Fabrication of tumor-targeting composites based on the triple helical ß-glucan through conjugation of aptamer.

Herein the nucleic acid aptamers were attached to the polydeoxyadenylic acid (poly(dA)) tail for improving the tumor-targetability and cellular internalization of s-LNT/poly(dA) composite composed of two single chains of triple helical ß-glucan lentinan (s-LNT) and one poly(dA) chain. The in vitro results demonstrate that the cellular uptake of s-LNT/poly(dA) composites in MCF-7 cancer cells was enhanced effectively after attaching the aptamer. The as-prepared fluorescin isothiocyanate (FITC)-labelled LNT (LNT-FITC) through grafting was used for tracing the enhanced tumor-targetability of the composites. As a result, the cellular internalization of the LNT-FITC into MCF-7 and 4T1 cancer cells was further increased by the aptamer conjugated to poly(dA). Meanwhile, the in vivo experiments further demonstrate more s-LNT/poly(dA)-aptamer composites were effectively accumulated at the tumor site compared with s-LNT alone. This work provides a novel strategy for fabricating triplex ß-glucan as delivery vectors with active tumor-targetability.

Microinjection free delivery of miRNA inhibitor into zygotes.

The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.

Scavenger receptor block as strategy for the identification of bone marrow homing phages by panning in vivo random peptide phage displayed libraries.

Surface molecules exclusively expressed by cells of the bone marrow (BM) are candidate targets for delivering therapeutic agents to this tissue. To identify ligands specific for the BM, we performed a series of pannings in vivo with random peptide phage displayed libraries (RPPDL). We could show that phages bind to bone marrow endothelium (BME) independently of the peptide insert, suggesting that the BM, similarly to spleen and liver, is part of the reticulo-endothelial system (RES). Furthermore, this strong "natural" affinity to the BME was abrogated by polyanions, indicating that phage trapping by this endothelium is mediated by scavenger receptors (SR). To circumvent interference by SR, polyinosinic acid was administered before phage panning in vivo. This led to the identification of a consensus motif that confers binding specificity for a subpopulation of hemopoietic marrow cells. Thus, SR inhibition, by avoiding phage trapping by the endothelium, seems to allow phage particles to extravasate and reach parenchymal cells. Accordingly, this panning strategy in vivo may be useful for the identification of targeting motifs specific for cells located in the extravascular space of various tissues.

Expression of the choroid plexus sodium-nucleoside cotransporter (N3) in Xenopus laevis oocytes.

In this study, we determined the expression of the Na(+)-nucleoside cotransporter (N3) in Xenopus laevis oocytes injected with poly(A)+ RNA isolated from rabbit choroid plexus. The Na(+)-dependent thymidine uptake in poly(A)+ RNA-injected oocytes (maximum 4-5 days after injection) increased proportionally with the injected dose of poly(A)+ RNA. Uptake was enhanced 4- to 5-fold in oocytes injected with 40 ng poly (A)+ RNA in comparison to water-injected oocytes. Consistent with the N3 Na(+)-nucleoside cotransporter, Na(+)-dependent thymidine uptake in poly(A)+ RNA-injected oocytes was inhibited significantly by both purine and pyrimidine nucleosides, but not by dideoxycytidine, a nucleoside analog modified on the ribose ring. These data suggest for the first time that the N3 Na(+)-nucleoside cotransporter in rabbit choroid plexus can be expressed in X. laevis oocytes.

Conjugated bile acid uptake by Xenopus laevis oocytes induced by microinjection with ileal Poly A+ mRNA.

Apical membranes of ileal enterocytes contain the major Na+/bile acid cotransporter activity in mammals. Microinjection of guinea pig ileal mucosal Poly A+ mRNA (25 ng) into Xenopus oocytes resulted in 22,23-3H-cholyltaurine uptake at day 3 after injection (453 fmol/oocyte-hr), while control viral mRNA (25 ng) gave an uptake rate of 133 fmol/oocyte-hr. The transport rate increased in direct relationship to the concentration of injected mRNA, cholyltaurine, or Na+ in the incubation media. Uptake of cholyltaurine using rabbit ileal mucosal Poly A+ mRNA was 3891 fmole/oocyte-hr compared to rabbit jejunal-mucosa Poly A+ mRNA (control) injections inducing 728 fmol/oocyte-hr. Such expression of the ileal Na+/bile acid cotransporter may facilitate cloning of this key mammalian gene.

