Per Flodby | University of Southern California (original) (raw)

Papers by Per Flodby

American Journal of Respiratory Cell and Molecular Biology, 2014

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that ... more Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

American journal of physiology. Lung cellular and molecular physiology, 2014

Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium an... more Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly ...

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for so... more Distal lung epithelium is maintained by proliferation of alveolar type II
(AT2) cells and, for some daughter AT2 cells, transdifferentiation into
alveolar type I (AT1) cells. We investigated if subpopulations of
alveolar epithelial cells (AEC) exist that represent various stages in
transdifferentiation from AT2 to AT1 cell phenotypes in normal adult
lung and if they can be identified using combinations of cell-specific
markers. Immunofluorescence microscopy showed that, in distal rat
and mouse lungs, 20–30% of NKX2.1 (or thyroid transcription
factor 1) cells did not colocalize with pro-surfactant protein C
(pro-SP-C), a highly specific AT2 cell marker. In distal rat lung,
NKX2.1 cells coexpressed either pro-SP-C or the AT1 cell marker
homeodomain only protein x (HOPX). Not all HOPX cells colocalize
with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5
cells were NKX2.1. HOPX was expressed earlier than AQP5 during
transdifferentiation in rat AEC primary culture, with robust expression
of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize
in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate
or transitional cells, and HOPX and AQP5 are expressed without
NKX2.1 in AT1 cells. These findings suggest marked heterogeneity
among cells previously identified as exclusively AT1 or AT2 cells,
implying the presence of subpopulations of intermediate or transitional
AEC in normal adult lung.

Journal of Biological Chemistry, 2003

Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzyme... more Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss.

American Journal of Respiratory Cell and Molecular Biology, Dec 20, 2012

Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell ... more Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell types, alveolar epithelial type (AT) I and AT2 cells. Genetically modified mice with cell-specific Cre/loxPmediated knockouts of relevant genes in each respective cell type would be useful to help elucidate the relative contributions of AT1 versus AT2 cells to alveolar homeostasis. Cre has previously been efficiently expressed in AT2 cells in mouse lung with the surfactant protein (SP)-C promoter; however, no transgenic mouse expressing Cre in AT1 cells has so far been available. To develop an AT1 cellspecific transgenic Cre mouse, we generated a knockin of a Cre-IRES-DsRed cassette into exon 1 of the endogenous aquaporin 5 (Aqp5) gene, a gene expressed specifically in AT1 cells in the distal lung epithelium, resulting in the mouse line, Aqp5-Cre-IRES-DsRed (ACID). Endogenous Aqp5 and transgenic Cre in ACID mice showed a very similar pattern of tissue distribution by RT-PCR. To analyze Cre activity, ACID was crossed to two Cre reporter strains, R26LacZ and mT/mG. Double-transgenic offspring demonstrated reporter gene expression in a very high fraction of AT1 cells in the distal lung, whereas AT2 cells were negative. As expected, variable reporter expression was detected in several other tissues where endogenous Aqp5 is expressed (e.g., submandibular salivary gland and stomach). ACID mice should be of major utility in analyzing the functional contribution of AT1 cells to alveolar epithelial properties in vivo with Cre/loxP-mediated gene deletion technology.

American journal of respiratory cell and molecular biology, Jan 11, 2016

Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fl... more Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and relative contributions of alveolar epithelial type II (AT2) vs type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which β1 subunit was knocked out in either AT1 cells or in both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing for the first time that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung wei...

Proceedings of the National Academy of Sciences of the United States of America, 1988

A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the... more A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium. Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.

Annals of the American Thoracic Society, Apr 1, 2015

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tr... more The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure. tripartite motif protein 72; caveolin 1; plasma membrane wounding and repair; alveolar epithelial cells; acute lung injury * S. C. Kim and T. Kellet made equal contribution to this work. Address for reprint requests and other correspondence: X. Zhao, 442 Riffe Bldg., 496 12 th Ave

American journal of respiratory cell and molecular biology, Jan 27, 2016

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endop... more Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated mice (cGrp78f/f) with lung epithelial cell-specific knockout (KO) of Grp78, a gene encoding the ER chaperone 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78f/f pups died immediately after birth, likely due to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes e...

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for so... more Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, approximately 20-30% of NKX2.1(+) (or thyroid transcription factor 1(+) (TTF1(+))) cells did not co-localize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1(+) cells co-expressed either pro-SP-C or the AT1 cell marker HOPX. Not all HOPX(+) cells co-localize with the AT1 cell marker aquaporin 5 (AQP5) and some AQP5(+) cells were NKX2.1(+). HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C co-localize in AT2 cells, NKX2.1 and HOPX or AQP5 co-localize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung.

