CORD-19-Sentences  

CORD-19:d0edffac8d4922de52cd50a59bc3b72b5fd946e3 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-161 Sentence denotes Enhanced expression of 70-kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome * NIH Public Access
TextSentencer_T2 163-171 Sentence denotes Abstract
TextSentencer_T3 172-258 Sentence denotes Objective-Fibrotic changes are initiated early in acute respiratory distress syndrome.
TextSentencer_T4 259-320 Sentence denotes This may involve overproliferation of alveolar type II cells.
TextSentencer_T5 321-529 Sentence denotes In an animal model of acute respiratory distress syndrome, we have shown that the administration of an adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) limited pathophysiological changes.
TextSentencer_T6 530-672 Sentence denotes We hypothesized that this improvement may be modulated, in part, by an early AdHSP-induced attenuation of alveolar type II cell proliferation.
TextSentencer_T7 673-759 Sentence denotes Setting-Hadassah-Hebrew University and University of Pennsylvania animal laboratories.
TextSentencer_T8 760-865 Sentence denotes Interventions-Lung injury was induced in male Sprague-Dawley rats via cecal ligation and double puncture.
TextSentencer_T9 866-1060 Sentence denotes At the time of cecal ligation and double puncture, we injected phosphate-buffered saline, AdHSP, or AdGFP (an adenoviral vector expressing the marker green fluorescent protein) into the trachea.
TextSentencer_T10 1061-1111 Sentence denotes Rats then received subcutaneous bromodeoxyuridine.
TextSentencer_T11 1112-1192 Sentence denotes In separate experiments, A549 cells were incubated with medium, AdHSP, or AdGFP.
TextSentencer_T12 1193-1254 Sentence denotes Some cells were also stimulated with tumor necrosis factor-α.
TextSentencer_T13 1255-1346 Sentence denotes After 48 hrs, cytosolic and nuclear proteins from rat lungs or cell cultures were isolated.
TextSentencer_T14 1347-1507 Sentence denotes These were subjected to immunoblotting, immunoprecipitation, electrophoretic mobility shift assay, fluorescent immunohistochemistry, and Northern blot analysis.
TextSentencer_T15 1508-1610 Sentence denotes Results-Alveolar type I cells were lost within 48 hrs of inducing acute respiratory distress syndrome.
TextSentencer_T16 1611-1671 Sentence denotes This was accompanied by alveolar type II cell proliferation.
TextSentencer_T17 1672-1773 Sentence denotes Treatment with AdHSP preserved alveolar type I cells and limited alveolar type II cell proliferation.
TextSentencer_T18 1774-1920 Sentence denotes Heat shock protein 70 prevented overexuberant cell division, in part, by inhibiting hyperphosphorylation of the regulatory retinoblastoma protein.
TextSentencer_T19 1921-2106 Sentence denotes This prevented retinoblastoma protein ubiquitination and degradation and, thus, stabilized the interaction of retinoblastoma protein with E2F1, a key cell division transcription factor.
TextSentencer_T20 2108-2319 Sentence denotes Conclusions-Heat shock protein 70-induced attenuation of cell proliferation may be a useful strategy for limiting lung injury when treating acute respiratory distress syndrome if consistent in later time points.
TextSentencer_T21 2320-2506 Sentence denotes Keywords acute respiratory distress syndrome; lung injury; sepsis; 70-kd heat shock proteins; cell cycle; regeneration Cell regeneration is a fundamental biological response in the lung.
TextSentencer_T22 2507-2589 Sentence denotes In most cases, pulmonary injury predominantly affects alveolar type I (ATI) cells.
TextSentencer_T23 2590-2761 Sentence denotes In contrast, the cuboidal, metabolically active alveolar type II (ATII) cells that produce surfactant and other products essential to pulmonary function tend to be spared.
TextSentencer_T24 2762-2916 Sentence denotes Damaged alveolar epithelial cells are replaced via an organized paradigm in which ATII cells appear to function as progenitor cells for ATI cells (1, 2) .
TextSentencer_T25 2917-3057 Sentence denotes Damage to type I cells stimulates type II cells to reenter the cell cycle, differentiate into type I cells, and spread along alveolar septa.
TextSentencer_T26 3058-3169 Sentence denotes This results in coverage of denuded basement membrane and re-establishment of epithelial continuity (1, 3, 4) .
TextSentencer_T27 3170-3252 Sentence denotes In severe inflammation, however, proliferation of ATII cells may become excessive.
TextSentencer_T28 3253-3381 Sentence denotes This can attenuate appropriate replacement of ATI cells in a manner that can lead to fibrosis and scarring (4) (5) (6) (7) (8) .
TextSentencer_T29 3382-3538 Sentence denotes Novel approaches to therapy might involve modulation of the various phases of epithelial cell repair to reduce fibrosis and restore function in lung injury.
TextSentencer_T30 3539-3630 Sentence denotes Study of acute respiratory distress syndrome (ARDS) (9) (10) (11) illustrates these issues.
TextSentencer_T31 3631-3728 Sentence denotes This inflammatory disorder damages and destroys ATI and, to a lesser degree, ATII cells (3, 12) .
TextSentencer_T32 3729-3797 Sentence denotes In ARDS, cell proliferation may be either beneficial or detrimental.
TextSentencer_T33 3798-3929 Sentence denotes Early in the disease, when loss of pulmonary epithelial cells contributes to pathology, enhancing cell division likely is of value.
TextSentencer_T34 3930-4055 Sentence denotes However, unchecked cell division also enhances coverage of the intra-alveolar exudate by ATII cells and can lead to fibrosis.
TextSentencer_T35 4056-4167 Sentence denotes Furthermore, the overabundance of ATII cells might interfere with differentiation of ATII cells into ATI cells.
TextSentencer_T36 4168-4285 Sentence denotes The rate at which ATII cells proliferate appears to be a key determinant of this balance between repair and fibrosis.
TextSentencer_T37 4286-4443 Sentence denotes Therefore, investigations into the determinants of pulmonary epithelial cell regeneration in ARDS and the factors that modulate the cell cycle are essential.
TextSentencer_T38 4444-4534 Sentence denotes In eukaryotic organisms, the different phases of the cell cycle are precisely coordinated.
TextSentencer_T39 4535-4591 Sentence denotes During G1, the first phase of the cycle, the cell grows.
TextSentencer_T40 4592-4678 Sentence denotes Once it reaches a certain size, it enters the S phase, during which DNA is replicated.
TextSentencer_T41 4679-4779 Sentence denotes In the next phase (G2), the cell checks that DNA replication is completed and prepares for division.
TextSentencer_T42 4780-4857 Sentence denotes Finally, in the mitosis phase (M), chromosomes separate and the cell divides.
TextSentencer_T43 4858-4936 Sentence denotes After division, most cells exit the cell cycle and enter a resting stage (G0).
TextSentencer_T44 4937-4985 Sentence denotes Some, however, immediately reenter the G1 phase.
TextSentencer_T45 4986-5196 Sentence denotes In G1, diverse metabolic stress and environmental cues are integrated and interpreted to influence the deployment of specific cellular programs that lead to tissue regeneration, differentiation, or death (13) .
TextSentencer_T46 5197-5295 Sentence denotes The key point at which a commitment to division occurs is referred to as the G1 restriction point.
TextSentencer_T47 5296-5464 Sentence denotes Control of progression past the G1 restriction point is determined primarily by the phosphorylation state of the retinoblastoma (Rb) family of proteins (14) (15) (16) .
