Howard G. Gratzner

Bio: Howard G. Gratzner is an academic researcher from University of Miami. The author has contributed to research in topics: Bromodeoxyuridine & DNA synthesis. The author has an hindex of 13, co-authored 20 publications receiving 3555 citations.

Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication

Abstract: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

Bromodeoxyuridine in tumors and chromosomes detected with a monoclonal antibody.

Abstract: Using a monoclonal antibody to bromodeoxyuridine (BUdR) and immunohistochemistry, we measured the incorporation of this thymidine analogue into the DNA of human normal and malignant cells exposed in vivo. BUdR given as a constant intravenous infusion for 12 or 24 h daily for up to 13 d resulted in a steady-state plasma level of 10(-6) M during the infusion. We demonstrated extensive incorporation of BUdR into both normal skin, normal bone marrow, and malignant melanoma cells. In addition, this infusion of BUdR was adequate to identify sister chromatid exchanges from human marrow chromosomes exposed in vivo. Using this constant infusion, significant but reversible (acute) toxicity was observed with myelosuppression and skin photosensitivity. These techniques, which are considerably less cumbersome and time-consuming than the use of radioactive isotopes of thymidine, can be used for further human studies of cell kinetics and chromosomal replication in both normal and malignant cells.

Cell‐Cycle Analysis Using A Monoclonal Antibody to Brdurd

Abstract: The flow cytometric measurement of DNA distributions of cells has many applications in biomedical research. Phase fractions estimated (calculated) from such distributions are used to study the growth characteristics of various types of cells, particularly when the cells have been exposed to perturbing agents such as chemotherapeutic drugs. For more than 10 years many methods for resolving DNA distributions into the three cell subpopulations (G1, S and G2 + M) have been reported in the literature. A new method of analysis utilizing a monoclonal antibody to bromodeoxyuridine (BrdUrd) has been developed (Gratzner, 1982; Dolbeare et al., 1983) which makes it possible in most cases to accurately determine phase fractions without resorting to mathematical models. The procedure involves the incorporation of BrdUrd by growing (DNA synthesizing) S phase cells, labelling the BrdUrd with a fluorescent monoclonal antibody, and the bivariate measurement of the antibody and of total DNA content, the latter through propidium-iodide staining. The resulting bivariate distributions clearly and simply resolve the three subpopulations. This paper describes the method and illustrates its use in the analysis of various fractions of elutriated exponentially growing Chinese hamster ovary (CHO) cells.

An immunofluorescence method for monitoring DNA synthesis by flow cytometry.

Abstract: An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 × 105 cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of “labeled” cells can be followed through the cell cycle.

Cell Wall Alterations Associated with the Hyperproduction of Extracellular Enzymes in Neurospora crassa

Abstract: A pleiotropic mutation in Neurospora (exo-1), which confers derepression of alpha-amylase, glucoamylase, beta-fructofuranosidase, and trehalase, appears to also affect the composition of the cell wall. Segregants resulting from the backcross of exo-1 to the wild-type strain from which it derived are altered in the ratio of galactosamine to glucosamine in hydrolysates of isolated cell walls. Conidial cell walls exhibit a marked decrease in the amount of galactosamine in both exo-1 and exo-1(+) strains. Increased levels (approximately sevenfold) of amylase are found in conidia of exo-1, as compared with those of exo-1(+).

Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: A new reagent for detection of DNA replication

Abstract: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

Abstract: The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression.

Abstract: In cancer patients, dormant micrometastases are often asymptomatic and clinically undetectable, for months or years, until relapse. We have studied dormant lung metastases under angiogenesis suppression in mice. The metastases exhibited rapid growth when the inhibition of angiogenesis was removed. Tumour cell proliferation, as measured by bromodeoxyuridine incorporation and immunohistochemical staining proliferating cell nuclear antigen, was not significantly different in dormant and growing metastases. However, tumour cells of dormant metastases exhibited a more than threefold higher incidence of apoptosis. These data show that metastases remain dormant when tumour cell proliferation is balanced by an equivalent rate of cell death and suggest that angiogenesis inhibitors control metastatic growth by indirectly increasing apoptosis in tumour cells.

Adult neurogenesis in the mammalian central nervous system

Abstract: Forty years since the initial discovery of neurogenesis in the postnatal rat hippocampus, investigators have now firmly established that active neurogenesis from neural progenitors continues throughout life in discrete regions of the central nervous systems (CNS) of all mammals, including humans. Significant progress has been made over the past few years in understanding the developmental process and regulation of adult neurogenesis, including proliferation, fate specification, neuronal maturation, targeting, and synaptic integration of the newborn neurons. The function of this evolutionarily conserved phenomenon, however, remains elusive in mammals. Adult neurogenesis represents a striking example of structural plasticity in the mature CNS environment. Advances in our understanding of adult neurogenesis will not only shed light on the basic principles of adult plasticity, but also may lead to strategies for cell replacement therapy after injury or degenerative neurological diseases.