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Atsushi Maruyama - One of the best experts on this subject based on the ideXlab platform.
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nuclear localization and antisense effect of pna internalized by asgp r mediated endocytosis with protein dna conjugates
Journal of Controlled Release, 2011Co-Authors: Tsutomu Ishihara, Arihiro Kano, Kentaro Obara, Minako Saito, Xuesi Chen, Tae Gwan Park, Toshihiro Akaike, Atsushi MaruyamaAbstract:In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the Asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 degrees C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs. (C) 2010 Elsevier B.V. All rights reserved.
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Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates
Journal of Controlled Release, 2010Co-Authors: Tsutomu Ishihara, Arihiro Kano, Kentaro Obara, Minako Saito, Xuesi Chen, Tae Gwan Park, Toshihiro Akaike, Atsushi MaruyamaAbstract:In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the Asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 degrees C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs. (C) 2010 Elsevier B.V. All rights reserved.
Ravin Narain - One of the best experts on this subject based on the ideXlab platform.
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asialoglycoprotein receptor mediated gene delivery to hepatocytes using galactosylated polymers
Biomacromolecules, 2015Co-Authors: Bindu Thapa, Piyush Kumar, Hongbo Zeng, Ravin NarainAbstract:Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition–fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free Asialofetuin or by addi...
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Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers
2015Co-Authors: Bindu Thapa, Piyush Kumar, Hongbo Zeng, Ravin NarainAbstract:Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition–fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free Asialofetuin or by adding free Asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver
Sukhdev Singh Kamboj - One of the best experts on this subject based on the ideXlab platform.
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purification of colocasia esculenta lectin and determination of its anti insect potential towards bactrocera cucurbitae
Journal of Environmental Biology, 2013Co-Authors: Kshema Thakur, Amrit Pal Kaur, Manpreet Kaur, Sukhdev Singh Kamboj, Satwinder Kaur, Jatinder Vir SinghAbstract:The present study reports the purification of a lectin from Colocasia esculenta (L.) Schott corms and evaluation of its anti-insect potential towards Bactrocera cucurbitae (Coquilett). The lectin was found to be specific towards N-acetyl-D-lactosamine (LacNac), a disaccharide and Asialofetuin, a desialylated serum glycoprotein in hemagglutination inhibition assay. Asialofetuin was used as a ligand to purify Colocasia esculenta agglutinin (CEA) by affinity chromatography. The purity of CEA was ascertained by the presence of a single band in reducing SDS-PAGE at pH 8.3. The affinity purified CEA was employed in artificial diet bioassay of second instar larvae (64-72 hr old) of the B. cucurbitae at concentrations ranging between 10-160 microg ml(-1). The lectin significantly (p < 0.01) decreased the percent pupation and emergence with respect to control. Effect on various enzymes was studied by employing LC50 (51.6 microg ml(-1)) CEA in the artificial diet bioassay of second instar larvae. All the enzymes tested namely esterases, phosphatases (acid and alkaline), superoxide dismutases, catalase and glutathione-S-transferase showed a significant (p < 0.01, p < 0.05) increase in their enzyme and specific activities. These results showed that CEA affected normal growth and development and presented stress to the larvae, activating their detoxification and anti-oxidant systems. Thus, the lectin seems to be a useful candidate for the control measures of B. cucurbitae under the integrated pest management (IPM) system.
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isolation purification and characterization of an n acetyl d lactosamine binding mitogenic and anti proliferative lectin from tubers of a cobra lily arisaema utile schott
Advances in Bioscience and Biotechnology, 2010Co-Authors: Vikram Dhuna, Jatinder Singh, Ajit Kumar Saxena, Kshitija Dhuna, Satyam Kumar Agrawal, Sukhdev Singh KambojAbstract:Lectins are the carbohydrate-binding proteins of non-immune origin which have been the subject of intense investigation over the last few decades owing to the variety of interesting biological properties. Most of the lectins which have been purified and characterized from plants have been obtained from dicotyledons. In the present study a lectin was purified from tubers of a monocot plant Arisaema utile (AUL) Schott by affinity chromatography on Asialofetuin-linked amino activated silica beads. AUL gave a single band in SDS-PAGE at pH 8.3 corresponding to subunit Mr 13.5 kDa. The native molecular mass of AUL was 54 kDa suggesting a homotetrameric structure. AUL gave multiple bands in isoelectric focusing and in native PAGE at pH 8.3. AUL was inhibited by N-acetyl-D-lactosamine (Lac NAc), a disaccharide and Asialofetuin, a complex desialylated serum glycoprotein. When treated with denaturing agents, the lectin was stable in the presence of urea (3 M), thiourea (4 M) and guanidine HCl (4 M). AUL was a glycoprotein with a carbohydrate content of 1.2%. Complete loss of activity was observed upon modification of tryptophan residues of the lectin. The activity was reduced to 25% after modification of tyrosine. Chemical modification of arginine, histidine, serine and cysteine residues of AUL did not affect its activity. Using Far UV CD spectra the estimated secondary structure was 37% α-helix, 25% β-sheet and 38% random contributions. The lectin showed potent mitogenic response towards human lymphocytes. In vitro anti-proliferative assay using 11 human cancer cell lines resulted in 50% inhibition of six cell lines viz. SW-620, HCT-15, SK-N-SH, IMR-32, Colo-205 and HT-29 at 38, 42, 43, 49, 50 and 89 µg/ml, respectively.