TextSentencer_T48 5465-5611 Sentence denotes Before the G1 restriction point, hypophosphorylated Rb forms a complex with the E2F1 transcription factor and acts as a transcriptional repressor.
TextSentencer_T49 5612-5679 Sentence denotes Rb polyphosphorylation leads to disassembly of the Rb/E2F1 complex.
TextSentencer_T50 5680-5802 Sentence denotes Active E2F1 can induce the transcription of genes encoding enzymes, such as DNA polymerase, that catalyze DNA replication.
TextSentencer_T51 5803-5938 Sentence denotes Rb polyphosphorylation, in turn, requires the activity of several cyclin-dependent kinases, in particular cyclin-dependent kinases 4/6.
TextSentencer_T52 5939-6065 Sentence denotes Activation of cyclin-dependent kinases 4/6 is controlled by their assembly into complexes with members of the cyclin D family.
TextSentencer_T53 6066-6163 Sentence denotes Inflammation is known to effect phosphorylation and the expression of the cyclin D1 family (17) .
TextSentencer_T54 6164-6328 Sentence denotes Therefore, it is logical to evaluate the components of the cell replication apparatus and the factors influencing their activity in ATII cells in experimental ARDS.
TextSentencer_T55 6329-6544 Sentence denotes Modification of these factors might provide important information regarding the pathogenesis of hyperproliferation and fibrosis in ARDS and also might suggest novel therapeutic approaches to this deadly disease (4).
TextSentencer_T56 6545-6666 Sentence denotes The heat shock response is a highly conserved cellular mechanism that protects against injury and environmental stresses.
TextSentencer_T57 6667-6773 Sentence denotes Of particular importance is the expression of members of the 70kd heat shock protein (Hsp70) family (18) .
TextSentencer_T58 6774-6935 Sentence denotes Previously, in a model of sepsis, cecal ligation and double puncture (2CLP) in rats, we found an inappropriate failure of Hsp70 expression in the lung (19, 20) .
TextSentencer_T59 6936-7118 Sentence denotes Enhancing Hsp70 expression in pneumocytes using an adenoviral vector (AdHSP) expressing Hsp70 decreased histologic abnormalities and improved short-term outcome following 2CLP (21) .
TextSentencer_T60 7119-7263 Sentence denotes Subsequent work has revealed that AdHSP limits inflammation, in part, by attenuation of the nuclear factor (NF)-κB pathway in pneumocytes (22) .
TextSentencer_T61 7264-7347 Sentence denotes However, this represents only one mechanism by which AdHSP might protect the lungs.
TextSentencer_T62 7348-7607 Sentence denotes Pioneering work by other investigators indicates that cell cycle components, regulatory proteins, and proteins in the mitogenic signal cascade associate with and may be protected by Hsp70 during periods of stress (17, (22) (23) (24) (25) (26) (27) (28) (29) .
TextSentencer_T63 7608-7744 Sentence denotes In ARDS, the characteristic exuberant replication of pulmonary epithelial cells correlates in time with impaired Hsp70 expression (20) .
TextSentencer_T64 7745-7931 Sentence denotes Therefore, it is important to explore AdHSP-mediated changes in pulmonary epithelial cell replication to understand more fully the effects of Hsp70 on the pathophysiology of lung injury.
TextSentencer_T65 7932-8076 Sentence denotes In the present study, we investigate the effects of sepsis-induced experimental ARDS on pulmonary epithelial cell replication 48 hrs after 2CLP.
TextSentencer_T66 8077-8173 Sentence denotes These studies test the hypothesis that administration of AdHSP alters cell regeneration in ARDS.
TextSentencer_T67 8174-8350 Sentence denotes All studies were approved by the University Laboratory Animal Resources Committee in both collaborating institutions and conformed to National Institutes of Health's standards.
TextSentencer_T68 8351-8449 Sentence denotes Severe sepsis was induced by 2CLP in male, adolescent rats, as previously described (21, 17, 30) .
TextSentencer_T69 8450-8551 Sentence denotes Animals were fluid resuscitated (50 mL/kg) at the time of the surgery and every 24 hrs after surgery.
TextSentencer_T70 8552-8600 Sentence denotes Three animals were evaluated for each treatment.
TextSentencer_T71 8601-8887 Sentence denotes As previously described, 10 11 viral plaque-forming units of recombinant E1,E3-deleted adenoviruses expressing green fluorescent protein (AdGFP) or AdHSP (21) dissolved in phosphate-buffered saline (PBS; total volume, 300 μL) were administered via tracheal puncture at the time of 2CLP.
TextSentencer_T72 8888-9106 Sentence denotes A549 cells (ATCC, Manassas, VA) were grown to confluence in F-12 medium or Hites, correspondingly, containing 2% fecal calf serum, 100 units/mL of penicillin and 100 μg/mL of streptomycin (GIBCO BRL, Grand Island, NY).
TextSentencer_T73 9107-9331 Sentence denotes After incubation with medium alone, AdHSP70, or AdGFP (5 × 10 7 plaque-forming units/mL) for 24 hrs, cells were treated with tumor necrosis factor (TNF)-α (20 ng/mL) (R&D, Minneapolis, MN) for 20 mins or were left untreated.
TextSentencer_T74 9332-9514 Sentence denotes Rats were injected subcutaneously with 50 mg/kg bromodeoxyuridine (BrdU; 2 mg/mL in saline, Sigma, St. Louis, MO) at the time of the initial operation, as previously described (19) .
TextSentencer_T75 9515-9609 Sentence denotes In cells, treatment with TNF or medium alone was followed by incubation with BrdU for 30 mins.
TextSentencer_T76 9610-9693 Sentence denotes Cytosolic and nuclear proteins were isolated as previously described (17, 21, 30) .
TextSentencer_T77 9694-9836 Sentence denotes Protein concentration was determined in an aliquot of each lysate using a Bradford protein assay kit (Bio-Rad Laboratories, Manheim, Germany).
TextSentencer_T78 9837-9986 Sentence denotes Because some of the additional studies planned required the use of cell culture, we investigated cellular replication in TNF-α-stimulated A549 cells.
TextSentencer_T79 9987-10096 Sentence denotes Before stimulation, these human airway epithelial-like cells were treated with medium alone, AdGFP, or AdHSP.
TextSentencer_T80 10097-10139 Sentence denotes This was followed by incubation with BrdU.
TextSentencer_T81 10140-10495 Sentence denotes Cells were washed once with PBS and lysed with buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 0.1% nuclear protein-40, 1 mM dithiothreitol, 0.5 mM phenylmethyl sulfonyl fluoride, 1 mM sodium orthovanadate, 20 mM paranitrophenylphospate (Sigma), 20 mM β-glycerophosphate (Sigma), and a protease inhibitor mixture (Roche Diagnostics, Manheim, Germany).
TextSentencer_T82 10496-10587 Sentence denotes Lysates were incubated on ice for 10 mins and centrifuged at 15,300 × g for 30 mins at 4°C.
TextSentencer_T83 10588-10705 Sentence denotes The supernatants (cytoplasmic fraction) were collected for immunoprecipitation and immunoblotting as described below.
TextSentencer_T84 10706-10884 Sentence denotes A total of 30 μg of total protein lysate was separated on 9% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for immunoblotting as previously described (21) .
TextSentencer_T85 10885-10981 Sentence denotes All signals were detected by enhanced chemiluminescence and quantified by scanning densitometry.
TextSentencer_T86 10982-11155 Sentence denotes DNA polymerase-α and DNA polymerase-β were identified with primary polyclonal rabbit DNA polymerase-α and polymerase-β antibodies (Santa-Cruz Biotechnology, Santa-Cruz, CA).
