms↗
PubMed1987

Inhibition of staphylococcal alpha-toxin by covalent modification of an arginine residue.

The effects of 1,2-cyclohexanedione and phenylglyoxal on staphylococcal alpha-toxin were studied. Modification of one arginine residue in alpha-toxin was sufficient to render the toxin nonhemolytic with no conformational change. Modified alpha-toxin did not protect cells from hemolysis by native alpha-toxin. An arginine residue is therefore at or near the binding site of alpha-toxin. Trypsin digestion of modified alpha-toxin generated a 20 kDa fragment which was isolated using a boric acid gel column. Upon regeneration, this 20 kDa fragment was not recognized by a population of antibodies which prevented alpha-toxin binding. The fragment was recognized by antibodies directed against post-binding events. However, the antibinding antibodies recognized the intact modified toxin. This leads us to conclude that antibinding determinants are not found directly in the binding site or are conformationally masked.

Animals↗
PubMed1987

The Menu Workbench: an automatic menu generator for bio-medical programs.

A program development tool, The Menu Workbench, is described and applied to the computer screen architecture, menu design, and program management of bio-medical programs. Prerequisites for the approach to these problems are presented. An intuitive screen menu system, developed for the program, Immuno-Ligand Analysis System (ILAS), is described in the text. Standardized screen layout and keyboard interaction is explained. The programming of this system of menus was automated with the program, Menu Workbench, in the Turbo PASCAL language in PCDOS, MSDOS and CP/M operating systems. Use of the Menu Workbench program in other bio-medical applications is discussed.

Algorithms↗
PubMed1984

Effect of staphylococcal alpha-hemolysin upon anion transport in the rabbit erythrocyte.

Equilibrium exchange of SO4(2-) was measured prior to and during hemolysis in rabbit erythrocytes exposed to staphylococcal alpha-hemolysin. The anion-transport protein of the rabbit erythrocyte has also been identified. Equilibrium exchange of SO4(2-) was measured by both efflux and influx of 35SO4(2-). The rate of influx of SO4(2-) in rabbit erythrocytes exposed to alpha-hemolysin was twice that of the untreated cells. The rate of SO4(2-) efflux was unchanged by alpha-hemolysin. Inhibition of anion exchange with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) did not inhibit hemolysis, therefore, the increased influx of SO4(2-) may occur through a DIDS-insensitive pathway.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Enzyme-linked immunosorbent assay for detection of Staphylococcus aureus alpha-toxin.

A sandwich enzyme-linked immunosorbent assay was developed for measuring Staphylococcus aureus alpha-toxin. This assay was 500 to 1,000 times more sensitive than the commonly used hemolytic titration assay and was less variable. The binding of alpha-toxin to the adsorbed antibody was most effective after an overnight incubation at 27 degrees C. The toxin was detectable even at a log2 17 dilution of an S. aureus culture supernatant.

Bacterial Toxins↗
PubMed1982

Complement fixation by antibodies to the alpha toxin of Staphylococcus aureus.

When rabbits were injected with the heat-denatured alpha toxin (toxoid) of Staphylococcus aureus, the immune response was demonstrated by an increase in antitoxin that fixed complement. Such antitoxin was detected in 72% of normal human sera. After fractionation of the antitoxin into two types (the antibinding antibodies and the indirect hemagglutinating antibodies), both types of antibodies were found to fix complement in the standard serological complement fixation test. In addition, the indirect hemagglutinating antibodies were capable of fixing complement when the antigen (alpha toxin or toxoid) was covalently or noncovalently bound to erythrocyte membranes. The fixation of complement by membrane-bound immune complexes did not result in lysis of the carrier erythrocytes. The prevalence of complement-fixing antitoxin in normal humans and animals raised the concern that the outcome of in vivo experiments involving alpha toxin could be influenced by the immune status of the host.

Animals↗
PubMed1980

Rabbit erythrocyte band 3: a receptor for staphylococcal alpha toxin.

Enzymes known to specifically cleave the band 3 component of the rabbit erythrocyte membrane were found to reduce both the hemolytic sensitivity to and the binding of the alpha toxin of Staphylococcus aureus. Lectins which bind to band 3 also inhibited the toxin. Lectins which do not bind to band 3 have no effect. Purified band 3, isolated by affinity chromatography on a concanavalin A column, was homogeneous by polyacrylamide gel electrophoresis, had a molecular weight of 100 000, and inhibited the hemolytic activity of alpha toxin. Antibodies to the toxin-toxoid receptor were serologically indistinguishable from antiband 3.

