B reast cancer is one of the most common female cancers worldwide and a major cause of cancer dea... more B reast cancer is one of the most common female cancers worldwide and a major cause of cancer death in women. Advances in the understanding of the molecular mechanisms underlying the malignant transformations of breast cancer cells has enabled the possible regulation of the expression of specific genes that are linked to breast cancer. Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting and diverse new therapeutic approach. This molecular tool holds great potential for the treatment of diseases that have until now been considered incurable. However, poor stability and insufficient cellular uptake have limited its usefulness. Therefore, this study focused on the delivery of siRNA to an upregulated breast cancer gene via the use of novel cationic liposomes (non-steric and steric stabilized) which have been synthesized and chemically analyzed. The cholesteryl cytofectin, N,N-dimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09) was synthesized from cholesterol chloroformate. Cationic liposomes were constructed from near equimolar quantities of MS09, dioleoylphosphatidylethanolamine (DOPE) and polyethylene glycol (PEG 2000) forming submicron stable unilamellar liposomes. Gel retardation, ethidium displacement and nuclease digestion assays confirmed that siRNA was fully liposome-associated, stable and protected from serum nucleases. Transfection activity of the cationic lipoplexes was determined using the luciferase reporter gene assay and cytotoxicity in vitro was evaluated using the cell viability (MTT) assay. Preliminary results suggest that these new cationic liposomal systems have an immense potential to be used for efficient delivery of siRNA therapeutics to bring about silencing of oncogenes associated with breast cancer. Biography Adrian Bivol holds an M.D. degree from the
A simple procedure has been developed for studying the transfer of DNA into cells using the proce... more A simple procedure has been developed for studying the transfer of DNA into cells using the process of receptor-mediated endocytosis. Poly-L-lysine 460 was covalently attached to the carbohydrate chains of avidin via periodate oxidation and NaBH4 reduction to give avidin-pLys460. Following purification through Sephacryl S-300, the conjugate was reacted with biotinylated transferrin. The resulting avidin-pLys460-[bio-transferrin] could be either fractionated by Superose 12 gel chromatography or used directly in experiments with DNA. Determination of the interaction between avidin-pLys460-]bio-transferrin] and DNA was carried out by nitro cellulose filter binding and agarose gel retardation assays. The conjugate was shown to bind DNA strongly, giving stable complexes soluble in 0.15-0.2 M salt solutions. Gene transfer using avidin-pLys460-[bio-transferrin] and the luciferase plasmid pRSVL was accomplished with Hela cells, alpha T3 pituitary cells and a human melanoma cell line used in the present study. Transfection was dependent on bio-transferrin and stimulated by the lysosomotropic agent chloroquine. The results are consistent with a receptor-mediated endocytosis mechanism of DNA delivery for Hela cells and a combination of receptor and adsorptive endocytosis for the alpha T3 pituitary and melanoma T-5 cell lines.
DOAJ (DOAJ: Directory of Open Access Journals), Jul 1, 2010
Effect of N-tritylated amino-adenosine-3',5'-cyclic monophosphate The effecT of cerTain N-TriTyla... more Effect of N-tritylated amino-adenosine-3',5'-cyclic monophosphate The effecT of cerTain N-TriTylaTed phenylalanine conjugaTes of amino-adenosine-3',5'-cyclic monophosphaTe on moloney murine leukaemia virus reverse TranscripTase acTiviTy
The beta-alanyl and L-histidyl analogues of puromycin were synthesized chemically and tested for ... more The beta-alanyl and L-histidyl analogues of puromycin were synthesized chemically and tested for their ability to release [3H] N-acetylphenylalanine from its tRNA carrier in the rat liver and E. Coli ribosomal systems. Both analogues were found to be inactive in releasing [3H] N-acetylphenylalanine. Reasons for the inactivity of these compounds are discussed in relation to the structure of the puromycin molecule and the requirements for puromycin-like activity.