TextSentencer_T87 11156-11244 Sentence denotes E2F1 was identified using a primary mouse monoclonal antibody (Lab Vision, Fremont, CA).
TextSentencer_T88 11245-11348 Sentence denotes Rb was identified with a primary mouse monoclonal Rb antibody (Cell Signaling Technology, Beverly, MA).
TextSentencer_T89 11349-11455 Sentence denotes Phospho-Rb (pRb) was identified with a primary polyclonal rabbit pRb antibody (Cell Signaling Technology).
TextSentencer_T90 11456-11561 Sentence denotes Hsp70 was identified using a mouse monoclonal antibody (StressGen Biotechnologies, Victoria, BC, Canada).
TextSentencer_T91 11562-11717 Sentence denotes In all cases, the secondary antibody was mouse anti-rabbit immunoglobulin G or goat anti-rabbit immunoglobulin G (Jackson, Immunoresearch, West Grove, PA).
TextSentencer_T92 11718-11803 Sentence denotes Electrophoretic mobility shift assay was performed as previously described (27, 30) .
TextSentencer_T93 11804-11915 Sentence denotes Briefly, a double-stranded DNA oligonucleotide containing a consensus -E2F1 binding site was labeled with 32 P.
TextSentencer_T94 11916-11983 Sentence denotes The labeled oligonucleotide was purified on a G-25 Sephadex column.
TextSentencer_T95 11984-12331 Sentence denotes Nuclear extracts containing 10 μg of nuclear protein were incubated with binding buffer (20 mM 4-[2hydroxyethyl]-1-piperazineethanesulfonic acid [Hepes; pH 7.9], 40 mM KCl, 6 mM MgCl 2 , 1 mM phenylmethyl sulfonyl fluoride, 1 mM dithiothreitol, 0.5% nuclear protein-40), deoxyinosinic deoxycytidylic acid (1 μg/μL) for 20 mins at room temperature.
TextSentencer_T96 12332-12382 Sentence denotes The labeled oligonucleotide was added for 20 mins.
TextSentencer_T97 12383-12445 Sentence denotes Specificity of DNA binding was determined by cold competition.
TextSentencer_T98 12446-12683 Sentence denotes To demonstrate that prevention of Rb phosphorylation resulted, in part, from an interaction with Hsp70, we immunoprecipitated nuclear protein extracts with an antibody to Rb and performed immunoblotting with antibodies to Hsp70 and E2F1.
TextSentencer_T99 12684-12833 Sentence denotes Samples containing 500 μg of cytosolic extract were immunoprecipitated using mouse monoclonal Rb antibody (Cell Signaling Technology), diluted 1:100.
TextSentencer_T100 12834-12873 Sentence denotes Samples were agitated overnight at 4°C.
TextSentencer_T101 12874-13006 Sentence denotes Protein A/G beads (Sigma) were added, and the samples were agitated for 1 hr at 4°C and centrifuged at 14,000 rpm for 5 mins at 4°C.
TextSentencer_T102 13007-13124 Sentence denotes The pellet was washed three times with lysis buffer (31) , suspended in sample buffer, and boiled at 95°C for 5 mins.
TextSentencer_T103 13125-13179 Sentence denotes The resulting mixture was prepared for immunoblotting.
TextSentencer_T104 13180-13450 Sentence denotes As controls for the specificity of immunoprecipitation, we incubated the lysate from 2CLP AdHSP and 2CLP PBS nuclear lung extracts overnight at 4°C with an amount of mouse immunoglobulin G equal to the amount of mouse Rb monoclonal antibody used for immunoprecipitation.
TextSentencer_T105 13451-13557 Sentence denotes This mixture was prepared in the same manner as samples treated with antibodies to Hsp70 or E2F1 antibody.
TextSentencer_T106 13558-13654 Sentence denotes Because no precipitation was noted, these control data were not included in the Results section.
TextSentencer_T107 13655-13847 Sentence denotes Immunostaining was preformed as previously described (32) , using a primary mouse antibody to AQP5 (Almone, Jerusalem, Israel) diluted 1:100, a well known marker for ATI cells (33) (34) (35) .
TextSentencer_T108 13848-14026 Sentence denotes This was followed by proliferating cell nuclear antigen (PCNA; Santa-Cruz Biotechnology) immunostaining, which was performed using a primary mouse antibody of PCNA diluted 1:200.
TextSentencer_T109 14027-14150 Sentence denotes BrdU detection was performed using a primary mouse antibody to BrdU (EnVision System, DAKO, Carpinteria, CA) diluted 1:100.
TextSentencer_T110 14151-14218 Sentence denotes All detections used a secondary anti-mouse immunoglobulin G (DAKO).
TextSentencer_T111 14219-14350 Sentence denotes AQP abundance was quantified by examining 20 lung fields/slide at 100× magnification and counting the number of AQP-positive cells.
TextSentencer_T112 14351-14470 Sentence denotes Cell counts for BrdU nuclear uptake and PCNA nuclear staining were obtained by analyzing 20 20× fields from each slide.
TextSentencer_T113 14471-14523 Sentence denotes Three slides per animal were studied in this manner.
TextSentencer_T114 14524-14571 Sentence denotes Each experimental group included three animals.
TextSentencer_T115 14572-14650 Sentence denotes An expert pathologist in the field of pulmonary diseases evaluated the slides.
TextSentencer_T116 14651-14747 Sentence denotes Lung tissues and cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X.
TextSentencer_T117 14748-14935 Sentence denotes Tissue sections were treated with a pro-surfactant protein-C (ProSP-C) rabbit polyclonal antibody (Chemicon clone 3786, Temecula, CA), a well known marker for ATII cells (4, 29, 36, 37) .
TextSentencer_T118 14936-15012 Sentence denotes Cells were incubated with the primary mouse monoclonal BrdU antibody (DAKO).
TextSentencer_T119 15013-15128 Sentence denotes In either case, incubation (diluted 1:50 in PBS/1% bovine serum albumin) proceeded at room temperature for 40 mins.
TextSentencer_T120 15129-15342 Sentence denotes After the first incubation step, cells or tissue sections were treated with a corresponding fluorescein isothiobyanate-labeled anti-rabbit or anti-mouse antibody (1:100 in PBS/1% bovine serum albumin) for 40 mins.
TextSentencer_T121 15343-15431 Sentence denotes Cell nuclei were counterstained with 4′,6′-diamino-2-phenylindol dihydrochloride (DAPI).
TextSentencer_T122 15432-15498 Sentence denotes Fluorescence was analyzed using a Nikon camera and software (38) .
TextSentencer_T123 15499-15578 Sentence denotes For quantification of ProSP-C, we analyzed 20 100× lung fields from each slide.
TextSentencer_T124 15579-15691 Sentence denotes In each field, we counted the number of alveoli and identified the number of ProSP-C-positive cells per alveoli.
TextSentencer_T125 15692-15744 Sentence denotes Three slides per animal were studied in this manner.
TextSentencer_T126 15745-15792 Sentence denotes Each experimental group included three animals.
TextSentencer_T127 15793-15876 Sentence denotes Total RNA was prepared as previously described (20, 39) with several modifications:
TextSentencer_T128 15877-15953 Sentence denotes 20 μg of total RNA were denatured for 15 min at 60°C in denaturation buffer.
TextSentencer_T129 15954-16136 Sentence denotes RNA samples were fractionated electrophoretically on a 1.2% gel containing MOPS × 1 and 0.5% formaldehyde and blotted to an N + membrane in standard saline citrate (SSC) × 10 buffer.