Brazilian Journal of Medical and Biological Research

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficien... more 1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70%) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60%) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

C68. ALVEOLAR EPITHELIUM, 2009

C107. PREVALENCE AND REGULATION OF EPITHELIAL MESENCHYMAL TRANSITION, 2009

B67. ION TRANSPORT IN LUNG CELLS: IMPACT ON LUNG FUNCTION AND DISEASE, 2012

C59. STEM CELLS, PROGENITOR CELLS AND TISSUE REGENERATION, 2010

C28. CHANNEL SURFING: LUNG EPITHELIAL ION TRANSPORT MECHANISMS IN HEALTH AND DISEASE, 2011

Annals of the American Thoracic Society, 2015

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tr... more The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure. tripartite motif protein 72; caveolin 1; plasma membrane wounding and repair; alveolar epithelial cells; acute lung injury * S. C. Kim and T. Kellet made equal contribution to this work. Address for reprint requests and other correspondence: X. Zhao, 442 Riffe Bldg., 496 12 th Ave

C27. GENETIC PROGRAMS DRIVING LUNG DEVELOPMENT AND REGENERATION, 2011

Current Topics in Membranes

American Journal of Respiratory Cell and Molecular Biology, 2014

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that ... more Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.

American journal of physiology. Lung cellular and molecular physiology, 2014

Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium an... more Claudins are tight junction proteins that regulate paracellular ion permeability of epithelium and endothelium. Claudin 4 has been reported to function as a paracellular sodium barrier and is one of three major claudins expressed in lung alveolar epithelial cells (AEC). To directly assess the role of claudin 4 in regulation of alveolar epithelial barrier function and fluid homeostasis in vivo, we generated claudin 4 knockout (Cldn4 KO) mice. Unexpectedly, Cldn4 KO mice exhibited normal physiological phenotype although increased permeability to 5-carboxyfluorescein and decreased alveolar fluid clearance were noted. Cldn4 KO AEC monolayers exhibited unchanged ion permeability, higher solute permeability, and lower short-circuit current compared with monolayers from wild-type mice. Claudin 3 and 18 expression was similar between wild-type and Cldn4 KO alveolar epithelial type II cells. In response to either ventilator-induced lung injury or hyperoxia, claudin 4 expression was markedly ...

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for so... more Distal lung epithelium is maintained by proliferation of alveolar type II
(AT2) cells and, for some daughter AT2 cells, transdifferentiation into
alveolar type I (AT1) cells. We investigated if subpopulations of
alveolar epithelial cells (AEC) exist that represent various stages in
transdifferentiation from AT2 to AT1 cell phenotypes in normal adult
lung and if they can be identified using combinations of cell-specific
markers. Immunofluorescence microscopy showed that, in distal rat
and mouse lungs, 20–30% of NKX2.1 (or thyroid transcription
factor 1) cells did not colocalize with pro-surfactant protein C
(pro-SP-C), a highly specific AT2 cell marker. In distal rat lung,
NKX2.1 cells coexpressed either pro-SP-C or the AT1 cell marker
homeodomain only protein x (HOPX). Not all HOPX cells colocalize
with the AT1 cell marker aquaporin 5 (AQP5), and some AQP5
cells were NKX2.1. HOPX was expressed earlier than AQP5 during
transdifferentiation in rat AEC primary culture, with robust expression
of both by day 7. We speculate that NKX2.1 and pro-SP-C colocalize
in AT2 cells, NKX2.1 and HOPX or AQP5 colocalize in intermediate
or transitional cells, and HOPX and AQP5 are expressed without
NKX2.1 in AT1 cells. These findings suggest marked heterogeneity
among cells previously identified as exclusively AT1 or AT2 cells,
implying the presence of subpopulations of intermediate or transitional
AEC in normal adult lung.

Journal of Biological Chemistry, 2003

Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzyme... more Very little is known about the in vivo regulation of mammalian fatty acid chain elongation enzymes as well as the role of specific fatty acid chain length in cellular responses and developmental processes. Here, we report that the Elovl3 gene product, which belongs to a highly conserved family of microsomal enzymes involved in the formation of very long chain fatty acids, revealed a distinct expression in the skin that was restricted to the sebaceous glands and the epithelial cells of the hair follicles. By disruption of the Elovl3 gene by homologous recombination in mouse, we show that ELOVL3 participates in the formation of specific neutral lipids that are necessary for the function of the skin. The Elovl3-ablated mice displayed a sparse hair coat, the pilosebaceous system was hyperplastic, and the hair lipid content was disturbed with exceptionally high levels of eicosenoic acid (20:1). This was most prominent within the triglyceride fraction where fatty acids longer than 20 carbon atoms were almost undetectable. A functional consequence of this is that Elovl3-ablated mice exhibited a severe defect in water repulsion and increased trans-epidermal water loss.