Lipophilic N4-acetyl (1b-d) and N4-chloroacetyl (2b-d) derivatives of cytidine, 2'-deoxycytidine ... more Lipophilic N4-acetyl (1b-d) and N4-chloroacetyl (2b-d) derivatives of cytidine, 2'-deoxycytidine and cytosine arabinoside (ara-C) were synthesized and their toxicity for A(T1)Cl-3 hamster fibrosarcoma cells determined. 2b-d proved potent with no colonies surviving at concentrations of 10(-4), 10(-4) and 10(-6) M, respectively. lb-d showed comparatively poor cytotoxicity with 95, 77 and 87% survival of colonies respectively. N4-chloroacetyl 2'-deoxycytidine (2c) and N4-chloroacetyl ara-C (2d) were shown to undergo hydrolytic deprotection in phosphate buffered saline at 50 degrees C to yield the parent nucleosides (circa 85%) and the N3-carboxymethyl derivatives (5c,d) via 1-H-2,3 dihydro-2,5-dioxoimidazo [1,2-c] pyrimidine intermediates (4c,d). This treatment abolished the toxicity of 2c at 10(-4) M whilst the potency of 2d remained undiminished at 10(-6) M. These results indicate that further investigation of N(-4)-chloroacetyl-ara C (2d) as a potential pro-drug of ara-C is warranted.
The formation of transferrin-DNA complexes intended for l&and-directed transfection studies has b... more The formation of transferrin-DNA complexes intended for l&and-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT), obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'Gmidazolides which on incubation with transferrin yielded the S'linked phosphoramidates (pT),-5'-transferrin. Homopolymeric chain extension of (pT),-5'transferrin by terminal transferase and dTI'P at 30" for 30 min yielded (pT),,,-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37" was pronounced after 30min although at 25" hydrolysis was <5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37" suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.
1. Cyclohexylpuromycin, an analogue of puromycin in which a cyclohexane ring replaces the aromati... more 1. Cyclohexylpuromycin, an analogue of puromycin in which a cyclohexane ring replaces the aromatic benzene ring of the L-phenylalanyl moeity of the nucleoside, has been synthesized and examined for its ability to release N-acetylphenylalanine from tRNA attached to rat liver ribosomes. 2. DL-Cyclohexylpuromycin was active in reacting with N-f3H]acetylphenylalanyl-tRNA on rat liver ribosomes to form N-[3H]acetylphenylalanylcyclohexylpuromycin. 3. The reaction product N-acetylphenylalanylcyclohexylpuromycin and the corresponding analogue N-acetylphenylalanylpuromycin were chemically synthesized for evaluation of the structure of the released N-acetylphenylalanyl-containing material. 4. The results obtained suggest that the model of Raacke (1971) for puromycin reactivity needs further examination with regard to the role played by the aromatic ring system of the L-phenylalanyl moiety of the nucleoside. Experimental Chemicals and reagents Puromycin dihydrochloride, puromycin aminonucleoside, GTP (disocium salt) and benzyloxycarbonyl chloride were obtained from the Sigma Chemical Co., St. Louis, U.S.A. Phenylalanyl-tRNA and ATP (disodium salt) were obtained from C. F. Boehringer und Soehne G.m.b.H., Mannheim, Germany. Yeast tRNA was supplied by Schwarz BioResearch Inc., Orangeburg, N.Y., U.S.A. Triethylamine and t.l.c. plates (silca gel 60F254, with fluorescent indicator) were supplied by E. Merck
Advances in Natural Sciences: Nanoscience and Nanotechnology, Mar 1, 2022
Inorganic nanoparticles (NPs) have found extensive application in medicine and pharmaceutics. Alt... more Inorganic nanoparticles (NPs) have found extensive application in medicine and pharmaceutics. Although chemical synthesis of NPs is the most commonly employed technique, it is often associated with toxicities due to the nature of the precursors and the experimental conditions used. Hence, there is a need for a safer biosynthetic approach. The current study involves the green synthesis of silver (Ag) and selenium (Se) NPs using an aqueous Ocimum tenuiflorum inflorescence extract. Total phenol and HPLC-MS based phytochemical analysis of the extract was performed. NPs were analysed using UV-visible, and Fourier transform infrared (FTIR) spectroscopy, electron microscopy (EM), x-ray diffraction (XRD) and nanoparticle tracking analysis (NTA). Surface plasmon resonance bands at 433 nm and 285 nm confirmed the synthesis of the Ag and SeNPs, respectively. NPs were monodisperse, small (&lt;65 nm), with good stability and significant antioxidant activity. Cytotoxicity evaluated in the human embryonic kidney (HEK293), cervical carcinoma (HeLa) and breast adenocarcinoma (MCF-7) cells showed a dose-dependent trend with Se possessing better biocompatibility in the normal HEK293 cells than Ag. Density functional theory identified anthocyanins (delphinidin-5-O-beta-d-glucoside and delphinidin-3-O-glucoside) to have the most favourable NP-reducing and stabilising potential from the identified plant compounds.
Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to stre... more Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to streptavidin have been prepared and found to transfer luciferase plasmid DNA very efficiently to HeLa cells in the presence of chloroquine. Transfection was dependent on (i) use of biotinylated short chain polylysine containing 70 lysine residues, (ii) biotinylated transferrin containing 1-2 biotin moieties, (iii) reaction of biotinylated transferrin with streptavidin followed by isolation of the resulting conjugate on Sephadex G-200 and (iv) interaction of streptavidin-biotinylated transferrin with biotinylated polylysine giving a complex suitable for DNA transfection. It was found that if the above sequence of steps resulting in the formation of streptavidin-biotinylated transferrin/biotinylated polylysine was followed without isolation of intermediate conjugates by Sephadex G-200 chromatography, pRSVL DNA transfer was still very efficient. Transfer of luciferase DNA by the streptavidin conjugates and subsequent expression of luciferase activity was almost completely inhibited by excess free transferrin, showing that gene transfer was through the transferrin receptor pathway via receptor-mediated endocytosis. The streptavidin (bio2-transferrin) bio10-pLys70 conjugate used in the present experiments was approximately one hundred times more efficient in pRSVL DNA transfection with the HeLa cells than the previously described avidin-pLys460 (bio-transferrin) complex.
Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl... more Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl-N-(3-trimethylpropylammonium) carbodiimide iodide at a reagent: lipoprotein mole ratio of 10 000:1. This was shown to increase the innate DNA-binding capacity of LDL 10-fold. [ 125 I]-labeled carbodiimide-modified LDL ([ 125 I])-labeled ECDI-LDL) appeared to recognize the LDL receptor on normal human skin fibroblasts, although some nonspecific binding also was detected. To demonstrate the large ionic component in the lipoprotein-DNA interactions, ε-NH 2 amino groups on the apolipoprotein B-100 (apoB-100) component of LDL were acetylated with acetic anhydride. A nitrocellulose filter-binding assay revealed that acetylated LDL bound approximately 25% of the [ 3 H]-labeled pBR322 plasmid DNA bound by native LDL under the same conditions. ECDI-LDL-[ 3 H]-labeled plasmid DNA complexes were considerably more stable to NaCl challenge than complexes formed between [ 3 H]-labeled plasmid DNA and native LDL. Thus, the half dissociation of ECDI-LDL containing complexes was achieved at 0.28 M NaCl, whereas for LDL-plasmid DNA complexes this was reached at 0.18 M NaCl. Displacement studies with native LDL studies showed that ECDI-LDL-[ 3 H]-labeled plasmid DNA complexes retained the ability to recognize the LDL receptor on normal skin fibroblasts. Finally, ECDI-LDL complexes with pSV2CAT expression plasmid were shown to transfect CV-1 fibroblasts, a cell line known to specifically recognize apoB-liposome conjugates.
Evidence is presented for targeted gene delivery to HepG2 cells via the endocytotic pathway under... more Evidence is presented for targeted gene delivery to HepG2 cells via the endocytotic pathway under tl~e direction of insulin. Serum albumin was treated with the water-soluble carbodiimide N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and the resultant positively charged N-acylurea albumin covalently conjugated to insulin by glutaraldehyde cross-linkage. The conjugated protein is shown by nitrocellulose filter binding and gel band shift assays to bind DNA, and by competitive displacement of [t25I]insulin to bind to the insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro system of transfection, G418 resistant clones developed at frequencies of 2.0-5.5 x 10-5. Subsequently, a 923bp PstI fragment within the neo sequence was identified by Southern transfer in genomic DNA from transfected cell populations which had been maintained on a G418 regime for 44 days.
