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Cook, J Michael

Overview:

I am interested in all aspects of flow cytometric analysis and cell sorting, especially the application of these techniques to elucidation of the development of the immune system and understanding primary immunodeficiencies. I am committed to assisting the Duke research community in the maximum utilization of the cytometers and techniques available to them through our shared reasource, including training of individuals to perform their own cytometry. My goal is to insure that their flow cytometry experiments will lead to peer reviewed publications and strong fundable RO1 applications.

Positions:

Assistant Research Professor of Immunology

Immunology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1984

Ph.D. — University of Virginia

Publications:

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor.

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.

Authors
Wang, Y; Chandra, R; Samsa, LA; Gooch, B; Fee, BE; Cook, JM; Vigna, SR; Grant, AO; Liddle, RA
MLA Citation
Wang, Y, Chandra, R, Samsa, LA, Gooch, B, Fee, BE, Cook, JM, Vigna, SR, Grant, AO, and Liddle, RA. "Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor." Am J Physiol Gastrointest Liver Physiol 300.4 (April 2011): G528-G537.
PMID
21183662
Source
pubmed
Published In
American journal of physiology. Gastrointestinal and liver physiology
Volume
300
Issue
4
Publish Date
2011
Start Page
G528
End Page
G537
DOI
10.1152/ajpgi.00387.2010

Plasmonic flow cytometry by immunolabeled nanorods.

Fluorescence-based flow cytometry measures multiple cellular characteristics, including levels of receptor expression, by assessing the fluorescence intensity from a population of cells whose cell surface receptors are bound by a fluorescently labeled antibody or ligand for that receptor. Functionalized noble metal nanoparticles provide a complementary method of receptor labeling based on plasmonics for population analysis by flow cytometry. The potential benefits of using plasmonic nanoparticles to label cell surface receptors in flow cytometry include scattering intensity from a single particle that is equivalent to fluorescence intensity of 10⁵ fluorescein molecules, biocompatibility and low cytotoxicity, and nonquenching optical properties. The large spectral tunability of nanorods also provides convenient access to plasmonic markers with peak surface plasmon resonances ranging from 600 to 2,200 nm, unlike gold nanosphere markers that are limited to visible wavelengths. Gold nanorod-based plasmonic flow cytometry is demonstrated herein by comparing the scattering of cells bound to anti-epidermal growth factor receptor (EGFR)-conjugated nanorods to the emission of cells bound to anti-EGFR-conjugated fluorescent labels. EGFR-expressing cells exhibited a statistically significant six-fold increase in scattering when labeled with anti-EGFR-conjugated nanorods compared with labeling with IgG1-conjugated nanorods. Large scattering intensities were observed despite using a 1,000-fold lower concentration of nanorod-conjugated antibody relative to the fluorescently labeled antibody.

Authors
Crow, MJ; Marinakos, SM; Cook, JM; Chilkoti, A; Wax, A
MLA Citation
Crow, MJ, Marinakos, SM, Cook, JM, Chilkoti, A, and Wax, A. "Plasmonic flow cytometry by immunolabeled nanorods." Cytometry A 79.1 (January 2011): 57-65.
PMID
21182183
Source
pubmed
Published In
Cytometry
Volume
79
Issue
1
Publish Date
2011
Start Page
57
End Page
65
DOI
10.1002/cyto.a.20994

Loss of beta-Catenin Impairs the Renewal of Normal and CML Stem Cells In Vivo.

A key characteristic of stem cells and cancer cells is their ability to self-renew. To test if Wnt signaling can regulate the self-renewal of both stem cells and cancer cells in the hematopoietic system, we developed mice that lack beta-catenin in their hematopoietic cells. Here we show that beta-catenin-deficient mice can form HSCs, but that these cells are deficient in long-term growth and maintenance. Moreover, beta-catenin deletion causes a profound reduction in the ability of mice to develop BCR-ABL-induced chronic myelogenous leukemia (CML), while allowing progression of acute lymphocytic leukemia (ALL). These studies demonstrate that Wnt signaling is required for the self-renewal of normal and neoplastic stem cells in the hematopoietic system.

Authors
Zhao, C; Blum, J; Chen, A; Kwon, HY; Jung, SH; Cook, JM; Lagoo, A; Reya, T
MLA Citation
Zhao, C, Blum, J, Chen, A, Kwon, HY, Jung, SH, Cook, JM, Lagoo, A, and Reya, T. "Loss of beta-Catenin Impairs the Renewal of Normal and CML Stem Cells In Vivo." Cancer cell 12.6 (December 2007): 528-541. (Academic Article)
PMID
18068630
Source
manual
Published In
Cancer Cell
Volume
12
Issue
6
Publish Date
2007
Start Page
528
End Page
541

Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance.

