Design and synthesis of a Tat-related gene transporter: a tool for carrying the adenovirus vector into cells

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.     

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Controlled release of DNA/polyamine complex by photoirradiation of a solid phase presenting o-nitrobenzyl ether tethered spermine or polyethyleneimine

Various gene transfer and automated/monitorized analytical applications require the controlled release of nucleic acid. A solid phase with spermine or polyethyleneimines (PEI, 600 MW) tethered by o-nitrobenzyl linkages was synthesized with polyethylene oxide beads (ArgoGel-NH(2)). The photolysis of test compound O-2-nitrophenethyl O,O-diethyl phosphate or solid phase with o-nitrobenzyl group as synthetic linker was completely degradable with photoirradiation at 365 nm for 10-18 min at 3.5 mW/cm(2). DNA binding with polyamine of the solid phase and releasing of DNA/polyamine were monitored by UV measurement and gel electrophoresis. The potential exists to employ a DNA-loaded solid phase for spatially, temporally, or dose-controlled release of DNA, at extracellular or intracellular sites.     

Total chemical synthesis of human activin beta(A)[12-116] and related large-loop polypeptides.

We report here the synthesis, purification, and characterization of several large polypeptides related to the human activin beta(A) subunit and their cyclic counterparts. In particular, we describe for the first time the total chemical synthesis of a 105-mer polypeptide, des[1-11] activin beta(A), and related large-loop polypeptide, by an optimized solid phase synthetic protocol based on 9-flouroenylmethyoxycarbonyl (Fmoc) chemistry. These studies show that automated chemical synthesis utilizing Fmoc-based solid phase synthetic strategies provides a practical alternative to recombinant DNA technology for the production of activin-related subunits, with the opportunity to rapidly provide different analogues and structural variants for subsequent structure-function and associated biophysical investigations.     

Studies on desorption of individual textile dyes and a synthetic dye effluent from dye-adsorbed agricultural residues using solvents

Two solvents, A and B (A: methanol, chloroform, water in the ratio 1:1:1; B: 50% methanol), were used to extract textile dyes adsorbed onto substrates for the purpose of future analyses of the amount of dyes degraded through solid state fermentation (SSF) using white rot fungi. Barley husk, apple pommace and corncob were separately soaked in five different dye solutions and a synthetic textile effluent. A maximum value of 93% desorption of Cibacron Red from corncob was achieved using solvent A. Barley husk was the only substrate from which the synthetic textile effluent could be desorbed, with 82% being recovered using solvent A.     

Argifin; efficient solid phase total synthesis and evalution of analogues of acyclic peptide

An effective solid phase synthesis of Argifin, providing subsequent access to effective synthesis of analogues, was developed in 13% overall yield, as well as elucidating structure–activity relationships. The novel acyclic peptide 18b, prepared from a synthetic intermediate of Argifin, was found to be 70 times more potent as an inhibitor of Serratia marcescens chitinases B than Argifin itself     

Synthesis of a peptide with full somatostatin activity

A peptide has been synthesized according to the structure proposed for somatostatin by the solid phase method. The synthetic product was assayed and found to possess full somatostatin activity as compared with the natural material.     

Solid phase synthesis of 5'-diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp-oligoribonucleotides for use as primers for influenza A virus RNA polymerase in vitro.

We have synthesized four different 5'-diphosphorylated oligoribonucleotides, varying in length from 11 to 13 nucleotides by a new solid phase method. After deprotection and partial purification the 5'-diphosphorylated oligoribonucleotides could be converted to capped (m7Gppp) oligoribonucleotides using guanylyl transferase. Radiolabelled capped oligoribonucleotides acted as primers for the influenza A virus RNA polymerase in vitro. The solid phase method described here should also allow the addition of 5'-diphosphates to synthetic oligodeoxyribonucleotides and be capable automation.     

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4-23, 31-46, and 56-77; these studies suggested that an antigenic site might be restricted to residues 60-77 in the 56-77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61-77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu60, thus confirming our suggestion that a major antigenic site is located in residues 60-77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60-77 indicating that the antibodies were specifically bound to the resin.     