A CC linked uracil dinucleoside analogue of tunicaminyl uracil has been evaluated in HeLa cells, ... more A CC linked uracil dinucleoside analogue of tunicaminyl uracil has been evaluated in HeLa cells, the 'nick-translation' assay and the Klenow reaction. The title compound-1 a symmetrical uracil dinucleoside was prepared from the synthetic galactosyl dimer 6,6'-bis(lI2:3,4 di-0isopropylidene-6-deoxy-aD -galactopyranose) by deacetalation peracetylation, treatment with 2,4-(trimethylsilyl)uracil in the presence of stannic chloride and subsequent removal of acetyl groups. A preliminary screening of the dimer shows it to be weakly cytotoxic and a weak inhibitor of glucosamine incorporation (17% inhibition at 10-5M) in the HeLa cell system. inhibitor of DNA replication (25% at 10-5M) and RNA biosynthesis
It has been observed that various glutaraldehyde linked conjugates of insulin and bovine serum al... more It has been observed that various glutaraldehyde linked conjugates of insulin and bovine serum albumin display enhanced binding to the insulin receptor. High molecular weight covalent complexes resulting from the conjugation of insulin to both N-acylurea albumin and unmodified albumin have been shown to displace (l*sl-Tyr*14) human insulin from HepG2 cell receptors successfully. Results indicate that their affinity for the insulin receptor is greater than that of insulin itself by an order of magnitude. The relationship between these results and similar insulin binding phenomena reported by other workers ('super-activity') is examined, particularly with regard to possible alterations to the insulin binding site brought about by the respective coinjugation procedures. It is suggested that modifications to the B29 lysine residue might play a crucial role in stabilising the interaction of conjugated insulin with its cognate receptor. The powerful potential of insulin to act as carrier in the intracellular delivery of drugs and other molecules is discussed.
Following ligand binding a number of cell-surface receptors become phosphorylated at tyrosine res... more Following ligand binding a number of cell-surface receptors become phosphorylated at tyrosine residues of their cytosolic domains. These phosphorylations are associated with initiation of a signalling programme involving a sequence of tyrosine-phosphorylated protein-protein interactions. In the recognition process between phosphorylated proteins, electrostatic interactions between negatively charged phosphorylated tyrosines, serine and threonine residues and positively charged lysines play an important role as well as hydrophobic and H-bonding reactions. We suggest in this paper that the fairly high-energy phosphate bond of certain protein phosphorylated tyrosines are possibly involved in inducing transitory protein cross-linking reactions. Through a process involving transfer of an activated phosphate of phosphorylated tyrosine to a side-chain carboxyl group of the receptor or next protein of the signalling sequence, an acyl phosphate is formed. This then acylates a hydroxyl group on a serine, threonine or tyrosine residue of the protein not carrying the carboxyl phosphate to give an ester linkage, thus cross-linking the two proteins of the signalling pathway. The covalent ester linkage is labile to hydrolysis and depending on the protein-protein molecular environment it might have a finite half-life. On hydrolysis, the transitory covalent linkage is broken with separation of the proteins. It is suggested therefore that formation of a protein-protein ester linkage introduces a type of timing device into the system. Breakdown of the original protein-phosphorylated tyrosine in this case therefore does not involve a phosphatase enzyme.
The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly ... more The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly evolving area of study, which may find application in corrective gene therapy for the treatment of neoplasms and other disorders of a genetic origin. Here, a step-wise account of the assembly and characterization of hepatocellular carcinoma cell-specific DNA lipoplexes and their cytotoxicity assessment in and delivery to the human hepatocellular carcinoma cell line HepG2 is given.
