Antineoplastic mechanism of antimicrobial peptides: Selective membrane destruction and non-membrane dissolution / 中国组织工程研究

BACKGROUND: Antimicrobial peptides, an extract from nature, are a basic component of host immunity that make toxic effect on highly proliferative cells. They have attracted extensive attention of scientists. The understanding of the antineoplastic mechanism of antimicrobial peptides can contribute to its application in clinical practice. OBJECTIVE: To summarize the research advances in antineoplastic mechanism of antimicrobial peptides. METHODS: The first author conducted a computer-based retrieval of PubMed, Springerlink, Web of Science, and ScienceDirect databases for relevant articles published from January 2015 to May 2019. The keywords were “antimicrobial, peptide, antitumor mechanisms, antitumor activity and anti-neoplastic”. The articles concerning antineoplastic mechanism of antimicrobial peptides and research progress were selected. RESULTS AND CONCLUSION: Cationic antimicrobial peptides synthesized by ribosomes and the host defense peptides can interact with the membrane of bacteria, which showed a broad-spectrum antimicrobial activity. Compared with normal cells, the proportion of phosphatidylserine on the surface of cancer cells, which is negatively charged, is increased dramatically. As a result, the cationic amphiphilic peptides are good candidate for the antineoplastic drugs, and possess a high selectivity. There are two major antitumor mechanism of antimicrobial peptides, which are selective membrane destruction and non-membrane dissolution (α-defensin-1 and lactoferrin B). The clinical application of antimicrobial peptides against tumors is mainly restricted by their stability and the ways to administration. By optimizing its structure and drug delivery systems, these antimicrobial peptides will play a critical role in the cancer treatment.

Construction and Verification of High-capacity Ribosome Display Single-chain Fv Library / 中国实用内科杂志

Objective To construct high-capacity ribosome display single-chain Fv library for selection of high affinity ScFv antibody.Methods We isolate human lymphocyte from peripheralblood(2 normal,3 gastric cancer,3 colonic cancer,1 pancreatic cancer,each 5 mL and 2 newborn,each 2 mL)and extract RNA for cloning whole human heavy chain and light chain gene by RT-PCR.VH and VL were rearranged randomly by SOEing(splicing by overlap extension,SOEing).Finally,the elements for in vitro screening such as T7 promoter and ribosome binding site were introduced while the SOEing products were amplified.Moreover,ribosome display template were verified by blue/white screening and further sequencing.Results We successfully constructed ribosome display ScFv library with a volume of 1.1?1013.Conclusion The construction of high-capacity ScFv library shed light on multiple therapeutic ScFv screening.

Control of coexpression plasmid of carcinoembryonic antigen and IL-2 gene by internal ribosome entrying site / 中国普通外科杂志

ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.

Quantitive analysis of the inhibition of HCV IRES mediated HCV core protein expression in cells by inhibitor RNA / 中华传染病杂志

Objective To study the inhibition of HCV IRES mediated HCV core protein expression in cells by inhibitor RNA. Methods Plasmid pcRz-IRNA, a eukaryotic expression vector with IRNA and two self cleavage ribozyme overhang at both sides respectively, was constructed and co-transfected with pcHCVcluc (containing HCV NCR, core and Luc genome) into the HHCC cell line (Human Hepatocellular Carcinoma cell line). Immunoflurescence tests were applied to detect the co-transfected cells, which were thereafter analysed with confocal microscope quantitatively. Luciferase activity was valued using Luc Assay System (Promega). Results The cotransfected cells expressed HCV core protein, and the fluorecein in which was reduced significantly in comparison with control. Conclusions IRNA can inhibit the expression of HCV IRES mediated core protein in the cotransfected cells.

Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.

Conformational change of 50S ribosomes takes place during protein synthesis. The primary change is most likely in the secondary or tertiary structure of rRNA in the L7/L12 stalk region. In order to throw further light on this conformational change, the change in fluorescence of tight couple 50S ribosomes on conversion to loose couple 50S ribosomes containing 5-(iodoacetamido ethyl)-aminonaphthalene-l-sulphonic acid-labelled L7/L12, following the treatment with elongation factor-G and 5'-guanylyl methylene diphosphate was measured. It was enhanced in agreement with the results reported earlier. Further, the quenching of fluorescence of 50S ribosomes containing 5-(iodoacetamido ethyl)- aminonaphthalene-1-sulphonic acid-labelled L7/L12 by acrylamide was studied. The quenching is more in case of loose couples. On conversion of loose couple 50S ribosomes to tight couple ones the quenching becomes less whereas the reverse happens on conversion of tight couple 70S ribosomes to loose couples. These results indicate the conformational change of L7/L12 stalk in the different functional states of 50S ribosomes.