A fundamental question in hematopoietic stem cell (HSC) biology is how self-renewal is controlled. Here we show that the molecular regulation of two critical elements of self-renewal, inhibition of differentiation and induction of proliferation, can be uncoupled, and we identify Notch signaling as a key factor in inhibiting differentiation. Using transgenic Notch reporter mice, we found that Notch signaling was active in HSCs in vivo and downregulated as HSCs differentiated. Inhibition of Notch signaling led to accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo. Finally, intact Notch signaling was required for Wnt-mediated maintenance of undifferentiated HSCs but not for survival or entry into the cell cycle in vitro. These data suggest that Notch signaling has a dominant function in inhibiting differentiation and provide a model for how HSCs may integrate multiple signals to maintain the stem cell state.

Authors
Duncan, AW; Rattis, FM; DiMascio, LN; Congdon, KL; Pazianos, G; Zhao, C; Yoon, K; Cook, JM; Willert, K; Gaiano, N; Reya, T
MLA Citation
Duncan, AW, Rattis, FM, DiMascio, LN, Congdon, KL, Pazianos, G, Zhao, C, Yoon, K, Cook, JM, Willert, K, Gaiano, N, and Reya, T. "Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance." Nat Immunol 6.3 (March 2005): 314-322.
PMID
15665828
Source
pubmed
Published In
Nature Immunology
Volume
6
Issue
3
Publish Date
2005
Start Page
314
End Page
322
DOI
10.1038/ni1164

Notch as a target to modulate hematopoietic stem cell differentiation

Authors
DiMascio, L; Congdon, K; Pazianos, G; Zhao, C; Cook, JM; Willert, K
MLA Citation
DiMascio, L, Congdon, K, Pazianos, G, Zhao, C, Cook, JM, and Willert, K. "Notch as a target to modulate hematopoietic stem cell differentiation." Cancer Biology and Therapy 4.2 (2005).
Source
scival
Published In
Cancer Biology and Therapy
Volume
4
Issue
2
Publish Date
2005

Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells.

BACKGROUND: Diethylstillbestrol (DES) and diethylstilbestrol diphosphate (DESdP) are effective agents for the treatment of advanced prostate cancers. Tumor-inhibiting effects of DES and DESdP are presumed secondary to suppression of androgen production in vivo. Little is known, however, about the direct cellular mechanisms of the tumor inhibition. Estrogens have been reported not only to stimulate growth but also to disrupt microtubule formation in prostate cancer cells. PURPOSE: The study was designed to examine and compare mechanisms of in vitro growth inhibition of DES and DESdP in human androgen-insensitive prostate cancer cells (DU145, 1-LN, and PC-3) and human androgen-sensitive prostate cancer cells (LNCaP) and to examine estrogen receptor modulation of such effects. METHODS: The cytotoxic effects of DES and DESdP were examined in vitro by use of a standard microculture tetrazolium assay to quantitate numbers of viable cells. Immunofluorescence microscopy, DNA fragmentation analysis, and fluorescence flow cytometry were used to investigate microtubules, the induction of apoptosis, and changes in cell cycle distribution. The degree of estrogen receptor positivity of untreated and treated cells was determined by immunohistochemistry and quantitative image analysis. RESULTS: LD50 levels (the dose at which 50% of cells are no longer viable) in the concentration range of 19-25 microM were observed for both DES and DESdP in all cell lines examined. DESdP-induced growth inhibition was found to be dependent on heat-labile phosphatases present in fetal calf serum. DES-induced cytotoxicity was not affected by the presence of 17 beta-estradiol, and it was not dependent on the presence of estrogen receptor. Estrogen receptor-positive cells and estrogen receptor-negative cells were equally responsive to DES. PC-3 cells stained with fluorescent anti-tubulin, phalloidin (actin stain), and 4',6-diamidino-2-phenylindole (DNA stain) showed no inhibition of microtubules or actin filaments but revealed the presence of apoptotic bodies in the nuclei. Fluorescence flow cytometry of nuclear DNA content of propidium iodide-stained nuclei from androgen-insensitive prostate cancer cells treated with 15 or 30 microM DES or DESdP revealed an increase in relative numbers of hypodiploid (apoptotic) nuclei, a depletion of G1- and S-phase cells, and an accumulation of cells in G2/M phase. Conversely, androgen-sensitive cells contained a lower percentage of hypodiploid nuclei but no accumulation of cells in G2/M phase. CONCLUSIONS: Direct cytotoxic effects of DES in prostate cancer cells are estrogen receptor independent and do not involve disruption of microtubule architecture but do involve the promotion of cell cycle arrest and apoptosis. These are the first data confirming direct cytotoxic effects of DES and DESdP in prostate cancer cells via an apoptotic mechanism. IMPLICATIONS. These results suggest that DES and DESdP have potential value as agents against androgen-insensitive prostate neoplasms through induction of an apoptotic cascade.