精子肽的固相合成及应用初探

目的 :探讨将多肽固相合成技术用于检测抗精子抗体的ELISA试剂盒制备。方法 :以多肽固相合成法合成特异性精子肽 ,并经高效液相纯化分析及质谱分析。以此合成精子肽包板制备检测抗精子抗体的ELISA试剂盒 ,检测血清标本的AsAb。结果 :HPLC结果显示 ,合成的精子肽纯度达 98.26%;质谱分析结果主峰分子质量与理论值一致。采用合成多肽抗原建立了检测抗精子抗体的酶联免疫吸附测定方法 ;不明原因不育患者组与对照组间AsAb发生率呈非常显著差异(P <0,005 )。结论 :本固相合成法可获得高纯度特异性精子肽 ;该精子肽包板的ELISA试剂盒可靠简便。     

The use of solid waste of a nylon-6 plant as a nutrient for bacterial decolourisation of dyes

Three caprolactam-degrading bacterial isolates grew in liquid synthetic medium containing solubilised solid waste of a nylon-6 production plant as the sole source of carbon and nitrogen. Typically, the caprolactam content of solid waste was decreased by 95% in 72 h by Alcaligenes faecalis. A. faecalis was the most potent caprolactam-degrading bacterium out of the three isolates. The biomass of the bacteria obtained by growth in the solubilised solid waste medium had the ability to decolourise some synthetic azo and triphenylmethane dyes. Decolourisation of dyes was obtained in static condition, in synthetic medium which contained only the components of the solid waste as the sole sources of carbon and nitrogen and also in nutritionally rich medium. The supplementation of yeast extract to solid waste medium did not increase the efficiency of decolourisation in case of two of the bacterial cultures. Depending on the dye, medium and bacteria used, decolourisation in the range of 35–94% was achieved in 48–96 h. The decolourisation was not due to the adsorption of the dyes by the bacterial biomass except in case of Procion Blue MR and Black B. Based on these observations, the simultaneous biological treatment of the solid waste of nylon-6 plant and the decolourisation of synthetic dyes present in wastewater or solid waste is envisaged.     

Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.     

A synthetic method for peptide-PEG-lipid conjugates: application of octreotide-PEG-DSPE synthesis

A solid phase synthesis method was established for the synthesis of peptide-poly(ethylene glycol)-lipid (peptide-PEG-lipid) conjugates. Octreotide-PEG(2000)-DSPE (OPD(2000)) was used as an example to demonstrate the synthetic approach. The OPD(2000) obtained had confirmed structure, activity, and purity providing a targeting molecule for preparation of well-defined drug delivery systems, such as targeted liposomes, for further studies.     

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.     

Chemical synthesis of a neurotoxic polypeptide from the sea anemone Stichodactyla helianthus

The sea anemone Stichodactyla helianthus neurotoxin I, a 48-residue polypeptide, was synthesized by automated solid phase methodology. The fully reduced polypeptide was subsequently refolded in the presence of a glutathione oxidoreduction buffer to the biologically active species containing three disulfide bonds. The overall yield after rigorous purification was 12.5%. The circular dichroism (CD), and proton nuclear magnetic resonance (1H NMR) spectra of the HPLC-purified synthetic toxin were indistinguishable from those obtained concurrently with the natural toxin. A subtilisin digest of the synthetic neurotoxin generated peptide fragments identical to that of a sample of the natural toxin subjected to the same treatment. The toxicity of the synthetic polypeptide was identical to that of the natural toxin (crab LD50, 3.1 micrograms/kg). The equilibrium dissociation constant (28 nM) for interaction of the synthetic toxin with crab axolemma vesicles was nearly identical to that of the natural toxin (25 nM).     

An antibody-lectin sandwich assay for the determination of CA125 antigen in ovarian cancer patients

A two-step forward sandwich assay was developed for the determination of the ovarian tumour associated glycoconjugate antigen CA125 with anti-CA125 Monoclonal antibody B27.1 on the solid phase and¹²⁵I-labelled wheat germ lectin as tracer in the solution phase. This Mab-lectin heterosandwich assay was optimized and the clinical utility was evaluated in sera from healthy volunteers and ovarian cancer patients. A correlation was established between Mab-lectin assay and the dual monoclonal antibody sandwich assay, TRUQUANT®OV2 RIA, that uses the same MAb B27.1 on the solid phase and a second¹²⁵I-labelled B43.13 MAb in the solution phase. A potentially improved clinical utility is suggested for the Mab-lectin assay. The unique format seems to identify novel isoforms of CA125 with different carbohydrate side chains that would otherwise be undetectable in the MAb-MAb sandwich assay wherein the paratopes are likely directed to protein determinants.     

