Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.
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In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells. (+info)
Translational control of specific genes during differentiation of HL-60 cells.
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Eukaryotic gene expression can be regulated through selective translation of specific mRNA species. Nevertheless, the limited number of known examples hampers the identification of common mechanisms that regulate translation of specific groups of genes in mammalian cells. We developed a method to identify translationally regulated genes. This method was used to examine the regulation of protein synthesis in HL-60 cells undergoing monocytic differentiation. A partial screening of cellular mRNAs identified five mRNAs whose translation was specifically inhibited and five others that were activated as was indicated by their mobilization onto polysomes. The specifically inhibited mRNAs encoded ribosomal proteins, identified as members of the 5'-terminal oligopyrimidine tract mRNA family. Most of the activated transcripts represented uncharacterized genes. The most actively mobilized transcript (termed TA-40) was an untranslated 1.3-kilobase polyadenylated RNA with unusual structural features, including two Alu-like elements. Following differentiation, a significant change in the cytoplasmic distribution of Alu-containing mRNAs was observed, namely, the enhancement of Alu-containing mRNAs in the polysomes. Our findings support the notion that protein synthesis is regulated during differentiation of HL-60 cells by both global and gene-specific mechanisms and that Alu-like sequences within cytoplasmic mRNAs are involved in such specific regulation. (+info)
Characterization of lipid efflux particles generated by seminal phospholipid-binding proteins.
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We reported recently that the choline phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa) of bovine seminal plasma (BSP) stimulate cholesterol and choline phospholipid efflux from fibroblasts. In this study, we characterized the lipid efflux particles generated by BSP proteins. The density gradient ultracentrifugation of the efflux medium from radiolabeled fibroblasts incubated with BSP proteins showed a single peak of [3H]cholesterol between density (d) 1.12 and 1.14 g/ml, which is in the range of high-density lipoproteins. Size-exclusion chromatographic and immunoblot analysis revealed that the efflux particles have a large size equal to or bigger than very low-density lipoproteins and contained BSP proteins. Lipid analysis of density gradient and gel filtration fractions from efflux medium of simultaneously labeled fibroblasts ([3H]cholesterol and [3H]choline) incubated with BSP proteins showed that the efflux particles were homogeneous and composed of cholesterol and choline phospholipids. The lipid particles contained BSP proteins, cholesterol and choline phospholipids in molar ratio of 0.05:1.21:1, respectively. Agarose gel electrophoresis showed that the BSP-generated lipid particles had a gamma migration pattern which is slower than low-density lipoproteins. The sonication of cholesterol and BSP proteins followed by gel filtration chromatographic analysis indicated no direct binding of cholesterol to BSP proteins. These results taken together indicate that BSP proteins induce a concomitant cholesterol and choline phospholipid efflux and generate large protein-lipid particles. (+info)
Replication of African swine fever virus DNA in infected cells.
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We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells by in situ hybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments ( approximately 6-12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of approximately 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by a de novo start mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy et al. (B. M. Baroudy, S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47, 723-729) for the replication of vaccinia virus DNA. (+info)
RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA.
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In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked. In contrast, 3' maturation is essentially unaffected. The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G. (+info)
Transcriptional repression by XPc1, a new Polycomb homolog in Xenopus laevis embryos, is independent of histone deacetylase.
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The Polycomb group (Pc-G) genes encode proteins that assemble into complexes implicated in the epigenetic maintenance of heritable patterns of expression of developmental genes, a function largely conserved from Drosophila to mammals and plants. The Pc-G is thought to act at the chromatin level to silence expression of target genes; however, little is known about the molecular basis of this repression. In keeping with the evidence that Pc-G homologs in higher vertebrates exist in related pairs, we report here the isolation of XPc1, a second Polycomb homolog in Xenopus laevis. We show that XPc1 message is maternally deposited in a translationally masked form in Xenopus oocytes, with XPc1 protein first appearing in embryonic nuclei shortly after the blastula stage. XPc1 acts as a transcriptional repressor in vivo when tethered to a promoter in Xenopus embryos. We find that XPc1-mediated repression can be only partially alleviated by an increase in transcription factor dosage and that inhibition of deacetylase activity by trichostatin A treatment has no effect on XPc1 repression, suggesting that histone deacetylation does not form the basis for Pc-G-mediated repression in our assay. (+info)
Heterogeneity of native ribosomal 60-S subunits in Ehrlich ascites tumor cells cultured in vitro.
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Native large ribosomal subunits in cultured Ehrlich ascites tumor cells analyzed by high-resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. A wash with 0.5 M KCl converts LI into particles with the density of LII particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCl wash of reticulocyte ribosomes leads to the formation of particles with the density of LI particles. A protein with a molecular weight of 57000 present in the high-KCl wash of 60-S native subunits was virtually absent in the KCl wash of 40-S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60-S subunits. Possible functions of these protein factors are discussed. (+info)
A quantitative sucrose gradient analysis of the translational activity of 18 mRNA species in testes from adult mice.
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Sucrose gradients have been widely used to study the translational activity of mRNA species in meiotic and haploid spermatogenic cells in mammals. Unfortunately, the results of these studies have been very inconsistent. The purpose of the present study was to obtain accurate and reproducible measurements of the translational activity of a large number of testicular mRNA in sucrose gradients. Extracts of adult testes and cultured seminiferous tubules were sedimented on sucrose gradients, and the distribution of 18 mRNA species was quantified by phosphoimaging. The proportions of various mRNA species sedimenting with polysomes in meiotic and haploid cells (approximately 6-74%) is less than typical of efficiently translated mRNAs (85-90%), demonstrating that the initiation of translation of virtually all mRNA species is at least partially inhibited and that the extent of inhibition is mRNA-specific. Most mRNA species in meiotic and early haploid spermatogenic cells are translated on polysomes in which the ribosome spacing is somewhat wider than in somatic cells, 100-150 verses 80-100 bases. However, the ribosome spacing on protamine mRNAs is unusually close (40-50 bases), and the spacing on poly(A) binding protein mRNA is unusually wide (212-272 bases), thus suggesting that the rate of translational initiation, termination and/or elongation is regulated on translationally active forms of certain mRNA. (+info)