Effects of general anesthetics and pressure on mammalian excitatory receptors expressed in Xenopus oocytes.

The effects of general anesthetics and pressure on receptors from the mammalian central nervous system have been investigated using oocyte expression techniques. Poly A+ mRNA extracted from rat whole brain was injected into mature Xenopus oocytes producing depolarizing responses to the fast excitatory neurotransmitters NMDA and kainate and the inhibitory neurotransmitters GABA and glycine. An apparatus was constructed to allow agonist dose-response curves to be determined at high pressures using voltage-clamped oocytes. This was used to investigate the excitatory transmitter kainate. It was found that anesthetics depress the current induced by kainate whereas pressure does not appear to affect the responses associated with this transmitter. Furthermore it was found that pressure does not reverse (or modify in any way) the changes in response brought about by application of anesthetics.

Reprogramming of the transcriptional machinery in Xenopus oocytes by injection of mouse poly(A)+ RNA.

We have devised an assay for species-specific transcription factors for the mouse rRNA gene by exploiting the ability of Xenopus oocytes to transcribe injected DNA and translate mRNAs. When mouse rRNA genes are microinjected into Xenopus oocytes, they are not transcribed. We show here that transcription of mouse rRNA genes is supported when mouse mRNAs are injected before the transcription template is injected, indicating that the necessary transcription factors are translated in the oocyte and are available to transcribe an appropriate template. The use of this assay in cloning genes for transcription factors is discussed.

Expression of thermotolerance following microinjection of poly(A)RNA isolated from thermotolerant CHO cells.

Poly(A)RNA was isolated from thermotolerant cells and microinjected into recipient non-tolerant Chinese hamster ovary (CHO) cells. The injected cells expressed thermotolerance to a subsequent test heat treatment both in terms of the end-points of colony formation (cell survival) and resumption of protein synthesis after test heating (translational labelling). The magnitude of thermotolerance expression was dependent on the experimental end-point (increase up to 3.8-fold for translational labelling and approximately 2-fold for survival) and on the time between microinjection and the test heat treatment. Control experiments showed that poly(A)RNA from non-tolerant cells did not alter the heat response of microinjected cells. Proteins corresponding to the poly(A)RNA from thermotolerant cells were analysed by in vitro translation and by labelling of microinjected cells, followed by SDS-PAGE. In vitro translations showed high levels of transcripts for classical heat-shock proteins (HSP 70/72, 89, 110) in poly(A)RNA from thermotolerant versus control cells. However, proteins synthesized in intact cells showed no detectable differences when cells were microinjected with poly(A)RNA from thermotolerant versus control cells, or not injected at all. In principle the data show that microinjection of specific poly(A)RNA fractions can be used for defining the contribution of individual gene products to the cellular heat response.

Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes.

In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mI15) receptor. When injected with sucrose gradient fractionated polyA+ RNA derived from the murine 115-dependent pre B cell line B13, these oocytes could specifically bind 35S-methionine labeled mI15. A size of approximately 4000 nucleotides (25S) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the MI15-receptor, suggesting that the oocytes express a different form of this receptor.

Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected with rat and chick brain RNA.

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

Activation by serotonin of starfish eggs expressing the rat serotonin 1c receptor.

Starfish oocytes were injected with mRNA for the serotonin 1c receptor or with rat brain poly A+ mRNA, incubated to allow expression of the membrane protein, then matured to eggs by addition of 1-methyladenine. Applying serotonin to these eggs caused cortical granule exocytosis like that occurring at fertilization. Because the serotonin 1c receptor specifically activates a G-protein, these results provide support for the hypothesis that sperm activate eggs by way of a receptor-G-protein interaction. The starfish oocyte may be a generally useful system for expression of exogenous mRNA for membrane proteins.

A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel.

Sodium channels encoded by the rat IIA cDNA clone [Auld, V. J., Goldin, A. L., Krafte, D. S., Marshall, J., Dunn, J., Catterall, W. A., Lester, H. A., Davidson, N. & Dunn, R. J. (1988) Neuron 1, 449-461] differ at seven amino acid residues from those encoded by the rat II cDNA [Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H. & Numa, S. (1986) Nature (London) 320, 188-192]. When expressed in Xenopus oocytes, rat IIA channels display a current-voltage relationship that is shifted 20-25 mV in the depolarizing direction relative to channels expressed from rat II cDNA or rat brain poly(A)+ mRNA. By modifying each variant residue in rat IIA to the corresponding residue in rat II, we demonstrate that a single Phe----Leu substitution at position 860 in the S4 segment of domain II is sufficient to shift the current-voltage relationship to that observed for channels expressed from rat brain poly(A)+ RNA or rat II cDNA. Rat genomic DNA encodes leucine but not phenylalanine at position 860, indicating that the phenylalanine at this position in rat IIA cDNA likely results from reverse transcriptase error.

