Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Isolation, purification and chemical composition of insoluble collagen from Guerin epithelioma.

1. The insoluble collagen from Guerin epithelioma was isolated and its chemical composition was determined. The unusually high histidine content is accompanied in tumour collagen by a relatively small amount of lysine and arginine. 2. The isolated protein was strongly bound to glycoprotein, which could not be removed by EDTA treatment unless this procedure was preceded by digestion of the complex with trypsin.     

Purification and properties of the heterogeneous subunits of elongation factor EF-1 from Guerin epithelioma cells.

Elongation factor EF-1 from Guerin epithelioma was separated into two subunit forms EF-1A and EF-1B by chromatography in the presence of 25% glycerol, successively on CM-Sephadex and DEAE-Sephadex. It was shown that EF-1A is a thermolabile, single polypeptide which catalyses the binding of aminoacyl-tRNA to ribosomes, similarly as eukaryotic EF-1 alpha or prokaryotic EF-Tu. EF-1B was characterized as a complex composed of at least two polypeptides. One of them is EF-1A, the other EF-1C, which stimulates EF-1A activity and protects this elongation factor from thermal inactivation.     

Construction of arginine-rich peptide displaying bionanocapsules

Bionanocapsule (BNC) is a hollow nanoparticle composed of L-protein of the hepatitis B virus surface antigen. BNC can deliver genes or drugs into specific human hepatocytes, but delivery is limited to hepatocytes. In this study, we attempted to alter the specificity of BNCs by genetically introducing cell-penetrating peptides (CPPs), such as arginine-rich peptides, into BNCs. The CPP-fused BNC was efficiently internalized into various cell lines in a short period without significant cytotoxicity. These results show that CPP-BNC could be applied as an efficient carrier for gene and drug delivery.     

Molecular basis for proline- and arginine-rich peptide inhibition of proteasome

PR39, a naturally occurring and cell-permeable proline- and arginine-rich peptide, blocks the degradation of inhibitor of nuclear factor κB (IκBα), thereby attenuating inflammation. It is a noncompetitive and reversible inhibitor of 20S proteasome. To identify its basis of action, we used solution NMR spectroscopy and mutational analyses of the active fragment, PR11, which identified amino acids required for human 20S proteasome inhibiting activity. We then examined PR11-mediated changes in the expression of nuclear factor κB-dependent genes in situ. The results provide prerequisites for proteasome inhibition by proline- and arginine-rich peptides, providing a powerful new tool to investigate inflammatory processes. These findings offer new leads in developing drugs to treat heart diseases or stroke.     

An intracellular delivery method for siRNA by an arginine-rich peptide

RNA interference has recently become a useful research tool for the studies of gene functions, regulations, and therapies. The double-stranded RNA is utilized to induce the sequence-specific gene silencing. To achieve this goal of specific gene silencing, a proper delivery system of siRNA is highly demanded. A number of approaches for delivering siRNA have been explored over the last few years. In the present study, we demonstrated a simple peptide-based siRNA delivery system in mammalian cells. A GC-EGFP cell line stably expressing enhanced green fluorescent protein was established from stable transfection of human gastric carcinoma cells. The synthetic nona-arginine peptide, an arginine-rich intracellular delivery peptide, or called protein transduction domain peptide, could noncovalently form stable complexes with EGFP siRNA and deliver these mixtures into cells. After entry, siRNA appeared to stay in perinuclear regions within cell, and ultimately fulfilled its targeted egfp gene silencing. These data were in consonance with that RNA-induced silencing complex components could be also localized to these perinuclear regions, creating a focal point for RNA interference factories. In the future, this non-toxic peptide may be proved to be a useful tool for the delivery of exogenous siRNA in RNA interference research.     

An intracellular delivery method for siRNA by an arginine-rich peptide

RNA interference has recently become a useful research tool for the studies of gene functions, regulations, and therapies. The double-stranded RNA is utilized to induce the sequence-specific gene silencing. To achieve this goal of specific gene silencing, a proper delivery system of siRNA is highly demanded. A number of approaches for delivering siRNA have been explored over the last few years. In the present study, we demonstrated a simple peptide-based siRNA delivery system in mammalian cells. A GC-EGFP cell line stably expressing enhanced green fluorescent protein was established from stable transfection of human gastric carcinoma cells. The synthetic nona-arginine peptide, an arginine-rich intracellular delivery peptide, or called protein transduction domain peptide, could noncovalently form stable complexes with EGFP siRNA and deliver these mixtures into cells. After entry, siRNA appeared to stay in perinuclear regions within cell, and ultimately fulfilled its targeted egfp gene silencing. These data were in consonance with that RNA-induced silencing complex components could be also localized to these perinuclear regions, creating a focal point for RNA interference factories. In the future, this non-toxic peptide may be proved to be a useful tool for the delivery of exogenous siRNA in RNA interference research.     

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide-PNA conjugates showed poor activity compared to a linear (R-Ahx-R)(4)-PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide-PNA conjugates showed activity similar to that of the corresponding linear peptide-PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence.     

Alkaline ribonuclease from rye germ cytosol.

1. Alkaline ribonuclease (pH optimum 7.6) was isolated from rye (Secale cereale L) germ cytosol and partially purified; the preparation was devoid of other nucleolytic activities. 2. The enzyme is a typical endonuclease hydrolysing all phosphodiester bonds in RNA, yielding ultimately purine and pyrimidine nucleoside 2',3'-cyclic phosphates and the corresponding 3'-phosphates. Upon extensive digestion of synthetic polyribonucleotides, pyrimidine, but not purine, nucleoside 3'-phosphates are formed. The enzyme does not hydrolyse synthetic purine cyclic nucleotides. 3. The enzyme does not depolymerize double-stranded complexes of poly(A) and poly(U). 4. Susceptibility to photooxidation and inhibition by 2-hydroxy-5-nitrobenzyl bromide and N-bromosuccinimide implies the involvement of tryptophan residue in the active centre of the enzyme.     

Endoribonuclease from bovine adrenal cortex cytosol.

An endoribonuclease which digests a variety of synthetic homoribopolymers and poly(A)-rich mRNA has been identified and purified greater than 500-fold with respect to specific activity from bovine adrenal cortex cytosol. Enzymatic digestion of synthetic poly(riboadenylic acid) was stimulated by Mn-2+ and Mg-2+ and the enzyme exhibited broad pH and salt optima. Poly(cytidylic acid) and poly(uridylic acid), but not poly(guanylic acid), served as substrates for the enzyme preparation; double-stranded RNA, DNA, and DNA-RNA hybrids were not digested by the enzyme. Digestion generated oligonucleotides with 3-hydroxyl and 5'-monophosphoester termini. On isoelectric focusing, the enzymatic activity banded at pH 8.3 plus or minus 0.2. An initial preferential cleavage of the poly(A) tract of poly(A)-rich RNA is suggested by the rapid appearance of a 4-6S digestion product highly enriched for adenylic acid; however, progressive digestion of the RNA occurs with additional incubation.     

A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide

Peptide-oligonucleotide conjugates were synthesized using two strategies: a mimetic signal peptide-conjugated oligonucleotide was assembled stepwise on CPG by using 2,2-dimethyl-3-hydroxypropionic acid as a linker. To solve the precipitation problem in the coupling reaction caused by the electrostatic interaction of arginine-rich peptides and oligonucleotide, oligonucleotides were absorbed on an anion-exchange resin, and then the on-resin fragment was applied for the conjugation with arginine-rich peptide. The peptide-antisense oligonucleotides showed permeability to the cell membrane of HepG-2 cells.     

Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells

The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.