TextSentencer_T130 16137-16423 Sentence denotes The membrane was prehybridized overnight at 42°C in hybridization solution and hybridized in the same buffer with 32 P-labeled probe of human polymerase-β complementary DNA (gift from Dr. Samuel Wilson, Laboratory of Structural Biology, National Institutes of Health) overnight at 42°C.
TextSentencer_T131 16424-16557 Sentence denotes The membrane was washed with 2 × SSC/0.05% SDS for 10 mins at room temperature and twice with 0.1 × SSC/0.1% SDS for 15 mins at 50°C.
TextSentencer_T132 16558-16600 Sentence denotes Signal was detected using autoradiography.
TextSentencer_T133 16601-16733 Sentence denotes It has been hypothesized that Hsp70 directly interferes with phosphorylation of a number of different proteins (40) (41) (42) (43) .
TextSentencer_T134 16734-16826 Sentence denotes When polyphosphorylated, Rb becomes a target for ubiquitination and proteasomal degradation.
TextSentencer_T135 16827-16938 Sentence denotes If AdHSP prevents Rb polyphosphorylation, it also should reduce polyubiquitination and proteasomal degradation.
TextSentencer_T136 16939-17070 Sentence denotes To investigate this, we treated cultured A549 cells with a proteasomal inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal, MG-132.
TextSentencer_T137 17071-17249 Sentence denotes A549 cells were incubated for 24 hrs with either medium alone or AdHSP70 (5 × 10 7 plaqueforming units/mL) and then treated with 20 μM of MG-132 (Sigma) in F-12 medium for 4 hrs.
TextSentencer_T138 17250-17322 Sentence denotes This was followed by TNF-α (20 ng/mL) (R&D) for 20 mins or no treatment.
TextSentencer_T139 17323-17472 Sentence denotes Nuclear extracts were isolated and immunoprecipitated with anti-Rb subjected to SDS-PAGE electrophoresis and probed with a monoclonal antibody to Rb.
TextSentencer_T140 17473-17542 Sentence denotes This allows detection of Rb-like proteins that are polyubiquitinated.
TextSentencer_T141 17543-17601 Sentence denotes Molecular weight markers appear to the right of the panel.
TextSentencer_T142 17602-17748 Sentence denotes A nonparametric analysis of variance test (Kruskal-Wallis) was performed, followed by multiple pair-wise comparisons with Bonferroni's correction.
TextSentencer_T143 17749-17791 Sentence denotes The significance level was set at p < .05.
TextSentencer_T144 17792-17829 Sentence denotes Bars in the graphs represent the SEM.
TextSentencer_T145 17830-17933 Sentence denotes Sepsis-induced ARDS is known to be associated with a loss of ATI cells and proliferation of ATII cells.
TextSentencer_T146 17934-18015 Sentence denotes We hypothesize that this process could be altered by the administration of AdHSP.
TextSentencer_T147 18016-18174 Sentence denotes To investigate this postulate, we examined the relative distribution of pulmonary epithelial cell type in lung sections obtained 48 hrs after a septic insult.
TextSentencer_T148 18175-18346 Sentence denotes These sections were stained with antibodies to specific proteins found exclusively in either ATI (Aquaporin 5, AQP5) (33) (34) (35) or ATII ProSP-C cells (4, 29, 36, 37) .
TextSentencer_T149 18347-18458 Sentence denotes Studies of sections from untreated (T0) animals revealed AQP-positive cells lining the alveoli (Fig. 1, left) .
TextSentencer_T150 18459-18597 Sentence denotes Immunostaining with an antibody to ProSP-C at the baseline demonstrated up to three positively stained cells per alveolus (Fig. 2, left) .
TextSentencer_T151 18598-18677 Sentence denotes Sepsis-induced ARDS changed the distribution and number of both types of cells.
TextSentencer_T152 18678-18846 Sentence denotes Animals subjected to 2CLP and treated with PBS (2CLPPBS) had diminished AQP in affected lung regions (Fig. 1, center) , indicating a loss of ATI cells (Fig. 1, graph) .
TextSentencer_T153 18847-18982 Sentence denotes In the same rats, there was a dramatic increase in the abundance of ProSP-C-positive cells, presumably in ATII cells (Fig. 2, center) .
TextSentencer_T154 18983-19081 Sentence denotes In contrast, treatment with AdHSP (2CLPHSP) revealed a pattern similar to that seen in T0 animals.
TextSentencer_T155 19082-19240 Sentence denotes That is, staining for AQP was prominent (Fig. 1, right) , whereas no more than three cells per alveolus appeared to contain ProSP-C (Fig. 2, left and graph) .
TextSentencer_T156 19241-19379 Sentence denotes Overall, these findings demonstrated a CLP-induced loss of ATI cells and proliferation of ATII 48 hrs following the initial septic insult.
TextSentencer_T157 19380-19428 Sentence denotes AdHSP treatment reversed or limited this change.
TextSentencer_T158 19429-19582 Sentence denotes To assure that expression of Hsp70 and not the adenoviral vector itself caused the observed changes, we investigated the effects of treatment with AdGFP.
TextSentencer_T159 19583-19660 Sentence denotes This was associated with only nine ProSP-C-positive cells/alveolus (Fig. 2) .
TextSentencer_T160 19661-19773 Sentence denotes It is logical to postulate that increased cellularity reflects an increase in cell division and differentiation.
TextSentencer_T161 19774-19847 Sentence denotes Others have shown that heat shock modulates the cell cycle (22, 26, 27) .
TextSentencer_T162 19848-19985 Sentence denotes Therefore, we examined the effects of AdHSP-enhanced expression of Hsp70 on different aspects of cell replication in sepsis-induced ARDS.
TextSentencer_T163 19986-20064 Sentence denotes To initiate this investigation, we examined two markers of cell proliferation.
TextSentencer_T164 20065-20116 Sentence denotes The first, BrdU, incorporates into replicating DNA.
TextSentencer_T165 20117-20204 Sentence denotes The second, PCNA, is an essential regulatory factor for DNA replication and DNA repair.
TextSentencer_T166 20205-20343 Sentence denotes When compared with T0, we found increased BrdU incorporation into ATII cells in PBS-treated septic animals (Table 1 ; Fig. 3A and graph) .
TextSentencer_T167 20344-20453 Sentence denotes Similarly, PBS-treated septic rats had increased PCNA staining relative to T0 (Table 1 ; Fig. 4A and right) .
TextSentencer_T168 20454-20576 Sentence denotes BrdU and PCNA-positive ATII cells were cuboidal and characterized by relatively abundant cytoplasm and large nuclei (Figs.
TextSentencer_T169 20577-20588 Sentence denotes 3A and 4A).
TextSentencer_T170 20589-20739 Sentence denotes Septic animals given the control vector AdGFP developed changes in PCNA expression similar to those observed in animals given PBS (Table 1; Fig. 4A ).
TextSentencer_T171 20740-20824 Sentence denotes This implies that the adenoviral vector itself does not contribute to these changes.
TextSentencer_T172 20825-20932 Sentence denotes The findings in PBS-and AdGFP-treated animals contrasted sharply with those observed in AdHSP-treated rats.
TextSentencer_T173 20933-21078 Sentence denotes In septic animals treated with AdHSP, we found decreased BrdU and PCNA staining relative to septic animals receiving PBS or AdGFP (Table 1; Figs.
TextSentencer_T174 21079-21092 Sentence denotes 3 A and 4A ).
