Ronald M. Hamelik

Purification of dextran-binding protein from cariogenic Streptococcus mutans

Abstract An extracellular protein produced by Streptococcus mutans was purified to electrophoretic homogeneity by affinity chromatography on Sephadex G50 followed by gel filtration. The protein is devoid of both dextransucrase and dextranase activity but binds dextran and therefore probably is implicated in the adherence of S. mutans cells to the host tooth surface. The presence of the dextran-binding protein may be a determinant of the pathogenicity of such cariogenic micro-organisms.

Click-iT assay with improved DNA distribution histograms.

The Click‐iT™ Assay developed and commercialized by Invitrogen is based on incorporation of a new 5‐bromo‐2′‐deoxyuridine analog, 5‐ethynyl‐2′‐deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated “click” reaction. This relatively convenient and useful procedure depends on fixation of cells with paraformaldehyde and staining of the DNA with 7‐aminoactinomycin‐D (7‐AAD). Both of these procedures result in DNA histograms with broad coefficients of variation (CVs). In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click‐iT™ Assay and stained with propidium iodide for generation of DNA histograms with low CVs. This modified procedure results in better DNA histograms by replacing 7‐AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps.

DNA index, genome size, and electronic nuclear volume of vertebrates from the Miami Metro Zoo.

Flow cytometry is a rapid and reliable method for measuring nuclear DNA content and genome size. Fluorochrome binding characteristics, sample preparation and differences in DNA condensation, and availability of binding sites can cause variations in results obtained.

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

Abstract An inhibitor of Streptococcus , mutans endodextranase was detected in proteins prepared from batch cultures of S. , mutans strains representing serotypes a through g . Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of S. , mutans known to produce this enzyme.

Multiple Forms of Dextran-Binding Proteins from Streptococcus Mutans

We have isolated a series of five proteins which appear to possess characteristic individual capacities for synthesizing dextrans and binding dextrans. Our suggestion that these proteins comprise an isozyme-like distribution of lectin and enzyme activities is, of course, very speculative and remains to be rigourously confirmed. However, the very identification of these several dextran binding proteins provides a biochemical basis to explain numerous observations suggesting that more than one mechanism for dextran binding is possessed by S. mutans (for instance: 24-27), especially the observations with mutants (24). These proteins probably are the molecular determinants of host infection by S. mutans and may prove to be potent immunogens for use in a vaccine. The presence of a dextran-binding lectin in S. mutans implicates this bacterial lectin in the earliest stage of infection: Attachment to host tissues. The multiplicity of proteins possessing characteristic dextran-synthesizing and dextran-binding capacities indicates the complexity of the adherence mechanisms evolved in S. mutans. Experiments with other bacteria (10-12, 28) suggest that bacterial lectins, in concert with host tissue carbohydrates, may be the molecular mediators of host recognition and subsequent initial attachment of bacterial cells to host tissues in non-pathogenic as well as pathogenic bacteria.

Flow Immunocytochemistry of Marker Expression in Cells from Body Cavity Fluids

Diagnostic cytology based on the examination of cells from body cavity fluids misses ∼50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy‐nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following markers: Ber‐EP4, progesterone (PR), MUC4, and thyroid transcription factor‐1 (TTF‐1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Statistical analysis of 79 samples in which data from cytology, DNA aneuploidy, and expression of at least one of the markers was available showed that by combining data from positive marker expression with that of aneuploidy, the sensitivity was increased from 58.5 to 100%. In contrast, out of the 38 samples designated as non‐malignant by diagnostic cytology, nine had aneuploid DNA content and 16 of the diploid samples had a positive marker expression. Specificity was reduced from 74.7 to 31.6% due to the presence of aneuploidy and marker expression in these samples. ALDH1pos/CD44pos/CD24neg expression has been reported to be associated with human breast tumor stem cells. Some of our samples had cells with this phenotype. Flow cytometry offers the advantage of rapid multiparametric analysis of DNA aneuploidy and marker expression in cells from body cavity fluids based on the analysis of a large number of cells without observer bias. By further developing the use of specific markers and aneuploidy, it may be possible to refine flow cytometric analysis for rapid detection of malignant cells in body cavity fluids.

Flow cytometric monitoring of fluorescent drug retention and efflux.

Laser flow cytometry has been used for monitoring cellular retention of fluorescent drugs such as fluorescent anticancer antibiotics (e.g., doxorubicin) and fluorochromes used for the detection of cellular drug efflux and resistance (e.g., rhodamine 123, Hoechst 33342). Multiparametric flow cytometry can be used for identification of tumor cell subpopulations based on their drug retention profiles with or without the presence of an efflux blocker. This rapid procedure can be used for identification of tumor cells with the drug-resistance phenotype based on drug efflux as well as for efflux blockers that may block efflux of a chemotherapeutic agent and thus increase cellular retention and sensitivity. It has been reported recently that some of the bone marrow stem cells (SP cells) efflux the Hoechst 33342 fluorochrome and thus can be rapidly identified by comparing red vs blue fluorescence in the presence or absence of an efflux blocker such as verapamil. The present chapter discusses some of the flow cytometric methods used for the study of cellular drug retention and the artifacts that may arise in such analysis.

An enzyme from Streptococcus mutans forms branches on dextran in the absence of sucrose

An enzyme in glucosyltransferase preparations from Streptococcus mutans catalyzed the transfer of [14C]glucopyranoside from purified isomaltosaccharides, each containing [14C]glucopyranoside at its non-reducing terminus, to acceptor dextran, in the absence of sucrose. Half of the radioactivity present in the resulting [14C]dextrans was resistant to hydrolysis by amylo-1,6-glucosidase. Treatment of the [14C]dextrans with endodextranase resulted in extensive hydrolysis and produced [14C]-labeled limit oligosaccharides containing branch sites. Acetolysis of the [14C]-labeled limit oligosaccharides yielded [14C]nigerose, thus indicating the formation of branch sites on dextran in the absence of sucrose. The enzyme catalyzing this reaction has not been identified but appears to be independent of the major extracellular glucosyltransferases of S. mutans.

Click-iT proliferation assay with improved DNA histograms.

The Click‐iT EdU cell proliferation assay (Invitrogen) for detection of replicating cells is based on incorporation of EdU into newly synthesized DNA and its recognition by azide dyes via a copper mediated “click” reaction. In the protocol provided by Invitrogen, cells are fixed with paraformaldehyde and stained with 7‐aminoactinomycin D (7‐AAD) for DNA content analysis. Both of these procedures result in DNA histograms with a broad coefficient of variation. We have modified this protocol and show that after EdU incorporation, nuclei isolated by hypotonic lysis of cells can be directly labeled using the Click‐iT Alexa Fluor 488 Assay kit and stained with propidium iodide. This modified procedure using isolated nuclei and propidium iodide staining results in DNA histograms with better resolution (lower coefficient of variation of the G1 peak) and shorter processing time by eliminating the fixation and permeabilization steps. Curr. Protoc. Cytom. 52:7.36.1‐7.36.7.

ALDH+/CD44+/CD24− expression in cells from body cavity fluids

Enhanced expression of aldehyde dehydrogenase 1 (ALDH1) and phenotypic markers (CD44+/CD24−) in stem cells from breast tumors has been reported. This study was undertaken to monitor expression of these markers in cells from body cavity fluids of female patients suspected to have a malignancy.