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a tuber lectin from arisaema jacquemontii blume with anti insect and anti proliferative properties
Journal of Biochemistry and Molecular Biology, 2006Co-Authors: Manpreet Kaur, Sukhdev Singh Kamboj, Ajit Kumar Saxena, K J Singh, Pushpinder J Rup, M I D Sharma, Madhulika Bhagat, Sarvesh Kumar Sood, Jatinder SinghAbstract:A tuber lectin from Arisaema jacquemontii Blume belonging to family Araceae was purified by employing a single step affinity chromatography using column of Asialofetuin-linked amino activated silica beads and the bound lectin was eluted with 100 mM glycine-HCl buffer pH 2.5. The purified A. jacquemontii lectin (AJL) showed a single protein band with an apparent molecular mass of 13.4 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The native molecular mass of AJL determined by gel filtration on a Biogel P-200 column was 52 kDa and its carbohydrate content was estimated to be 3.40%. Thus AJL is a tetrameric glycoprotein. The purified lectin agglutinated erythrocytes from rabbit but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested except N-acetyl-D-lactosamine having minimal inhibitory sugar concentration (MIC) 25 mM. Among the glycoproteins tested only Asialofetuin was found to be inhibitory (MIC125 microg/mL). A single band was obtained in native PAGE at pH 4.5 while PAGE at pH 8.3 showed two bands. Isoelectric focusing of AJL gave multiple bands in the pI range of 4.6-5.5. When incorporated in artificial diet AJL significantly affected the development of Bactrocera cucurbitae (Coquillett) larvae indicating the possibility of using this lectin in a biotechnological strategy for insect management of cucurbits. Larvae fed on artificial diet containing sublethal dose of AJL showed a significant decrease in acid phosphatase and alkaline phosphatase activity while esterase activity markedly increased as compared to larvae fed on diet without lectin. Out of various human cancer cell lines employed in sulphorhodamine B (SRB) assay, this lectin was found to have appreciable inhibitory effect on the in vitro proliferation of HCT-15, HOP-62, SW-620, HT-29, IMR-32, SKOV-3, Colo-205, PC-3, HEP-2 and A-549 cancer cell lines by 82, 77, 73, 70, 41, 41, 37, 29, 21 and 21% respectively.
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isolation of a novel n acetyl d lactosamine specific lectin from alocasia cucullata schott
Biotechnology Letters, 2005Co-Authors: Amandeep Kaur, Jatinder Singh, Sukhdev Singh Kamboj, A Saxena, Vikram DhunaAbstract:An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on Asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca2+ and Mn2+ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.