TextSentencer_T175 21093-21173 Sentence denotes In fact, data were similar to values observed in untreated (T0) rats (Table 1) .
TextSentencer_T176 21174-21323 Sentence denotes Because some of the additional studies planned required the use of cell culture, we investigated cellular replication in TNF-α-stimulated A549 cells.
TextSentencer_T177 21324-21444 Sentence denotes Before stimulation, these human airway epithelial-like cells were treated with medium alone, AdGFP, or AdHSP (Fig. 3B ).
TextSentencer_T178 21445-21487 Sentence denotes This was followed by incubation with BrdU.
TextSentencer_T179 21488-21630 Sentence denotes Although stimulation of cells with or without AdGFP increased BrdU staining, AdHSP administration limited incorporation into cells (Fig. 3B) .
TextSentencer_T180 21631-21738 Sentence denotes Thus, the effects of AdHSP on cell division during inflammation could be studied both in vivo and in vitro.
TextSentencer_T181 21739-21878 Sentence denotes Previous studies have shown that sepsis can cause either accelerated or impaired transcription in cells of a number of organ systems (20) .
TextSentencer_T182 21879-21944 Sentence denotes This might extend to genes encoding important cell cycle enzymes.
TextSentencer_T183 21945-22129 Sentence denotes For example, the ability of Hsp70 to attenuate sepsis-induced proliferation of ATII cells might result from an effect on DNA polymerase-β, an enzyme of key importance in DNA synthesis.
TextSentencer_T184 22130-22261 Sentence denotes Increased expression of this enzyme will occur once the cell has passed the G1 restriction point and entered the S phase (39, 40) .
TextSentencer_T185 22262-22358 Sentence denotes Therefore, we examined the effects of sepsis-induced ARDS on the expression of DNA polymerase-β.
TextSentencer_T186 22359-22615 Sentence denotes When compared with normal (T0) controls, septic animals receiving either PBS or AdGFP increased the abundance of the messenger RNA encoding DNA polymerase-β (Fig. 4B) , a change that also resulted in an increased abundance of the protein itself (Fig. 4C ).
TextSentencer_T187 22616-22689 Sentence denotes This change was not detected following AdHSP treatment of septic animals.
TextSentencer_T188 22690-22786 Sentence denotes Thus, Hsp70 expression limited the expression of this key enzyme in the DNA replication pathway.
TextSentencer_T189 22787-22889 Sentence denotes Heat shock limits the progression past the G1 restriction point by altering the E2F-pRb pathway (27) .
TextSentencer_T190 22890-23019 Sentence denotes Although our findings can only confirm an association in time, it is reasonable to hypothesize that Hsp70 causes this limitation.
TextSentencer_T191 23020-23150 Sentence denotes To investigate this possibility, we examined E2F1 binding activity in nuclear extracts using electrophoretic mobility shift assay.
TextSentencer_T192 23151-23321 Sentence denotes Sepsisinduced ARDS, whether untreated or treated with AdGFP, induced a statistically significant increase in E2F1 DNA binding activity relative to T0 controls (Fig. 5A) .
TextSentencer_T193 23322-23422 Sentence denotes However, the administration of AdHSP to septic rats significantly reduced E2F1 DNA binding activity.
TextSentencer_T194 23423-23510 Sentence denotes The activity of E2F1 is controlled, in part, by the 110-kd retinoblastoma gene product.
TextSentencer_T195 23511-23613 Sentence denotes This nuclear phosphoprotein binds to E2F1 and limits its ability to activate E2F1-dependent promoters.
TextSentencer_T196 23614-23774 Sentence denotes In contrast to the unphosphorylated or hypophosphorylated forms of Rb, which predominate during G1, polyphosphorylated Rb has a low affinity for E2F1 (40, 41) .
TextSentencer_T197 23775-23857 Sentence denotes In vitro studies suggest that heat shock stabilizes unphosphorylated Rb (40, 41) .
TextSentencer_T198 23858-24036 Sentence denotes Therefore, we tested the hypothesis that the sepsis-induced decrease in E2F1 binding activity reflected increased Rb phosphorylation and that AdHSP attenuated Rb phosphorylation.
TextSentencer_T199 24037-24228 Sentence denotes Using nuclear proteins isolated from control animals (T0), septic animals treated with PBS, septic animals treated with AdGFP, or septic animals treated with AdHSP, we examined Rb (Fig. 5B) .
TextSentencer_T200 24229-24456 Sentence denotes These studies revealed that the intranuclear abundance of Rb protein in septic animals receiving PBS or AdGFP was significantly lower than in the T0 controls and than in AdHSP-treated septic animals ( Fig. 5B and upper panel) .
TextSentencer_T201 24457-24624 Sentence denotes The abundance of nuclear pRb was significantly higher than control in septic animals treated with PBS but not in those treated with AdHSP ( Fig. 5B and lower panel) .
TextSentencer_T202 24625-24687 Sentence denotes These data indicate that sepsis induced phosphorylation of Rb.
TextSentencer_T203 24688-24953 Sentence denotes To determine whether the prevention of Rb phosphorylation might result, in part, from an interaction with Hsp70, we immunoprecipitated nuclear protein extracts with an antibody to Rb and performed immunoblotting with an antibody to Hsp70 (Fig. 5C and upper panel) .
TextSentencer_T204 24954-25170 Sentence denotes Our data confirmed that Hsp70 associates with the Rb complex in septic animals treated with AdHSP and that no Hsp70-Rb association occurred in septic animals receiving either PBS or AdGFP (Fig. 5C and upper panel) .
TextSentencer_T205 25171-25368 Sentence denotes Additionally, immunoprecipitation with antibodies to Rb followed by immunoblotting to E2F1 (Fig. 5C and lower panel) demonstrated an interaction between E2F1 and Rb in AdHSP-treated septic animals.
TextSentencer_T206 25369-25421 Sentence denotes Thus, AdHSP treatment preserved the E2F1/Rb complex.
TextSentencer_T207 25422-25626 Sentence denotes Controls for the specificity of immunoprecipitation demonstrated that this interaction is specific for Hsp70, Rb, and E2F1 and does not represent a non-specific antibody antigen reaction (data not shown).
TextSentencer_T208 25627-25763 Sentence denotes It has been hypothesized that Hsp70 directly interferes with the phosphorylation of a number of different proteins (40) (41) (42) (43) .
TextSentencer_T209 25764-25856 Sentence denotes When polyphosphorylated, Rb becomes a target for ubiquitination and proteasomal degradation.
TextSentencer_T210 25857-25968 Sentence denotes If AdHSP prevents Rb polyphosphorylation, it also should reduce polyubiquitination and proteasomal degradation.
TextSentencer_T211 25969-26100 Sentence denotes To investigate this, we treated cultured A549 cells with a proteasomal inhibitor, carbobenzoxyl-leucinyl-leucinyl-leucinal, MG-132.
TextSentencer_T212 26101-26226 Sentence denotes Stimulating MG-132-treated A549 cells with TNF-α induced Rb polyphosphorylation and polyubiquitination ( Fig. 5D and right) .
TextSentencer_T213 26227-26362 Sentence denotes However, Rb ubiquitination was absent when TNFstimulated, MG132-treated cells in cultures were treated with AdHSP ( Fig. 5D and left) .
TextSentencer_T214 26363-26497 Sentence denotes Previous studies in lung injury models have proposed that ATII cells are essential to repair damaged alveolar epithelium (1, 12, 44) .
