Aliphatic Poly(ester-carbonate)s Bearing Amino Groups and Its RGD Peptide Grafting

This article deals with (1) synthesis of novel cyclic carbonate monomer (2-oxo [1,3]dioxan-5-yl)carbamic acid benzyl ester (CAB) containing protected amino groups; (2) ring-opening copolymerization of the cyclic monomer with L-lactide (LA) to provide novel degradable poly(ester-carbonate)s with functional groups; (3) removal of the protective benzyloxycarbonyl (Cbz) groups by catalytic hydrogenation to afford the corresponding poly(ester-co-carbonate)s with free amino groups; (4) grafting of oligopeptide Gly-Arg-Gly-Asp-Ser-Tyr (GRGDSY, abbreviated as RGD) onto the copolymer pendant amino groups in the presence of 1,1'-carbonyldiimidazole (CDI).

Rapid label-free detection of metal-induced Alzheimer's amyloid beta peptide aggregation by electrochemical method

Amyloid beta peptide plays a critical role in the pathogenesis of Alzheimer's disease (AD). Metal ions are highly enriched in cerebral amyloid deposits in AD and are proposed to be able to mediate A beta conformation. Therefore, a rapid, low-cost, and sensitive detection of metal-induced A beta aggregation and their relation to AD is clearly needed for the clinical diagnosis and treatment. In this report, we study metal-induced A beta aggregation by a rapid, label-free electrochemical method and monitor both the aggregation kinetics and the morphology in the absence or presence of Zn (II) and Cu (II).

Investigation of Calmodulin-Peptide Interactions Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Time-dependent DNA condensation induced by amyloid beta-peptide

The major protein component of the amyloid deposition in Alzheimer's disease is a 39-43 residue peptide, amyloid beta (A beta). A beta is toxic to neurons, although the mechanism of neurodegeneration is uncertain. Evidence exists for non-B DNA conformation in the hippocampus of Alzheimer's disease brains, and A beta was reportedly able to transform DNA conformation in vitro. In this study, we found that DNA conformation was altered in the presence of A beta, and A beta induced DNA condensation in a time-dependent manner. Furthermore, A beta sheets, serving as condensation nuclei, were crucial for DNA condensation, and Cu2+ and Zn2+ ions inhibited A beta sheet-induced DNA condensation. Our results suggest DNA condensation as a mechanism of A beta toxicity.

RGD peptide grafted biodegradable amphiphilic triblock copolymer poly(glutamic acid)-b-poly(L-lactide)-b-poly(glutamic acid): Synthesis and self-assembly

A novel amphiphilic biodegradable triblock copolymer (PGL-PLA-PGL) with polylactide (PLA) as hydrophobic middle block and poly(glutamic acid) (PGL) as hydrophilic lateral blocks was successfully synthesized by ring-opening polymerization (ROP) Of L-lactide (LA) and N-carboxy anhydride (NCA) consecutively and by subsequent catalytic hydrogenation. The results of cell experiment of PGL-PLA-PGL suggested that PGL could improve biocompatibility of polyester obviously. The copolymer could form micelles of spindly shape easily in aqueous solution. The pendant carboxyl groups of the triblock copolymer were further activated with N-hydroxysuccinimide and combined with a cell-adhesive peptide GRGI)SY Incorporation of the oligopeptide further enhanced the hydrophilicity and led to formation of spherical micelles. PGL-PLAPGL showed better cell adhesion and spreading ability than pure PLA and the GRGDSY-containing copolymer exhibited even further improvement in cell adhesion and spreading ability, indicating that the copolymer could find a promising application in drug delivery or tissue engineering.

A biodegradable triblock copolymer poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine): Synthesis, self-assembly and RGD peptide modification

A novel biodegradable triblock copolymer poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-lysine) (PEG-PLA-PLL) was synthesized by acidolysis of poly(ethylene glycol)-b-poly(L-lactide)-b-poly(F-benzyloxycarbonyl-L-lysine) (PEG-PLA-PZLL) obtained by the ring-opening polymerization (ROP) of epsilon-benzyloxycarbonyl-L-lysine N-carboxyanhydride (ZLys NCA) with amino-terminated PEG-PLA-NH2 as a macro-initiator, and the pendant amino groups of the lysine residues were modified with a peptide known to modulate cellular functions, Gly-Arg-Gly-Asp-Ser-Tyr (GRGDSY, abbreviated as RGD) in the presence of 1,1'-carbonyldiimidazole (CDI). The structures of PEG-PLA-PLL/RGD and its precursors were confirmed by H-1 NMR, FT-IR, amino acid analysis and XPS analysis. The cell adhesion and cell spread on the PEG-PLA-PLL/RGD film were enhanced compared to those on pure PLA film. Therefore, the novel RGD-grafted triblock copolymer is promising for cell or tissue engineering applications. Both copolymers PEG-PLA-PZLL and PEG-PLA-PLL showed an amphiphilic nature and could self-assemble into micelles of homogeneous spherical morphology. The micelles were determined by fluorescence technique, dynamic light scattering (DLS), and field emission scanning electron microscopy (ESEM) and could be expected to find application in drug and gene delivery systems.

Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer

Advances in tissue engineering require biofunctional scaffolds that can provide not only physical support for cells but also chemical and biological cues needed in forming functional tissues. To achieve this goal, a novel RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) (PEG-PLA-PGL/RGD) was synthesized in four steps (1) to prepare diblock copolymer PEG-PLA-OH and to convert its -OH end group into -NH2 (to obtain PEG-PLA-NH2), (2) to prepare triblock copolymer PEG-PLA-PBGL by ring-opening polymerization of NCA (N-carboxyanhydride) derived from benzyl glutamate with diblock copolymer PEG-PLA-NH2 as macroinitiator, (3) to remove the protective benzyl groups by catalytic hydrogenation of PEGPLA-PBGL to obtain PEG-PLA-PGL, and (4) to react RGD (arginine-glycine-(aspartic amide)) with the carboxyl groups of the PEG-PLA-PGL. The structures of PEG-PLA-PGL/RGD and its precursors were confirmed by H-1 NMR, FT-IR, amino acid analysis, and XPS analysis. Addition of 5 wt % PEG-PLA-PGL/RGD into a PLGA matrix significantly improved the surface wettability of the blend films and the adhesion and proliferation behavior of human chondrocytes and 3T3 cells on the blend films. Therefore, the novel RGD-grafted triblock copolymer is expected to find application in cell or tissue engineering.

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H2O2 was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H2O2 without electron mediator or promoter, at a potential of +0.10 V versus Ag \ AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N = 3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.