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PubMed1984

Rhodopseudomonas blastica atp operon. Nucleotide sequence and transcription.

The nucleotide sequence has been determined of a 12,368 base-pair region of DNA cloned from the non-sulphur photosynthetic bacterium Rhodopseudomonas blastica. It contains a cluster of six genes of which five encode the subunits of F1-ATPase; the sixth codes for an unknown protein. The genes are arranged in the same order as in the Escherichia coli unc operon, except that the unknown gene is placed between those for gamma and beta subunits. Neither the genes for F0 subunits, nor a homologue of the E. coli uncI gene is associated with this locus. The six genes are transcribed from a single promoter and we have designated this region the R. blastica atp operon. The two distal genes, beta and epsilon, may also be transcribed from a second promoter. Initiation and termination points for transcription have been identified by primer extensions and S1 nuclease mapping experiments. Signals involved in initiation of translation (Shine and Dalgarno sequences) and termination of transcription in the photosynthetic bacterium resemble those in E. coli. However, no common features can be identified in these two bacteria between 5' regions adjacent to sites of initiation of transcription. The sequence also contains a gene that encodes a protein homologous to discoidin, a cell surface lectin of Dictyostelium discoideum thought to be involved in cell--cell aggregation. Seven other reading frames have not been identified.

Amino Acid Sequence↗
PubMed2006

Phagocytotic removal of apoptotic endocrine cells by folliculostellate cells and its functional implications in clusterin accumulation in pituitary colloids in helmeted guinea fowl (Numida meleagris).

The different cell types in the anterior pituitary behave as dynamic populations. The gland maintains a continuous renewal of cells to ensure a dynamic balance between cell division, differentiation, growth arrest and apoptosis. Apoptosis is a frequent event in the anterior pituitary in which unwanted cells are eliminated without affecting neighboring cells. We examined the link between apoptosis and the occurrence of colloids in the guinea fowl (Numida meleagris) pituitary gland and the relationship of clusterin accumulation in the colloids. S-100 positive folliculostellate (FS) cells were found surrounding colloids. Apoptotic cells detected by single stranded DNA (ssDNA) immunohistochemistry were observed in the whole anterior pituitary and preferentially near colloid masses. Clusterin protein was detected in endocrine cells, FS cells and in the colloids. In situ hybridization showed clusterin mRNA in endocrine cells and FS cells. Simultaneous localization was performed to determine whether clusterin mRNA and ssDNA within anterior pituitary was present within the same cell. Clusterin mRNA was not detected in apoptotic cells but was present in neighboring surviving cells. At the ultrastructural level, numerous endocrine cells at different stages of apoptosis were found phagocytosed by FS cells. Our results suggest that clusterin is produced by endocrine cells for cytoprotection before death. Apoptotic endocrine cells are phagocytosed by FS cells and digested by their lysosomal enzymes. In FS cells, clusterin interacts and aggregates with by-products of digestion that subsequently become stored in colloid as a residual body.

Animals↗
PubMed2006

Isolation and characterization of novel marine Roseobacter clade members producing unique intracellular chromium-rich aggregates.

The marine Roseobacter clade comprises one of the largest fractions of heterotrophic marine bacteria and accounts for about 16% of 16S rRNA gene clones retrieved from marine bacterioplankton. Their global distribution seems to be related to oceanic water masses and their environmental and biogeochemical properties. In this study, we report isolation and characterization of novel Roseobacter clade members from the Yellow Sea, China. Phylogenetic analysis of 16S rRNA gene sequences reveals that the new isolates (YSCB1, YSCB2, YSCB3 and YSCB4) are closely related to uncultured Arctic seawater bacterium R7967 (99.57-100% sequence identity) and to the cultured Roseobacter sp. DSS-1 (99.27-99.76% sequence identity) isolated from the southeastern coastal water of the USA. Interestingly, YSCB strains possess unique intracellular chromium-containing aggregates. Therefore, these novel Roseobacter clade members exhibit a peculiar property in mineral biogeneration.

Chromium↗
PubMed1996

A kinesin-like protein is required for germ plasm aggregation in Xenopus.