TextSentencer_T215 26498-26631 Sentence denotes In this paradigm, ATII cells replicate, migrate across the denuded alveolar basement membrane, and then differentiate into ATI cells.
TextSentencer_T216 26632-26716 Sentence denotes Furthermore, it has been postulated that this process becomes dysfunctional in ARDS.
TextSentencer_T217 26717-26842 Sentence denotes In this syndrome, ATII cells migrate not across a denuded basement membrane but rather cover a collection of cellular debris.
TextSentencer_T218 26843-26972 Sentence denotes This stimulates ongoing ATII or pleuripotential cell proliferation and limits differentiation of these precursors into ATI cells.
TextSentencer_T219 26973-27072 Sentence denotes The studies presented here were designed to test aspects of this hypothesis in sepsis-induced ARDS.
TextSentencer_T220 27073-27189 Sentence denotes We first demonstrated that this model stimulates both a loss of ATI cells and excessive proliferation of ATII cells.
TextSentencer_T221 27190-27246 Sentence denotes We then reversed this pathology by treatment with AdHSP.
TextSentencer_T222 27247-27415 Sentence denotes Our data indicate that the ability of Hsp70 to attenuate sepsis-induced replication of ATII cells results, at least in part, from an effect on the G1 restriction point.
TextSentencer_T223 27416-27608 Sentence denotes Specifically, we showed that Hsp70 treatment attenuates the activity of E2F1, a key cell division transcription factor that is inactive when complexed to the regulatory retinoblastoma protein.
TextSentencer_T224 27609-27760 Sentence denotes Our findings reveal that Hsp70 interferes with phosphorylation of Rb, preventing its degradation by the proteasome and, thus, limiting E2F1 activation.
TextSentencer_T225 27761-27902 Sentence denotes The expression of one of the principal target genes for E2F1, DNA polymerase-β, was increased by 2CLP and attenuated by treatment with AdHSP.
TextSentencer_T226 27903-27974 Sentence denotes These data may have important mechanistic and therapeutic implications.
TextSentencer_T227 27975-28161 Sentence denotes Our previous work demonstrated that the intratracheal administration of AdHSP reduced pathologic changes and significantly decreased mortality 48 hrs after the induction of sepsis (21) .
TextSentencer_T228 28162-28266 Sentence denotes This study explored one of a number of potential mechanisms by which this protection might be conferred.
TextSentencer_T229 28267-28345 Sentence denotes In this report, we examine another way in which AdHSP might limit lung injury.
TextSentencer_T230 28346-28388 Sentence denotes Specifically, we present two key findings.
TextSentencer_T231 28389-28545 Sentence denotes First, we have confirmed, by staining for BrdU and PCNA, that the loss of ATI cells in lung injury is associated with extensive proliferation of ATII cells.
TextSentencer_T232 28546-28668 Sentence denotes To demonstrate, we stained sections with an antibody to ProSP-C, a protein specific and characteristic for this cell type.
TextSentencer_T233 28669-28778 Sentence denotes This is essential because 2CLP is associated with a massive infiltration of inflammatory cells into the lung.
TextSentencer_T234 28779-28881 Sentence denotes While the majority of these migrating cells are neutrophils, there is also an increase in macrophages.
TextSentencer_T235 28882-28943 Sentence denotes These latter can be difficult to distinguish from ATII cells.
TextSentencer_T236 28944-29046 Sentence denotes However, ProSP-C, the precursor of surfactant protein-C, is uniquely expressed in ATII cells (2, 45) .
TextSentencer_T237 29047-29162 Sentence denotes To identify the lost cells, we stained sections with AQP, an apical transmembrane protein found in ATI cells (46) .
TextSentencer_T238 29163-29243 Sentence denotes This demonstrates a decrease in the number of these cells per microscopic field.
TextSentencer_T239 29244-29414 Sentence denotes While we attribute the decrease in AQP abundance observed in septic animals, receiving either PBS or AdGFP, to this loss of ATI cells, there are alternative explanations.
TextSentencer_T240 29415-29537 Sentence denotes Inflammation down-regulates the expression of the AQP5 receptor (47) , and thus, the expression per cell may be decreased.
TextSentencer_T241 29538-29641 Sentence denotes Alternatively, inflammatory cell infiltration makes it difficult to distinguish alveoli histologically.
TextSentencer_T242 29642-29685 Sentence denotes This may result in fewer alveoli per field.
TextSentencer_T243 29686-29791 Sentence denotes Thus, while the number of ATI cells per field may be decreased, the number per alveolus may be unchanged.
TextSentencer_T244 29792-29989 Sentence denotes Careful examination of Figure 1B clearly demonstrated that the number of ATI cells per high-powered field is reduced after 2CLP but preserved when septic rats were administered intratracheal AdHSP.
TextSentencer_T245 29990-30145 Sentence denotes The use of ProSP-C and AQP5 to study the distribution of ATI and ATII cells various models of acute inflammation is a well-validated approach (1, 12, 44) .
TextSentencer_T246 30146-30385 Sentence denotes More importantly, our data provide a possible mechanistic explanation for the absence of a hypercellular ATII cell response in animals subjected to 2CLP and those treated with AdHSP as opposed to the response seen with either PBS or AdGFP.
TextSentencer_T247 30386-30579 Sentence denotes We found that AdHSP treatment reduced the sepsis-induced increase in the DNA binding activity of E2F1 and in the expression of DNA polymerase β, processes that are essential to DNA replication.
TextSentencer_T248 30580-30697 Sentence denotes The activity of E2F1 has been touted as key to the control of cell cycle progression beyond the G1 restriction point.
TextSentencer_T249 30698-30798 Sentence denotes Inhibition of E2F1 binding activity would explain why induced Hsp70 expression limits cell division.
TextSentencer_T250 30799-30906 Sentence denotes A similar role for Hsp70 has been proposed by others based on in vitro data (22) (23) (24) (25) (26) (27) .
TextSentencer_T251 30907-31068 Sentence denotes In their experiments, cultured cells subjected to heat shock demonstrated decreased cyclin D1 and E2F1 levels and diminished E2F1 DNA binding activity (27, 28) .
TextSentencer_T252 31069-31281 Sentence denotes Our study represents the first in vivo demonstration of Hsp70-mediated inhibition of cell proliferation and identifies an effect on proteins involved with progression from the G1 to the S phase of the cell cycle.
TextSentencer_T253 31282-31380 Sentence denotes Further mechanistic information is provided by examination of the abundance of pRb following 2CLP.
TextSentencer_T254 31381-31461 Sentence denotes We observed that AdHSP attenuated the sepsisinduced hyperphosphorylation of pRb.
TextSentencer_T255 31462-31520 Sentence denotes This allowed for the stabilization of the Rb/E2F1 complex.
TextSentencer_T256 31521-31690 Sentence denotes In other pathways, Hsp70 prevents or limits protein phosphorylation of a number of key intracellular regulatory proteins, such as heat shock factor-1 and IκBα (42, 43) .
TextSentencer_T257 31691-31809 Sentence denotes This suggests that one mechanism by which Hsp70 may limit pathologic changes is via the modulation of phosphorylation.
TextSentencer_T258 31810-31913 Sentence denotes However, our previous study on the NF-kB pathway suggests that this is an incomplete explanation (22) .
TextSentencer_T259 31914-32055 Sentence denotes The effects of Hsp70 on this inflammatory pathway appears to involve the stabilization of multi-component protein complexes, such as Rb/E2F1.
TextSentencer_T260 32056-32141 Sentence denotes Indeed, this is consistent with the activity of Hsp70 in some, but not all, pathways.