Animals↗
PubMed1979

Immunologic evidence that staphylococcal alpha toxin is oriented on membranes.

Antibodies to staphylococcal alpha toxin were separated into two distinct populations. One population prevented binding of alpha toxin onto erythrocyte membranes. The other population neutralized after the toxin was bound onto erythrocytes and thereby brought about an indirect hemagglutination reaction. The data suggest that alpha toxin has a membrane-binding region.

Animals↗
PubMed1979

The binding of fluorescein-labelled stapbylococcal alpha toxoid to erythrocytes.

Erythrocytes of different animal species have variable hemolytic sensitivity to staphylococcal alpha toxin. Specific and non-specific binding of toxin was measured using fluorescein-labelled toxoid. These studies indicate that toxoid binding to erythrocytes increases with concentration for all species tested. Scatchard plot analyses of 35 animals representing seven species indicate that rabbit, pig, cow, and chicken erythrocytes possess 125 980, 103 920, 82 500, and 41 200 receptors per cell, respectively. The number of receptors remains constant over a period of at least 10 days. No detectable receptors were found for human, rat, and guinea pig erythrocytes. A correlation coefficient of 0.992 exists between receptor number and hemolytic sensitivity for those species having receptors. Variation in hemolytic sensitivity is governed by receptor number and not by variation in the dissociation constant. A threshold sensitivity of 37 000 receptors per cell has been calculated. Since species lacking detectable receptors have considerable sensitivity to hemolysis, it is proposed that two binding mechanisms, specific and non-specific, exist which prepare erythrocytes for destruction.

Animals↗
PubMed1976

Production and purification of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'.

The gamma haemolysin of Staphylococcus aureus 'Smith 5R' was produced on Dolman-Wilson agar overlain with cellophane. Maximal yields of crude lysin with titres of 2000 to 4000 haemolytic units/ml were obtained within 24 h at 37 degrees C in 10% (v/v) CO2 in air, on medium adjusted to pH 7-0. The crude lysin was purified 2700-fold (with 75% recovery) by ultrafiltration, gel filtration and ammonium sulphate fractionation. The specific activity of the lysin was 10(5) haemolytic units/mg protein after the dialysed active precipitate was extracted with NaCl and reprecipitated with ammonium sulphate. Purified gamma lysin was homogeneous by disc electrophoresis and immunoelectrophoresis.

Ammonium Sulfate↗
PubMed1976

Properties of the gamma haemolysin of Staphylococcus aureus 'Smith 5R'.

Purified gamma haemolysin of Staphylococcus aureus was characterized in relation to the alpha, beta and delta haemolysins. The sedimentation coefficient of the gamma lysin was 2-65, somewhat higher than the S20,w values of 1-4 for freshly purified alpha lysin and 1-8 for the beta lysin. The molecular weight of gamma lysin determined by gel filtration was 45000 daltons. The pI of gamma lysin was 6-0, while that of the alpha, beta and delta lysins ranged from 8-5 to 9-6. The amino acid analysis of gamma lysin was characterized by low levels of methionine and histidine. Methionine was, however, the N-terminus, which suggested that all of the amino acid might be involved in the N-terminal group. The gamma lysin was immunologically distinct from the alpha, beta and delta lysins by quantitative precipitin tests; in Ouchterlony agar gel diffusion tests, single lines of precipitation were observed which showed no evidence of cross-reactions amongst the four haemolysins. Gamma, beta and delta lysins had no affect in mice when injected at increasing doses ranging from 0 to 100 mug. The alpha lysin killed mice, the LD50 dose being 0-60 +/- 0-12 mug, or 27 to 34 mug/kg mouse tissue. Gamma lysin was, however, lethal for guinea pigs when 50 mug quantities were injected intracradially. Gamma lysin also lysed human leucocytes and destroyed C-6 (human lymphoblast) cells.

Amino Acids↗
PubMed1975

Trypsin-mediated activation of the alpha-haemolysin of Staphylococcus aureus.

Alpha protoxin of Staphylococcus aureus "Wood 46" was activated by trypsin which had been coupled to carboxymethylcellulose, as indicated by the toxin's ability to hydrolyse tosyl-arginine methylester (TAME). A Lineweaver-Burk plot of the degradation of TAME by toxin and trypsin showed that toxin had a greater affinity for the substrate than had trypsin. N-terminal amino-acid analyses of activated toxin suggested that leucine or isoleucine is the N-terminus, in contrast to protoxin, the N-terminus of which is histidine.

Amino Acid Sequence↗