In embryos of X. laevis, and many other species, early development requires targeted movements of molecules and molecular aggregates within the oocyte or egg cytoplasm. One well-known example in Xenopus is the aggregation of germ plasm, a group of cytoplasmic islands that become distributed during the first few cell cycles to cells that will give rise to the germ line. Nothing is known about the cytoskeletal motor proteins that may drive these movements. We show here that a recently identified Xenopus kinesin-like protein, Xklp1, is required for the aggregation of germ plasm in early Xenopus embryos, thus assigning this protein a role in a developmentally important cytoplasmic localization.

Animals↗
PubMed2006

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.

Animals↗
PubMed2001

Isolation of viral coat protein mutants with altered assembly and aggregation properties.

A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant.

Amino Acid Substitution↗
PubMed2002

Replication of a cytopathic strain of bovine viral diarrhea virus activates PERK and induces endoplasmic reticulum stress-mediated apoptosis of MDBK cells.

Endoplasmic reticulum (ER) stress signaling is an adaptive cellular response to the loss of ER Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen. ER stress-activated signaling pathways regulate protein synthesis initiation and can also trigger apoptosis through the ER-associated caspase 12. Viruses that utilize the host cell ER as an integral part of their life cycle would be predicted to cause some level of ER stress. Bovine viral diarrhea virus (BVDV) is a positive-stranded RNA virus of the Flaviviridae family. BVDV and related flaviviruses use the host ER as the primary site of envelope glycoprotein biogenesis, genomic replication, and particle assembly. We are using a cytopathic strain of BVDV (cpBVDV) that causes cellular apoptosis as a model system to determine how virus-induced ER stress contributes to pathogenesis. We show that, in a natural infection of MDBK cells, cpBVDV activates the ER transmembrane kinase PERK (PKR-like ER kinase) and causes hyperphosphorylation of the translation initiation factor eIF2 alpha, consistent with the induction of an ER stress response. Additionally, we show that initiation of cellular apoptosis correlates with downregulation of the antiapoptotic Bcl-2 protein, induced expression of caspase 12, and a decrease in intracellular glutathione levels. Defining the molecular stress pathways leading to cpBVDV-induced apoptosis provides the basis to study how other ER-tropic viruses, such as hepatitis C and B viruses, modulate the host cell ER stress response during the course of persistent infection.

Animals↗
PubMed2003

AlphaB-crystallin modulates protein aggregation of abnormal desmin.

AlphaB-crystallin (CryAB) is the most abundant small heat shock protein in the heart. Upregulation of CryAB in desmin-related myopathy and its downregulation in end-stage congestive heart failure have both been reported. We previously demonstrated via cardiac-specific transgenesis that modest increases in normal CryAB are not detrimental to the heart, whereas expression of the R120G mutation of CryAB caused a desminopathy. It is generally believed that CryAB plays an important role in protecting the intermediate filaments, but the underlying mechanism is unclear. We hypothesized that CryAB protects the desmin filaments via preventing abnormal desmin protein from aggregating adversely. To test this hypothesis in vivo, mice expressing a desmin mutation that causes a desmin-related cardiomyopathy (D7) were bred into the R120G-CryAB transgenic (TG) background to examine the accumulation and aberrant aggregation of desmin protein. Despite lower mRNA expression of D7-des than in the D7-des TG hearts, the double-TG myocardium exhibited significantly higher desmin protein levels and dramatically more aberrant desmin aggregates than the D7-des TG hearts. The double-TG mice displayed a significantly stronger cardiac hypertrophic response, with the mice dying of congestive heart failure before 7 weeks. To explore the ability of wild-type (WT) CryAB to protect against mutant desmin, a desmin mutant was expressed in both the conventional and WT-CryAB stably transfected HEK cells. Significantly less aberrant desmin aggregation was observed in the WT-CryAB-overexpressing cells than in the HEK cells. The results suggest that CryAB modulates abnormal desmin aggregation and can serve a cardioprotective role.

Animals↗
PubMed2000

Pulmonary immune responses during primary mycobacterium bovis- Calmette-Guerin bacillus infection in C57Bl/6 mice.