B reast cancer is one of the most common female cancers worldwide and a major cause of cancer dea... more B reast cancer is one of the most common female cancers worldwide and a major cause of cancer death in women. Advances in the understanding of the molecular mechanisms underlying the malignant transformations of breast cancer cells has enabled the possible regulation of the expression of specific genes that are linked to breast cancer. Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting and diverse new therapeutic approach. This molecular tool holds great potential for the treatment of diseases that have until now been considered incurable. However, poor stability and insufficient cellular uptake have limited its usefulness. Therefore, this study focused on the delivery of siRNA to an upregulated breast cancer gene via the use of novel cationic liposomes (non-steric and steric stabilized) which have been synthesized and chemically analyzed. The cholesteryl cytofectin, N,N-dimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09) was synthesized from cholesterol chloroformate. Cationic liposomes were constructed from near equimolar quantities of MS09, dioleoylphosphatidylethanolamine (DOPE) and polyethylene glycol (PEG 2000) forming submicron stable unilamellar liposomes. Gel retardation, ethidium displacement and nuclease digestion assays confirmed that siRNA was fully liposome-associated, stable and protected from serum nucleases. Transfection activity of the cationic lipoplexes was determined using the luciferase reporter gene assay and cytotoxicity in vitro was evaluated using the cell viability (MTT) assay. Preliminary results suggest that these new cationic liposomal systems have an immense potential to be used for efficient delivery of siRNA therapeutics to bring about silencing of oncogenes associated with breast cancer. Biography Adrian Bivol holds an M.D. degree from the
A simple procedure has been developed for studying the transfer of DNA into cells using the proce... more A simple procedure has been developed for studying the transfer of DNA into cells using the process of receptor-mediated endocytosis. Poly-L-lysine 460 was covalently attached to the carbohydrate chains of avidin via periodate oxidation and NaBH4 reduction to give avidin-pLys460. Following purification through Sephacryl S-300, the conjugate was reacted with biotinylated transferrin. The resulting avidin-pLys460-[bio-transferrin] could be either fractionated by Superose 12 gel chromatography or used directly in experiments with DNA. Determination of the interaction between avidin-pLys460-]bio-transferrin] and DNA was carried out by nitro cellulose filter binding and agarose gel retardation assays. The conjugate was shown to bind DNA strongly, giving stable complexes soluble in 0.15-0.2 M salt solutions. Gene transfer using avidin-pLys460-[bio-transferrin] and the luciferase plasmid pRSVL was accomplished with Hela cells, alpha T3 pituitary cells and a human melanoma cell line used in the present study. Transfection was dependent on bio-transferrin and stimulated by the lysosomotropic agent chloroquine. The results are consistent with a receptor-mediated endocytosis mechanism of DNA delivery for Hela cells and a combination of receptor and adsorptive endocytosis for the alpha T3 pituitary and melanoma T-5 cell lines.
DOAJ (DOAJ: Directory of Open Access Journals), Jul 1, 2010
Effect of N-tritylated amino-adenosine-3',5'-cyclic monophosphate The effecT of cerTain N-TriTyla... more Effect of N-tritylated amino-adenosine-3',5'-cyclic monophosphate The effecT of cerTain N-TriTylaTed phenylalanine conjugaTes of amino-adenosine-3',5'-cyclic monophosphaTe on moloney murine leukaemia virus reverse TranscripTase acTiviTy
The beta-alanyl and L-histidyl analogues of puromycin were synthesized chemically and tested for ... more The beta-alanyl and L-histidyl analogues of puromycin were synthesized chemically and tested for their ability to release [3H] N-acetylphenylalanine from its tRNA carrier in the rat liver and E. Coli ribosomal systems. Both analogues were found to be inactive in releasing [3H] N-acetylphenylalanine. Reasons for the inactivity of these compounds are discussed in relation to the structure of the puromycin molecule and the requirements for puromycin-like activity.
