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2. Proteases and Fibrosis

Author

Heightman, Melissa, Ort, Tatiana, de Garavilla, Lawrence, Kilgore, Ken, Laurent, Geoffrey J., Vergnolle, Nathalie, editor, and Chignard, Michel, editor

Published
2011
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3. Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity

Author

Fu, Zhirong, Akula, Srinivas, Qiao, Chang, Ryu, Jinhye, Chahal, Gurdeep, de Garavilla, Lawrence, Kervinen, Jukka, Thorpe, Michael, Hellman, Lars, Fu, Zhirong, Akula, Srinivas, Qiao, Chang, Ryu, Jinhye, Chahal, Gurdeep, de Garavilla, Lawrence, Kervinen, Jukka, Thorpe, Michael, and Hellman, Lars

Abstract

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.

Published
2021
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6. Urotensin II is a nitric oxide-dependent vasodilator and natriuretic peptide in the rat kidney

Author

Zhang, Andrew Y., Chen, Ya-Fei, Zhang, David X., Yi, Fu-Xian, Qi, Jenson, Andrade-Gordon, Patricia, de Garavilla, Lawrence, Li, Pin-Lan, and Zou, Al-Ping

Subjects
Hemodynamics -- Research, Nitric oxide -- Research, Biological sciences
Abstract

Recent studies have indicated that urotensin II (UII), a cyclic peptide, is vasoactive and may be involved in cardiovascular dysfunctions. It remains unknown, however, whether UII plays a role in the control of renal vascular tone and tubular function. In the present study, a continuous infusion of synthetic human UII (hUII) into the renal artery (RA) in anesthetized rats was found to increase renal blood flow (RBF) and urinary water and sodium excretion (UV and [U.sub.Na.]V) in a dose-dependent manner. At a dose of 20 ng*[kg.sup.-1]. [min.sup.-1], it increased RBF by 20% and UV and [U.sub.Na]V by 94 and 109%, respectively. Nitric oxide (NO) synthase inhibitor [N.sup.G]-nitro-L-arginine methyl ester (L-NAME) completely abolished hUII-induced increases in RBF and water/sodium excretion. In isolated, pressurized, and phenylephrine-precontracted small RA with internal diameter of ~200 [micro]m, hUII produced a concentration-dependent vasodilation with a maximal response of 55% at 1.5 [micro]M. L-NAME significantly blocked this hUII-induced vasodilation by 60%. In denuded RA, hUII had neither vasodilator nor vasoconstrictor effect. With the use of 4,5-diaminofluorescein diacetate-based fluorescence imaging analysis of NO levels, hUII (1 [micro]M) was shown to double the NO levels within the endothelium of freshly dissected small RA, and L-NAME blocked this UII-induced production of endothelial NO. These results indicate that UII produces vasodilator and natriuretic effects in the kidney and that UII-induced vasodilation is associated with increased endothelial NO in the RA. natriuretic factor; renal circulation; sodium reabsorption; renal tubule; renal hemodynamics

Published
2003

15. Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases : Two Highly Specific Leu-Ases

Author

Zhongwei, Yuan, Akula, Srinivas, Fu, Zhirong, de Garavilla, Lawrence, Kervinen, Jukka, Thorpe, Michael, Hellman, Lars, Zhongwei, Yuan, Akula, Srinivas, Fu, Zhirong, de Garavilla, Lawrence, Kervinen, Jukka, Thorpe, Michael, and Hellman, Lars

Abstract

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit beta-chymase and a guinea pig alpha-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species.

Published
2019
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17. Extended cleavage specificities of mast cell proteases 1 and 2 from golden hamster: Classical chymase and an elastolytic protease comparable to rat and mouse MCP-5

Author

Thorpe, Michael, Fu, Zhirong, Albat, Emanuelle, Akula, Srinivas, de Garavilla, Lawrence, Kervinen, Jukka, and Hellman, Lars

