Emmanuelle Fantino - Academia.edu (original) (raw)

Papers by Emmanuelle Fantino

Research paper thumbnail of Downstream activating sequence within the coding region of a yeast gene: specific binding in vitro of RAP1 protein

Molecular & general genetics : MGG, 1992

Using a gel retardation assay, a protein factor that specifically interacts with a 33 bp intragen... more Using a gel retardation assay, a protein factor that specifically interacts with a 33 bp intragenic sequence of the highly expressed and glucose-inducible SRP1 gene of Saccharomyces cerevisiae has been detected. This binding site is located in a transcribed region and within the open reading frame (positions +710 to +743 relative to the first base of the initiation codon). A mutant strain carrying a deletion of this binding site showed a dramatic decrease in steady-state levels of SRP1 transcripts. This decline is not the result of a decrease in mRNA stability, since expression of hybrid genes in which the SRP1 promoter was replaced by the heterologous CYC1 promoter was not affected by the binding site deletion. These findings suggest that the 33 bp sequence contains a cis-acting downstream activating element which is involved in the transcriptional activation of the SRP1 promoter. Sequence comparisons showed similarities between a site located within the 33 bp sequence and the high...

Research paper thumbnail of Gene cloning from yeast chromosome-specific mini-library Isolation of the SRP1 -related DNA sequence located on chromosome XV

FEBS Letters, 1988

We have isolated and purified chromosome XV DNA molecules from the yeast S. cerevisiae using cont... more We have isolated and purified chromosome XV DNA molecules from the yeast S. cerevisiae using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. A chromosome-specific mini-library was constructed and the element of the SRPI (serine-rich protein) related sequence family located within chromosome XV was isolated by in situ colony hybridization with an SRPl probe. Results indicated that (i) a single-copy sequence homologous to SRPI is present within chromosome XV; (ii) this sequence lies within the 2.3 kb Hind111 fragment of the plasmid pXVAc6; (iii) the lack of a specific transcript from this SRPI-related element suggests that it could be considered as a pseudogene.

Research paper thumbnail of Airway, but not serum or urinary, levels of YKL-40 reflect inflammation in early cystic fibrosis lung disease

BMC Pulmonary Medicine, 2014

Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk fac... more Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk factor being an exaggerated inflammatory response. Currently, assessment of neutrophilic inflammation in early cystic fibrosis (CF) lung disease relies on bronchoalveolar lavage (BAL). The chitinase-like protein YKL-40 is raised in sputum and serum of adults with CF. We investigated YKL-40 in BAL, serum and urine to determine whether this reflected inflammation and infection in young children with CF. YKL-40 was measured in matched samples of BAL, serum and urine obtained from 36 infants and young children with CF participating in an early surveillance program. Levels were compared to clinical data and markers of inflammation detected in the lung. YKL-40 in BAL correlated with pulmonary infection [β=1.30 (SE 0.34), p < 0.001] and BAL markers of inflammation [macrophage number: r2 = 0.34, p < 0.001; neutrophil number: r2 = 0.74, p < 0.001; neutrophil elastase: r2 = 0.47, p < 0.001; CXCL8: r2 = 0.45, p < 0.001; IL-β: r2 = 0.62, p < 0.001]. YKL-40 was detectable in serum but levels did not correlate with BAL levels in the same individuals (r2 = 0.04, p = 0.14) or with inflammatory markers. YKL-40 was below the limit of detection in urine (30 pg/ml). This study demonstrates that levels of the chitinase-like protein YKL-40 reflect airway inflammation and infection in early CF lung disease. The lack of increased YKL-40 in serum in the absence of systemic inflammation limits the benefit of this potential biomarker in early disease.

Research paper thumbnail of Hanging drop online supplimentary (3)

Research paper thumbnail of Phenotypic, Functional and Plasticity Features of Classical and Alternatively Activated Human Macrophages

American Journal of Respiratory Cell and Molecular Biology, 2015

Macrophages are dynamic cells that mature under the influence of signals from the local microenvi... more Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-g and IL-4/IL-13, respectively. M1 and M2 were identified as CD64 1 CD80 1 and CD11b 1 CD209 1 , respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-g, IL-8, TNF-a, IL-1b, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b 1 CD209 1 and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.