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone.

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.

Taurine and beta-alanine act on both GABA and glycine receptors in Xenopus oocyte injected with mouse brain messenger RNA.

The responding pathway (process from agonist binding to channel opening) of taurine and beta-alanine was investigated in Xenopus oocytes injected with mouse brain poly(A)+ RNA. Responses to gamma-aminobutyric acid (GABA), glycine, taurine and beta-alanine were induced in oocytes injected with poly(A)+ RNA extracted from 3 regions, cerebrum, cerebellum and brainstem of the mouse brain. From comparison, responses to these 4 inhibitory amino acids in each regional poly(A)+ RNA-injected oocytes were categorized into at least 3 groups: (1) GABA, (2) glycine, and (3) taurine and beta-alanine. No cross-desensitization was observed between GABA response and glycine response, but taurine and beta-alanine responses cross-desensitized both the GABA and glycine responses. Taurine and beta-alanine responses were partially inhibited by the GABA antagonist, bicuculline, and also by the glycine antagonist, strychnine. The results suggest that the taurine or the beta-alanine response in the brain is caused through both the GABA receptor and the glycine receptor.

Synthesis of biologically active fibroblast-activating factor (FAF) by xenopus oocytes injected with T lymphocyte mRNA.

Activation of human peripheral blood T lymphocytes results in the production of fibroblast-activating factor (FAF), a mediator which stimulates fibroblast proliferation. This lymphokine, which may provide a molecular link between cell-mediated immune reactions and fibroplasia, has been identified as a T-cell product both in vitro and in vivo. In order to study the mechanisms of synthesis and activity of FAF, poly(A) RNA was isolated from concanavalin A-stimulated T lymphocytes and injected into Xenopus oocytes. The injected oocytes translated the messenger RNA and produced a material with the biological and biochemical properties of human FAF. The oocyte product induced proliferation in serum-free quiescent fibroblast monolayers and exhibited the same molecular weight and charge as the T-cell-derived factor. Oocytes injected with poly(A)-RNA from unstimulated T lymphocytes produced little, if any, FAF activity. We conclude that activation of T lymphocytes enhances transcription of FAF mRNA as detected in the oocyte translation assay. This translated material has biological activity and biochemical characteristics consistent with FAF and is suitable for further studies on the expression and synthesis of FAF (poly)peptides.

Translation of mRNA for human lymphotoxin in microinjected Xenopus oocytes.

Synthesis and secretion of biologically active human lymphotoxin (LT) can be detected in Xenopus laevis oocytes following their inoculation with poly(A+) RNA from human stimulated peripheral blood lymphocytes, but not in oocytes inoculated with RNA from unstimulated lymphocytes or from fibroblastoid cells. In size-fractionating mRNA of stimulated lymphocytes most LT activity is found to be coded for by RNA with an approximate sedimentation value of 19 S.

Continued studies on the adjuvancy effect of natural and synthetic double-stranded RNA preparations with inactivated Newcastle disease vaccines in fowls.

The adjuvant effect of the natural ds-RNA, BRL 5907, with inactivated Newcastle disease vaccines was confirmed using different oil-based formulations and could be given either as single injections or separately at adjacent sites. A minimum dose level of 0-04 mg BRL 5907 per bird was required for a significant enhancement of antibody levels following vaccination. While the synthetic ds-RNA Poly IC gave a similar response to that observed with BRL 5907, no significant effects could be detected with two other natural ds-RNA materials.

Studies on hypoxia: XII. Detrimental effects of synthetic polyribonucleotides on epiphyseal plates of rats exposed to hypoxia.

The effect of different doses of polyadenylic and polyuridylic acids (poly A:U) was studied in control rats and in rats exposed to hypoxia. In the control rats, administration of different doses of poly A:U did not change the thickness of the epiphyseal plate or increase the incorporation of 3H-phenylalanine as judged using radioautography. Rats exposed to hypoxia showed a significant dose-related reduction in the thickness of the epiphyseal plate and 3H-phenylalanine incorporation.