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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Loss of nucleolar localization of NAT10 promotes cell migration and invasion in hepatocellular carcinoma

Highlights: • Residues 68–75 and 989–1018 are complete NuLSs of NAT10. • NuLSs-deleted NAT10 translocates from nucleolus to cytoplasm and membrane. • NuLSs-deleted NAT10 facilitates α-tubulin acetylation, stabilizes microtubule and promotes HCC cell migration. • Cytoplasmic and membranous NAT10 is correlated with poor survival of HCC patients. NAT10, a nucleolar acetyltransferase, participates in a variety of cellular processes including ribosome biogenesis and DNA damage response. Immunohistochemistry staining showed that cytoplasmic and membranous NAT10 is related to the clinical pathologic characteristics in human cancer tissues. However, the mechanism about how NAT10 translocates from the nucleolus to cytoplasm and membrane is unclear. Here, we obtain a NAT10 deletion mutant localizing in cytoplasm and membrane. Bioinformatics analysis showed that residues 68–75 and 989–1018 are two potential nuclear localization signals (NLS) of NAT10. GFP-NAT10 deletion mutant (Δ989-1018) predominantly translocates into cytoplasm with faint signal retained in the nucleolus, while GFP-NAT10(Δ68-75) still remains in the nucleolus and nucleoplasm, indicating residues 989–1018 is the main nucleolar localization signal (NuLS). GFP-NAT10-D3, with both fragments (residues 68–75 and 989–1018) deleted, completely excludes from the nucleolus and translocates to cytoplasm and membrane. Therefore, complete NuLSs of NAT10 should include residues 68–75 and 989–1018. The cytoplasmic and membranous NAT10 mutant (Flag-NAT10-D3) colocalizes with α-tubulin in cytoplasm and with integrin on cell membrane. Importantly, Flag-NAT10-D3 promotes α-tubulin acetylation and stabilizes microtubules. Consequently, Flag-NAT10-D3 promotes migration and invasion in hepatocellular carcinoma (HCC) cells. Statistical analysis of immunohistochemistry staining of NAT10 in HCC tissues demonstrates that the cytoplasmic NAT10 is correlated with poorer prognosis compared with nuclear NAT10, while the membranous NAT10 predicts the poorest clinical outcome of the patients. We thus provide the evidence for the function of cytoplasmic and membranous NAT10 in the metastasis and prognosis of HCC patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Leptomycin B alters the subcellular distribution of CRM1 (Exportin 1)

CRM1 (chromosome maintenance region 1, Exportin 1) binds to nuclear export signals and is required for nucleocytoplasmic transport of a large variety of proteins and RNP complexes. Leptomycin B (LMB), the first specific inhibitor of CRM1 identified, binds covalently to cysteine 528 in the nuclear export signal binding region of CRM1 leading to the inhibition of protein nuclear export. Although the biochemical mechanisms of action of CRM1 inhibitors such as LMB are well studied, the subcellular effects of inhibition on CRM1 are unknown. We have found that LMB causes CRM1 to redistribute from the nucleus to the cytoplasm in A549 cells. A significant decrease in nuclear CRM1 coupled with an increase in cytoplasmic CRM1 was sustained for up to 4 h, while there was no change in total CRM1 protein in fractionated cells. Cells expressing an LMB insensitive HA-tagged CRM1-C528S protein were unaffected by LMB treatment, whereas HA-tagged wildtype CRM1 redistributed from the nucleus to the cytoplasm with LMB treatment, similar to endogenous CRM1. GFP-tagged CRM1 protein microinjected into the cytoplasm of A549 cells distributed throughout the cell in untreated cells remained primarily cytoplasmic in LMB-treated cells. Upon nuclear microinjection, GFP-CRM1 translocated to and accumulated in the cytoplasm of LMB-treated cells. Thus, LMB binds to CRM1 and causes its redistribution to the cytoplasm by inhibiting its nuclear import. Decreasing the nuclear availability of CRM1 likely contributes to the accumulation of CRM1 cargo proteins in the nucleus, suggesting a new mechanism of action for LMB.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Oestrogen regulates SOX9 bioavailability by rapidly activating ERK1/2 and stabilising microtubules in a human testis-derived cell line

