Localization Of Poly Research Articles

Localization of Poly(glycidyl methacrylate) Grafted on Reduced Graphene Oxide in Poly(lactic acid)/Poly(trimethylene terephthalate) Blends for Composites with Enhanced Electrical and Thermal Conductivities

Interfacial localization of conductive fillers in a cocontinuous immiscible polymer blend is an efficient way of improving the electrical and thermal conductivities of the composite. Conductive path formation at the interface of a cocontinuous structure is expected to provide high conductivity by a smaller amount of the filler, which can be used for applications as conductive materials. In this study, biobased poly(lactic acid) (PLA) was blended with poly(trimethylene terephthalate) (PTT) to make the cocontinuous immiscible polymer blend. Poly(glycidyl methacrylate) (PGMA) was grafted on reduced graphene oxide (rGO) to make a PGMA-grafted rGO (rGO-PGMA). The epoxy group of GMA on rGO-PGMA reacted with the end groups of both PLA and PTT and localized at the interface between PLA and PTT by a two-step blending procedure to form the conductive path between PLA and PTT. From transmission electron microscopy observation, it was found that rGO-PGMA localized between the interface of PLA and PTT. Both electrical and thermal conductivities of the composite were improved, which was confirmed by the electrical volume resistivity and thermal diffusivity measurements, compared with neat polymers and other blends.

Mitochondria are devoid of poly(ADP-ribose)polymerase-1, but harbor its product oligo(ADP-ribose).

There are conflicting data about localization of poly(ADP-ribose)polymerase-1 and its product poly(ADP-ribose) in mitochondria. To finally clarify the discussion, we investigated with biochemical and cell biological methods the potential presence of poly(ADP-ribose) polymerase-1 in these organelles. Our data show that endogenous and overexpressed poly(ADP-ribose)polymerase 1 is only localized to the nucleus with a clear exclusion of cytosolic compartments. In addition, highly purified mitochondria devoid of nuclear contaminations do not contain poly(ADP-ribose)polymerase-1. Although no poly(ADP-ribose)polymerase-1 enzyme is detectable in mitochondria, a shorter variant of its product poly(ADP-ribose) is present, associated specifically with a small subset of mitochondrial proteins as revealed by immunoprecipitation and protein fingerprint analysis. These proteins are located at key-points of the Krebs-cycle, are chaperones involved in mitochondrial functionality and quality-control, and are RNA-binding proteins important for transcript stability, respectively. Of note, despite the fact that especially poly(ADP-ribose)polymerase-1 is its own major target for modification, we could not detect this enzyme by mass spectrometry in these organelles. These data suggests a new way of targeted nuclear-mitochondrial signaling, mediated by nuclear poly(ADP-ribosyl)ation dependent on poly(ADP-ribose)polymerase-1.

Inter‐spherulitic/inner‐spherulitic localization of PBSU during crystallization of PVDF in PVDF/PBSU blend

AbstractIn this work, the miscibility effect on the localization of poly(butylene succinate) (PBSU) during the crystallization of PVDF in their blend has been investigated. After annealed at 200 °C and 240 °C, homogeneous and phase‐separated structures can be obtained respectively, which was followed by isothermal crystallization at 141 °C. In the case of 200°C, PBSU tends to enrich in inner‐spherulitic regions because of the excellent miscibility of the blend and the higher growth rate of PVDF crystals. When the specimen was annealed at 240 °C, phase separation produces PVDF and PBSU domains. Upon cooling to 141 °C, one part of PBSU is miscible with , while the other part of it remains as phase‐separated domains due to the high viscosity and slow relaxation of them. The former accounts for the distribution of PBSU in inner‐spherulitic regions. In the latter, however, phase‐separated structures depress the diffusion of PVDF during its crystallization, leading to the lower magnitude of growth rate of spherulites. Both of them contribute to the localization of PBSU in inter‐spherulitic regions. The distribution of PBSU among PVDF spherulites has been validated by long periods, pore size, and mechanical performance of the porous PVDF membranes.

