An MRI-Visible Non-Viral Vector Bearing GD2 Single Chain Antibody for Targeted Gene Delivery to Human Bone Marrow Mesenchymal Stem Cells

The neural ganglioside GD2 has recently been reported to be a novel surface marker that is only expressed on human bone marrow mesenchymal stem cells within normal marrow. In this study, an MRI-visible, targeted, non-viral vector for effective gene delivery to human bone marrow mesenchymal stem cells was first synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAb_GD2), to the distal ends of PEG-g-PEI-SPION. The targeted vector was then used to condense plasmid DNA to form nanoparticles showing stable small size, low cytotoxicity, and good biocompatibility. Based on a reporter gene assay, the transfection efficiency of targeting complex reached the highest value at 59.6% ± 4.5% in human bone marrow mesenchymal stem cells, which was higher than those obtained using nontargeting complex and lipofectamine/pDNA (17.7% ± 2.9% and 34.9% ± 3.6%, respectively) (P<0.01). Consequently, compared with the nontargeting group, more in vivo gene expression was observed in the fibrotic rat livers of the targeting group. Furthermore, the targeting capacity of scAb_GD2-PEG-g-PEI-SPION was successfully verified in vitro by confocal laser scanning microscopy, Prussian blue staining, and magnetic resonance imaging. Our results indicate that scAb_GD2-PEG-g-PEI-SPION is a promising MRI-visible non-viral vector for targeted gene delivery to human bone marrow mesenchymal stem cells.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).     

Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H⁺-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.     

Long-Term Inactivation Study of Three Enteroviruses in Artificial Surface and Groundwaters, Using PCR and Cell Culture

Since the transmission of pathogenic viruses via water is indistinguishable from the transmission via other routes and since the levels in drinking water, although significant for health, may be too low for detection, quantitative viral risk assessment is a useful tool for assessing disease risk due to consumption of drinking water. Quantitative viral risk assessment requires information concerning the ability of viruses detected in drinking water to infect their host. To obtain insight into the infectivity of viruses in relation to the presence of virus genomes, inactivation of three different enteroviruses in artificial ground and surface waters under different controlled pH, temperature, and salt conditions was studied by using both PCR and cell culture over time. In salt-peptone medium, the estimated ratio of RNA genomes to infectious poliovirus 1 in freshly prepared suspensions was about 10⁰. At 4°C this ratio was 10³ after 600 days, and at 22°C it was 10⁴ after 200 days. For poliovirus 1 and 2 the RNA/infectious virus ratio was higher in artificial groundwater than in artificial surface water, but this was not the case for coxsackievirus B4. When molecular detection is used for virus enumeration, it is important that the fraction of infectious virus (based on all virus genomes detected) decays with time, especially at temperatures near 22°C.     

Three-way interactions between mosquito population,viral strain and temperature underlying chikungunya virus transmission potential

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.     

GLUT4 and Transferrin Receptor Are Differentially Sorted Along the Endocytic Pathway in CHO Cells

The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750–12754.). In this study, we demonstrate that at 37°C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37°C, cell surface–labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15°C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37°C after accumulating labeled GLUT4 at 15°C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15°C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37°C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at both 15° and 37°C. Thus, GLUT4 is sorted differently from the transferrin receptor as evidenced by the targeting of each protein to distinct early endosomal compartments and by the formation of GSVs. These results suggest that the sorting of GLUT4 from TfR may occur primarily at the level of the plasma membrane into distinct endosomes and that the organization of the endocytic system in CHO cells more closely resembles that of neuroendocrine cells than previously appreciated.     

OBSERVATIONS ON SELECTED ISOLATED RETINAL ELEMENTS AND AN ANALYSIS OF ROD CELL RNA OF THE RABBIT

RNA was analyzed in the whole rod cell and in the rod cell nucleus of the rabbit retina. The analysis was performed on rod cells or rod cell nuclei after they were isolated by microdissection and collected. The nuclei were denuded by selectively lysing inner and outer segments chemically. The rod cell contained an average of 0.65 µµg RNA. About 60% of the total RNA was nuclear. RNA concentration was of the order of 0.4% w/v. RNA base composition was determined for the whole rod cell and for the rod cell nucleus, and from it the base composition of cytoplasmic RNA was derived also. Microdissection of the retina revealed that each Müller cell had firmly attached to it a certain complement of visual cells, forming a bouquet-like arrangement. The unusual susceptibility of the inner and outer segments to lysis was regarded as an indication of an unique composition of the plasma membrane.     

Exosomes mediate an epithelial‐mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR‐543 was found in exosomes from EMTed RPE cells, and miR‐543‐enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.     

Conditional,Temperature-Induced Proteolytic Regulation of Cyanobacterial RNA Helicase Expression

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.     

A COMPARISON BETWEEN HETEROGENEOUS NUCLEAR RNA AND POLYSOMAL MESSENGER RNA IN HELA CELLS BY RNA-DNA HYBRIDIZATION

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 µg of HeLa cell DNA binds 0.05–0.10 µg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.     

CHANGES IN THE BASE COMPOSITION OF NUCLEAR RIBONUCLEIC ACID OF NEURONS DURING A SHORT PERIOD OF ENHANCED PROTEIN PRODUCTION

Nuclei from isolated nerve cells were sampled by microdissection. The content and composition of the nuclear RNA was studied and compared with that of the cytoplasmic RNA of Deiters' nerve cells of rabbits. Analyses were made of control nerve cells and of cells in which an enhanced RNA and protein production had been induced by chemical means, tricyano-amino-propene, for 60 minutes. The nuclear RNA content of the control nerve cells was 56 µµg, i.e. 3 per cent of the total RNA content of the nerve cell. The base ratios were: adenine 21.3, guanine 26.6, cytosine 30.8, uracil 21.3. Purine-pyrimidine analyses showed that the nuclear RNA differed significantly from the cytoplasmic RNA in having higher adenine and uracil values. The guanine and cytosine values were high, however, and the ratio G/C was 0.86 as compared with 1.16 for the cytoplasmic RNA. The composition of the nuclear RNA was interpreted as reflecting the extraordinarily strong development of the nucleolus in these neurons. During the 60 minutes of enhanced neuronal RNA production (+25 per cent) the guanine value increased and the uracil value decreased significantly in the nuclear RNA. In the cytoplasmic RNA the guanine value also increased although not so much as the nuclear guanine. The cytoplasmic cytosine value decreased. The result indicated that the production of the characteristic cytoplasmic RNA had been influenced by the change in the nuclear RNA     

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale. There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation. However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons. Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms. Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation. We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons. This approach is likely to have broad utility in the field of neuronal endocytic trafficking.     

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

Effect of Chilling Temperatures upon Cell Cultures of Tomato

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv `VF36,' and cv `VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No growth was observed at 8°C, indicating an abrupt limit to growth between 8 and 12°C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8°C for up to 2 weeks. When cultures kept at 8°C for up to 30 days were transferred to 28°C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0°C, and for cells of `VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9°C but additional growth at 8°C did not occur, nor could growth be maintained by subculture at 8 or 9°C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10°C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.     

PANCREATIC MICROSOMES : AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, (~15 mµ), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mµ), limited by a thin membrane (7 mµ) which bears small (~15 mµ) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules (~150 mµ) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small (~15 mµ), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.