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81.
We have previously shown that pH-sensitive immunoliposomes can mediate a target-specific delivery of plasmid DNA to tumor cells grown in a mouse model [Wang, C.-Y., & Huang, L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7851-7855]. The efficiency of delivery in terms of the target cell transformation frequency has now been characterized for both short- and long-term gene expression in a tissue culture system. Herpes simplex virus thymidine kinase (TK) gene was used as a reporter gene. It was placed under the control of the promoter for the rat phosphoenolpyruvate carboxykinase gene, which contains a cAMP regulatory element. Therefore, the expression of the exogenous gene in the target cell, mouse Ltk- cells, can be regulated by cAMP drugs. The plasmid DNA was encapsulated in liposomes using a detergent dialysis method. The efficiency of gene delivery was optimized with respect to the time course and dose of liposome-associated DNA. The existence of antibody of the liposomes was essential for the maximal level of DNA delivery. Delivery was also dependent on the lipid composition of the liposome. The pH-sensitive lipid composition gave 8-fold higher efficiency than the corresponding pH-insensitive composition. The transformation efficiency of the target cell also depended on the regulation of gene expression; cells incubated with dibutyryl-cAMP and theophylline showed a much higher level of transformation frequency than cells incubated without the drugs. When all liposome and incubation parameters are optimized, the Ltk- cells showed a 47% efficiency for the short-term transformation, and 2% for the long-term transformation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
82.
Y Hashimoto K Chida M Huang M Katayama T Nishihira T Kuroki 《Biochemical and biophysical research communications》1989,163(1):406-411
The protein kinase C (PKC) activities of tumor tissue and adjacent normal mucosa of human cancers of the esophagus (8 cases), stomach (1 case) and colon (3 cases) were measured. Considerable variations were found in the activity of PKC and in its subcellular distribution in these cancers. The PKC activities of the membrane and cytosolic fractions of the eight esophageal cancers were, however, similar to those of the adjacent normal mucosa: the average PKC activities of the tumor tissues and normal mucosa were 7.5 and 8.3 pmol/min/mg protein, respectively, in their membrane fractions and 7.9 and 7.8 pmol/min/mg protein, respectively, in their cytosolic fractions. 相似文献
83.
Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets 总被引:9,自引:0,他引:9
Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus (T. stejnegeri formosensis) snake venom, inhibits platelet aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets (Ki = 2 X 10(-8) M) without affecting the platelet-release reaction. 125I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. 125I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS) [Huang et al. (1987) J. Biol. Chem. 262, 161]. We determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acid sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen alpha 1 (I) and laminin B1, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. Reduction of trigramin resulted in a marked decrease in its ability to block vWF binding to human platelets.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
84.
Immunoliposomes with different acid sensitivities as probes for the cellular endocytic pathway 总被引:1,自引:0,他引:1
By combining dioleoylphosphatidylethanolamine (DOPE) with oleic acid (OA), palmitoylhomocysteine (PHC) or dipalmitoylsuccinylglycerol (DPSG) we have prepared pH-sensitive liposomes with different acid sensitivities. DOPE/OA liposomes are the most acid sensitive, while DOPE/DPSG liposomes are the least acid sensitive. Incubation of DOPE/OA liposomes with mouse L929 cells reduces the pH-sensitivity of these liposomes by altering the lipid composition. Using diphtheria toxin fragment A as a marker for cytoplasmic delivery, we find that the delivery kinetics of pH-sensitive immunoliposomes closely correlates with the modified acid sensitivities of the liposomes. Immunoliposomes encounter pH 6-6.2 with a t1/2 of 5-15 min after internalization. By contrast, acidification of the endosomes to pH 5.0 takes longer (t1/2 approximately 25 min). We also used a whole cell null point technique (Yamishiro and Maxfield (1987) J. Cell Biol. 105, 2713-2721) to directly determine the average pH encountered by the endocytosed immunoliposomes. We find that acidification determined by the null point method proceeds less rapidly than that estimated from DTA delivery data. This is likely due to the fact that the measured DTA delivery is done by those liposomes which first arrive at the endosomes with sufficient acidity. Our data suggests that DOPE/PHC immunoliposomes deliver at the early endosome while DOPE/DPSG immunoliposomes deliver at the late endosomes. The DOPE/OA immunoliposomes, with the altered composition and acid sensitivity, deliver with a kinetics intermediate between the other two immunoliposomes. Thus, pH-sensitive liposomes represent useful probes for studying the kinetics of endosome acidification. 相似文献
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88.
P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay. 总被引:1,自引:0,他引:1
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Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments. 相似文献
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