Subjects
Biochemistry, Substrate Specificity, Database and Informatics Methods, Mice, Phage Display, Animal Cells, Cricetinae, Medicine and Health Sciences, Bacteriophages, Mast Cells, Phylogeny, Connective Tissue Cells, Mammals, Biochemistry and Molecular Biology, Eukaryota, Proteases, Recombinant Proteins, Enzymes, Molecular Biology Display Techniques, Connective Tissue, Vertebrates, Viruses, Immunologi, Hamsters, Amino Acid Analysis, Medicine, Cellular Types, Anatomy, Sequence Analysis, Research Article, Bioinformatics, Science, Immunology, Research and Analysis Methods, Rodents, Chymases, Species Specificity, Consensus Sequence, Animals, Humans, Amino Acid Sequence, Molecular Biology Techniques, Molecular Biology, Molecular Biology Assays and Analysis Techniques, Mesocricetus, Sequence Homology, Amino Acid, Secretory Vesicles, Organisms, Biology and Life Sciences, Proteins, Cell Biology, Rats, Biological Tissue, Amniotes, Enzymology, Serine Proteases, Cell Surface Display Techniques, Sequence Alignment, Biokemi och molekylärbiologi
Abstract

Serine proteases constitute the major protein content of mast cell secretory granules. Here we present the extended cleavage specificity of two such proteases from the golden hamster, Mesocricetus auratus. Analysis by phage display technique showed that one of them (HAM1) is a classical chymase with a specificity similar to the human mast cell chymase. However, in contrast to the human chymase, it does not seem to have a particular preference for any of the three aromatic amino acids, Phe, Tyr and Trp, in the P1 position of substrates. HAM1 also efficiently cleaved after Leu similarly to human and many other mast cell chymases. We observed only a 3-fold lower cleavage activity on Leu compared to substrates with P1 aromatic amino acids. Chymotryptic enzymes seem to be characteristic for connective tissue mast cells in mammalian species from opossums to humans, which indicates a very central role of these enzymes in mast cell biology. HAM1 also seems to have the strongest preference for negatively charged amino acids in the P2 position of all mast cell chymases so far characterized. The second hamster chymase, HAM2, is an elastolytic in its activity, similarly to the alpha-chymases in rats and mice (rMCP-5 and mMCP-5, respectively). The presence of an alpha-chymase that developed elastase activity thereby seems to be a relatively early modification of the alpha-chymase within the rodent branch of the mammalian evolutionary tree.

Published
2018

18. Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities

Author

Thorpe, Michael, Fu, Zhirong, Chahal, Gurdeep, Akula, Srinivas, Kervinen, Jukka, de Garavilla, Lawrence, Hellman, Lars, and Publica

Subjects
Proteomics, Cathepsin G, Neutrophils, Bioinformatics, Immune Cells, Immunology, lcsh:Medicine, Research and Analysis Methods, Biochemistry, Substrate Specificity, White Blood Cells, Database and Informatics Methods, Chymases, Phage Display, Animal Cells, Medicine and Health Sciences, Humans, Bacteriophages, Amino Acid Sequence, Mast Cells, Molecular Biology Techniques, Peptide Libraries, lcsh:Science, Molecular Biology, Conserved Sequence, Molecular Biology Assays and Analysis Techniques, Blood Cells, lcsh:R, Organisms, Biology and Life Sciences, Proteins, Proteases, Cell Biology, Enzymes, Molecular Biology Display Techniques, Enzyme Activation, Kinetics, Viruses, Proteolysis, Enzymology, Amino Acid Analysis, Tryptases, lcsh:Q, Cellular Types, Serine Proteases, Sequence Analysis, Sequence Alignment, Research Article
Abstract

Human neutrophils express at least four active serine proteases, cathepsin G, N-elastase, proteinase 3 and neutrophil serine protease 4 (NSP4). They have all been extensively studied due to their importance in neutrophil biology and immunity. However, their extended cleavage specificities have never been determined in detail. Here we present a detailed cleavage specificity analysis of human cathepsin G (hCG). The specificity was determined by phage display analysis and the importance of individual amino acids in and around the cleavage site was then validated using novel recombinant substrates. To provide a broader context to this serine protease, a comparison was made to the related mast cell protease, human chymase (HC). hCG showed similar characteristics to HC including both the primary and extended specificities. As expected, Phe, Tyr, Trp and Leu were preferred in the P1 position. In addition, both proteases showed a preference for negatively charged amino acids in the P2´ position of substrates and a preference for aliphatic amino acids both upstream and downstream of the cleavage site. However, overall the catalytic activity of hCG was ~10-fold lower than HC. hCG has previously been reported to have a dual specificity consisting of chymase and tryptase-type activities. In our analysis, tryptase activity against substrates with Lys in P1 cleavage position was indeed only 2-fold less efficient as compared to optimal chymase substrates supporting strong dual-type specificity. We hope the information presented here on extended cleavage specificities of hCG and HC will assist in the search for novel in vivo substrates for these proteases as well as aid in the efforts to better understand the role of hCG in immunity and bacterial defence.

