Boris Rotman

Transport systems for galactose and galactosides in Escherichia coli: II. Substrate and inducer specificities

Abstract Genetic and biochemical studies have shown Escherichia coli K12 to have four transport systems for galactose and galaetosides: galactose permease, methylgalactoside permease, TMG I and TMG II permeases. Biochemical evidence suggested that they have some substrates and inducers in common. In order to define more clearly the specificities of the individual transport systems, particularly those of the galactose and methylgalaetoside permeases, we examined and compared mutants of E. coli K12, which, by their patterns of accumulation of various sugars, appear to lack one or more of the permeases. From these studies we concluded that the four permeases do show some similarities in the sugars which they transport, and which act as inducers. However, the galactose and methylgalactoside permeases can be distinguished from each other and from the TMG I and TMG II permeases by their substrate and inducer preferences. The only compound found to be transported by the galactose permease was d -galactose. d -Galactose is also the most effective inducer of this transport system. The methylgalactoside permease transported methyl-β- d -galactopyranoside, d -fucose and ethyl-β- d -galactopyranoside, as well as d -galactose. The most effective inducer of this system is d -fucose

Distribution of suboptimally induced β-d-galactosidase in Escherichia coli: The enzyme content of individual cells

Abstract Novick & Weiner (1957) proposed a model in which induction of the lac operon with suboptimal concentrations of inducer generates a population containing both uninduced and fully induced cells. The latter arise as cells acquire the galactoside transport system, thus initiating an autocatalytic cycle of induction since this permease can transport an inducer for its own synthesis. Evidence in favor of this model has been obtained from direct measurements of the enzyme content of individual cells, using a fluorogenic assay sensitive to one molecule of β- d -galactosidase. Fully induced cells, at the predicted frequency, were found in suboptimally induced populations of wild type strains, and of a strain lacking thiogalactoside transacetylase, but not of a strain lacking galactoside permease. In the wild type, the frequency of cells with an enzyme content intermediate between uninduced and fully induced levels was greater than the frequency predicted for cells within the autocatalytic cycle of induction. According to the model, then, in some of these cells, induction of β- d -galactosidase has occurred without formation of the permease necessary to initiate accumulation of inducer.

A galactosidase immunosorbent test for human immunoglobulin E

We report here the development of a galactosidase-immunosorbent test (GIST) for immunoglobulin E (IgE) antibodies in which the amount of galactosidase adsorbed to a cellulose disc is a single valued function of IgE concentration in human serum. Rabbit anti-IgE immunoglobulin insolubilized on cellulose discs is incubated sequentially with human serum, sheep anti-IgE serum, and a covalent conjugate of rabbit antisheep immunoglobulin with the enzyme beta-D-galactoside galactohydrolase (E.C.) 3.2.1.23). Colorimetric assay of enzyme conjugate adsorbed to discs permits quantitation of 1.0 to 25 ng of IgE per test. Concentrations of IgE in 48 sera as measured by the GIST gave a linear correlation coefficient of 0.97 with IgE concentrations as determined by radioimmunoassay. Preliminary studies indicate that the GIST makes possible nonisotopic measurement of ragweed-specific IgE antibiotics in human serum. The GIST for IgE is simple to perform and requires neither short-lived radioisotopes, expensive scintillation detection equipment, nor scarce, purified IgE.

Application of a real-time biosensor to detect bacteria in platelet concentrates ☆

A spore-based biosensor for detecting low levels of bacteria in real-time has been recently developed. The system (termed LEXSAS, label-free exponential signal-amplification system) exploits spores ability to produce fluorescence when sensing neighboring bacterial cells. We studied the LEXSAS as a possible approach for identifying bacterially contaminated platelet concentrates prior to transfusion because the system offers rapid analysis, high sensitivity, and low cost. If successful, this approach could reduce the risk of morbidity and mortality from transfusion-related bacteremia and sepsis. In this study, we used the LEXSAS for detecting bacteria in platelet concentrates spiked with Bacillus cereus, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Streptococcus pyogenes. Bacteria were separated from platelets using a 2-min procedure based on bacterial resistance to detergents and osmotic shock. The results indicate that the LEXSAS could be used to design a practical biosensor for identifying bacterially contaminated platelets in real-time.

Predictive Value of the Fluorescent Cytoprint Assay (FCA): A Retrospective Correlation Study of In Vitro Chemosensitivity and Individual Responses to Chemotherapy

The potential clinical usefulness of the fluorescent cytoprint assay (FCA) was assessed retrospectively in 73 cancer patients by correlating individual tumor chemosensitivity in vitro with responses to chemotherapy. The data show that the FCA has a sensitivity of 98%, specificity of 81%, and predictive accuracies of 85% and 97% for positive and negative clinical responses, respectively.