Mechanisms of protective immunity to mycobacterial infection in the lung remain poorly defined. In this study, T-cell subset expansion and cytokine expression in bronchoalveolar spaces, lung parenchyma, and mediastinal lymph nodes of mice infected intratracheally with Mycobacterium bovis-Calmette-Guerin bacillus (BCG) were analyzed in parallel with histopathology and bacterial burden. M. bovis-BCG was cleared rapidly from bronchoalveolar spaces without evidence for persistence. In lung parenchyma bacteria grew during the first 4 wk followed by gradual clearance with less than 0.1% of the original inoculum persisting for more than 8 mo. Clearance of M. bovis-BCG from bronchoalveolar lavage was associated with recruitment of both neutrophils and lymphocytes. Lung CD4(+), CD8(+), and gammadelta T-cell receptor-positive T cells expanded maximally by Week 4, and declined by Week 8 to control values despite bacterial persistence. Both CD4(+) and CD8(+) lung T cells produced interferon (IFN)-gamma in response to M. bovis-BCG. Four distinct pathologic states of lung parenchymal infection were noted. Early focal sub-bronchial inflammation with transmigration of cells into airways was followed by diffuse peribronchitis, perivasculitis, and alveolitis with activated macrophages, lymphoblasts, and occasional giant cells. The latter stage corresponded to maximal M. bovis-BCG growth. Resolving infection consisted of small lymphocytes and foamy macrophages, which coincided with decreasing M. bovis-BCG colony-forming units, T-cell infiltration, and IFN-gamma expression. A final quiescent phase consisted of residual lymphoid aggregates and perivasculitis associated with persistent spontaneous IFN-gamma production. Bacterial dissemination to lymph node and spleen occurred by Week 4 and declined in parallel to lung. In contrast to lung, IFN-gamma secretion was detected only late despite early expansion of CD4(+) and CD8(+) T cells. By reverse transcriptase/polymerase chain reaction, IFN-gamma and interleukin (IL)-12 p40 messenger RNA (mRNA) in lung paralleled IFN-gamma protein production. Tumor necrosis factor-alpha, IL-4 and IL-10 mRNA expression was not increased during M. bovis-BCG lung infection. Thus, protective immunity to M. bovis-BCG in the lung evolved differently in air space, lung, and lymph node.

Animals↗
PubMed1997

Control of cell-type specific gene expression in Dictyostelium by the general transcription factor GBF.

To understand how positional information within an organism specifies patterning during development, we are analyzing spatially regulated gene expression in Dictyostelium. CAR3 is a member of the cAMP, 7-span receptor family which directs the transition from unicellular to multicellular organism and regulates cellular differentiation and pattern formation. CAR3 mRNA is expressed maximally at 8-10 hours of development, as individual cells aggregate and differentiate, and is accumulated to equivalent levels in all cells. CAR3 is also induced in shaking cultures by response to extracellular cAMP. We now show, by extensive mutagenesis, that the maximum length of contiguous sequences required for accurate spatiotemporal regulation of CAR3 is approx. 350 bp. These sequences include three significant elements located in upstream and transcribed regions. Arrays of G-boxes (GBF regulatory sites) are centered near positions -165 and +50 and, although either is sufficient for induction by cAMP and expression in prespore cells, both are required for expression in prestalk cells. Another GC-rich element near position -80 is required for maximal expression of prespore-specific constructs, although full-length promoters carrying clustered mutations through the -80 region are still expressed in all cells, but with slightly reduced expression. Spatiotemporal expression of CAR3 during development, thus, requires cell-specific combinatorial interactions of multiple but redundant regulatory components. These essential elements are located in upstream and transcribed regions. However, most surprisingly, a primary control for spatial patterning of CAR3 expression appears to be mediated by GBF, a general transcription factor expressed ubiquitously during Dictyostelium development following early aggregation.

Animals↗
PubMed1999

Prothrombin gene expression in rat kidneys provides an opportunity to examine its role in urinary stone pathogenesis.

Urinary form of prothrombin (PT) fragment 1 is the most abundant protein in calcium oxalate crystals generated in human urine. The protein has also been detected in human calcium-containing stones. In its purified form, the protein inhibits calcium oxalate crystal growth and, more importantly, aggregation in aqueous inorganic solutions and undiluted human urine. Recently, PT gene expression has been reported in human kidneys. However, access to human renal tissue for studies is limited, and it is not possible to easily manipulate PT biosynthesis in human subjects. The aim of this investigation, therefore, was to determine whether PT gene expression is present in rat kidneys. Samples of total RNA were isolated from the kidneys and livers (positive controls) of 12 male hooded Wistar rats. Using reverse transcription-PCR, mRNA corresponding to the thrombin and F1+2 regions of PT was analyzed by agarose gel electrophoresis. The expression of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase was also examined, to determine the availability of amplifiable substrate in each specimen. The amplified products were also sequenced, to determine their identities. All rat kidneys displayed evidence of expression of the thrombin and F1+2 domains of the PT gene. This similarity between human and rat kidneys allows the possibility of using established rat models of stone disease to evaluate therapeutic strategies to reduce stone formation.