Expanded polyglutamine impairs normal nuclear distribution of fused in sarcoma and poly (rC)-binding protein 1 in Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Immunohistochemical studies using the 1C2 antibody for polyglutamine expansion have detected characteristic intranuclear inclusions (INIs) in affected neurons in HD. Further, in vitro and mouse models of HD have shown that the INIs recruit several proteins relating to RNA splicing and translation. In the present study, we immunohistochemically investigated the association of INIs with various heterogeneous nuclear ribonucleoproteins in the cerebral cortex of four autopsy cases of HD. Fused in sarcoma (FUS) was colocalized with 1C2-positive nuclear inclusions in all examined cases. Localization of poly (rC)-binding protein 1 (PCBP1) in 1C2-positive nuclear inclusions was also observed. Double immunofluorescence revealed complete or partial loss of the normal, diffuse nuclear distribution of FUS or PCBP1 in neurons with 1C2-positive nuclear inclusions. This maldistribution of FUS in cortical neurons suggests a severe disturbance of messenger RNA processing, which may be a common pathogenetic mechanism of FUS-related familial amyotrophic lateral sclerosis.

Passive and active hepatoma tumor targeting of new N-(2-hydroxypropyl)methacrylamide copolymer conjugates: synthesis, characterization, and evaluation in vitro and in vivo

Human hepatocellular carcinoma (HCC) is one of the major causes of death worldwide. To investigate the relative importance of active and passive targeting strategies, the synthesis, characterization, in vitro uptake, and in vivo biodistribution of specific sulfapyridine HPMA (HPMA: N-(2-hydroxypropyl methacrylamide)) copolymer (sulfapyridine: SPD) conjugates, nonspecific HPMA copolymer conjugates, and DTPA are described in this study. The poly(HPMA)-SPD-DTPA (DTPA: diethylenetriaminepentaacetic acid), poly(HPMA)-DTPA, and DTPA conjugates were radiolabeled with the radionuclide 99mTc and tested for uptake by cultured H22 cells. The cellular accumulation of poly(HPMA)-SPD-DTPA-99mTc complex was found to be time-dependent. The poly(HPMA)-SPD-DTPA-99mTc tracer exhibited rapid uptake kinetics in cell culture with a t 1/2 of ∼5 min. The uptake of poly(HPMA)-SPD-DTPA-99mTc was significantly higher than that of poly(HPMA)-DTPA-99mTc, indicating that the uptake of the poly(HPMA)-SPD-DTPA-99mT was active binding. The uptake of poly(HPMA)-DTPA-99mTc was significantly higher than that of DTPA-99mTc, suggesting that the uptake of the poly(HPMA)-DTPA-99mT was passive binding. Twenty-four hour necropsy data in the hepatocellular carcinoma tumor model showed significantly higher (p < 0.001) tumor localization for poly(HPMA)-SPD-DTPA-99mTc (4.98 ± 0.48%ID/g [percentage injected dose per gram tissue]) compared with poly(HPMA)-DTPA-99mTc (2.69 ± 0.15% ID/g) and DTPA-99mTc (0.83 ± 0.03%ID/g). Moreover, higher T/B for poly(HPMA)-SPD-DTPA-99mTc indicated reduced extravazation of the targeted polymeric conjugates in normal tissues. Specific molecular targeting and nonspecific vascular permeability are both significant in the relative tumor localization of poly(HPMA)-SPD-DTPA-99mTc. Extravascular leak in nonspecific organs appears to be a major factor in reducing the T/B for the sulfapyridine molecules. Thus, the poly(HPMA)-SPD-DTPA is expected to be used as the potential macromolecular targeting carrier for hepatoma carcinoma in mice.