Solid phase synthesis of mRNA by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl protection and its stability in E. coli system.

The decaoligoribonucleotides containing initiation codon AUG of the phage O beta-A protein mRNA were synthesized on a solid phase by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl (Cee) protection. The Cee group is completely stable under the acidic conditions required to remove the 5'-terminal protecting groups in oligoribonucleotide synthesis on a solid support, and yet is easily removable at pH 2.0 for the final unblocking step. Stabilization of the synthetic mRNA to in the cell-free translation system from E. coli A19 was measured. It was found that the oligomers with selected 2'-O-methylation were degraded completely in the translation system.     

Effect of cosolvents on solubility of protein in equilibrium between different states

This paper describes a theoretical work on the solubility of a protein in equilibrium between different forms such as isomerization (A ? B) and dimerization (2A ? B). Under the assumptions that A and B have different solubilities and form their own solid phases (in the form of either precipitation or crystal), it was shownthat only either A or B precipitates at a given condition, another form being in equilibrium in solution with the precipitating form, provided that the solubility ratio of A to B is not identical to their equilibrium composition in the solution phase determined by the equilibrium constant. It follows, then, that the total concentration of the protein in the solution phase can be calculated by summing the solubility of the precipitating form and the concentration in solution of another form in equilibrium with the precipitating form. Assuming various preferential protein-solvent interactions for the two forms in solution as well as solid phases, dependences of the total protein solubility on the additive concentration were examined. It was shown that this dependence may be complex, showing maximum, minimum, or inflection point, depending on the preferential protein interactions with solvent components.     

A new synthesis of the tripeptide Gly-His-Lys with antimicrobial activity

Summary A new solid phase synthesis of the growth-modulating tripeptide Gly-His-Lys is described. 2-Chlorotrityl chloride resin and 9-fluorenylmethoxycarbonyl-(Fmoc), 4-methyltrityl-(Mtt) protecting groups were used. The synthetic tripeptide was tested for its activity against bacteria, yeast and fungi. The in vitro effect of the tripeptide on DNA, RNA and protein synthesis was studied as well.     

Binding characteristics and cross-reactivity of three different antilipid A monoclonal antibodies

A detailed characterization of binding specificity and cross-reactivity of three antilipid A murine mAb was performed. Binding characteristics of these three mAb were investigated against Ag (ReLPS, lipid A, derivatives of lipid A) in solid phase (ELISA) and in fluid phase (C consumption, inhibition studies), and upon incorporation in membranes (E: passive hemolysis assay, and liposomes: inhibition studies). Cross-reactivity with heterologous Ag was investigated in ELISA (LPS, Gram-negative bacteria) and immunoblot experiments (LPS). The binding specificity of mAb 26-5 (IgG2b), raised against synthetic lipid A, was located in the hydrophilic region of biphospholipid A and was also exposed after membrane incorporation of lipid A or after preincubation of lipid A with polymyxin B (PMX). mAb 26-20 (IgM), also raised against synthetic lipid A, showed binding specificity for the hydrophobic region of lipid A: no binding to membrane-associated lipid A could be demonstrated, and binding in ELISA could be blocked very efficiently by PMX. The reaction pattern of mAb 8-2 (IgM), raised against the heat-killed Re mutant of Salmonella typhimurium, was in part similar to that of mAb 26-20. However, inhibition of binding with PMX was less efficient and a high specificity for ReLPS, also after membrane incorporation of this Ag, was demonstrated. In contrast to mAb 26-5 and 26-20, mAb 8-2 showed extensive cross-reactivity with heterologous LPS preparations and heat-killed as well as live Gram-negative bacteria. It is concluded that each of the three mAb binds to a different antigenic epitope in lipid A and that exposure of those epitopes for antibody binding is restricted in a differential manner, depending on mode of Ag presentation. The here defined reaction patterns provide a basis for the interpretation of potential inhibitory effects on in vitro and in vivo biologic (and toxic) activities of endotoxins and Gram-negative bacteria.