Changes in the Membrane Permeability of Human Leukocytes Measured by Fluorochromasia in a Rapid Flow Fluorometer

Fluorochromasia is a property of cells to accumulate fluorescein intracellularly as a result of the enzymatic hydrolysis of a fluorogenic substrate, thereby becoming brightly fluorescent under blue light (Rotman and Papermaster 1966). The f luorochromatic reaction requires a nonpolar fluorogenic substrate, such as diesters of fluorescein and aliphatic acids. This type of substrate can penetrate the cell membrane and can be hydrolyzed intracellularly, thus liberating fluorescein, a negatively charged molecule, which does not diffuse readily across the cytoplasmic membrane of normal cells. If the rate of hydrolysis of the substrate exceeds the rate of fluorescein excretion, the intracellular concentration of fluorescein builds up until it reaches a steady state (in about seven minutes in our experiments) in which the rate of substrate hydrolysis is equal to the rate of fluorescein excretion.

Measurements of Single Molecules of Antibody by their Ability to Activate a Defective Enzyme

Certain defective enzymes produced by genetically altered bacteria show a substantial and specific activation in the presence of antibodies directed against the corresponding normal enzyme, Pollock et al. 1967; Rotman and Celada 1968; Arnon and Cinader 1971). This type of response has inherent capabilities for measuring antibodies with ease and sensitivity since it transduces the antigen-antibody binding into a catalytic reaction. The increases in enzymatic activity mediated by antibodies are generally modest except for those of a penicillinase (Pollock 1967) and a β-D-galactosidase (Rotman and Cinader 1968; Messer and Melchers 1970) which are 70- and 550-fold, respectively. The β-D-galactosidase reaction was chosen for our studies not only because of its high activation factor, but also for the fluorogenic assay of this enzyme, which measures quantitatively the activity of a single β-D-galactosidase molecule, (Rotman 1961;1970). The expectation was that the sensitivity of the fluorogenic assay would serve to measure individual molecules of activating antibody. The results of the experiments presented here fulfilled this expectation and, furthermore, demonstrated that one molecule of monovalent antibody (Fab) is sufficient for activating a molecule of a defective β-D-galactosidase (AMEF). The latter conclusion could not be reached a priori, because AMEF is a tetramer with four identical sub-units (Rotman and Celada 1968) and, therefore, it could have required binding to each of the subunits for activation.

A quantitative cytotoxicity assay using whole blood: application to h-2 tissue typing.

A fluorochromatic cytotoxicity assay featuring direct use of whole blood and quantitative evaluation is described. Its application to tissue typing for H-2 antigens together with data indicating reproducibility specificity of the assay are presented. Procedures for visual and automated quantification are included.

Dissociation of the tetrameric form of β-D-galactosidase by inactivating antibodies

Abstract From the following results, we have concluded that inactivation of β-D-galactosidase (EC 3.2.1.23) by specific antibodies occurs through dissociation of the 16S enzyme into 10S subunits: (a) 10s material is only observed when inactivation has occurred; (b) in enzyme preparations inactivated to different levels, the fraction of β-D-galactosidase protein present in 10S form corresponds to the level of inactivation in the given preparation; (c) inactivation is accompanied by a loss of enzymatically active 16S material equivalent to the amount of 10S material formed.

Constructing fluorogenic Bacillus spores (F-spores) via hydrophobic decoration of coat proteins.

Background Bacterial spores are protected by a coat consisting of about 60 different proteins assembled as a biochemically complex structure with intriguing morphological and mechanical properties. Historically, the coat has been considered a static structure providing rigidity and mainly acting as a sieve to exclude exogenous large toxic molecules, such as lytic enzymes. Over recent years, however, new information about the coats architecture and function have emerged from experiments using innovative tools such as automated scanning microscopy, and high resolution atomic force microscopy. Principal Findings Using thin-section electron microscopy, we found that the coat of Bacillus spores has topologically specific proteins forming a layer that is identifiable because it spontaneously becomes decorated with hydrophobic fluorogenic probes from the milieu. Moreover, spores with decorated coat proteins (termed F-spores) have the unexpected attribute of responding to external germination signals by generating intense fluorescence. Fluorescence data from diverse experimental designs, including F-spores constructed from five different Bacilli species, indicated that the fluorogenic ability of F-spores is under control of a putative germination-dependent mechanism. Conclusions This work uncovers a novel attribute of spore-coat proteins that we exploited to decorate a specific layer imparting germination-dependent fluorogenicity to F-spores. We expect that F-spores will provide a model system to gain new insights into structure/function dynamics of spore-coat proteins.