Lipophilic N4-acetyl (1b-d) and N4-chloroacetyl (2b-d) derivatives of cytidine, 2'-deoxycytidine ... more Lipophilic N4-acetyl (1b-d) and N4-chloroacetyl (2b-d) derivatives of cytidine, 2'-deoxycytidine and cytosine arabinoside (ara-C) were synthesized and their toxicity for A(T1)Cl-3 hamster fibrosarcoma cells determined. 2b-d proved potent with no colonies surviving at concentrations of 10(-4), 10(-4) and 10(-6) M, respectively. lb-d showed comparatively poor cytotoxicity with 95, 77 and 87% survival of colonies respectively. N4-chloroacetyl 2'-deoxycytidine (2c) and N4-chloroacetyl ara-C (2d) were shown to undergo hydrolytic deprotection in phosphate buffered saline at 50 degrees C to yield the parent nucleosides (circa 85%) and the N3-carboxymethyl derivatives (5c,d) via 1-H-2,3 dihydro-2,5-dioxoimidazo [1,2-c] pyrimidine intermediates (4c,d). This treatment abolished the toxicity of 2c at 10(-4) M whilst the potency of 2d remained undiminished at 10(-6) M. These results indicate that further investigation of N(-4)-chloroacetyl-ara C (2d) as a potential pro-drug of ara-C is warranted.
The formation of transferrin-DNA complexes intended for l&and-directed transfection studies has b... more The formation of transferrin-DNA complexes intended for l&and-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT), obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'Gmidazolides which on incubation with transferrin yielded the S'linked phosphoramidates (pT),-5'-transferrin. Homopolymeric chain extension of (pT),-5'transferrin by terminal transferase and dTI'P at 30" for 30 min yielded (pT),,,-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37" was pronounced after 30min although at 25" hydrolysis was <5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37" suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.
1. Cyclohexylpuromycin, an analogue of puromycin in which a cyclohexane ring replaces the aromati... more 1. Cyclohexylpuromycin, an analogue of puromycin in which a cyclohexane ring replaces the aromatic benzene ring of the L-phenylalanyl moeity of the nucleoside, has been synthesized and examined for its ability to release N-acetylphenylalanine from tRNA attached to rat liver ribosomes. 2. DL-Cyclohexylpuromycin was active in reacting with N-f3H]acetylphenylalanyl-tRNA on rat liver ribosomes to form N-[3H]acetylphenylalanylcyclohexylpuromycin. 3. The reaction product N-acetylphenylalanylcyclohexylpuromycin and the corresponding analogue N-acetylphenylalanylpuromycin were chemically synthesized for evaluation of the structure of the released N-acetylphenylalanyl-containing material. 4. The results obtained suggest that the model of Raacke (1971) for puromycin reactivity needs further examination with regard to the role played by the aromatic ring system of the L-phenylalanyl moiety of the nucleoside. Experimental Chemicals and reagents Puromycin dihydrochloride, puromycin aminonucleoside, GTP (disocium salt) and benzyloxycarbonyl chloride were obtained from the Sigma Chemical Co., St. Louis, U.S.A. Phenylalanyl-tRNA and ATP (disodium salt) were obtained from C. F. Boehringer und Soehne G.m.b.H., Mannheim, Germany. Yeast tRNA was supplied by Schwarz BioResearch Inc., Orangeburg, N.Y., U.S.A. Triethylamine and t.l.c. plates (silca gel 60F254, with fluorescent indicator) were supplied by E. Merck
Advances in Natural Sciences: Nanoscience and Nanotechnology, Mar 1, 2022
Inorganic nanoparticles (NPs) have found extensive application in medicine and pharmaceutics. Alt... more Inorganic nanoparticles (NPs) have found extensive application in medicine and pharmaceutics. Although chemical synthesis of NPs is the most commonly employed technique, it is often associated with toxicities due to the nature of the precursors and the experimental conditions used. Hence, there is a need for a safer biosynthetic approach. The current study involves the green synthesis of silver (Ag) and selenium (Se) NPs using an aqueous Ocimum tenuiflorum inflorescence extract. Total phenol and HPLC-MS based phytochemical analysis of the extract was performed. NPs were analysed using UV-visible, and Fourier transform infrared (FTIR) spectroscopy, electron microscopy (EM), x-ray diffraction (XRD) and nanoparticle tracking analysis (NTA). Surface plasmon resonance bands at 433 nm and 285 nm confirmed the synthesis of the Ag and SeNPs, respectively. NPs were monodisperse, small (&lt;65 nm), with good stability and significant antioxidant activity. Cytotoxicity evaluated in the human embryonic kidney (HEK293), cervical carcinoma (HeLa) and breast adenocarcinoma (MCF-7) cells showed a dose-dependent trend with Se possessing better biocompatibility in the normal HEK293 cells than Ag. Density functional theory identified anthocyanins (delphinidin-5-O-beta-d-glucoside and delphinidin-3-O-glucoside) to have the most favourable NP-reducing and stabilising potential from the identified plant compounds.
Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to stre... more Conjugates consisting of biotinylated transferrin and biotinylated poly-L-lysine attached to streptavidin have been prepared and found to transfer luciferase plasmid DNA very efficiently to HeLa cells in the presence of chloroquine. Transfection was dependent on (i) use of biotinylated short chain polylysine containing 70 lysine residues, (ii) biotinylated transferrin containing 1-2 biotin moieties, (iii) reaction of biotinylated transferrin with streptavidin followed by isolation of the resulting conjugate on Sephadex G-200 and (iv) interaction of streptavidin-biotinylated transferrin with biotinylated polylysine giving a complex suitable for DNA transfection. It was found that if the above sequence of steps resulting in the formation of streptavidin-biotinylated transferrin/biotinylated polylysine was followed without isolation of intermediate conjugates by Sephadex G-200 chromatography, pRSVL DNA transfer was still very efficient. Transfer of luciferase DNA by the streptavidin conjugates and subsequent expression of luciferase activity was almost completely inhibited by excess free transferrin, showing that gene transfer was through the transferrin receptor pathway via receptor-mediated endocytosis. The streptavidin (bio2-transferrin) bio10-pLys70 conjugate used in the present experiments was approximately one hundred times more efficient in pRSVL DNA transfection with the HeLa cells than the previously described avidin-pLys460 (bio-transferrin) complex.
Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl... more Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl-N-(3-trimethylpropylammonium) carbodiimide iodide at a reagent: lipoprotein mole ratio of 10 000:1. This was shown to increase the innate DNA-binding capacity of LDL 10-fold. [ 125 I]-labeled carbodiimide-modified LDL ([ 125 I])-labeled ECDI-LDL) appeared to recognize the LDL receptor on normal human skin fibroblasts, although some nonspecific binding also was detected. To demonstrate the large ionic component in the lipoprotein-DNA interactions, ε-NH 2 amino groups on the apolipoprotein B-100 (apoB-100) component of LDL were acetylated with acetic anhydride. A nitrocellulose filter-binding assay revealed that acetylated LDL bound approximately 25% of the [ 3 H]-labeled pBR322 plasmid DNA bound by native LDL under the same conditions. ECDI-LDL-[ 3 H]-labeled plasmid DNA complexes were considerably more stable to NaCl challenge than complexes formed between [ 3 H]-labeled plasmid DNA and native LDL. Thus, the half dissociation of ECDI-LDL containing complexes was achieved at 0.28 M NaCl, whereas for LDL-plasmid DNA complexes this was reached at 0.18 M NaCl. Displacement studies with native LDL studies showed that ECDI-LDL-[ 3 H]-labeled plasmid DNA complexes retained the ability to recognize the LDL receptor on normal skin fibroblasts. Finally, ECDI-LDL complexes with pSV2CAT expression plasmid were shown to transfect CV-1 fibroblasts, a cell line known to specifically recognize apoB-liposome conjugates.
Evidence is presented for targeted gene delivery to HepG2 cells via the endocytotic pathway under... more Evidence is presented for targeted gene delivery to HepG2 cells via the endocytotic pathway under tl~e direction of insulin. Serum albumin was treated with the water-soluble carbodiimide N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and the resultant positively charged N-acylurea albumin covalently conjugated to insulin by glutaraldehyde cross-linkage. The conjugated protein is shown by nitrocellulose filter binding and gel band shift assays to bind DNA, and by competitive displacement of [t25I]insulin to bind to the insulin receptor. When the expression vectors ptkNEO and pAL-8 which incorporate the neo gene were complexed to the conjugate in an in vitro system of transfection, G418 resistant clones developed at frequencies of 2.0-5.5 x 10-5. Subsequently, a 923bp PstI fragment within the neo sequence was identified by Southern transfer in genomic DNA from transfected cell populations which had been maintained on a G418 regime for 44 days.
