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Search Results: 1 - 10 of 313735 matches for " James J Caffrey "
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Measurements of the acid-binding capacity of ingredients used in pig diets
Peadar G Lawlor, P Brendan Lynch, Patrick J Caffrey, James J O'Reilly, M Karen O'Connell
Irish Veterinary Journal , 2005, DOI: 10.1186/2046-0481-58-8-447
Abstract: In the pig, protein digestion begins in the stomach with the action of pepsins, secreted as the enzyme precursors - pepsinogens - by stomach mucosa. Conversion of pepsinogen to pepsin occurs rapidly at pH 2.0 but only slowly at pH 5.0 to 6.0. In turn, pepsins work best in an acidic environment, pH 2.0 to 3.5, and activity declines rapidly above this pH. Carbohydrate hydrolysis in the stomach occurs by the action of salivary amylase, which, in contrast to pepsin, is inactivated once pH falls to 3.5 [14,18,22].In the suckling pig, acid secretion is low and the principal source of acidity is bacterial fermentation of lactose from sows milk to lactic acid [9,10,14]. A high level of lactate in the stomach tends to inhibit HCl secretion [10,22]. Ingestion of solid feed reduces the level of lactic acid in the stomach [22] and stimulates HCl production [10,7] but, in practice, creep feed consumption is low and variable at least up to four weeks of age [15].At weaning, a combination of low acid secretion, lack of lactose substrate, and consumption of large meals at infrequent intervals can result in elevated pH, often to over 5.0 and it may remain high for several days [14]. The high acid-binding/buffering capacity of the feed (its ability to neutralise feed acid) helps to further raise the stomach pH [20,13,6]. Inclusion of whey or lactose in the starter diet ensures continuation of bacterial fermentation and some, though reduced, lactic acid production [14,11]. Development of HCl secretory capacity occurs more rapidly in the weaned pig than in the suckling pig [8].Lowering the acid-binding capacity of diets for newly-weaned pigs can help ease the transition from milk to solid food at weaning.Raised stomach pH after weaning results in reduced digestion of feed which will then be fermented in the hind gut and may provoke diarrhoea. A high gastric pH will also allow pathogens to survive and allow them greater opportunity to colonise the digestive tract [6,22].The concept of m
Combined histomorphometric and gene-expression profiling applied to toxicology
Andres Kriete, Mary K Anderson, Brad Love, John Freund, James J Caffrey, M Brook Young, Timothy J Sendera, Scott R Magnuson, J Mark Braughler
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-5-r32
Abstract: Recent reports describe the use of gene-expression profiling for the identification of molecular markers of toxicity [1-3]. This technique alone does not account for morphological changes in tissues that have traditionally been used by pathologists to discriminate between types and severity of toxicological responses [4-6]. For a comprehensive approach to toxicological evaluation, we developed a unique methodology that uses histomorphometric profiles, derived from machine vision, in conjunction with gene-expression profiles, termed extensible morphometric relational gene-expression analysis (EMeRGE). This novel method was evaluated on an established, extreme model of liver toxicity using carbon tetrachloride (CCl4) in rats that were dosed for 3 days and allowed to recover. Liver is relevant in toxicology as the primary organ of metabolism and detoxification; it is a recurrent target of chronic drug toxicity.A fully automated analytical microscope equipped with machine-vision hardware and software was used to generate quantitative information about the structure and heterogeneity of liver. The histomorphometric profiles could be used to evaluate tissue heterogeneity across the tissue including regions of hepatocellular necrosis. Representative images of tissue sections from control and treated tissues are shown in Figure 1. Examples of processed sample image tiles are shown in Figure 2, where a control liver (Figure 2a) can be compared to a treated liver (Figure 2b), illustrating the significant structural damage induced by treatment with CCl4. Gene-expression profiles were generated from the same livers using DNA microarrays. The microarrays measured mRNA transcription levels of genes important in adsorption, distribution, metabolism and excretion (ADME). Previous studies of these genes, including markers of toxic stress, apoptosis, growth regulation and repair, were consistent with documented toxicologic responses to CCl4, where expression of components of cytochro
A Comparative Chemogenomics Strategy to Predict Potential Drug Targets in the Metazoan Pathogen, Schistosoma mansoni
Conor R. Caffrey, Andreas Rohwer, Frank Oellien, Richard J. Marh?fer, Simon Braschi, Guilherme Oliveira, James H. McKerrow, Paul M. Selzer
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004413
Abstract: Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for ‘druggable’ protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen.
Prolyl Oligopeptidase from the Blood Fluke Schistosoma mansoni: From Functional Analysis to Anti-schistosomal Inhibitors
Pavla Fajtová?,Sa?a ?tefani??,Martin Hradilek?,Jan Dvo?ák?,Ji?í Vondrá?ek?,Adéla Jílková?,Lenka Ulrychová?,James H. McKerrow?,Conor R. Caffrey,Michael Mare?
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0003827
Abstract: Background Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. Methodology/Principal Findings We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. Recombinant SmPOP was produced in Escherichia coli and its active site specificity investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. The inhibition profile is analogous to those for mammalian POPs. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin I and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. Conclusions/Significance We provide the first localization and functional analysis of SmPOP together with chemical tools for measuring its activity. We briefly discuss the notion that SmPOP, operating at the host-parasite interface to cleave host bioactive peptides, may contribute to the survival of the parasite. If substantiated, SmPOP could be a new target for the development of anti-schistosomal drugs.
Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica
Ileana Corvo,Anthony J. O'Donoghue,Lucía Pastro,Natalia Pi-Denis,Alegra Eroy-Reveles,Leda Roche,James H. McKerrow,John P. Dalton,Charles S. Craik,Conor R. Caffrey,José F. Tort
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002269
Abstract: Background A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. Methodology/Principal Findings Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. Conclusions/Significance These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases.
Cure of Hookworm Infection with a Cysteine Protease Inhibitor
Jon J. Vermeire,Lorine D. Lantz,Conor R. Caffrey
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001680
Abstract: Background Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed. Methods and Findings Using various life-cycle stages of the hookworm Ancylostoma ceylanicum in vitro and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homop?henylalanyl-vinyl sulfone phenyl) and K11777 (N-methylpiperazine-phenylalanyl-homopheny?lalanyl-vinylsulfonephenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. In vitro, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in ex vivo adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 b.i.d. (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose. Conclusion A new, oral single-dose anthelmintic that is active in an animal model of hookworm infection and that possesses a distinct mechanism of action from current anthelmintics is discovered. The data highlight both the possibility of repurposing the anti-chagasic K11777 as a treatment for hookworm infection and the opportunity to further develop CPIs as a novel anthelmintic class to target hookworms and, possibly, other helminths.
Prediction of specificity-determining residues for small-molecule kinase inhibitors
Daniel R Caffrey, Elizabeth A Lunney, Deborah J Moshinsky
BMC Bioinformatics , 2008, DOI: 10.1186/1471-2105-9-491
Abstract: Here we describe S-Filter, a method that combines sequence and structural information to predict specificity-determining residues for a small molecule and its kinase selectivity profile. Analysis was performed on seven selective kinase inhibitors where a structural basis for selectivity is known. S-Filter correctly predicts specificity determinants that were described by independent groups. S-Filter also predicts a number of novel specificity determinants that can often be justified by further structural comparison.S-Filter is a valuable tool for analyzing kinase selectivity profiles. The method identifies potential specificity determinants that are not readily apparent, and provokes further investigation at the structural level.The human genome contains approximately 500 protein kinases that regulate numerous cellular processes via protein phosphorylation [1]. Protein kinases mediate cell signaling pathways that are important for metabolism, development, apoptosis, immune responses, cell proliferation, and differentiation. Several of these pathways have been implicated in cancer, inflammation, and metabolic diseases. Thus, a number of protein kinases have been proposed as drug targets for these diseases [2]. Designing selective kinase inhibitors is a major challenge in drug discovery and development. The gene family is large and most kinases domains are similar in sequence and structure. The selectivity issues associated with small molecules that bind to the ATP catalytic binding site are particularly challenging as most kinases have the same active-site chemistry.Understanding the basis of kinase inhibitor selectivity is crucial to the design of safe and efficacious drugs. Ideally, a drug will inhibit a small set of kinases that are relevant to the disease while avoiding the inhibition of kinases that may lead to toxic side effects. For example, imatinib inhibits a number of kinases that are believed to be important for certain cancer types [3]. However, most kina
Proteomic Identification of IPSE/alpha-1 as a Major Hepatotoxin Secreted by Schistosoma mansoni Eggs
Maha-Hamadien Abdulla ,Kee-Chong Lim,James H. McKerrow,Conor R. Caffrey
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001368
Abstract: Background Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. Methodology/Principal Findings Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. Conclusions We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.
Schistosomiasis Mansoni: Novel Chemotherapy Using a Cysteine Protease Inhibitor
Maha-Hamadien Abdulla,Kee-Chong Lim,Mohammed Sajid,James H McKerrow,Conor R Caffrey
PLOS Medicine , 2007, DOI: 10.1371/journal.pmed.0040014
Abstract: Background Schistosomiasis is a chronic, debilitating parasitic disease infecting more than 200 million people and is second only to malaria in terms of public health importance. Due to the lack of a vaccine, patient therapy is heavily reliant on chemotherapy with praziquantel as the World Health Organization–recommended drug, but concerns over drug resistance encourage the search for new drug leads. Methods and Findings The efficacy of the vinyl sulfone cysteine protease inhibitor K11777 was tested in the murine model of schistosomiasis mansoni. Disease parameters measured were worm and egg burdens, and organ pathology including hepato- and splenomegaly, presence of parasite egg–induced granulomas in the liver, and levels of circulating alanine aminotransferase activity as a marker of hepatocellular function. K11777 (25 mg/kg twice daily [BID]), administered intraperitoneally at the time of parasite migration through the skin and lungs (days 1–14 postinfection [p.i.]), resulted in parasitologic cure (elimination of parasite eggs) in five of seven cases and a resolution of other disease parameters. K11777 (50 mg/kg BID), administered at the commencement of egg-laying by mature parasites (days 30–37 p.i.), reduced worm and egg burdens, and ameliorated organ pathology. Using protease class-specific substrates and active-site labeling, one molecular target of K11777 was identified as the gut-associated cathepsin B1 cysteine protease, although other cysteine protease targets are not excluded. In rodents, dogs, and primates, K11777 is nonmutagenic with satisfactory safety and pharmacokinetic profiles. Conclusions The significant reduction in parasite burden and pathology by this vinyl sulfone cysteine protease inhibitor validates schistosome cysteine proteases as drug targets and offers the potential of a new direction for chemotherapy of human schistosomiasis.
A Global Comparison of the Human and T. brucei Degradomes Gives Insights about Possible Parasite Drug Targets
Susan T. Mashiyama,Kyriacos Koupparis,Conor R. Caffrey,James H. McKerrow ,Patricia C. Babbitt
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001942
Abstract: We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups (“M32” and “C51”) that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at http://babbittlab.ucsf.edu/resources.htm?l.
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