TextSentencer_T261 32142-32296 Sentence denotes Morimoto (23) has proposed that the specific effects of chaperones are determined by the associated co-factors and not the activity of the protein itself.
TextSentencer_T262 32297-32366 Sentence denotes Therefore, Hsp70 may inhibit certain processes and facilitate others.
TextSentencer_T263 32367-32453 Sentence denotes This is consistent with the different effects on the ATI and ATII cells reported here.
TextSentencer_T264 32454-32551 Sentence denotes Additional studies will investigate the contributions of potential co-factors to these processes.
TextSentencer_T265 32552-32680 Sentence denotes Others have demonstrated that adenoviruses may encode proteins that specifically induce cells to enter the cell cycle (48, 49) .
TextSentencer_T266 32681-32776 Sentence denotes In our studies, however, AdGFP did not alter the proliferative response to sepsis-induced ARDS.
TextSentencer_T267 32777-32919 Sentence denotes Because AdHSP limited cell division whereas AdGFP did not, we can attribute the inhibition of cell division solely to the expression of Hsp70.
TextSentencer_T268 32920-32989 Sentence denotes The present study evaluated only the 48-hr time point following 2CLP.
TextSentencer_T269 32990-33059 Sentence denotes This time point was chosen based on previous studies (19) (20) (21) .
TextSentencer_T270 33060-33242 Sentence denotes Evaluation at time points further removed from CLP will be needed to evaluate fully the effect of ATII cell proliferation on fibrosis and of Hsp70 overexpression on the injured lung.
TextSentencer_T271 33243-33360 Sentence denotes Our findings support the conclusion that Hsp70 alters one pathway critical to regulation of the cell cycle (19, 21) .
TextSentencer_T272 33361-33442 Sentence denotes This effect on proliferation may have longterm beneficial or detrimental effects.
TextSentencer_T273 33443-33542 Sentence denotes ARDS is an inflammatory syndrome that is associated with significant morbidity and mortality (50) .
TextSentencer_T274 33543-33692 Sentence denotes In part, these finding at the 48-hr time point may represent the early changes that later on culminate in the development of pulmonary fibrosis (9) .
TextSentencer_T275 33693-33795 Sentence denotes Therefore, interventions that attenuate ATII proliferation may limit this process and reduce fibrosis.
TextSentencer_T276 33796-33884 Sentence denotes Additional studies will be required to examine the effects of Hsp70 on fibrosis in ARDS.
TextSentencer_T277 33885-34050 Sentence denotes In addition, ongoing investigations will help confirm the value of pharmacologic, genetic, or mechanical induction of Hsp70 expression in this highly lethal disease.
TextSentencer_T278 34051-34183 Sentence denotes Effects of sepsis and adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) on alveolar type I (ATI) cell abundance.
TextSentencer_T279 34184-34306 Sentence denotes Sections from the lungs of septic animals treated with phosphate-buffered saline (2CLPPBS) or AdHSP (2CLPAdHSP) are shown.
TextSentencer_T280 34307-34403 Sentence denotes Tissue was harvested at T0 (controls) or 48 hrs after cecal ligation and double puncture (2CLP).
TextSentencer_T281 34404-34511 Sentence denotes Upper panels, AQP; immunostaining for A panels is 40× magnification and for B panels is 100× magnification.
TextSentencer_T282 34512-34550 Sentence denotes AQP-containing cells stain dark brown.
TextSentencer_T283 34551-34607 Sentence denotes Lower panels, graph shows AQP5 ATI cells uptake/alveoli.
TextSentencer_T284 34608-34683 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .02 relative to T0 and AdHSP.
TextSentencer_T285 34684-34818 Sentence denotes Effects of sepsis and adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) on alveolar type II (ATII) cell abundance.
TextSentencer_T286 34819-35037 Sentence denotes Immunofluorescence (green) staining of sections from lungs of septic animals treated with phosphate-buffered saline (2CLPPBS), AdHSP, or an adenoviral vector expressing the marker green fluorescent protein (2CLPAdGFP).
TextSentencer_T287 35038-35134 Sentence denotes Tissue was harvested at T0 (controls) or 48 hrs after cecal ligation and double puncture (2CLP).
TextSentencer_T288 35135-35203 Sentence denotes Upper panels: pro-surfactant protein-C (ProSP-C), 40× magnification.
TextSentencer_T289 35204-35246 Sentence denotes Lower panels, ProSP-C, 100× magnification.
TextSentencer_T290 35247-35293 Sentence denotes Graph shows ProSP-C ATII cells uptake/alveoli.
TextSentencer_T291 35294-35369 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .03 relative to T0 and AdHSP.
TextSentencer_T292 35370-35475 Sentence denotes The slides are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T293 35476-35654 Sentence denotes Effects of sepsis/inflammation and adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) on bromodeoxyuridine (BrdU) incorporation into tissue and cultured cells.
TextSentencer_T294 35655-35689 Sentence denotes A, BrdU staining of lung sections.
TextSentencer_T295 35690-35778 Sentence denotes Fixed tissue sections from rats with sepsis-induced acute respiratory distress syndrome.
TextSentencer_T296 35779-35858 Sentence denotes Tissue was harvested at 48 hrs after cecal ligation and double puncture (2CLP).
TextSentencer_T297 35859-35924 Sentence denotes Top panels, septic animals treated with AdHSP, 40× magnification.
TextSentencer_T298 35925-36023 Sentence denotes Middle panels, septic animals treated with phosphate-buffered saline (PBS), 20× 40× magnification.
TextSentencer_T299 36024-36091 Sentence denotes Bottom panels, septic animals treated with PBS, 100× magnification.
TextSentencer_T300 36092-36165 Sentence denotes Two separate fields showing BrdU incorporation into the nuclei are shown.
TextSentencer_T301 36166-36208 Sentence denotes Black arrows indicate BrdU-stained nuclei.
TextSentencer_T302 36209-36257 Sentence denotes Graph, BrdU nuclear uptake per field per animal.
TextSentencer_T303 36258-36326 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .04 relative to AdHSP.
TextSentencer_T304 36327-36432 Sentence denotes The slides are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T305 36433-36484 Sentence denotes B, tumor necrosis factor (TNF)-αtreated A549 cells.
TextSentencer_T306 36485-36650 Sentence denotes Cultured cell monolayers were stimulated with TNF-α, treated with either adenoviruses expressing green fluorescent protein (AdGFP) or AdHSP, and incubated with BrdU.
TextSentencer_T307 36651-36747 Sentence denotes Top panels, TNF-α plus medium; middle panels, TNF-α plus AdGFP; bottom panels, TNF-α plus AdHSP.
TextSentencer_T308 36748-36766 Sentence denotes 40× magnification.
TextSentencer_T309 36767-36832 Sentence denotes White arrows indicate green intranuclear BrdU immunofluorescence.
TextSentencer_T310 36833-37024 Sentence denotes Effects of sepsis and adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) on proliferating cell nuclear antigen (PCNA) staining and DNA polymerase expression in lung tissue.
TextSentencer_T311 37025-37048 Sentence denotes A, PCNA immunostaining.
TextSentencer_T312 37049-37150 Sentence denotes Fixed tissue sections were obtained from rats with sepsisinduced acute respiratory distress syndrome.
TextSentencer_T313 37151-37230 Sentence denotes Tissue was harvested at 48 hrs after cecal ligation and double puncture (2CLP).
TextSentencer_T314 37231-37362 Sentence denotes Left, T0; center left, septic animals treated with phosphatebuffered saline (PBS); center right, septic animals treated with AdHSP.
