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.
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.
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 (<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.
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.
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.
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.
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 (<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.
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