American Journal of Respiratory Cell and Molecular Biology, Dec 20, 2012

Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell ... more Pulmonary alveolar epithelium is comprised of two morphologically and functionally distinct cell types, alveolar epithelial type (AT) I and AT2 cells. Genetically modified mice with cell-specific Cre/loxPmediated knockouts of relevant genes in each respective cell type would be useful to help elucidate the relative contributions of AT1 versus AT2 cells to alveolar homeostasis. Cre has previously been efficiently expressed in AT2 cells in mouse lung with the surfactant protein (SP)-C promoter; however, no transgenic mouse expressing Cre in AT1 cells has so far been available. To develop an AT1 cellspecific transgenic Cre mouse, we generated a knockin of a Cre-IRES-DsRed cassette into exon 1 of the endogenous aquaporin 5 (Aqp5) gene, a gene expressed specifically in AT1 cells in the distal lung epithelium, resulting in the mouse line, Aqp5-Cre-IRES-DsRed (ACID). Endogenous Aqp5 and transgenic Cre in ACID mice showed a very similar pattern of tissue distribution by RT-PCR. To analyze Cre activity, ACID was crossed to two Cre reporter strains, R26LacZ and mT/mG. Double-transgenic offspring demonstrated reporter gene expression in a very high fraction of AT1 cells in the distal lung, whereas AT2 cells were negative. As expected, variable reporter expression was detected in several other tissues where endogenous Aqp5 is expressed (e.g., submandibular salivary gland and stomach). ACID mice should be of major utility in analyzing the functional contribution of AT1 cells to alveolar epithelial properties in vivo with Cre/loxP-mediated gene deletion technology.

American journal of respiratory cell and molecular biology, Jan 11, 2016

Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fl... more Active ion transport by basolateral Na-K-ATPase (Na pump) creates a Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and β1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the β1 subunit and relative contributions of alveolar epithelial type II (AT2) vs type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which β1 subunit was knocked out in either AT1 cells or in both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing for the first time that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung wei...

Proceedings of the National Academy of Sciences of the United States of America, 1988

A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the... more A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium. Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.

Annals of the American Thoracic Society, Apr 1, 2015

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tr... more The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure. tripartite motif protein 72; caveolin 1; plasma membrane wounding and repair; alveolar epithelial cells; acute lung injury * S. C. Kim and T. Kellet made equal contribution to this work. Address for reprint requests and other correspondence: X. Zhao, 442 Riffe Bldg., 496 12 th Ave

American journal of respiratory cell and molecular biology, Jan 27, 2016

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endop... more Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated mice (cGrp78f/f) with lung epithelial cell-specific knockout (KO) of Grp78, a gene encoding the ER chaperone 78-kDa glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78f/f pups died immediately after birth, likely due to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes e...

American Journal of Physiology - Lung Cellular and Molecular Physiology, 2015

Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for so... more Distal lung epithelium is maintained by proliferation of alveolar type II (AT2) cells and, for some daughter AT2 cells, transdifferentiation into alveolar type I (AT1) cells. We investigated if subpopulations of alveolar epithelial cells (AEC) exist that represent various stages in transdifferentiation from AT2 to AT1 cell phenotypes in normal adult lung and if they can be identified using combinations of cell-specific markers. Immunofluorescence microscopy showed that, in distal rat and mouse lungs, approximately 20-30% of NKX2.1(+) (or thyroid transcription factor 1(+) (TTF1(+))) cells did not co-localize with pro-surfactant protein C (pro-SP-C), a highly specific AT2 cell marker. In distal rat lung, NKX2.1(+) cells co-expressed either pro-SP-C or the AT1 cell marker HOPX. Not all HOPX(+) cells co-localize with the AT1 cell marker aquaporin 5 (AQP5) and some AQP5(+) cells were NKX2.1(+). HOPX was expressed earlier than AQP5 during transdifferentiation in rat AEC primary culture, with robust expression of both by day 7. We speculate that NKX2.1 and pro-SP-C co-localize in AT2 cells, NKX2.1 and HOPX or AQP5 co-localize in intermediate or transitional cells, and HOPX and AQP5 are expressed without NKX2.1 in AT1 cells. These findings suggest marked heterogeneity among cells previously identified as exclusively AT1 or AT2 cells, implying the presence of subpopulations of intermediate or transitional AEC in normal adult lung.

Brazilian Journal of Medical and Biological Research

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficien... more 1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70%) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60%) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

C68. ALVEOLAR EPITHELIUM, 2009

C107. PREVALENCE AND REGULATION OF EPITHELIAL MESENCHYMAL TRANSITION, 2009

B67. ION TRANSPORT IN LUNG CELLS: IMPACT ON LUNG FUNCTION AND DISEASE, 2012

C59. STEM CELLS, PROGENITOR CELLS AND TISSUE REGENERATION, 2010

C28. CHANNEL SURFING: LUNG EPITHELIAL ION TRANSPORT MECHANISMS IN HEALTH AND DISEASE, 2011

Annals of the American Thoracic Society, 2015

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tr... more The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure. tripartite motif protein 72; caveolin 1; plasma membrane wounding and repair; alveolar epithelial cells; acute lung injury * S. C. Kim and T. Kellet made equal contribution to this work. Address for reprint requests and other correspondence: X. Zhao, 442 Riffe Bldg., 496 12 th Ave

C27. GENETIC PROGRAMS DRIVING LUNG DEVELOPMENT AND REGENERATION, 2011

Current Topics in Membranes