Dennis R Voelker

Yale-New Haven Hospital, New Haven, Connecticut, United States

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Publications (169)857.98 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC-MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.
    Journal of Proteome Research 07/2014; · 5.06 Impact Factor
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    ABSTRACT: Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) is a phosphorus-free betaine-lipid analog of phosphatidylcholine (PtdCho) synthesized by many soil bacteria, algae, and non-vascular plants. Synthesis of DGTS and other phosphorus-free lipids in bacteria occurs in response to phosphorus (P) deprivation, and results in the replacement of phospholipids by non-phosphorous lipids. The genes encoding DGTS biosynthetic enzymes have previously been identified and characterized in bacteria and the alga Chlamydomonas reinhardtii. We now report that many fungal genomes, including those of plant and animal pathogens, encode the enzymatic machinery for DGTS biosynthesis, and that fungi synthesize DGTS during P-limitation. This finding demonstrates that replacement of phospholipids by non-phosphorous lipids is a strategy used in divergent eukaryotic lineages for the conservation of P under P-limiting conditions. Mutants of Neurospora crassa were used to show that DGTS synthase encoded by the BTA1 gene is solely responsible for DGTS biosynthesis, and is under the control of the fungal phosphorus-deprivation regulon, mediated by the NUC-1/Pho4p transcription factor. Furthermore, we describe the rational re-engineering of lipid metabolism in the yeast S. cerevisiae such that PtdCho is completely replaced by DGTS, and demonstrate that essential processes of membrane biogenesis and organelle assembly are functional and support growth in the engineered strain.
    Eukaryotic Cell 04/2014; · 3.59 Impact Factor
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    ABSTRACT: Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a coenzyme A (CoA)-dependent mechanism, and play an important role in controlling levels of free arachidonic acid (AA) and production of lipid mediators of inflammation. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. A facile LPAT assay relevant to multiple substrates can provide a more realistic view of the regulation of arachidonic acid (AA) metabolism and phospholipid composition. Microsome preparations from RAW 264.7 cells were used to compare traditional LPAT assays, using individual lysophospholipids and AA-CoA, vs. a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using liquid chromatography coupled to tandem mass spectrometry. Both types of assays provided similar results, but the choice assay provided far more information relevant to multiple fatty acyl chain incorporation into various phospholipid classes. The dual choice assay was successfully used to validate engineered suppression of LPCAT3 activity in RAW 264.7 cells. These findings demonstrate that this assay provides much richer biochemical detail about the in vivo selectivity of LPATs.
    The Journal of Lipid Research 02/2014; · 4.39 Impact Factor
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    ABSTRACT: Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa that also includes other parasites of clinical/veterinary importance, such as Plasmodium, Cryptosporidium and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells, and requires synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer), and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a PtdSer decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N-terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% impairment in the parasite growth. The PtdEtn content of the parasite mutant was unaffected however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant strain, and depletion of nutrient exacerbated the growth defect (~56%), which was partially restored by ethanolamine. Further, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium indicated additional routes of PtdEtn biogenesis such as acquisition of host-derived lipid. Collectively, these results demonstrate a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
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    ABSTRACT: Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis phosphatidylethanolamine (PtdEtn). One route involves the synthesis of phosphatidylserine (PtdSer) in the ER, the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce PtdEtn. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns-4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein:protein and protein:lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, that we name Pbi1p (PstB2p interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N-terminus of Psd2p (C2-1), as well the previously characterized C2 domain of Psd2p (C2-2.) The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein:protein and protein:lipid interactions. Specifically, our model predicts this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.
    Journal of Biological Chemistry 12/2013; · 4.65 Impact Factor
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    ABSTRACT: Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.