Research paper thumbnail of Influence Of Growth Factors And Glucocorticoids In Media On Epithelial Cell Responses

C36. LUNG EPITHELIUM, SUBMUCOSAL GLANDS AND SMOOTH MUSCLE, 2012

Research paper thumbnail of Brief exposure to cigarette smoke impairs airway epithelial cell innate anti-viral defence

Toxicology in Vitro, 2014

Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma ex... more Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma exacerbations. Environmental cigarette smoke exposure is associated with a significant increase in the risk for these infections in children. To determine the impact of short-term exposure to cigarette smoke on innate immune responses of airway epithelial cells infected with hRV. A human bronchial epithelial cell line (HBEC-3KT) was exposed to cigarette smoke extract (CSE) for 30 min and subsequently infected with hRV serotype 1B. Viral-induced cytokine release was measured with AlphaLISA and viral replication quantified by shed viral titer and intracellular viral copy number 24h post-infection. CSE induced a concentration-dependent decrease in CXCL10 (p<0.001) and IFN-β (p<0.001), with a 79% reduction at the highest dose with an associated 3-fold increase in shed virus. These effects were maintained when infection was delayed up to 24h post CSE exposure. Exogenous IFN-β treatment at t=0 after infection blunts the effects of CSE on viral replication (p<0.05). A single exposure of 30 min to cigarette smoke has a lasting impact on epithelial innate defence providing a plausible mechanism for the increase in respiratory infections seen in children exposed to second-hand tobacco smoke.

Research paper thumbnail of A piece in the puzzle: an ion channel candidate gene for schizophrenia

Molecular Medicine Today, 1998

Mutations in ion channels have been found to cause a variety of mendelian genetic diseases, and p... more Mutations in ion channels have been found to cause a variety of mendelian genetic diseases, and polyglutamine repeat expansion is a newly recognized pathogenic mechanism that causes several rare, genetic, late-onset neurological syndromes. Polymorphic polyglutamine tracts are present in a recently described human, calcium-activated potassium channel, KCNN3 (also known as hKCa3), and alleles of this gene that contain longer repeats have been associated with schizophrenia. The physiological function of the channel is consistent with an etiological role in this disease; drugs designed to target this channel might therefore provide novel psychotherapeutics.

Research paper thumbnail of Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy

Research paper thumbnail of The Microtubule Depolymerizing Agent CYT997 Causes Extensive Ablation of Tumor Vasculature In Vivo

Journal of Pharmacology and Experimental Therapeutics, 2011

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridi... more The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Research paper thumbnail of Environmental Contributions to the Leading Causes of Disease Burden Among Australian Children

Journal of Pediatric Gastroenterology and Nutrition, 2013

Research paper thumbnail of The 2.7 Å Crystal Structure of the Autoinhibited Human c-Fms Kinase Domain

Journal of Molecular Biology, 2007

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine... more c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.

Research paper thumbnail of Dissecting Specificity in the Janus Kinases: The Structures of JAK-Specific Inhibitors Complexed to the JAK1 and JAK2 Protein Tyrosine Kinase Domains

Journal of Molecular Biology, 2009

The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play promin... more The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play prominent roles in numerous cytokine signaling pathways, with aberrant JAK activity associated with a variety of hematopoietic malignancies, cardiovascular diseases and immune-related disorders. Whereas the structures of the JAK2 and JAK3 PTK domains have been determined, the structure of the JAK1 PTK domain is unknown. Here, we report the high-resolution crystal structures of the "active form" of the JAK1 PTK domain in complex with two JAK inhibitors, a tetracyclic pyridone 2-tbutyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one (CMP6) and (3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile (CP-690,550), and compare them with the corresponding JAK2 PTK inhibitor complexes. Both inhibitors bound in a similar manner to JAK1, namely buried deep within a constricted ATP-binding site, thereby providing a basis for the potent inhibition of JAK1. As expected, the mode of inhibitor binding in JAK1 was very similar to that observed in JAK2, highlighting the challenges in developing JAK-specific inhibitors that target the ATP-binding site. Nevertheless, differences surrounding the JAK1 and JAK2 ATP-binding sites were apparent, thereby providing a platform for the rational design of JAK2-and JAK1-specific inhibitors.