Authors
Robertson, CN; Roberson, KM; Padilla, GM; O'Brien, ET; Cook, JM; Kim, CS; Fine, RL
MLA Citation
Robertson, CN, Roberson, KM, Padilla, GM, O'Brien, ET, Cook, JM, Kim, CS, and Fine, RL. "Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells." J Natl Cancer Inst 88.13 (July 3, 1996): 908-917.
PMID
8656443
Source
pubmed
Published In
Journal of the National Cancer Institute
Volume
88
Issue
13
Publish Date
1996
Start Page
908
End Page
917

A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastatic breast cancer in the bone marrow.

A significant percentage of women with primary breast cancer have micrometastatic disease in the bone marrow. Bone marrow involvement may be an adverse prognostic factor, and more aggressive therapy may be indicated for these patients. There are a number of different techniques and antibodies used to detect tumor cells in the bone marrow. We used the same panels of four antibreast cancer antibodies and compared three immunodetection techniques: two-color immunofluorescence, immunohistochemical staining, and fluorescence-activated cell sorting with cytologic examination of the sorted cells. The two-color immunofluorescence technique was superior and consistently detected one tumor cell contaminating one million normal bone marrow cells and had no reactivity with normal bone marrow.

Authors
Vredenburgh, JJ; Silva, O; Tyer, C; DeSombre, K; Abou-Ghalia, A; Cook, M; Layfield, L; Peters, WP; Bast, RC
MLA Citation
Vredenburgh, JJ, Silva, O, Tyer, C, DeSombre, K, Abou-Ghalia, A, Cook, M, Layfield, L, Peters, WP, and Bast, RC. "A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastatic breast cancer in the bone marrow." J Hematother 5.1 (February 1996): 57-62.
PMID
8646482
Source
pubmed
Published In
Stem Cells and Development
Volume
5
Issue
1
Publish Date
1996
Start Page
57
End Page
62
DOI
10.1089/scd.1.1996.5.57

Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes.

We have used electron microscopy and flow cytofluorimetry to study endocytosis and intracellular transport of fluid phase bovine serum albumen gold complexes and membrane bound concanavalin A through endosomal compartments of bloodstream forms of Trypanosoma brucei rhodesiense. Both markers were rapidly endocytosed from the flagellar pocket. Within 20 minutes at 37 degrees C the markers reached a large, vesicular, perinuclear compartment that stained heavily with the CB1 monoclonal antibody. Neither marker left the flagellar pocket and entered cells at 4 degrees C. When cells were incubated at 12 degrees C, both markers entered the cell and were transported to collecting tubules, a tubular endosomal compartment that receives endocytosed material from coated endocytic vesicles. However, no material was transported from collecting tubules to the late, perinuclear compartment at 12 degrees C. The morphology of collecting tubule membranes was specifically altered at 12 degrees C; tubules became shorter and were arrayed near the flagellar pocket. The morphological alteration and the block in transport of endocytic markers to the perinuclear compartment seen at 12 degrees C were reversed 10 minutes after cells were returned to 37 degrees C. We also used flow cytofluorimetric measurements of pH dependent fluorescence quenching to measure the pH of the terminal endocytic compartment. Fluoresceinated lectins accumulated in a terminal compartment with a pH of 6.0-6.1, a value considerably higher than that of mammalian lysosomes. Fluorescence from fluoresceinated lectins in this terminal endocytic compartment was dequenched when bloodstream forms were incubated in the presence of chloroquine.

Authors
Brickman, MJ; Cook, JM; Balber, AE
MLA Citation
Brickman, MJ, Cook, JM, and Balber, AE. "Low temperature reversibly inhibits transport from tubular endosomes to a perinuclear, acidic compartment in African trypanosomes." J Cell Sci 108 ( Pt 11) (November 1995): 3611-3621.
PMID
8586672
Source
pubmed
Published In
Journal of cell science
Volume
108 ( Pt 11)
Publish Date
1995
Start Page
3611
End Page
3621

A comparison of tumor and normal tissue microvascular hematocrits and red cell fluxes in a rat window chamber model.