Highlights: • Brief oestrogen treatment causes the cytoplasmic retention of the testis factor SOX9. • The cytoplasmic retention of SOX9 by oestrogen is associated with the rapid stabilisation of microtubules. • ERK1/2 is required for the stabilisation of microtubules by activated oestrogen receptors. • Oestrogen can act on both a non-genomic and genomic level to impact gonad somatic cell fate. Nuclear SOX9 is essential for Sertoli cell differentiation and the development of a testis. Exposure of Sertoli cells to exogenous oestrogen causes cytoplasmic retention of SOX9, inhibiting testis development and promoting ovarian development. The cytoplasmic localisation of SOX9 requires a stabilised microtubule network and a key MAPK complex, ERK1/2, is responsive to oestrogen and known to affect the microtubule network. We hypothesised that oestrogen could stabilise microtubules through the activation of ERK1/2 to promote the cytoplasmic retention of SOX9. Treatment of human testis-derived NT2/D1 cells for 30 min with oestrogen rapidly activated ERK1/2, stabilised the microtubule network and increased cytoplasmic localisation of SOX9. The effects of oestrogen on SOX9 and tubulin were blocked by the ERK1/2 inhibitor U0126, demonstrating that ERK1/2 mediates the stabilisation of microtubules and cytoplasmic retention of SOX9 by oestrogen. Together, these data revealed a previously unknown mechanism for oestrogen in impacting MAPK signalling to block SOX9 bioavailability and the differentiation of Sertoli cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells

Highlights: • Oleic acid (OA) induces cytoplasmic, but not nucleoplasmic, LDs in HeLa cells. • Cells harboring OA-induced cytoplasmic LDs exhibit deformity of the nucleus. • Multinucleation by paclitaxel and reversine treatment coincides with LD formation. • LD formation and nuclear deformation are mutually affectable. Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Characterization of a beta-catenin nuclear localization defect in MCF-7 breast cancer cells

Beta-catenin plays a key role in transducing Wnt signals from the plasma membrane to the nucleus. Here we characterize an unusual subcellular distribution of beta-catenin in MCF-7 breast cancer cells, wherein beta-catenin localizes to the cytoplasm and membrane but atypically did not relocate to the nucleus after Wnt treatment. The inability of Wnt or the Wnt agonist LiCl to induce nuclear localization of beta-catenin was not due to defective nuclear transport, as the transport machinery was intact and ectopic GFP-beta-catenin displayed rapid nuclear entry in living cells. The mislocalization is explained by a shift in the retention of beta-catenin from nucleus to cytoplasm. The reduced nuclear retention is caused by unusually low expression of lymphoid enhancer factor/T-cell factor (LEF/TCF) transcription factors. The reconstitution of LEF-1 or TCF4 expression rescued nuclear localization of beta-catenin in Wnt treated cells. In the cytoplasm, beta-catenin accumulated in recycling endosomes, golgi and beta-COP-positive coatomer complexes. The peripheral association with endosomes diminished after Wnt treatment, potentially releasing β-catenin into the cytoplasm for nuclear entry. We propose that in MCF-7 and perhaps other breast cancer cells, beta-catenin may contribute to cytoplasmic functions such as ER-golgi transport, in addition to its transactivation role in the nucleus. - Highlights: • MCF-7 breast cancer cells are defective for beta-catenin nuclear accumulation. • Beta-catenin nuclear retention is lost in MCF-7 cells due to poor LEF1/TCF expression. • Beta-catenin accumulates in the cytoplasm at endosomes and golgi in MCF-7 cells. • Wnt signaling can stimulate release of beta-catenin from endosomes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Heat shock induces the nuclear accumulation of YAP1 via SRC