Published
2018

20. Highly Selective Cleavage of Cytokines and Chemokines by the Human Mast Cell Chymase and Neutrophil Cathepsin G

Author

Fu, Zhirong, Thorpe, Michael, Alemayehu, Rahel, Roy, Ananya, Kervinen, Jukka, de Garavilla, Lawrence, Abrink, Magnus, Hellman, Lars, Fu, Zhirong, Thorpe, Michael, Alemayehu, Rahel, Roy, Ananya, Kervinen, Jukka, de Garavilla, Lawrence, Abrink, Magnus, and Hellman, Lars

Abstract

Human mast cell chymase (HC) and human neutrophil cathepsin G (hCG) show relatively similar cleavage specificities: they both have chymotryptic activity but can also cleave efficiently after leucine. Their relatively broad specificity suggests that they may cleave almost any substrate if present in high enough concentrations or for a sufficiently long time. A number of potential substrates have been identified for these enzymes and, recently, these enzymes have also been implicated in regulating cytokine activity by cleaving numerous cytokines and chemokines. To obtain a better understanding of their selectivity for various potential in vivo substrates, we analyzed the cleavage of a panel of 51 active recombinant cytokines and chemokines. Surprisingly, our results showed a high selectivity of HC; only 4 of 51 of these proteins were substantially cleaved. hCG cleaved a few additional proteins, although this occurred after adding almost equimolar amounts of enzyme to target. The explanation for this wide difference in activity against peptides or other linear substrates compared with native proteins is most likely related to the reduced accessibility of the enzymes to potential cleavage sites in folded proteins. In this article, we present evidence that sites not exposed on the surface of the protein are not cleaved by the enzyme. Interestingly, both enzymes readily cleaved IL-18 and IL-33, two IL-1-related alarmins, as well as the cytokine IL-15, which is important for T cell and NK cell homeostasis. Cleavage of the alarmins by HC and hCG suggests a function in regulating excessive inflammation.

Published
2017
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22. Nonpeptide inhibitors of cathepsin G: optimization of a novel beta-ketophosphonic acid lead by structure-based drug design

Author

Greco, Michael N., Hawkins, Michael J., Powell, Eugene T., Almond, Harold R., Jr., Corcoran, Thomas W., De Garavilla, Lawrence, Kauffman, Jack A., Recacha, Rosario, Chattopadhyay, Debashish, Andrade-Gordon, Patricia, and Maryanoff, Bruce E.

Subjects
Chemical research -- Analysis, Acids -- Physiological aspects, Ketones -- Physiological aspects, Chemical inhibitors -- Physiological aspects, Cathepsins -- Physiological aspects, Chemistry
Abstract

Research has been conducted on the serine protease cathepsin G. The nonpeptide inhibitors of this cathepsin have been investigated via the use of the structure-based drug design approach and the results indicate that they possess beta-ketophosphonic acid subunit.

Published
2002

23. Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis) : an interesting animal model for the analysis of the function of the human mast cell chymase

Author

Thorpe, Michael, Yu, Jing, Boinapally, Vamsi, Ahooghalandari, Parvin, Kervinen, Jukka, de Garavilla, Lawrence, Hellman, Lars, Thorpe, Michael, Yu, Jing, Boinapally, Vamsi, Ahooghalandari, Parvin, Kervinen, Jukka, de Garavilla, Lawrence, and Hellman, Lars