A CC linked uracil dinucleoside analogue of tunicaminyl uracil has been evaluated in HeLa cells, ... more A CC linked uracil dinucleoside analogue of tunicaminyl uracil has been evaluated in HeLa cells, the 'nick-translation' assay and the Klenow reaction. The title compound-1 a symmetrical uracil dinucleoside was prepared from the synthetic galactosyl dimer 6,6'-bis(lI2:3,4 di-0isopropylidene-6-deoxy-aD -galactopyranose) by deacetalation peracetylation, treatment with 2,4-(trimethylsilyl)uracil in the presence of stannic chloride and subsequent removal of acetyl groups. A preliminary screening of the dimer shows it to be weakly cytotoxic and a weak inhibitor of glucosamine incorporation (17% inhibition at 10-5M) in the HeLa cell system. inhibitor of DNA replication (25% at 10-5M) and RNA biosynthesis
It has been observed that various glutaraldehyde linked conjugates of insulin and bovine serum al... more It has been observed that various glutaraldehyde linked conjugates of insulin and bovine serum albumin display enhanced binding to the insulin receptor. High molecular weight covalent complexes resulting from the conjugation of insulin to both N-acylurea albumin and unmodified albumin have been shown to displace (l*sl-Tyr*14) human insulin from HepG2 cell receptors successfully. Results indicate that their affinity for the insulin receptor is greater than that of insulin itself by an order of magnitude. The relationship between these results and similar insulin binding phenomena reported by other workers ('super-activity') is examined, particularly with regard to possible alterations to the insulin binding site brought about by the respective coinjugation procedures. It is suggested that modifications to the B29 lysine residue might play a crucial role in stabilising the interaction of conjugated insulin with its cognate receptor. The powerful potential of insulin to act as carrier in the intracellular delivery of drugs and other molecules is discussed.
Following ligand binding a number of cell-surface receptors become phosphorylated at tyrosine res... more Following ligand binding a number of cell-surface receptors become phosphorylated at tyrosine residues of their cytosolic domains. These phosphorylations are associated with initiation of a signalling programme involving a sequence of tyrosine-phosphorylated protein-protein interactions. In the recognition process between phosphorylated proteins, electrostatic interactions between negatively charged phosphorylated tyrosines, serine and threonine residues and positively charged lysines play an important role as well as hydrophobic and H-bonding reactions. We suggest in this paper that the fairly high-energy phosphate bond of certain protein phosphorylated tyrosines are possibly involved in inducing transitory protein cross-linking reactions. Through a process involving transfer of an activated phosphate of phosphorylated tyrosine to a side-chain carboxyl group of the receptor or next protein of the signalling sequence, an acyl phosphate is formed. This then acylates a hydroxyl group on a serine, threonine or tyrosine residue of the protein not carrying the carboxyl phosphate to give an ester linkage, thus cross-linking the two proteins of the signalling pathway. The covalent ester linkage is labile to hydrolysis and depending on the protein-protein molecular environment it might have a finite half-life. On hydrolysis, the transitory covalent linkage is broken with separation of the proteins. It is suggested therefore that formation of a protein-protein ester linkage introduces a type of timing device into the system. Breakdown of the original protein-phosphorylated tyrosine in this case therefore does not involve a phosphatase enzyme.
The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly ... more The application of homing peptides to direct DNA and RNA lipoplexes to target cells is a rapidly evolving area of study, which may find application in corrective gene therapy for the treatment of neoplasms and other disorders of a genetic origin. Here, a step-wise account of the assembly and characterization of hepatocellular carcinoma cell-specific DNA lipoplexes and their cytotoxicity assessment in and delivery to the human hepatocellular carcinoma cell line HepG2 is given.
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Papers by Mario Ariatti