Highlights: • YAP1 enters the nucleus in response to heat shock. • YAP1 spontaneously returns to the cytoplasm after heat shock. • SRC is involved in the nuclear accumulation of YAP1. • HSP90 is involved in the return to the cytoplasm of YAP1. • YAP1 cross-talks with NF-κB signaling in response to heat shock. Yes-associated protein 1 (YAP1), a co-transcription activator, shuttles between the cytoplasm and the nucleus. Phosphorylation by large tumor suppressor kinases (LATS1/2) is the major determinant of YAP1 subcellular localization. Unphosphorylated YAP1 interacts with transcription factors in the nucleus and regulates gene transcription, while phosphorylated YAP1 is trapped in the cytoplasm and is degraded. We found that when U2OS and HeLa cells are exposed to 42 °C, YAP1 enters the nucleus within 30 min and returns to the cytoplasm at 4 h. SRC and HSP90 are involved in nuclear accumulation and return to the cytoplasm, respectively. Upon heat shock, LATS2 forms aggregates including protein phosphatase 1 and is dephosphorylated and inactivated. SRC activation is necessary for the formation of aggregates, while HSP90 is required for their dissociation. YAP1 is involved in heat shock-induced NF-κB signaling. Mechanistically, YAP1 is implicated in strengthening the interaction between RELA and DPF3, a component of SWI/SNF chromatin remodeling complex, in response to heat shock. Thus, YAP1 plays a role as a thermosensor.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Isolation of human monoclonal antibodies that bind to two different antigens and are encoded by germline VH and VL genes

This paper reports isolation of two monoclonal antibodies (mAbs) that bind to both a membrane protein and a cytoplasmic protein. Most Abs established as markers for autoimmune disease bind to cytoplasmic or nuclear substances. However, it remains unknown how these Abs are produced. On the other hand, there were examples where clones originally isolated as Abs that bind to membrane proteins also showed binding activity to cytoplasmic or nuclear substances. Based on these results, the following hypothesis has been proposed. The Abs that had been originally produced against a membrane protein showed cross-reactivity against cytoplasmic or nuclear substances. In the present study we reported isolation of Abs that bound to both a membrane protein, CADM1, and a cytoplasmic protein, α-actinin-4. The method adopted in the present study could be generally applicable to isolation of Abs showing such dual specificity. Firstly, we constructed a huge human Ab library using various organs including naïve B-cell-rich organs such as bone marrow and umbilical cords. Then, we developed a comprehensive screening method for isolation of Abs that bound to cell surface antigens. Through extensive screenings with many kinds of cell we newly obtained a library composed of around 4000 independent clones that bind to membrane proteins. We screened this library with α-actinin-4 and succeeded in isolating two Abs. They bound to α-actinin-4 and a membrane protein CADM1. Furthermore, they are encoded by naïve heavy and light chain variable genes (V{sub H} & V{sub L}). These results suggested that cross-reactive Abs to both a membrane protein and a cytoplasmic protein could be present in germline repertoire of Ab in humans. This methodology adopted in the present study could be applied to isolation of cross-reactive Abs possibly involved in autoimmune diseases.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fused with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1998

Development of the endolymphatic sac and duct in the Japanese red-bellied newt, Cynops pyrrhogaster

The development and maturation of the endolymphatic sac (ES) and duct (ED) were studied in the newt Cynops pyrrhogaster. The ES first appears as an oval capsule at the dorsal-medial tip of the otic vesicle at stage 39, about 11 days after oviposition. The ES consists of polymorphous epithelial cells with a minimum of cytoplasm. The intercellular space (IS) between the epithelial cells is narrow and has a smooth surface. At stage 44, the size of the ES increases as many vacuoles in the IS become filled. At stage 46, 18 days after oviposition, the ES elongates markedly and a slit-like lumen is found in the ES. The epithelium contains a few cell organelles which are scattered in the cytoplasm. The vacuoles in the IS are fused, which expands the IS. Two days later (stage 48), floccular material (endolymph) is present in the expanded lumen. The IS dilates and has a wide and irregular appearance. At stage 50, approximately 26 days after oviposition, the ES extends and expands significantly and crystals (otoconia) can now be seen in the widened lumen of the ES. The cytoplasm of the cuboidal epithelial cells contains an abundance of vesicles surrounded by ribosomes and Golgi complexes. Intercellular digitations are formed in the expanded IS. At stage 54, the ES forms a large bellow-like pouch. Numerous otoconia accumulate in the lumen. Free floating cells and cell debris can be seen in the lumen at this stage. The epithelial cells contain numerous cytoplasmic organelles which are evenly distributed in the cytoplasm. Granules are found in the apical and lateral cytoplasm. The IS is loose and displays a labyrinthine appearance. The primitive ED first appears as a connection between the ES and the saccule but no lumen is present inside at stage 39. At stage 46, a narrow lumen is formed in the ED, which corresponds to the formation of the ES lumen. At stage 50, as the ED extends, floccular material is seen in the lumen. At stage 54, the ED bears numerous microvilli on its luminal surface. Otoconia and endolymph are present in the ED. Tight junctions between the epithelial cells are formed at stage 46. A fully developed intercellular junctional complex is produced at stage 54. Based on the development of the ES and ED, the maturation of function of the ES and ED are discussed.