Animals↗
PubMed1995

[Pathologic observations of hepatitis C].

The morphological changes in liver biopsies from 70 patients with a clinical diagnosis of hepatitis C were studied. Sixty-two of the patients had history of blood transfusions. All patients were seropositive for anti-HCV by first and second generation test and/or were seropositive for HCV RNA by PCR method. Clinically, twenty cases diagnosed as acute hepatitis, fifty were chronic including 7 cases with both HBV and HCV infection. The main morphological changes in acute cases being 1. Focal degeneration of liver cells, including large and small droplet fatty changes. 2. Focal necrosis and acidophilic bodies usually being surrounded by T lymphocytes. 3. Sinusoidal inflammatory cell infiltration. 4. Lymphoid aggregation with bile duct epithelial damage was found in 60% of portal tracts. The chronic cases included 17 chronic persistent hepatitis cases (CPH) and 33 chronic active hepatitis cases (CAH). The main histopathologic changes in CAH included dense clusters of lymphocytes (lymphoid aggregates) present in the enlarged portal tracts with bridging necrosis and fibrosis, lobular inflammation including acidophilic bodies, focal necrosis and fatty degeneration. The sinusoidal cells were often hyperplastic. These changes may provide useful diagnostic clues for hepatitis C.

Adult↗
PubMed1988

The Fc gamma receptor of natural killer cells is a phospholipid-linked membrane protein.

Three types of receptor for the Fc (constant) region of human immunoglobulin G have been described; FcRI, a high-affinity (Ka approximately equal to 10(8) M-1) receptor expressed on monocytes; FcRII (CD32), a low-affinity (Ka approximately equal to 10(6) M-1) receptor expressed on B cells, granulocytes, macrophages and platelets; and FcRIII (CD16, FcRIo), a low-affinity receptor expressed on macrophages, neutrophils, eosinophils, natural killer cells and a subset of T cells believed to comprise the suppressor cells. Anti-CD16 antibodies block natural killer-cell mediated antibody dependent cellular cytotoxicity (ADCC). Binding of aggregated IgG to CD16 on natural killer cells leads to the expression of lymphocyte activation antigens, mediator release, morphological changes and lytic activity. We report here the isolation of a complementary DNA clone encoding CD16 determinants which gave rise to IgG binding of the expected affinity and subtype specificity in COS cells, and which proved to encode a phospholipid anchored protein. A single messenger RNA transcript was found in all positive RNA samples, and N-glycanase treatment showed the form found in COS cells was identical to the form present on peripheral blood mononuclear cells (PBMCs). We also show that CD16 is most closely related to the alpha-form of the murine IgG 2b/1 receptor and propose that extracellular contacts mediate the signal initiated by IgG binding.

Amino Acid Sequence↗
PubMed2003

Molecular, ultrastructural and functional characterization of a Spanish family with Hermansky-Pudlak syndrome: role of insC974 in platelet function and clinical relevance.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder, which is genetically heterogeneous. In humans, mutations associated with this syndrome have been identified that affect four genes, most of them located in the HPS-1 gene. We evaluated the clinical, molecular, platelet ultrastructure and platelet function data obtained from one Spanish HPS patient and his relatives. The proband was compound heterozygous for a de novo nonsense mutation (Arg-131Stop), which has not been described previously, and for a common frameshift mutation (insC974). These two mutations were also identified by reverse transcription polymerase chain reaction (RT-PCR) in half the RNA, supporting the premise that they have minor effects on either transcription or RNA stability. The patient had an almost complete absence of platelet-dense granules. Accordingly, his platelets showed a small aggregatory response, reduced CD63 surface expression after platelet activation and minor serotonin uptake. Interestingly, despite the absence of clinical symptoms, two relatives carrying only one HPS-1 mutation (insC974) presented a decreased content of platelet-dense granules and showed significant reductions in platelet aggregation, expression of CD63 after platelet activation and serotonin uptake. Data show that the presence of a single mutation affecting one allele of the HPS-1 gene might have relevance in the organogenesis of platelet-dense granules, affecting platelet function. However, these functional defects were not of a great enough magnitude to have clinical significance and, thus, these subjects were clinically asymptomatic.

