Gregory E. Conner, Ph.D.

Department of Cell Biology


This research program focuses on airway cell biology with a main emphasis on the function and composition of secretions in normal and diseased airways. The research area provides opportunities for graduate students and postdoctoral fellows with diverse interests to develop their own research programs. A variety of techniques are available for use on projects including recombinant DNA/molecular biology, immunochemistry, various types of microscopy, cell culture and subcellular fractionation.


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Airway Cell Biology

Reactive Oxygen Species

Thiocyanate and Cystic Fibrosis

Cathepsin D Reagents and Protocols

Biographical Information

Recent Publications

Contact Information/Mailing and Shipping Addresses

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BIOGRAPHICAL INFORMATION

BA

Molecular Biology

1972

Vanderbilt University

Nashville

TN

Ph.D.

Biochemistry

1978

University of Florida

Gainesville

FL

Post-Doctoral

Cell Biology

1978-1981

The Rockefeller University

New York

NY

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SOME RECENT PUBLICATIONS

Gattas MV, Forteza R, Fragoso MA, Fregien N, Salas P, Salathe M, Conner GE. Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli. Free Free Radic Biol Med 10: 1450-1458, 2009

Fragoso MA, Torbati A, Fregien N, Conner GE. Molecular heterogeneity and alternative splicing of human lactoperoxidase. Arch Biochem Biophys. 482(1-2):52-7 (2009)

Ransford GA, Fregien N, Qiu F, Dahl G, Conner GE, Salathe M. Pannexin 1 Contributes to ATP Release in Airway Epithelia. Am J Respir Cell Mol Biol. Feb 12. [Epub ahead of print] (2009)

Conner, G. E., C. Wijkstrom-Frei, S. H. Randell, V. E. Fernandez and M. Salathe. The lactoperoxidase system links anion transport to host defense in cystic fibrosis. FEBS letters 581: 271-278 (2007)

Schmid, A., G. Bai, N. Schmid, M. Zaccolo, L. E. Ostrowski, G. E. Conner, N. Fregien and M. Salathe. Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells. J Cell Sci 119: 4176-4186 (2006)

Forteza, R., M. Salathe, F. Miot, R. Forteza and G. E. Conner. Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am J Respir Cell Mol Biol 32: 462-469 (2005)

Campos, M. A., A. R. Abreu, M. C. Nlend, M. A. Cobas, G. E. Conner and P. L. Whitney. Purification and characterization of PLUNC from human tracheobronchial secretions. Am J Respir Cell Mol Biol 30: 184-192 (2004)

Fragoso, M. A., V. Fernandez, R. Forteza, S. H. Randell, M. Salathe and G. E. Conner. Transcellular thiocyanate transport by human airway epithelia. J Physiol 561: 183-194 (2004)

Sutto, Z., G. E. Conner and M. Salathe. Regulation of human airway ciliary beat frequency by intracellular pH. J Physiol 560: 519-532 (2004)

El-Chemaly S, Salathe M, Baier S, Conner GE, Forteza R. Hydrogen peroxide-scavenging properties of normal human airway secretions. Am J Respir Crit Care Med 167:425-30 (2003)

Wijkstrom-Frei, C., S. El-Chemaly, R. Ali-Rachedi, C. Gerson, M. A. Cobas, R. Forteza, M. Salathe and G. E. Conner. Lactoperoxidase and Human Airway Host Defense. Am J Respir Cell Mol Biol 29: 206-212 (2003)

Conner G.E., Salathe M., Forteza R. Lactoperoxidase and H2O2 metabolism in the airway. Am. J. Resp. Crit. Care Med. 166:S57-61 (2002)

Nlend M.C., Bookman R.J., Conner G.E., and Salathe M. Regulator of G-protein signaling protein 2 modulates purinergic calcium and ciliary beat frequency responses in airway epithelia. Am. J. Respir. Cell Mol. Biol. 27: 436-445 (2002).

Salathe M., Forteza R. Conner G.E. Post-secretory fate of host defense components in mucus. Novartis Foundation Symposium 248:20-6 (2002).

Gerson, C., J. Sabater, M. Scuri, A. Torbati, R. Coffey, J.W. Abraham, I. Lauredo, R. Forteza, A. Wanner, M. Salathe, W. M. Abraham, and G. E. Conner. The Lactoperoxidase System Functions in Bacterial Clearance of Airways. Am. J. Resp. Cell Mol. Biol. 22: 665-671, 2000

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AIRWAY CELL BIOLOGY

Protection of the Respiratory System
Secreted Enzymes of the Airway Mucosa

The airway mucosa represents one of the most important interfaces between an animal and its environment. The mucosa must provide a sophisticated defense against airborne material of a variety of sizes and composition. Unsuccessful or inappropriate response of the airway mucosa is detrimental to the airway and underlying tissues. Our program in pulmonary cell biology studies components of airway secretions that may play a role in respiratory diseases.