TextSentencer_T315 37363-37475 Sentence denotes Right, septic animals treated with adenoviruses expressing green fluorescent protein (AdGFP), 40× magnification.
TextSentencer_T316 37476-37510 Sentence denotes Black arrows, PCNA-stained nuclei.
TextSentencer_T317 37511-37559 Sentence denotes Graph, PCNA nuclear uptake per field per animal.
TextSentencer_T318 37560-37628 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .04 relative to AdHSP.
TextSentencer_T319 37629-37734 Sentence denotes The slides are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T320 37735-37799 Sentence denotes B, steady-state DNA polymerase-β messenger RNA (mRNA) abundance.
TextSentencer_T321 37800-37879 Sentence denotes Left, representative Northern blot analysis of DNA polymerase-β mRNA abundance.
TextSentencer_T322 37880-37962 Sentence denotes The total RNA isolated from the lungs of rats at T0 or 48 hrs after 2CLP is shown.
TextSentencer_T323 37963-38042 Sentence denotes A total of 20 μg RNA per lane was separated on a 1.2% agarose/formaldehyde gel.
TextSentencer_T324 38043-38118 Sentence denotes Probe consisted of 32 P-labeled DNA complementary to DNA polymerase-β mRNA.
TextSentencer_T325 38119-38186 Sentence denotes Right panels, densitometric analysis of steady-state RNA abundance.
TextSentencer_T326 38187-38262 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .04 relative to T0 and AdHSP.
TextSentencer_T327 38263-38367 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T328 38368-38419 Sentence denotes C, intranuclear DNA polymerase-β protein abundance.
TextSentencer_T329 38420-38494 Sentence denotes Left: representative immunoblot analysis of intranuclear DNA polymerase-β.
TextSentencer_T330 38495-38566 Sentence denotes Protein was isolated from the lungs of rats at T0 or 48 hrs after 2CLP.
TextSentencer_T331 38567-38682 Sentence denotes A total of 30 μg of protein per lane was separated on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
TextSentencer_T332 38683-38749 Sentence denotes Probe consisted of polyclonal rabbit antibody to DNA polymerase-β.
TextSentencer_T333 38750-38814 Sentence denotes Right, densitometric analysis of steady-state protein abundance.
TextSentencer_T334 38815-38890 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .03 relative to T0 and AdHSP.
TextSentencer_T335 38891-38995 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T336 38996-39150 Sentence denotes Effects of sepsis and adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) on the E2F-phospho-retinoblastoma (Rb) pathway in lung tissue.
TextSentencer_T337 39151-39226 Sentence denotes A, top, electrophoretic mobility shift assay for E2F1 DNA binding activity.
TextSentencer_T338 39227-39478 Sentence denotes Nuclear protein was isolated from the lungs of untreated controls (T0) or 48 hrs after cecal ligation and double puncture (2CLP) in rats treated with phosphate-buffered saline (PBS), adenoviruses expressing green fluorescent protein (AdGFP), or AdHSP.
TextSentencer_T339 39479-39658 Sentence denotes A total of 10 μg of nuclear protein was incubated for 20 mins at room temperature with a 32 P-labeled double-stranded DNA oligonucleotide containing a consensus-E2F1 binding site.
TextSentencer_T340 39659-39763 Sentence denotes Cold competition (CC)-2CLPPBS samples with unlabeled oligonucleotide were added to the reaction mixture.
TextSentencer_T341 39764-39835 Sentence denotes Bottom, densitometric analysis of electrophoretic mobility shift assay.
TextSentencer_T342 39836-39911 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .03 relative to T0 and AdHSP.
TextSentencer_T343 39912-40016 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T344 40017-40105 Sentence denotes Bottom: E2F1 nuclear extracts loading control of 30 μg of total protein lysate per lane.
TextSentencer_T345 40106-40147 Sentence denotes B, immunoblotting for Rb protein species.
TextSentencer_T346 40148-40278 Sentence denotes Nuclear protein isolated from the lungs of untreated controls (T0) or 48 hrs after 2CLP in rats treated with PBS, AdGFP, or AdHSP.
TextSentencer_T347 40279-40329 Sentence denotes A total of 30 μg of total protein lysate per lane.
TextSentencer_T348 40330-40387 Sentence denotes Top, the probe consisted of mouse monoclonal Rb antibody.
TextSentencer_T349 40388-40439 Sentence denotes Right, densitometric analysis of protein abundance.
TextSentencer_T350 40440-40515 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .03 relative to T0 and AdHSP.
TextSentencer_T351 40516-40620 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T352 40621-40628 Sentence denotes Bottom.
TextSentencer_T353 40629-40686 Sentence denotes The probe consisted of rabbit polyclonal antibody to pRb.
TextSentencer_T354 40687-40742 Sentence denotes Right, densitometric analysis of the protein abundance.
TextSentencer_T355 40743-40818 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .03 relative to T0 and AdHSP.
TextSentencer_T356 40819-40923 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T357 40924-41010 Sentence denotes C, effects of the 70-kd heat shock protein (Hsp70) on the E2F1-Rb protein interaction.
TextSentencer_T358 41011-41141 Sentence denotes The nuclear protein was isolated from the lungs of untreated controls (T0) or 48 hrs after 2CLP in rats treated with PBS or AdHSP.
TextSentencer_T359 41142-41215 Sentence denotes Immunoprecipitation (IP) was performed using 500 μg of cytosolic extract.
TextSentencer_T360 41216-41277 Sentence denotes Precipitate was resuspended and immunoblotting was performed.
TextSentencer_T361 41278-41337 Sentence denotes A total of 30 μg of total protein lysate per lane is shown.
TextSentencer_T362 41338-41396 Sentence denotes Densitometric analysis of protein abundance was performed.
TextSentencer_T363 41397-41465 Sentence denotes Data are mean ± SE, n = 3 at each point. *p < .04 relative to AdHSP.
TextSentencer_T364 41466-41570 Sentence denotes The blots are representative samples, and the graph combines data from multiple animals and experiments.
TextSentencer_T365 41571-41653 Sentence denotes Top, immunoprecipitation with mouse monoclonal anti-Rb, detection with anti-Hsp70.
TextSentencer_T366 41654-41756 Sentence denotes Bottom, immunoprecipitation with mouse monoclonal anti-Rb, detection with monoclonal antibody to E2F1.
TextSentencer_T367 41757-41873 Sentence denotes D, treatment consisted of tumor necrosis factor (TNF)-α-stimulated cultured A549 cells with a proteasomal inhibitor.
TextSentencer_T368 41874-42051 Sentence denotes Anti-Rb immunoprecipitated protein was isolated from the nuclei of A549 cells incubated with MG-132, a specific inhibitor of the 26S proteasome, to preserve ubiquitinated forms.
TextSentencer_T369 42052-42248 Sentence denotes A549cells were incubated with medium alone; MG cells were incubated with medium plus MG-132; TNF-α were stimulated with TNF-α; MG plus TNF, cells were treated with TNF-α and incubated with MG-132.
TextSentencer_T370 42249-42296 Sentence denotes Right, A549 plus medium, treatment with medium.
TextSentencer_T371 42297-42341 Sentence denotes Left, A549 plus AdHSP, treatment with AdHSP.
TextSentencer_T372 42342-42422 Sentence denotes Molecular mass markers (in kilodaltons) are indicated to the left of the panels.
TextSentencer_T373 42423-42573 Sentence denotes Table 1 Effects of different treatments on number of bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and positive pulmonary cells