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Localization of poly(A)+ RNA and mRNA export factors in interchromatin granule clusters of two-cell mouse embryos

Interchromatin granule clusters (IGCs), also known as nuclear speckles, splicing factor compartments, or SC35-domains, are one of the most universal nuclear organelles of the cell. We have used two-cell mouse embryos as an experimental system to study the possible association of poly(A)(+) RNA and factors involved in RNA export (Tip-associated protein [TAP] and heterogenous nuclear ribonucleoprotein A/B [hnRNP A/B]) with IGCs. Poly(A)(+) RNA was localized by microinjections of 2'-O-Me(U)(22) probes conjugated with tetramethylrhodamine. RNA export proteins were detected by immunofluorescence confocal laser microscopy and immunogold-labeling electron microscopy. We found that poly(A)(+) RNA was located in IGCs regardless of the transcriptional state of the nuclei. hnRNP A/B was also found to characterize IGCs of the embryo nuclei with various levels of transcription activity. In transcriptionally active embryo nuclei, TAP was detected in the vicinity of IGCs rather than inside them; however, when transcription was inhibited by drugs, TAP was localized to IGCs. Our data support the idea that IGCs not only serve as splicing factor reservoirs, but also take part in mRNA retention and export.

Distribution of poly(A) RNA and splicing machinery elements in mature Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro

The localization of poly(A) mRNA and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and the SC35 protein, in mature Hyacinthus orientalis L. pollen grains before anthesis and pollen tubes germinating in vitro were analyzed. The observations indicated a pattern of poly(A) mRNA distribution in mature pollen grains before anthesis which differed from that in germinating pollen grains. Directly before anthesis, poly(A) mRNA was homogeneously distributed throughout the whole cytoplasm, whereas after rehydration, it accumulated at one of the pollen poles. In the pollen tube, poly(A) mRNA was present in the cytoplasm, mainly in the areas beneath the cell membrane and the apical zone. Both before anthesis and during growth of the pollen tube, splicing snRNPs and SC35 protein were localized mainly in the area of the pollen nuclei. During anthesis and just after rehydration of the pollen grains, the pattern of labeling and the levels of the investigated antigens in the areas of the vegetative and generative nuclei were similar. During growth of the pollen tube, a change was observed in the distribution and an increase in the levels of trimethylguanosine snRNA and SC35 protein in the vegetative nucleus. Such a pattern of localization of the splicing machinery suggests resumption of transcription and/or maturation of pre-mRNA in the growing pollen tube.

Poly(A)+ RNA during vegetative development of Acetabularia peniculus.

In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)+ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)+ RNA in the juvenile stage of the Acetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)+ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)+ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)+ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.

Localization of poly(A)-binding protein 2 (PABP2) in nuclear speckles is independent of import into the nucleus and requires binding to poly(A) RNA.

The nuclei of mammalian cells contain domains, termed nuclear speckles, which are enriched in splicing factors and poly(A) RNA. Although nuclear speckles are thought to represent reservoirs from which splicing factors are recruited to sites of transcription and splicing, the presence of poly(A) RNA in these structures remains enigmatic. An additional component of the speckles is poly(A) binding protein 2 (PABP2), a protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In this work we investigated whether PABP2 contributes to the targeting of poly(A) RNA to the speckles. The results show that localization of PABP2 in speckles is independent of import of the protein into the nucleus. Inhibition of transcription or poly(A) synthesis at the end of mitosis does not affect nuclear import of PABP2 but prevents its localization to speckles. Furthermore, PABP2 mutants with decreased ability to bind to poly(A) fail to localize to speckles. Taken together the results show that PABP2 localizes to the nuclear speckles as a consequence of its binding to poly(A) RNA, contrasting to splicing factors which assemble into speckles in the absence of newly synthesized transcripts.