Research paper thumbnail of Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

Journal of Hazardous Materials, 2013

Research paper thumbnail of Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex

Research paper thumbnail of Biological Evaluation of ent -Narciclasine, ent -Lycoricidine, and Certain Enantiomerically-Related Congeners

Chemistry & Biodiversity, 2009

Research paper thumbnail of Expression of truncated presenilin 2 splice variant in Alzheimer's disease, bipolar disorder, and schizophrenia brain cortex

Molecular Brain Research, 2004

We have analysed the expression of a truncated variant presenilin 2 protein (PS2V) in frontal cor... more We have analysed the expression of a truncated variant presenilin 2 protein (PS2V) in frontal cortex from subjects with Alzheimer's disease (AD) and age-matched controls, and compared these results with cortex from bipolar disorder (BP), schizophrenia (SZ) and controls in a second brain bank collection. PS2V protein was detected as a 14 kDa species with antibodies directed to the PS2 N-terminal region and to the new C-terminus created by alternative transcription. PS2V protein levels were significantly increased by two-fold in AD cortex, as compared to age-matched controls. In tissue from the second collection, levels of PS2V were markedly elevated in some BP and SZ cases, but there was no overall difference between diagnostic groups. Our findings support previous evidence for increased expression of this variant PS2 isoform in sporadic AD and suggest this isoform may contribute to neurodegeneration.

Research paper thumbnail of CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

Blood, 2010

Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis o... more Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

Research paper thumbnail of The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor

Blood, 2006

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an importa... more JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 Å crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family.

Research paper thumbnail of Discovery of 2-(α-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS

Bioorganic & Medicinal Chemistry Letters, 2009

A series of 2-(a-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyros... more A series of 2-(a-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Research paper thumbnail of Downstream activating sequence within the coding region of a yeast gene: specific binding in vitro of RAP1 protein

Molecular & general genetics : MGG, 1992

Using a gel retardation assay, a protein factor that specifically interacts with a 33 bp intragen... more Using a gel retardation assay, a protein factor that specifically interacts with a 33 bp intragenic sequence of the highly expressed and glucose-inducible SRP1 gene of Saccharomyces cerevisiae has been detected. This binding site is located in a transcribed region and within the open reading frame (positions +710 to +743 relative to the first base of the initiation codon). A mutant strain carrying a deletion of this binding site showed a dramatic decrease in steady-state levels of SRP1 transcripts. This decline is not the result of a decrease in mRNA stability, since expression of hybrid genes in which the SRP1 promoter was replaced by the heterologous CYC1 promoter was not affected by the binding site deletion. These findings suggest that the 33 bp sequence contains a cis-acting downstream activating element which is involved in the transcriptional activation of the SRP1 promoter. Sequence comparisons showed similarities between a site located within the 33 bp sequence and the high...

Research paper thumbnail of Gene cloning from yeast chromosome-specific mini-library Isolation of the SRP1 -related DNA sequence located on chromosome XV

FEBS Letters, 1988

We have isolated and purified chromosome XV DNA molecules from the yeast S. cerevisiae using cont... more We have isolated and purified chromosome XV DNA molecules from the yeast S. cerevisiae using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. A chromosome-specific mini-library was constructed and the element of the SRPI (serine-rich protein) related sequence family located within chromosome XV was isolated by in situ colony hybridization with an SRPl probe. Results indicated that (i) a single-copy sequence homologous to SRPI is present within chromosome XV; (ii) this sequence lies within the 2.3 kb Hind111 fragment of the plasmid pXVAc6; (iii) the lack of a specific transcript from this SRPI-related element suggests that it could be considered as a pseudogene.