Hydrogen peroxide has been shown by others to be elevated during airway inflammatory diseases such as asthma and is a major contributor to the inflammatory reactions associated with a variety of airway diseases. Our studies have identified the major hydrogen peroxide scavenging activity in airway secretions. We have now purified and characterized the activity and shown it to be a single protein that comprises 1% of the soluble secreted protein in sheep airways. Sequence analysis of the purified peroxidase together with cDNA cloning and enzymatic and spectral analysis show that the peroxidase is identical to lactoperoxidase (LPO) expressed in mammary gland. Airway LPO is secreted by goblet cells and functions both in a biocidal capacity and in controlling the reactive oxygen species in the airway. Other biochemically similar peroxidases produce biocidal compounds to protect against infection. We have shown that airway peroxidase has a similar function in the sheep and human respiratory tract.

Human airway secretions also contain peroxidase and we are studying levels of airway peroxidase and substrates in secretions of cystic fibrosis patients who have frequent bacterial infections and who have defects in ion transport that may also alter peroxidase substrate concentrations. We are also studying peroxidase in asthmatics who typically have elevated levels of hydrogen peroxide during periods of heightened inflammation. In addition, we are examining the regulation of the peroxidase system in cultures of differentiated airway epithelial cells. Finally we are working on the molecular sources of hydrogen peroxide in airway epithelia.

Tissue kallikrein is also secreted into the airway primarily by serous cells of the submucosal glands. This enzyme cleaves polypeptide hormones and precursors into active forms. Bronchial tissue kallikrein mediates bronchoconstriction in response to a variety of stimuli by cleaving high molecular weight kininogen to form lysyl-bradykinin that in tern causes bronchial smooth muscle contraction. Bronchial kallikrein is believed to mediate aspects of airway hyperresponsiveness and airway inflammation and thus is thought to play a role in asthma. This enzyme is currently being purified and characterized in order to better understand how it is regulated in normal and inflamed airways.

Hyaluronan is also secreted into the airway lumen and serves mutiple functions. Besides contributing to the viscoelastic properties of mucus, it also regulates enzymatic activity of tissue kallikrein and serves to immobilize kallikrein and airway LPO on the surface epithelial cells. This immobilization is mediated by hyaluronan binding to cell surface RHAMM and prevents the removal of the enzymes by the constant ciliary movement that clears the airways.

We use human airway epithelial cells growing in culture at an air-liquid interface as a model system. Cells are harvested from human lungs obtained from the Organ Procurement Organization through local IRB approved protocols. After dissection, airway mucosa is digested with proteases and cells are plated in normal submerged culture for expansion. For experiments, cells are plated on transwells consisting of a filter support that allows manipulation of separate apical and basolateral compartments. After growth to confluency, media is removed from the apical surface and the culture differentiates into an pseudostratified columnar epithelial layer with ciliated cells and goblet cells readily apparent. These cultures are then used to study synthesis and secretion of mucus components, transepithelial transport, ciliary beat frequency and other features specific to airway epithelia.

The beating of cilia on the surface of the cultures moves secreted mucus and trapped debris in a circular pattern reflecting the geometry of the culture system. These circular movements resemble "hurricanes" and can be seen in this movie.

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eroxidase

Cytochemical detection of airway peroxidase. Fixed sheep trachea were incubated in diaminobenzidine and hydrogen peroxide (panel a) or diaminobenzidine alone (panel c). The dark precipitated product of the airway peroxidase is visible in (arrow inpanel a). The same section in panel a was stained to show goblet cells in panelb.

 

 

 



ir-liquid interface culture of
                                airway epithelial cells

Air-liquid interface cultures of airway epithelial cells showing ciliated cells and goblet cells.