Blood Platelets↗
PubMed2005

Regulated cellular partitioning of SR protein-specific kinases in mammalian cells.

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.

Active Transport, Cell Nucleus↗
PubMed1993

Activation of Cyp1a1 and Cyp1a2 genes in adult mouse hepatocytes in primary culture.

Expression of Cyp1a1 and Cyp1a2 genes was investigated in adult C57BL/6NCrj mouse hepatocytes in primary culture for up to 5 days. When the cells were cultivated as monolayers on collagen-coated dishes, CYP1A1 mRNA species were prominently induced after treatment with 3-methylcholanthrene (MCA) throughout the observation period. Substantial induction of CYP1A2 mRNA by MCA was also observed at day 1 of cultivation, followed by a decrease to very low levels thereafter. In contrast, when cultivated on non-coated dishes, the hepatocytes formed multicellular aggregates (spheroids) and prominent induction of both mRNA species was found for up to 5 days. Constitutive expression of CYP1A2 mRNA in spheroid culture was maintained throughout the observation period, whereas that in monolayer culture decreased rapidly. The time-course of the induced CYP1A2 mRNA amounts after the treatment with MCA or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) followed the same pattern as that of CYP1A1 mRNA. Expressed amounts of CYP1A1 or CYP1A2 mRNA in spheroid culture were higher than or similar to the levels in the case of in vivo production, respectively. Induction of both mRNA species was also observed in hepatocytes from nonresponsive DBA/2NCrj mouse in spheroid culture, but the expressed amount after MCA treatment was far smaller than for C57BL/6NCrj cells, despite equivalent expression in the two strains after TCDD. Activities of aryl hydrocarbon hydroxylase (AHH) and acetanilide 4-hydroxylase (AAH) were elevated with either type of cultivation after treatment with MCA or TCDD. Ratios of AAH to AHH were not changed between the two cultures after 24 h treatment. However, the ratios in spheroid culture after 48 h treatment increased, whereas they did not change in monolayer culture. The present observations indicate that the spheroid culture is more suitable than the monolayer system for studying the mechanism of Cyp1a2 gene expression in adult mouse hepatocytes.

Animals↗
PubMed2006

Influence of freeze-thaw stress on the structure and function of microbial communities and denitrifying populations in soil.

Microbial N2O release during the course of thawing of soil was investigated in model experiment focusing on denitrification, since freeze-thaw has been shown to cause significant physical and biological changes in soil, including a surge of N2O and CO2. The origin of these is still controversially discussed. The increase in denitrification after thawing may be attributed to the diffusion of organic substrates newly available to denitrifiers from disrupted soil aggregates, leading to an increase in microbial activity. Laboratory experiments with upper soil layer of a grassland were conducted in microcosms for real-time gas measurements during the entire phase of freeze and thaw. Shifts in microbial communities were evident on resolution of 16S and 18S rRNA genes and transcripts by denaturing gradient gel electrophoresis (DGGE). Microbial expression profiles were compared by RNA-arbitrarily primed PCR technique and subsequent resolution of amplified products on acrylamide gels. Differences in expression levels of periplasmic nitrate reductase gene (napA) and cytochrome cd1 nitrite reductase (nirS) were observed by most-probable-number-reverse transcription-PCR, with higher levels of expression occurring just after thawing began, followed by a decrease. napA and nirS DGGE profiles showed no change in banding patterns with fingerprints derived from DNA, whereas those derived from cDNA showed a clear succession of denitrifying bacteria, with the most complex pattern being observed at the end of the N2O surge. This study provides insight into the structural community changes and expression dynamics of denitrifiers as a result of freeze-thaw stress. Also, the results presented here support the belief that the gas fluxes observed during thawing is a result of freezing initiated high microbial activity.

Bacteria↗
PubMed2004

Nuclear heat shock response and novel nuclear domain 10 reorganization in respiratory syncytial virus-infected a549 cells identified by high-resolution two-dimensional gel electrophoresis.

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by +/-2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection.

Cell Nucleus↗