Construction and expression of the pIRES-enchanced green fluorescent protein reporter gene vector carrying BMP_2 Gene / 重庆医科大学学报

Objective:To construct the pIRES2-enchanced green flurescent protein expression vector carrying BMP_2 gene in order to provide an ideal reporter gene for the expression and identify the location of portein in vitro and vivo.Methods:The BMP_2 cDNA sequence was excised,and subcloned on pUC18 vector.And then the BMP_2 sequence on the pUC18 plasmid was excised again and ligated into Sal Ⅰ site of pIRES2-EGFP treated by phosphorylase in sense and antisense direction.The correct recombinant plasmid was transfected into 293-T cells,the expression of BMP_2 was determined by FCM,LCM and RT-PCR.Results:The recombinant expression vector was verified correctly by enzyme digestion,sequence analysis.This vector was expressed in cells.Conclusion:A pIRES2 green fluorescent protein reporter gene vector containing BMP_2 has been constructed successfully,thus providing an important and convenient tool to study intracellular localization of BMP_2.

Do ribosomal RNAs act merely as scaffold for ribosomal proteins.

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-binding, (b) poly U-dependent binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (Ρ) site. A model has been proposed for translocation on the basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to be noncontroversial.

Studies on the structure and function of 16S ribosomal RNA using structure-specific chemical probes.

Recent technological developments permit us to examine the accessibility of specific atoms on any nucleotide in any large RNA molecule to certain chemical probes. This can provide detailed information about the higher order structure of large RNA molecules, including secondary and tertiary structure, protein-RNA contacts, binding sites for functional ligands and possible biologically significant conformational changes. Here, we summarize recent studies on (i) the conformation of naked 16S rRNA under a variety of ionic conditions, and (ii) the behaviour of 16S rRNA in active and inactive 30S subunits, as defined by Zamir, Elson and their colleagues. The latter study reveals a reciprocal conformational change in the vicinity of the decoding region of 16S rRNA in 30S ribosomal subunits. This conformational change appears to be a rearrangement of tertiary and/or quaternary structure involving several universally conserved nucleotides. No reproducible effects are seen elsewhere in the molecule, suggesting that the active-inactive transition is a result of the observed conformational change.

Conformational change of 23S RNA in 50S ribosome is responsible for translocation in protein synthesis.

Since the recognition of the ‘translocation’ phenomenon during protein synthesis several theories have been proposed, without much success, to explain the translocation of peptidyl tRNA from the aminoacyl site to the peptidyl site. The involvement of L7/L12 proteins and therefore the L7/L12 stalk region of 50S ribosomes in the translocation process has been widely accepted. The mobility of the stalk region, as recognised by many workers, must be of physiological significance. It has recently been shown in this laboratory that 50S ribosomes derived from tight and loose couple 70S ribosomes differ markedly in quite a few physical and biological properties and it appears that these differences are due to the different conformations of 23S RNAs. It has also been possible to interconvert tight and loose couple 50S ribosomes with the help of the agents, elongation factor –G, GTP (and its analogues) which are responsible for translocation. Thus loose couple 70S ribosomes so long thought to be inactive ribosomes are actually products of translocation. Further, the conformational change of 23S RNA appears to be responsible for the interconversion of tight and loose couple 50S ribosomes and thus the process of translocation. A model has been proposed for translocation on the basis of the direct experimental evidences obtained in this laboratory.

Presence of precursor ribosome in the ribosomal preparation from chloramphenicol-treated Escherichia coli ΑΒ301/Ι05 (RNase III – ).

On sucrose gradient centrifugation, the ribosomal preparation from chloramphenicoltreated 32P labelled Escherichia coli AB301/105 (RNase III¯ ) showed the presence of a radioactive peak moving slower than the 70S ribosome; this peak disappeared on treatment with RNase III. The presence of precursor 30S RNA was shown in such preparations by affinity chromatography on a lysine-sepharose 4B column as well as polyacrylamide gel electrophoresis. Dialysis against low Mg2± concentration followed by sucrose density gradient electrophoresis. Dialysis against dissociation of 70S ribosome into its subunits, did not lead to the dissociation of the precursor ribosome. However, the dissociation took place upon treatment with RNase III. A tentative model of coupled rRNA transcription and ribosome assembly has been presented.

Immunoprecipitation of 70S, 50S and 30S ribosomes of Escherichia coli.

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.

Alteration of the structure of the Escherichia coli ribosomes on treatment with Fab fragment of immunoglobulin raised against the ribosomes.

Rabbits were immunised against Escherichia coli ribosomes and the partially purified immunoglobulin G fraction had maximum ability to precipitate the ribosomes as well as the extracted ribosomal proteins. By digestion of immunoglobulin G with papain, monovalent Fab fragments were produced. The 70 S ribosome and its subunits (50 S and 30 S) were separately treated with Fab and then tested in the kinetic assay of degradation of ribosomes by ribonuclease I at various Mg2+ concentrations. Treated ribosomes and their subunits were degraded at faster rates than the nontreated ones; the rates in both the control and the treated cases were dependent on the concentration of Mg2+. These results indicate the unfolding of the structure of the ribosome on treatment with antibody fragments, which may be due to the weakening of the interaction between rRNAs and ribosomal proteins.