    Proceedings of the National Academy of Sciences 10/2013; · 9.81 Impact Factor
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    ABSTRACT: Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and significant morbidity and mortality in the elderly, imunosuppressed and immunocompromised patients, and patients with chronic lung diseases. Recently, we reported the pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), inhibited RSV infection in vitro and in vivo, by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice, markedly attenuated infection and associated inflammatory responses. Based on these findings, we expanded our studies to determine if POPG is effective for prophylaxis and post-infection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2-1.0 mg/ml at 24hrs after RSV infection of HEp-2 cells, suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2-6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making post-infection application impractical. Intranasal administration of POPG (0.8-3.0 mg), 45 min before RSV inoculation in mice, reduced viral infection by 1 log unit, suppressed inflammatory cell appearance in the lung, and suppressed virus elicited interferon-γ production. These findings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection, and has potential for application in humans.
    The Journal of Lipid Research 06/2013; · 4.39 Impact Factor
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    ABSTRACT: Nuclear factor-kappa B (NFkB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFkB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFkB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFkB in host defense in humans is not fully understood. We sought to examine the role of NFkB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFkB activation using BAY 11-7082 (BAY, an inhibitor of IkBa kinase) or an adenovirus construct with a dominant-negative IkBa significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFkB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFkB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.
    PLoS ONE 04/2013; · 3.53 Impact Factor
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    ABSTRACT: There is increasing interest in the application of nanotechnology to solve the difficult problem of therapeutic administration of pharmaceuticals. Nanodiscs, composed of a stable discoidal lipid bilayer encircled by an amphipathic membrane scaffold protein that is an engineered variant of the human Apo A-I constituent of high-density lipoproteins, have been a successful platform for providing a controlled lipid composition in particles that are especially useful for investigating membrane protein structure and function. In this communication, we demonstrate that nanodiscs are effective in suppressing respiratory syncytial viral (RSV) infection both in vitro and in vivo when self-assembled with the minor pulmonary surfactant phospholipid palmitoyloleoylphosphatidylglycerol (POPG). Preparations of nanodiscs containing POPG (nPOPG) antagonized interleukin-8 production from Beas2B epithelial cells challenged by RSV infection, with an IC50 of 19.3 μg/mL. In quantitative in vitro plaque assays, nPOPG reduced RSV infection by 93%. In vivo, nPOPG suppressed inflammatory cell infiltration into the lung, as well as IFN-γ production in response to RSV challenge. nPOPG also completely suppressed the histopathological changes in lung tissue elicited by RSV and reduced the amount of virus recovered from lung tissue by 96%. The turnover rate of nPOPG was estimated to have a halftime of 60-120 minutes (m), based upon quantification of the recovery of the human Apo A-I constituent. From these data, we conclude that nPOPG is a potent antagonist of RSV infection and its inflammatory sequelae both in vitro and in vivo.
    International Journal of Nanomedicine 01/2013; 8:1417-1427. · 4.20 Impact Factor
  • Mari Numata, Pitchaimani Kandasamy, Dennis R Voelker
    Expert Review of Respiratory Medicine 06/2012; 6(3):243-6.
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    ABSTRACT: Toxoplasma gondii is an obligate intracellular parasite capable of causing fatal infections in immunocompromised individuals and neonates. Examination of the phosphatidylserine (PtdSer) metabolism of T. gondii reveals that the parasite secretes a soluble form of PtdSer decarboxylase (TgPSD1), which preferentially decarboxylates liposomal PtdSer with an apparent K(m) of 67 μM. The specific enzyme activity increases by 3-fold during the replication of T. gondii, and soluble phosphatidylserine decarboxylase (PSD) accounts for ∼20% of the total PSD, prior to the parasite egress from the host cells. Extracellular T. gondii secreted ∼20% of its total PSD activity at 37 °C, and the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) inhibited the process by 50%. Cycloheximide, brefeldin A, ionic composition of the medium, and exogenous PtdSer did not modulate the enzyme secretion, which suggests a constitutive discharge of a presynthesized pool of PSD in axenic T. gondii. TgPSD1 consists of 968 amino acids with a 26-amino acid hydrophobic peptide at the N terminus and no predicted membrane domains. Parasites overexpressing TgPSD1-HA secreted 10-fold more activity compared with the parental strain. Exposure of apoptotic Jurkat cells to transgenic parasites demonstrated interfacial catalysis by secreted TgPSD1 that reduced host cell surface exposure of PtdSer. Immunolocalization experiments revealed that TgPSD1 resides in the dense granules of T. gondii and is also found in the parasitophorous vacuole of replicating parasites. Together, these findings demonstrate novel features of the parasite enzyme because a secreted, soluble, and interfacially active form of PSD has not been previously described for any organism.