Localization of poly(A)+-containing RNA during female gametophyte development in Medicago sativa and the diploid mutant Medicago sativa ssp. falcata using digoxigenin-labelled oligo-dT probes

The detection and distribution pattern of poly(A)+-containing RNA during embryo sac development in the wild type and in a diplosporic mutant of alfalfa was studied using digoxigenin-labelled oligo-dT probes. In the very early stage of development, the megaspore mother cell does not show any poly(A)+RNA accumulation. Subsequently, poly(A) + RNA appears concomitantly in the megaspore mother cell and the nucellus tissue, both in wild and mutant alfalfa. The distribution pattern of poly(A) + RNA reveals some differences. In the mutant, the absence of label in the chalazal part of the nucellus at the megaspore mother cell stage seems to be significant. Compared to the wild type, the hybridization signal at the functional megaspore as well as at the two-nucleate coenocytic stage was reduced. However, the late stages of embryo sac development in the two types were almost comparable, except that the central cell of the mutant accumulated more label. The significance of the results in relation to reduced and unreduced embryo sac development is discussed.

Three-dimensional localization of poly(A) RNA and splicing components in the nucleus

The physical distribution of active genes has long been a subject of interest and speculation, however technical limitations have necessitated that it be addressed only by indirect approaches with sometimes contradictory results. However, developments in fluorescence in situ hybridization methodologies allow the position of specific genes and RNAs to be visualized directly within intact cells, thus providing a more direct means to study such questions. To address whether compartmentalization occurs during the production and processing of pre-mRNA in mammalian somatic cells we have recently investigated the distribution of nuclear polyadenylated transcripts which represent approximately 90% of all pre-mRNA. We found that poly(A) RNA forms discrete nuclear “transcript domains” which are specifically positioned with respect to the underlying genome and contain snRNP antigens of the pre-mRNA splicing class. Several lines of evidence indicate a close spatial and temporal linkage between transcription and processing of pol II RNAs (reviewed in 5), therefore, it is possible that poly(A) RNA transcript domains reflect a clustering of active genes at these sites.

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.

Accumulation and Spatial Distribution of Poly(A)+ RNA in Oocytes and Early Embryos of Drosophila melanogaster: (Poly(A)+ RNA/in situ hybridization/oocytes and embryos/Drosophila melanogaster).

We describe the accumulation and distribution of poly (A)+ RNA during oogenesis and early embryogenesis as revealed by in situ hybridization with a radio-labeled poly (U) probe. The amount of poly (A)+ RNA in nurse cell cytoplasm continuously increased untill mid-vitellogenic stage (st. 10), then decreased with the rapid increase of poly (A)+ RNA in the oocyte (st. 11). The localization of poly (A)+ RNA at stage 10 was in the anterior region of the oocyte, where it is connected by cytoplasmic bridge to the nurse cells. These observations indicate that most of the poly (A)+ RNA synthesized in the nurse cells is transferred to the oocyte through the cytoplasmic bridges at stage 10-11. During the remainder of oogenesis (st. 11-14) and during preblastodermal embryogenesis, poly (A)+ RNA was evenly distributed over the cytoplasm of oocytes and embryos. At blastoderm stage, poly(A)+ RNA became concentrated in the peripheral region of embryos. Though the somatic nuclei of the blastoderm contained a detectable amount of poly (A)+ RNA, the pole cell nuclei did not. The cytoplasmic RNA visualised by acridine orange staining and the poly (A)+ RNA detected by hybridization with [3 H]poly (U) exhibited identical distributions during oogenesis and early embryogenesis. These observations provide a basis to assess the unique distributions of specific RNA sequences involved in early development.

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes.

Poly(A)+RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)+ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)+RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)+RNA and tubulin mRNA.

Localization of poly(A) Sequences that are Lost during Oocyte Maturation of &lt;I&gt;Xenopus laevis&lt;/I&gt;

Xenopus oocytes and eggs were fractionated into post-mito-chondrial supernatant and pellet fractions, after which the content of poly(A) sequences and their size distribution were analyzed. It was found that, while the poly(A) content in the post-mitochondrial supernatant fraction remained constant during oocyte maturation, that in the pellet fraction decreased signi-ficantly without any change in its sequence length. This decrease in poly(A)content as has recently been reported to occur during oocyte maturation can thus be confined to the pellet fraction.