NASA Discipline Neuroscience↗
DOE OSTI2014

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

PAK1 translocates into nucleus in response to prolactin but not to estrogen

Tyrosyl phosphorylation of the p21-activated serine–threonine kinase 1 (PAK1) has an essential role in regulating PAK1 functions in breast cancer cells. We previously demonstrated that PAK1 serves as a common node for estrogen (E2)- and prolactin (PRL)-dependent pathways. We hypothesize herein that intracellular localization of PAK1 is affected by PRL and E2 treatments differently. We demonstrate by immunocytochemical analysis that PAK1 nuclear translocation is ligand-dependent: only PRL but not E2 stimulated PAK1 nuclear translocation. Tyrosyl phosphorylation of PAK1 is essential for this nuclear translocation because phospho-tyrosyl-deficient PAK1 Y3F mutant is retained in the cytoplasm in response to PRL. We confirmed these data by Western blot analysis of subcellular fractions. In 30 min of PRL treatment, only 48% of pTyr-PAK1 is retained in the cytoplasm of PAK1 WT clone while 52% re-distributes into the nucleus and pTyr-PAK1 shuttles back to the cytoplasm by 60 min of PRL treatment. In contrast, PAK1 Y3F is retained in the cytoplasm. E2 treatment causes nuclear translocation of neither PAK1 WT nor PAK1 Y3F. Finally, we show by an in vitro kinase assay that PRL but not E2 stimulates PAK1 kinase activity in the nuclear fraction. Thus, PAK1 nuclear translocation is ligand-dependent: PRL activates PAK1 and induces translocation of activated pTyr-PAK1 into nucleus while E2 activates pTyr-PAK1 only in the cytoplasm. - Highlights: • Prolactin but not estrogen causes translocation of PAK1 into nucleus. • Tyrosyl phosphorylation of PAK1 is required for nuclear localization. • Prolactin but not estrogen stimulates PAK1 kinase activity in nucleus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Cellular localization of ATBF1 protein and its functional implication in breast epithelial cells

ATBF1, a large transcription factor, was normally localized in nuclei, and its mislocalization to cytoplasm was reported in multiple cancers. However, localization of ATBF1 in breast epithelial cells and its potential functions were unknown. Here, we investigated ATBF1 localization via immunofluorescence staining in different kinds of breast epithelial cells. In MCF10A cells and normal mice mammary gland tissues, ATBF1 was mainly localized in nuclei. Knockdown of ATBF1 expression in MCF10A cells by siRNA promoted cell proliferation. Moreover, ATBF1 was co-localized with chromosome during mitosis, indicating its potential function in mitosis. In an estrogen receptor (ER)-positive breast cancer cell line (MCF7), estrogen induced ATBF1 translocation from cytoplasm to nuclei in an ER dependent pathway. In ER-negative cells (Hs578T and MDA-MB-231), ATBF1 was co-localized with GM130 in cytoplasm, indicating ATBF1 localization was associated with protein modification in golgi body. The results were beneficial for intensive investigation of ATBF1's function with different cellular localization in breast cancer. - Highlights: • Cellular localization of ATBF1 is correlated with its function in breast cancer. • Nuclear ATBF1 was co-localized with chromosome during mitosis normally. • Estrogen induced translocation of cytoplasmic ATBF1 to nuclei in MCF7. • Translocation of ATBF1 in ER{sup +} breast cancer cells was ER dependent. • Cytoplasmic ATBF1 was co-localized with golgi body in ER{sup −} breast cancer cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Oxidized low-density lipoprotein promotes osteoclast differentiation from CD68 positive mononuclear cells by regulating HMGB1 release