This laboratory has previously used a window chamber model to measure red blood cell velocity in mammary tumors and normal granulation tissues of the F-344 rat. Because red cell flux and hematocrit more accurately reflect the oxygen carrying potential of blood, we used this model to measure these parameters. Red blood cells were labelled with fluorescein isothiocyanate, and 0.2 ml. packed cells were injected intravenously into rats bearing an 8 to 10 day old R-3230 mammary carcinoma. beta-phycoerythrin (0.15 mg.) was also injected and served as a plasma dye to outline the blood vessels. A sample of peripheral blood was then taken and analyzed by flow cytometry to determine the labeled fraction of red blood cells. Flowing tumor and normal tissue vessels were recorded onto a VCR, and these video images were used to determine vascular length and diameter, RBC flux and velocity, and hematocrit. Median vessel diameter and loge (red blood cell flux) were significantly greater in tumors than in normal tissues (p = 0.007 and p < 0.025, respectively). After controlling for these variables, the median tumor hematocrit of 19% was not significantly greater than the median normal tissue hematocrit of 15%. This technique provides a nontoxic and reproducible method that is now being used to assist in the in vivo definition of tumor oxygenation.

Authors
Brizel, DM; Klitzman, B; Cook, JM; Edwards, J; Rosner, G; Dewhirst, MW
MLA Citation
Brizel, DM, Klitzman, B, Cook, JM, Edwards, J, Rosner, G, and Dewhirst, MW. "A comparison of tumor and normal tissue microvascular hematocrits and red cell fluxes in a rat window chamber model." Int J Radiat Oncol Biol Phys 25.2 (January 15, 1993): 269-276.
PMID
8420874
Source
pubmed
Published In
International Journal of Radiation Oncology, Biology, Physics
Volume
25
Issue
2
Publish Date
1993
Start Page
269
End Page
276

The beta-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation.

The beta-phosphoro[35S]thioate analogue of UDP-glucose ((beta-35S)UDP-Glc) is utilized with approximately the same efficiency as the parent compound by the UDP-glucose:glycoprotein glucose-1-phosphotransferase (glucosyltransferase), which catalyzes the transfer of alpha Glc-1-P from UDP-Glc to mannose-containing oligosaccharides on acceptor glycoproteins. The same endogenous acceptor glycoproteins are labeled by the glucosyltransferase using [beta-32P]UDP-Glc and (beta-35S)UDP-Glc. However, in liver homogenates, incorporation from [beta-32P]UDP-Glc ceases to increase after about 4 min of incubation, while incorporation from (beta-35S)UDP-Glc persists for at least 1 h. This difference is due to an approx. 10-fold slower hydrolytic rate for the phosphorothioate analogue than for the parent compound, a finding similar to previous work showing that a variety of nucleases and phosphodiesterases are less efficient in cleaving phosphorothioate DNA than the native polymer.

Authors
Marchase, RB; Saunders, AM; Rivera, AA; Cook, JM
MLA Citation
Marchase, RB, Saunders, AM, Rivera, AA, and Cook, JM. "The beta-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation." Biochim Biophys Acta 916.2 (November 26, 1987): 157-162.
PMID
2823902
Source
pubmed
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
916
Issue
2
Publish Date
1987
Start Page
157
End Page
162

The β-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation

The β-phosphoro[35S]thioate analogue of UDP-glucose ((β-35S)UDP-Glc) is utilized with approximately the same efficiency as the parent compound by the UDP-glucose:glycoprotein glucose-1-phosphotransferase (glucosyltransferase), which catalyzes the transfer of αGlc-1-P from UDP-Glc to mannose-containing oligosaccharides on acceptor glycoproteins. The same endogenous acceptor glycoproteins are labeled by the glucosyltransferase using [β-32P]UDP-Glc and (β-35S)UDP-Glc. However, in liver homogenates, incorporation from [β-32P]UDP-Glc ceases to increases after about 4 min of incubation, while incorporation from (β-35S)UDP-Glc persists for at least 1 h. This difference is due to an approx. 10-fold slower hydrolytic rate for the phosphorothioate analogue than for the parent compound, a finding similar to previous work showing that a variety of nucleases and phosphodiesterases are less efficient in cleaving phosphorothioate DNA than the native polymer. © 1987.

Authors
Marchase, RB; Saunders, AM; Rivera, AA; Cook, JM
MLA Citation
Marchase, RB, Saunders, AM, Rivera, AA, and Cook, JM. "The β-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation." Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular 916.2 (1987): 157-162.
Source
scival
Published In
BBA - Protein Structure and Molecular Enzymology
Volume
916
Issue
2
Publish Date
1987
Start Page
157
End Page
162
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