Research paper thumbnail of Airway, but not serum or urinary, levels of YKL-40 reflect inflammation in early cystic fibrosis lung disease

BMC Pulmonary Medicine, 2014

Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk fac... more Cystic fibrosis (CF) lung disease begins in early life and is progressive with the major risk factor being an exaggerated inflammatory response. Currently, assessment of neutrophilic inflammation in early cystic fibrosis (CF) lung disease relies on bronchoalveolar lavage (BAL). The chitinase-like protein YKL-40 is raised in sputum and serum of adults with CF. We investigated YKL-40 in BAL, serum and urine to determine whether this reflected inflammation and infection in young children with CF. YKL-40 was measured in matched samples of BAL, serum and urine obtained from 36 infants and young children with CF participating in an early surveillance program. Levels were compared to clinical data and markers of inflammation detected in the lung. YKL-40 in BAL correlated with pulmonary infection [β=1.30 (SE 0.34), p < 0.001] and BAL markers of inflammation [macrophage number: r2 = 0.34, p < 0.001; neutrophil number: r2 = 0.74, p < 0.001; neutrophil elastase: r2 = 0.47, p < 0.001; CXCL8: r2 = 0.45, p < 0.001; IL-β: r2 = 0.62, p < 0.001]. YKL-40 was detectable in serum but levels did not correlate with BAL levels in the same individuals (r2 = 0.04, p = 0.14) or with inflammatory markers. YKL-40 was below the limit of detection in urine (30 pg/ml). This study demonstrates that levels of the chitinase-like protein YKL-40 reflect airway inflammation and infection in early CF lung disease. The lack of increased YKL-40 in serum in the absence of systemic inflammation limits the benefit of this potential biomarker in early disease.

Research paper thumbnail of Hanging drop online supplimentary (3)

Research paper thumbnail of Phenotypic, Functional and Plasticity Features of Classical and Alternatively Activated Human Macrophages

American Journal of Respiratory Cell and Molecular Biology, 2015

Macrophages are dynamic cells that mature under the influence of signals from the local microenvi... more Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-g and IL-4/IL-13, respectively. M1 and M2 were identified as CD64 1 CD80 1 and CD11b 1 CD209 1 , respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-g, IL-8, TNF-a, IL-1b, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b 1 CD209 1 and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.

Research paper thumbnail of Influence Of Growth Factors And Glucocorticoids In Media On Epithelial Cell Responses

C36. LUNG EPITHELIUM, SUBMUCOSAL GLANDS AND SMOOTH MUSCLE, 2012

Research paper thumbnail of Brief exposure to cigarette smoke impairs airway epithelial cell innate anti-viral defence

Toxicology in Vitro, 2014

Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma ex... more Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma exacerbations. Environmental cigarette smoke exposure is associated with a significant increase in the risk for these infections in children. To determine the impact of short-term exposure to cigarette smoke on innate immune responses of airway epithelial cells infected with hRV. A human bronchial epithelial cell line (HBEC-3KT) was exposed to cigarette smoke extract (CSE) for 30 min and subsequently infected with hRV serotype 1B. Viral-induced cytokine release was measured with AlphaLISA and viral replication quantified by shed viral titer and intracellular viral copy number 24h post-infection. CSE induced a concentration-dependent decrease in CXCL10 (p<0.001) and IFN-β (p<0.001), with a 79% reduction at the highest dose with an associated 3-fold increase in shed virus. These effects were maintained when infection was delayed up to 24h post CSE exposure. Exogenous IFN-β treatment at t=0 after infection blunts the effects of CSE on viral replication (p<0.05). A single exposure of 30 min to cigarette smoke has a lasting impact on epithelial innate defence providing a plausible mechanism for the increase in respiratory infections seen in children exposed to second-hand tobacco smoke.

Research paper thumbnail of A piece in the puzzle: an ion channel candidate gene for schizophrenia

Molecular Medicine Today, 1998

Mutations in ion channels have been found to cause a variety of mendelian genetic diseases, and p... more Mutations in ion channels have been found to cause a variety of mendelian genetic diseases, and polyglutamine repeat expansion is a newly recognized pathogenic mechanism that causes several rare, genetic, late-onset neurological syndromes. Polymorphic polyglutamine tracts are present in a recently described human, calcium-activated potassium channel, KCNN3 (also known as hKCa3), and alleles of this gene that contain longer repeats have been associated with schizophrenia. The physiological function of the channel is consistent with an etiological role in this disease; drugs designed to target this channel might therefore provide novel psychotherapeutics.