Abstract

Serine proteases are the major protein constituents within mast cell secretory granules. These proteases are subdivided into chymases and tryptases depending on their primary cleavage specificity. Here, we present the extended cleavage specificity of the macaque mast cell chymase and compare the specificity with human chymase (HC) and dog chymase (DC) that were produced in the same insect cell expression host. The macaque chymase (MC) shows almost identical characteristics as the HC, including both primary and extended cleavage specificities as well as sensitivity to protease inhibitors, whereas the DC differs in several of these characteristics. Although previous studies have shown that mouse mast cell protease-4 (mMCP-4) is similar in its hydrolytic specificity to the HC, mouse mast cells contain several related enzymes. Thus mice may not be the most appropriate model organism for studying HC activity and inhibition. Importantly, macaques express only one chymase and, as primates, are closely related to human general physiology. In addition, the human and macaque enzymes both cleave angiotensin I (Ang I) in the same way, generating primarily angiotensin II (Ang II) and they do not further degrade the peptide like most rodent enzymes do. Both enzymes also cleave two additional potential in vivo substrates, fibronectin and secretory leukocyte protease inhibitor (SLPI) in a similar way. Given the fact that both HC and MC are encoded by a single gene with high sequence homology and that many physiological processes are similar between these species, the macaque may be a very interesting model to study the physiological role of the chymase and to determine the potency and potential side-effects of various chymase inhibitors designed for therapeutic human use.

Published
2012
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26. Potent, nonpeptide inhibitors of human mast cell tryptase. Synthesis and biological evaluation of novel spirocyclic piperidine amide derivatives

Author

Costanzo, Michael J., Yabut, Stephen C., Zhang, Han-Cheng, White, Kimberley B., de Garavilla, Lawrence, Wang, Yuanping, Minor, Lisa K., Tounge, Brett A., Barnakov, Alexander N., Lewandowski, Frank, Milligan, Cynthia, Spurlino, John C., Abraham, William M., Boswell-Smith, Victoria, Page, Clive P., and Maryanoff, Bruce E.

Published
2008
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30. Structural Basis for Elastolytic Substrate Specificity in Rodent α-Chymases

Author

Kervinen, Jukka, primary, Abad, Marta, additional, Crysler, Carl, additional, Kolpak, Michael, additional, Mahan, Andrew D., additional, Masucci, John A., additional, Bayoumy, Shariff, additional, Cummings, Maxwell D., additional, Yao, Xiang, additional, Olson, Matthew, additional, de Garavilla, Lawrence, additional, Kuo, Lawrence, additional, Deckman, Ingrid, additional, and Spurlino, John, additional

Published
2008
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31. In-Depth Study of Tripeptide-Based α-Ketoheterocycles as Inhibitors of Thrombin. Effective Utilization of the S1‘ Subsite and Its Implications to Structure-Based Drug Design.

Author

Costanzo, Michael J., primary, Almond, Harold R., additional, Hecker, Leonard R., additional, Schott, Mary R., additional, Yabut, Stephen C., additional, Zhang, Han-Cheng, additional, Andrade-Gordon, Patricia, additional, Corcoran, Thomas W., additional, Giardino, Edward C., additional, Kauffman, Jack A., additional, Lewis, Joan M., additional, de Garavilla, Lawrence, additional, Haertlein, Barbara J., additional, and Maryanoff, Bruce E., additional

Published
2007
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32. Discovery of Potent, Selective, Orally Active, Nonpeptide Inhibitors of Human Mast Cell Chymase

Author

Greco, Michael N., primary, Hawkins, Michael J., additional, Powell, Eugene T., additional, Almond,, Harold R., additional, de Garavilla, Lawrence, additional, Hall, Jeffrey, additional, Minor, Lisa K., additional, Wang, Yuanping, additional, Corcoran, Thomas W., additional, Di Cera, Enrico, additional, Cantwell, Angelene M., additional, Savvides, Savvas N., additional, Damiano, Bruce P., additional, and Maryanoff, Bruce E., additional

Published
2007
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33. Selection of a 2-azabicyclo[2.2.2]octane-based α4β1 integrin antagonist as an inhaled anti-asthmatic agent

Author

Lawson, Edward C., primary, Santulli, Rosemary J., additional, Dyatkin, Alexey B., additional, Ballentine, Scott A., additional, Abraham, William M., additional, Rudman, Sandra, additional, Page, Clive P., additional, de Garavilla, Lawrence, additional, Damiano, Bruce P., additional, Kinney, William A., additional, and Maryanoff, Bruce E., additional

Published
2006
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35. A Novel, Potent Dual Inhibitor of the Leukocyte Proteases Cathepsin G and Chymase