Highlights: • The ratio of ox-LDL to LDL was elevated in patients with ankylosing spondylitis. • The CD68{sup +}/RANK{sup +} cell number was increased in peripheral blood from AS patients. • 0.03% ox-LDL promoted cytoplasmic translocation and release of HMGB1. • CD68 mediated ox-LDL-induced release of HMGB1. • Ox-LDL-induced EGR1 expression promoted cytoplasmic translocation of HMGB1. Patients with ankylosing spondylitis (AS) have an increased risk for cardiovascular mortality. The circulating ox-LDL/LDL ratio is associated with subclinical atherosclerosis in patients with systemic lupus erythematosus. In this study, we found that the ox-LDL/LDL ratio was increased in AS patients. The levels of serum RANKL and HMGB1 were also elevated in AS patients, and the number of CD68{sup +}/RANK{sup +} cells was increased in peripheral blood from AS patients. 0.03% ox-LDL in LDL, similar to the ox-LDL/LDL ratio in peripheral blood from AS patients, promoted cytoplasmic translocation and release of HMGB1 as well as RANK expression. Further investigation evidenced that ox-LDL-induced EGR1 expression contributed to the cytoplasmic translocation of HMGB1 and CD68 assisted the secretion of HMGB1 from cytoplasm to extracellular matrix. Extracellular HMGB1 induced RANK expression in CD68{sup +} mononuclear cells, forming osteoclast precursors that were differentiated to osteoclasts in response to RANKL. Taken together, these results suggested that the changes, including ox-LDL/LDL ratio, CD68{sup +}/RANK{sup +} cells number, and the levels of RANKL and HMGB1 in AS patients, favored osteoclastogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Prolonged exposure to particulate chromate inhibits RAD51 nuclear import mediator proteins

Particulate hexavalent chromium (Cr(VI)) is a human lung carcinogen and a human health concern. The induction of structural chromosome instability is considered to be a driving mechanism of Cr(VI)-induced carcinogenesis. Homologous recombination repair protects against Cr(VI)-induced chromosome damage, due to its highly accurate repair of Cr(VI)-induced DNA double strand breaks. However, recent studies demonstrate Cr(VI) inhibits homologous recombination repair through the misregulation of RAD51. RAD51 is an essential protein in HR repair that facilitates the search for a homologous sequence. Recent studies show prolonged Cr(VI) exposure prevents proper RAD51 subcellular localization, causing it to accumulate in the cytoplasm. Since nuclear import of RAD51 is crucial to its function, this study investigated the effect of Cr(VI) on the RAD51 nuclear import mediators, RAD51C and BRCA2. We show acute (24 h) Cr(VI) exposure induces the proper localization of RAD51C and BRCA2. In contrast, prolonged (120 h) exposure increased the cytoplasmic localization of both proteins, although RAD51C localization was more severely impaired. These results correlate temporally with the previously reported Cr(VI)-induced RAD51 cytoplasmic accumulation. In addition, we found Cr(VI) does not inhibit interaction between RAD51 and its nuclear import mediators. Altogether, our results suggest prolonged Cr(VI) exposure inhibits the nuclear import of RAD51C, and to a lesser extent, BRCA2, which results in the cytoplasmic accumulation of RAD51. Cr(VI)-induced inhibition of nuclear import may play a key role in its carcinogenic mechanism since the nuclear import of many tumor suppressor proteins and DNA repair proteins is crucial to their function. - Highlights: • RAD51C nuclear localization is reduced after prolonged Cr(VI) exposure. • Prolonged Cr(VI) inhibits nuclear localization of BRCA2 less severely than RAD51C. • RAD51C deficiency induces the cytoplasmic accumulation of RAD51. • Cr(VI) does not inhibit interactions between RAD51 and its nuclear import partners.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