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Reactive Oxygen Species in the Airway

The Dual Oxidases (Duox 1 and Duox 2) have been identified as a major source of reactive oxygen species (ROS) including hydrogen peroxide in the airway. Lactoperoxidase has been identified as the major scavenger of hydrogen peroxide in airway secretions. Since lactoperoxidase requires hydrogen peroxide for its antibacterial activity, we are exploring the regualtion of Duox activity in the airway. We have shown that intracellular calcium concentrations regulate the activity of Duox in airway epithelia using a real-time fluorescence measurement of hydrogen peroxide production in response to stimuli that increase intracellular calcium, e.g. purinergic receptor stimulation. We are also studying the intracellular location of the Duox forms and have found that only a small portion of Duox is exposed at the cell surface

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Thiocyanate and Cystic Fibrosis

Since lactoperoxidase requires an anion for its activity, thiocyanate transport defects might provide a link between the defective anion channel (CFTR) in cystic fibrosis and chronic airway infection that is characteristic of this disease. We demonstrated defective thiocyanate transport in cystic fibrosis airway epithelia (Am. J. Respir. Crit. Care Med. 163: A490 (2001); J. Physiol. 561: 183-194 (2004); FEBS Letters 581: 271-278 (2007)) supporting the idea that a loss of thiocyanate may contribute to increased infection through lack of lactoperoxidase activity. Current studies are examining the actual levels of thiocyanate in normal and CF airways.

It is interesting to note that compuational modeling of airway surface liquid (FEBS Letters 581: 271-278 (2007)) suggests that CF airways might have increased hydrogen peroxide due to a loss of lactoperoxidase activity as lactoperoxidase is normally the major consumer of hydrogen peroxide in airway secretions (Am J Respir Crit Care Med 167: 425-430 (2003)). Elevated hdyrogen peroxide may contribute to the chronic inflammation seen in CF.


Structure and Function of Cathepsin D

Look here for structural, enzymatic, or genetic info on Cathepsin D.

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Cathepsin D Reagents and Protocols
Available Reagents (Contact info):

Human fibroblast cathepsin D cDNAs
pCPSD1 -full length preprocathepsin D with 80 nt 5' UTR, expressiblein mamalian cells
pTCPSD - procathepsin D without the signal peptide in a pET vector for bacterial expression
pVL1392CD- preprocathepsin D for homologous recombination using bacculovirus

Human fibroblast procathepsin D antiserum and recombinat human procathepsin inclusion bodies.

Here are the most frequently requested cathepsin D protocols:

Immunoprecipitation using our polyvalent antibodies against human fibroblast procathepsin D: -an example of immunoprecipitates from human endothelial cells .

Pepstatin Agarose Purification of Cathepsin D and Procathepsin D including tissues, cultured cells and media
Assay and Activation
Pepstatin Agarose Preparation

 CATHEPSIN D PUBLICATIONS:

Conner, G.E., J.A. Udey, C. Pinto, and J. Sola. Nonhuman Cells Correctly Sort and Process the Human Lysosomal Enzyme Cathepsin D. Biochemistry 28: 3530-3533, 1989.

Conner, G.E. Isolation of Procathepsin D from Cathepsin D by Pepstatin Affinity Chromatography:Autocatalytic Conversion of an inactive to active form of the enzyme. Biochem. J. 263: 601-604, 1989.

Conner GE, Udey JA. Expression and refolding of recombinant human fibroblast procathepsin D. DNA Cell Biol 9:1-9, 1990

Conner, G.E. The Role of the Procathepsin D Propeptide in Sorting to the Lysosome. J. Biol. Chem. 267: 21738-21745, 1992.

Scarborough, P.E., K. Guruprasad, C. Topham, G. icho, G.E. Conner, T.L. Blundell and B.M. Dunn. Exploration of Subsite Binding Specificity of Human Cathepsin D through Kinetics and Rule-based Molecular Modelling. Protein Science 2: 264-276, 1993

Zhu, Y. and G.E. Conner. Intermolecular Association of Lysosomal Protein Precursors During Biosynthesis. J. Biol. Chem. 269: 3846-3851, 1994

Richo, G. and G.E. Conner. Structural Requirements of Procathepsin D Activation and Maturation. J. Biol. Chem. 269: 14806-14812, 1994

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CONTACT INFORMATION

Shipping Address:

Room 7168A RMSB
Division of Pulmonary and Critical Care Medicine
University of Miami School ofMedicine
1600 N.W. 10th Ave.
Miami FL 33136

Mailing Address:

Department of Cell Biology
R-124
University of Miami School of Medicine
PO Box 016960
Miami FL 33101

EMAIL Address : gconner@miami.edu

Office: 305 243-6926
FAX: 305 243-6992

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RETURN TO

eturn

CDB Graduate Program


Copyright, Gregory E. Conner, 2000-2007,September 11, 2012 --> --> -->