    Journal of Biological Chemistry 05/2012; 287(27):22938-47. · 4.65 Impact Factor
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    ABSTRACT: Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.
    Journal of Biological Chemistry 04/2012; 287(23):19386-98. · 4.65 Impact Factor
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    ABSTRACT: In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.
    Traffic 03/2012; 13(6):880-90. · 4.65 Impact Factor
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    ABSTRACT: Human coronavirus strain 229E (HCoV-229E) commonly causes upper respiratory tract infections. However, lower respiratory tract infections can occur in some individuals, indicating that cells in the distal lung are susceptible to HCoV-229E. This study determined the virus susceptibility of primary cultures of human alveolar epithelial cells and alveolar macrophages (AMs). Fluorescent antibody staining indicated that HCoV-229E could readily infect AMs, but no evidence was found for infection in differentiated alveolar epithelial type II cells and only a very low level of infection in type II cells transitioning to the type I-like cell phenotype. However, a human bronchial epithelial cell line (16HBE) was readily infected. The innate immune response of AMs to HCoV-229E infection was evaluated for cytokine production and interferon (IFN) gene expression. AMs secreted significant amounts of tumour necrosis factor alpha (TNF-α), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein 1β (MIP-1β/CCL4) in response to HCoV-229E infection, but these cells exhibited no detectable increase in IFN-β or interleukin-29 in mRNA levels. AMs from smokers had reduced secretion of TNF-α compared with non-smokers in response to HCoV-229E infection. Surfactant protein A (SP-A) and SP-D are part of the innate immune system in the distal lung. Both surfactant proteins bound to HCoV-229E, and pre-treatment of HCoV-229E with SP-A or SP-D inhibited infection of 16HBE cells. In contrast, there was a modest reduction in infection in AMs by SP-A, but not by SP-D. In summary, AMs are an important target for HCoV-229E, and they can mount a pro-inflammatory innate immune response to infection.
    Journal of General Virology 11/2011; 93(Pt 3):494-503. · 3.13 Impact Factor
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    ABSTRACT: The 23-megabase genome of Plasmodium falciparum, the causative agent of severe human malaria, contains ∼5300 genes, most of unknown function or lacking homologs in other organisms. Identification of these gene functions will help in the discovery of novel targets for the development of antimalarial drugs and vaccines. The P. falciparum genome is unusually A+T-rich, which hampers cloning and expressing these genes in heterologous systems for functional analysis. The large repertoire of genetic tools available for Saccharomyces cerevisiae makes this yeast an ideal system for large scale functional complementation analyses of parasite genes. Here, we report the construction of a cDNA library from P. knowlesi, which has a lower A+T content compared with P. falciparum. This library was applied in a yeast complementation assay to identify malaria genes involved in the decarboxylation of phosphatidylserine. Transformation of a psd1Δpsd2Δdpl1Δ yeast strain, defective in phosphatidylethanolamine synthesis, with the P. knowlesi library led to identification of a new parasite phosphatidylserine decarboxylase (PkPSD). Unlike phosphatidylserine decarboxylase enzymes from other eukaryotes that are tightly associated with membranes, the PkPSD enzyme expressed in yeast was equally distributed between membrane and soluble fractions. In vitro studies reveal that truncated forms of PkPSD are soluble and undergo auto-endoproteolytic maturation in a phosphatidylserine-dependent reaction that is inhibited by other anionic phospholipids. This study defines a new system for probing the function of Plasmodium genes by library-based genetic complementation and its usefulness in revealing new biochemical properties of encoded proteins.