Author

de Garavilla, Lawrence, primary, Greco, Michael N., additional, Sukumar, Narayanasami, additional, Chen, Zhi-Wei, additional, Pineda, Agustin O., additional, Mathews, F. Scott, additional, Di Cera, Enrico, additional, Giardino, Edward C., additional, Wells, Grace I., additional, Haertlein, Barbara J., additional, Kauffman, Jack A., additional, Corcoran, Thomas W., additional, Derian, Claudia K., additional, Eckardt, Annette J., additional, Damiano, Bruce P., additional, Andrade-Gordon, Patricia, additional, and Maryanoff, Bruce E., additional

Published
2005
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36. In-Depth Study of Tripeptide-Based α-Ketoheterocycles as Inhibitors of Thrombin. Effective Utilization of the S1‘ Subsite and Its Implications to Structure-Based Drug Design

Author

Costanzo, Michael J., primary, Almond, Harold R., additional, Hecker, Leonard R., additional, Schott, Mary R., additional, Yabut, Stephen C., additional, Zhang, Han-Cheng, additional, Andrade-Gordon, Patricia, additional, Corcoran, Thomas W., additional, Giardino, Edward C., additional, Kauffman, Jack A., additional, Lewis, Joan M., additional, de Garavilla, Lawrence, additional, Haertlein, Barbara J., additional, and Maryanoff, Bruce E., additional

Published
2004
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38. Potent nonpeptide vasopressin receptor antagonists based on oxazino- and thiazinobenzodiazepine templates

Author

Matthews, Jay M., primary, Hoekstra, William J., additional, Dyatkin, Alexey B., additional, Hecker, Leonard R., additional, Hlasta, Dennis J., additional, Poulter, Brenda L., additional, Andrade-Gordon, Patricia, additional, de Garavilla, Lawrence, additional, Demarest, Keith T., additional, Ericson, Eric, additional, Gunnet, Joseph W., additional, Hageman, William, additional, Look, Richard, additional, Moore, John B., additional, Reynolds, Charles H., additional, and Maryanoff, Bruce E., additional

Published
2004
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40. Corrigendum to “Aza-bicyclic amino acid sulfonamides as α4β1/α4β7 integrin antagonists”

Author

Dyatkin, Alexey B, primary, Hoekstra, William J, additional, Kinney, William A, additional, Kontoyianni, Maria, additional, Santulli, Rosemary J, additional, Kimball, Edward S, additional, Carolyn Fisher, M, additional, Prouty, Stephen M, additional, Abraham, William M, additional, de Garavilla, Lawrence, additional, Andrade-Gordon, Patricia, additional, Hlasta, Dennis J, additional, He, Wei, additional, Hornby, Pamela J, additional, Damiano, Bruce P, additional, and Maryanoff, Bruce E, additional

Published
2004
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43. Potent, Small-Molecule Inhibitors of Human Mast Cell Tryptase. Antiasthmatic Action of a Dipeptide-Based Transition-State Analogue Containing a Benzothiazole Ketone

Author

Costanzo, Michael J., primary, Yabut, Stephen C., additional, Almond,, Harold R., additional, Andrade-Gordon, Patricia, additional, Corcoran, Thomas W., additional, de Garavilla, Lawrence, additional, Kauffman, Jack A., additional, Abraham, William M., additional, Recacha, Rosario, additional, Chattopadhyay, Debashish, additional, and Maryanoff, Bruce E., additional

Published
2003
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46. Structure–Function Analysis of Urotensin II and Its Use in the Construction of a Ligand–Receptor Working Model

Author

Kinney, William A., primary, Almond, Jr., Harold R., additional, Qi, Jenson, additional, Smith, Charles E., additional, Santulli, Rosemary J., additional, de Garavilla, Lawrence, additional, Andrade-Gordon, Patricia, additional, Cho, Daniel S., additional, Everson, Anita M., additional, Feinstein, Mark A., additional, Leung, Perry A., additional, and Maryanoff, Bruce E., additional

Published
2002
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47. Nonpeptide Inhibitors of Cathepsin G: Optimization of a Novel β-Ketophosphonic Acid Lead by Structure-Based Drug Design

Author

Greco, Michael N., primary, Hawkins, Michael J., additional, Powell, Eugene T., additional, Almond,, Harold R., additional, Corcoran, Thomas W., additional, de Garavilla, Lawrence, additional, Kauffman, Jack A., additional, Recacha, Rosario, additional, Chattopadhyay, Debashish, additional, Andrade-Gordon, Patricia, additional, and Maryanoff, Bruce E., additional

Published
2002
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