A screening system is described that can detect and confirm inhibitors of the late steps of cell ... more A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.
To estimate the effectiveness of a new high-performance Australian medical sheepskin (meeting Aus... more To estimate the effectiveness of a new high-performance Australian medical sheepskin (meeting Australian Standard 4480.1-1998) in preventing pressure ulcers in a general hospital population at low to moderate risk of these ulcers. Open-label randomised controlled clinical trial. A large metropolitan teaching hospital in Melbourne, Victoria, in 2000. 441 patients aged over 18 years admitted between 12 June and 30 November 2000, with expected length of stay over 2 days and assessed as at low to moderate risk of developing pressure ulcers. Patients were randomly allocated to receive a sheepskin mattress overlay for the duration of their hospital stay (218 patients) or usual treatment, as determined by ward staff (referent group, 223 patients). Incidence rate and cumulative incidence of pressure ulcers, assessed daily throughout hospital stay. 58 patients developed pressure ulcers (sheepskin group, 21; referent group, 37). Cumulative incidence risk was 9.6% in the sheepskin group (95% C...
At a time when hospital mortality for adult cardiac operations is continuing to fall, the combine... more At a time when hospital mortality for adult cardiac operations is continuing to fall, the combined mitral valve coronary bypass subset remains at relatively high risk. Efforts to improve results should be directed toward a more general application of mitral reconstruction in this population, tailoring the type of repair to the pathological anatomy of valve dysfunction. Other promising therapeutic measures include the liberal use of reperfusion therapy in the acute papillary muscle dysfunction group, better selection patients for operation, and perhaps operative recommendation to a greater proportion of the more stable patients that previously were treated medically. Finally increasing the general awareness of this problem should hasten the development of improved management strategies.
Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III col... more Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)
The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidan... more The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.
Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III col... more Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemical and Biophysical Research Communications, 1989
Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to a... more Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.
Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host.... more Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter. We cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcri...
Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibi... more Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system. The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P. aeruginosa is very different from that described for Escherichia coli with mexXY. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion...
The innate immune response plays an important but unknown role in host defense against Mycobacter... more The innate immune response plays an important but unknown role in host defense against Mycobacterium tuberculosis. To define the function of innate immunity during tuberculosis, we evaluated M. tuberculosis replication dynamics during murine infection. Our data show that the early pulmonary innate immune response limits M. tuberculosis replication in a MyD88-dependent manner. Strikingly, we found that little M. tuberculosis cell death occurs during the first 2 weeks of infection. In contrast, M. tuberculosis cells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficient M. tuberculosis was not alleviated by the absence of interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protects M. tuberculosis from an early innate host response that is independent of ...
Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here ... more Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing…
A screening system is described that can detect and confirm inhibitors of the late steps of cell ... more A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.
To estimate the effectiveness of a new high-performance Australian medical sheepskin (meeting Aus... more To estimate the effectiveness of a new high-performance Australian medical sheepskin (meeting Australian Standard 4480.1-1998) in preventing pressure ulcers in a general hospital population at low to moderate risk of these ulcers. Open-label randomised controlled clinical trial. A large metropolitan teaching hospital in Melbourne, Victoria, in 2000. 441 patients aged over 18 years admitted between 12 June and 30 November 2000, with expected length of stay over 2 days and assessed as at low to moderate risk of developing pressure ulcers. Patients were randomly allocated to receive a sheepskin mattress overlay for the duration of their hospital stay (218 patients) or usual treatment, as determined by ward staff (referent group, 223 patients). Incidence rate and cumulative incidence of pressure ulcers, assessed daily throughout hospital stay. 58 patients developed pressure ulcers (sheepskin group, 21; referent group, 37). Cumulative incidence risk was 9.6% in the sheepskin group (95% C...
At a time when hospital mortality for adult cardiac operations is continuing to fall, the combine... more At a time when hospital mortality for adult cardiac operations is continuing to fall, the combined mitral valve coronary bypass subset remains at relatively high risk. Efforts to improve results should be directed toward a more general application of mitral reconstruction in this population, tailoring the type of repair to the pathological anatomy of valve dysfunction. Other promising therapeutic measures include the liberal use of reperfusion therapy in the acute papillary muscle dysfunction group, better selection patients for operation, and perhaps operative recommendation to a greater proportion of the more stable patients that previously were treated medically. Finally increasing the general awareness of this problem should hasten the development of improved management strategies.
Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III col... more Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)
The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidan... more The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.
Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III col... more Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemical and Biophysical Research Communications, 1989
Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to a... more Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.
Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host.... more Mycobacterium tuberculosis senses and responds to the shifting and hostile landscape of the host. To characterize the underlying intertwined gene regulatory network governed by approximately 200 transcription factors of M. tuberculosis, we have assayed the global transcriptional consequences of overexpressing each transcription factor from an inducible promoter. We cloned and overexpressed 206 transcription factors in M. tuberculosis to identify the regulatory signature of each. We identified 9,335 regulatory consequences of overexpressing each of 183 transcription factors, providing evidence of regulation for 70% of the M. tuberculosis genome. These transcriptional signatures agree well with previously described M. tuberculosis regulons. The number of genes differentially regulated by transcription factor overexpression varied from hundreds of genes to none, with the majority of expression changes repressing basal transcription. Exploring the global transcriptional maps of transcri...
Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibi... more Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system. The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P. aeruginosa is very different from that described for Escherichia coli with mexXY. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion...
The innate immune response plays an important but unknown role in host defense against Mycobacter... more The innate immune response plays an important but unknown role in host defense against Mycobacterium tuberculosis. To define the function of innate immunity during tuberculosis, we evaluated M. tuberculosis replication dynamics during murine infection. Our data show that the early pulmonary innate immune response limits M. tuberculosis replication in a MyD88-dependent manner. Strikingly, we found that little M. tuberculosis cell death occurs during the first 2 weeks of infection. In contrast, M. tuberculosis cells deficient in the surface lipid phthiocerol dimycocerosate (PDIM) exhibited significant death rates, and consequently, total bacterial numbers were reduced. Host restriction of PDIM-deficient M. tuberculosis was not alleviated by the absence of interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), or the phagocyte oxidase subunit p47. Taken together, these data indicate that PDIM protects M. tuberculosis from an early innate host response that is independent of ...
Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here ... more Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing…
Uploads