SU-E-T-667: Radiosensitization Due to Gold Nanoparticles: A Monte Carlo Cellular Dosimetry Investigation of An Expansive Parameter Space

Purpose: To investigate dose enhancement to cellular compartments following gold nanoparticle (GNP) uptake in tissue, varying cell and tissue morphology, intra and extracellular GNP distribution, and source energy using Monte Carlo (MC) simulations. Methods: Models of single and multiple cells are developed for normal and cancerous tissues; cells (outer radii 5–10 µm) are modeled as concentric spheres comprising the nucleus (radii 2.5–7.5 µm) and cytoplasm. GNP distributions modeled include homogeneous distributions throughout the cytoplasm, variable numbers of GNP-containing endosomes within the cytoplasm, or distributed in a spherical shell about the nucleus. Gold concentrations range from 1 to 30 mg/g. Dose to nucleus and to cytoplasm for simulations including GNPs are compared to simulations without GNPs to compute Nuclear and Cytoplasm Dose Enhancement Factors (NDEF, CDEF). Photon source energies are between 20 keV and 1.25 MeV. Results: DEFs are highly sensitive to GNP intracellular distribution; for a 2.5 µm radius nucleus irradiated by a 30 keV source, NDEF varies from 1.2 for a single endosome containing all GNPs to 8.2 for GNPs distributed about the nucleus (7 mg/g). DEFs vary with cell dimensions and source energy: NDEFs vary from 2.5 (90 keV) to 8.2 (30 keV) for a 2.5 µm radius nucleus and from 1.1 (90 keV) to 1.3 (30 keV) for a 7.5 µm radius nucleus, both with GNPs in a spherical shell about the nucleus (7 mg/g). NDEF and CDEF are generally different within a single cell. For multicell models, the presence of gold within intervening tissues between source and target perturbs the fluence reaching cellular targets, resulting in DEF inhomogeneities within a population of irradiated cells. Conclusion: DEFs vary by an order of magnitude for different cell models, GNP distributions, and source energies, demonstrating the importance of detailed modelling for advancing GNP development for radiotherapy. Funding provided by the Natural Sciences and Engineering Council of Canada (NSERC), and the Canada Research Chairs Program (CRC)

61 RADIATION PROTECTION AND DOSIMETRY↗
NASA NTRS1985

Sulfate-reducing bacteria: Microbiology and physiology

The sulfate reducing bacteria, the first nonphotosynthetic anaerobic bacteria demonstrated to contain c type cytochromes, perform electron transfer coupled to phosphorylation. A new bioenergetic scheme for the formation of a proton gradient for growth of Desulfovibrio on organic substrates and sulfate involving vectors electron transfer and consistent with the cellular localization of enzymes and electron transfer components was proposed. Hydrogen is produced in the cytoplasm from organic substrates and, as a permease molecule diffuses rapidly across the cytoplasmic membrane, it is oxidized to protons and electrons by the periplasmic hydrogenase. The electrons only are transferred across the cytoplasmic membrane to the cytoplasm where they are used to reduce sulfate to sulfide. The protons are used for transport or to drive a reversible ATPOSE. The net effect is the transfer of protons across the cytoplasmic membrane with the intervention of a proton pump. This type of H2 cycling is relevant to the bioenergetics of other types of anaerobic microorganisms.

Peck, H. D.↗
NASA NTRS1986

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

NASA Center ARC↗
DOE OSTI2014

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor

Highlights: • So far, eRF3 has been thought to function exclusively in the cytoplasm. • eRF3 is a nucleo-cutoplasmic shuttling protein. • eRF3 has a leptomycin-sensitive nuclear export signal (NES). • Removal of NES by proteolytic cleavage allows eRF3 to translocate to the nucleus. • The processed eRF3 (p-eRF3) interacts with a nuclear tumor suppressor ARF. - Abstract: The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61–71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.

60 APPLIED LIFE SCIENCES↗