    Journal of Biological Chemistry 11/2011; 287(1):222-32. · 4.65 Impact Factor
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    ABSTRACT: Influenza A virus (IAV) is a worldwide public health problem causing 500,000 deaths each year. Palmitoyl-oleoyl-phosphatidylglycerol (POPG) is a minor component of pulmonary surfactant, which has recently been reported to exert potent regulatory functions upon the innate immune system. In this article, we demonstrate that POPG acts as a strong antiviral agent against IAV. POPG markedly attenuated IL-8 production and cell death induced by IAV in cultured human bronchial epithelial cells. The lipid also suppressed viral attachment to the plasma membrane and subsequent replication in Madin-Darby canine kidney cells. Two virus strains, H1N1-PR8-IAV and H3N2-IAV, bind to POPG with high affinity, but exhibit only low-affinity interactions with the structurally related lipid, palmitoyl-oleoyl-phosphatidylcholine. Intranasal inoculation of H1N1-PR8-IAV in mice, in the presence of POPG, markedly suppressed the development of inflammatory cell infiltrates, the induction of IFN-γ recovered in bronchoalveolar lavage, and viral titers recovered from the lungs after 5 days of infection. These findings identify supplementary POPG as a potentially important new approach for treatment of IAV infections.
    American Journal of Respiratory Cell and Molecular Biology 11/2011; 46(4):479-87. · 4.15 Impact Factor
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    ABSTRACT: Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae. IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae, but only in asthma. SP-A derived from asthmatic subjects did not significantly attenuate IL-8 and MUC5AC in the setting of M. pneumoniae infection compared with SP-A derived from normal subjects. We conclude that SP-A derived from asthmatic subjects does not abrogate inflammation effectively, and this dysfunction may be modulated by SP-A1/SP-A.
    AJP Lung Cellular and Molecular Physiology 07/2011; 301(4):L598-606. · 3.52 Impact Factor
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    ABSTRACT: FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E1 ↔ E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.
    Journal of Biological Chemistry 03/2011; 286(11):9699-9712. · 4.65 Impact Factor
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    Chassidy Johnson, Sohail Jahid, Dennis R Voelker, Hung Fan
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    ABSTRACT: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.
    Virology 02/2011; 412(2):349-56. · 3.35 Impact Factor
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    ABSTRACT: Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.
    The Journal of Infectious Diseases 02/2011; 203(9):1240-8. · 5.85 Impact Factor

Publication Stats

6k Citations
857.98 Total Impact Points


  • 2010–2013
    • Yale-New Haven Hospital
      New Haven, Connecticut, United States
  • 2009–2013
    • National Jewish Health
      • Department of Medicine
      Denver, Colorado, United States
    • University of Alberta
      Edmonton, Alberta, Canada
  • 2012
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany
  • 2011
    • Colorado State University
      • Microbiology, Immunology & Pathology
      Fort Collins, CO, United States
  • 2001–2008
    • Kanazawa Medical University
      • • Department of Respiratory Medicine
      • • Department of Internal Medicine (III)
      Kanazawa-shi, Ishikawa-ken, Japan
    • Moredun Research Institute
      Penicuik, Scotland, United Kingdom
  • 1994–2008
    • Sapporo Medical University
      • Department of Biochemistry
      Sapporo, Hokkaidō, Japan
  • 2007
    • The Ohio State University
      • Department of Internal Medicine
      Columbus, OH, United States
  • 2005
    • Kyushu University
      • Research Institute for Diseases of the Chest
      Fukuoka-shi, Fukuoka-ken, Japan
  • 2002
    • University of Iowa
      Iowa City, Iowa, United States
  • 1998–1999
    • University of Texas at Austin
      • Institute for Cellular and Molecular Biology
      Texas City, TX, United States
    • Rutgers, The State University of New Jersey
      • New Jersey Agricultural Experiment Station
      New Brunswick, NJ, United States
    • University of Colorado
      Denver, Colorado, United States
  • 1996
    • Emory University
      Atlanta, Georgia, United States