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purification and characterization of a lectin from arisaema tortuosum schott having in vitro anticancer activity against human cancer cell lines
Journal of Biochemistry and Molecular Biology, 2005Co-Authors: Vikram Dhuna, Jatinder Singh, Jagmohan Singh Bains, Sukhdev Singh Kamboj, Shanmugavel Kamboj, Ajit Kumar SaxenaAbstract:A lectin with in-vitro anticancer activity against established human cancer cell lines has been purified by affinity chromatography on Asialofetuin-linked amino activated silica beads from the tubers of Arisaema tortuosum, popularly known as Himalayan Cobra lily, a monocot plant from the family Araceae. The bound Arisaema tortuosum lectin (ATL) was eluted with glycine-HCl buffer, pH 2.5. ATL was effectively inhibited by Asialofetuin, a complex desialylated serum glycoprotein as well as by N-acetyl-D-lactosamine, a disaccharide. It gave a single band corresponding to a subunit molecular weight of 13.5 kDa in SDS-PAGE, pH 8.8 both under reducing and non-reducing conditions. When subjected to gel-filtration on Biogel P-200, it was found to have a molecular weight of 54 kDa, suggesting a homotetramer structure, in which individual polypeptides are not bound to each other with disulfide bonds. ATL is a glycoprotein with 0.9 % carbohydrate content, stable up to 55(o)C and at pH 2 to 10. The lectin had no requirement for divalent metal ions i.e. Ca(2+) and Mn(2+) for its activity. However, as reported for other monocot lectins, ATL gave multiple bands in isoelectric focusing and Native PAGE, pH 8.3. The lectin was found to inhibit in vitro proliferation of human cancer cell lines HT29, SiHa and OVCAR-5.
Hansdieter Klenk - One of the best experts on this subject based on the ideXlab platform.
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The asialoglycoprotein receptor is a potential liver-specific receptor for
2015Co-Authors: Marburg Virus, Stephan Becker, Martin Spiess, Hansdieter KlenkAbstract:The liver is one of the main target organs of Marburg virus (MBG), a filovirus causing severe haemorrhagic fever with a high fatality rate in humans and non-human primates. MBG grown in certain cells does not contain neuraminic acid, but has terminal galactose on its surface glycoprotein. This observation i dicated that the asialoglycoprotein receptor (ASGP-R) of hepatocytes may serve as a receptor for MBG in the liver. Binding studies revealed that the attachment of MBG to ASGP-R-expressing HepG2 cells, but not to ASGP-R-negative E6 Vero cells, has the characteristics ofligand binding to the ASGP-R: binding is dependent on calcium and is inhibited by excess Asialofetuin and by anti-ASGP-R antiserum. Asialofetuin and the specific antiserum also inhibited MBG infection of HepG2 cells. In addition, it was shown that expression of ASGP-R cDNA in NIH 3T3 cells enhanced the susceptibility of these cells to MBG infection 4'5-fold. Interaction of MBG with the hepatic ASGP-R could thus explain the marked hepato-tropism of the virus
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the asialoglycoprotein receptor is a potential liver specific receptor for marburg virus
Journal of General Virology, 1995Co-Authors: Stephan Becker, Martin Spiess, Hansdieter KlenkAbstract:The liver is one of the main target organs of Marburg virus (MBG), a filovirus causing severe haemorrhagic fever with a high fatality rate in humans and non-human primates. MBG grown in certain cells does not contain neuraminic acid, but has terminal galactose on its surface glycoprotein. This observation indicated that the asialoglycoprotein receptor (ASGP-R) of hepatocytes may serve as a receptor for MBG in the liver. Binding studies revealed that the attachment of MBG to ASGP-R-expressing HepG2 cells, but not to ASGP-R-negative E6 Vero cells, has the characteristics of ligand binding to the ASGP-R: binding is dependent on calcium and is inhibited by excess Asialofetuin and by anti-ASGP-R antiserum. Asialofetuin and the specific antiserum also inhibited MBG infection of HepG2 cells. In addition, it was shown that expression of ASGP-R cDNA in NIH 3T3 cells enhanced the susceptibility of these cells to MBG infection 4·5-fold. Interaction of MBG with the hepatic ASGP-R could thus explain the marked hepatotropism of the virus.
Tsutomu Ishihara - One of the best experts on this subject based on the ideXlab platform.
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nuclear localization and antisense effect of pna internalized by asgp r mediated endocytosis with protein dna conjugates
Journal of Controlled Release, 2011Co-Authors: Tsutomu Ishihara, Arihiro Kano, Kentaro Obara, Minako Saito, Xuesi Chen, Tae Gwan Park, Toshihiro Akaike, Atsushi MaruyamaAbstract:In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the Asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 degrees C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs. (C) 2010 Elsevier B.V. All rights reserved.
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Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates
Journal of Controlled Release, 2010Co-Authors: Tsutomu Ishihara, Arihiro Kano, Kentaro Obara, Minako Saito, Xuesi Chen, Tae Gwan Park, Toshihiro Akaike, Atsushi MaruyamaAbstract:In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the Asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37 degrees C for at least 3 h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs. (C) 2010 Elsevier B.V. All rights reserved.