Research paper thumbnail of Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy

Research paper thumbnail of The Microtubule Depolymerizing Agent CYT997 Causes Extensive Ablation of Tumor Vasculature In Vivo

Journal of Pharmacology and Experimental Therapeutics, 2011

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridi... more The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Research paper thumbnail of Environmental Contributions to the Leading Causes of Disease Burden Among Australian Children

Journal of Pediatric Gastroenterology and Nutrition, 2013

Research paper thumbnail of The 2.7 Å Crystal Structure of the Autoinhibited Human c-Fms Kinase Domain

Journal of Molecular Biology, 2007

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine... more c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 A resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.

Research paper thumbnail of Dissecting Specificity in the Janus Kinases: The Structures of JAK-Specific Inhibitors Complexed to the JAK1 and JAK2 Protein Tyrosine Kinase Domains

Journal of Molecular Biology, 2009

The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play promin... more The Janus kinases (JAKs) are a pivotal family of protein tyrosine kinases (PTKs) that play prominent roles in numerous cytokine signaling pathways, with aberrant JAK activity associated with a variety of hematopoietic malignancies, cardiovascular diseases and immune-related disorders. Whereas the structures of the JAK2 and JAK3 PTK domains have been determined, the structure of the JAK1 PTK domain is unknown. Here, we report the high-resolution crystal structures of the "active form" of the JAK1 PTK domain in complex with two JAK inhibitors, a tetracyclic pyridone 2-tbutyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one (CMP6) and (3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile (CP-690,550), and compare them with the corresponding JAK2 PTK inhibitor complexes. Both inhibitors bound in a similar manner to JAK1, namely buried deep within a constricted ATP-binding site, thereby providing a basis for the potent inhibition of JAK1. As expected, the mode of inhibitor binding in JAK1 was very similar to that observed in JAK2, highlighting the challenges in developing JAK-specific inhibitors that target the ATP-binding site. Nevertheless, differences surrounding the JAK1 and JAK2 ATP-binding sites were apparent, thereby providing a platform for the rational design of JAK2-and JAK1-specific inhibitors.

Research paper thumbnail of Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

Journal of Hazardous Materials, 2013

Research paper thumbnail of Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable preinitiation complex

Research paper thumbnail of Biological Evaluation of ent -Narciclasine, ent -Lycoricidine, and Certain Enantiomerically-Related Congeners

Chemistry & Biodiversity, 2009

Research paper thumbnail of Expression of truncated presenilin 2 splice variant in Alzheimer's disease, bipolar disorder, and schizophrenia brain cortex

Molecular Brain Research, 2004

We have analysed the expression of a truncated variant presenilin 2 protein (PS2V) in frontal cor... more We have analysed the expression of a truncated variant presenilin 2 protein (PS2V) in frontal cortex from subjects with Alzheimer's disease (AD) and age-matched controls, and compared these results with cortex from bipolar disorder (BP), schizophrenia (SZ) and controls in a second brain bank collection. PS2V protein was detected as a 14 kDa species with antibodies directed to the PS2 N-terminal region and to the new C-terminus created by alternative transcription. PS2V protein levels were significantly increased by two-fold in AD cortex, as compared to age-matched controls. In tissue from the second collection, levels of PS2V were markedly elevated in some BP and SZ cases, but there was no overall difference between diagnostic groups. Our findings support previous evidence for increased expression of this variant PS2 isoform in sporadic AD and suggest this isoform may contribute to neurodegeneration.

Research paper thumbnail of CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

Blood, 2010

Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis o... more Activating alleles of Janus kinase 2 (JAK2) such as JAK2(V617F) are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5muM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2(V617F) allele burden, JAK2(V617F) cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2(V617F) cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells.

Research paper thumbnail of The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor

Blood, 2006

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an importa... more JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 Å crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family.

Research paper thumbnail of Discovery of 2-(α-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS

Bioorganic & Medicinal Chemistry Letters, 2009

A series of 2-(a-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyros... more A series of 2-(a-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.