Human Ribonuclease with a Pendant Poly(Ethylene Glycol) Inhibits Tumor Growth in Mice

Human pancreatic ribonuclease (RNase 1) is a small secretory protein that catalyzes the cleavage of RNA. This highly cationic enzyme can enter human cells spontaneously but is removed rapidly from circulation by glomerular filtration. Here, this shortcoming is addressed by attaching a poly(ethylene glycol) (PEG) moiety to RNase 1. The pendant has no effect on ribonucleolytic activity but does increase persistence in circulation. The RNase 1-CPEG conjugates inhibit the growth of tumors in a xenograft mouse model of human lung cancer. Both retention in circulation and tumor growth inhibition correlate with the size of the pendant PEG. A weekly dose of the 60-kDa conjugate at 1 μmol/kg inhibited nearly all tumor growth without affecting body weight. Its molecular efficacy is ~5000-fold greater than that of erlotinib, which is a small molecule in clinical use for the treatment of lung cancer. These data demonstrate that the addition of a PEG moiety can enhance the in vivo efficacy of human proteins that act within cells and highlight a simple means of converting an endogenous human enzyme into a cytotoxin with potential clinical utility.     

NMR Structure of an Oligonucleotide Containing A Base Pair 3-(2-Hydroxyethyl) Deoxyuridine-Adenine

Abstract

It has been shown that ethylene oxide reacts with dC at the N3 position to produce a potentially mutagenic lesion, 3-(2-hydroxyethyl) deoxyuridine (3-HE-dU). In this article, we report NMR and Molecular Mechanic studies of a duplex containing the 3-HE-dU base with an adenine in front of the lesion which is in the sequence, 5′-GCAAGTC(3-HE-dU)AAAACG.     

Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore.     

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.     

Poly(Ethylene Glycol)-Cholesterol Inhibits L-Type Ca2+ Channel Currents and Augments Voltage-Dependent Inactivation in A7r5 Cells

Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol) cholesteryl ether (PEG-cholesterol) is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca²⁺ channel currents (I _Ca,L) recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of I _Ca,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-β-cyclodextrin (MβCD) is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MβCD:cholesterol complex (cholesterol (MβCD)) caused inhibition of I _Ca,L but did not augment voltage-dependent inactivation. Incubation with MβCD increased I _Ca,L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MβCD of the cells initially pretreated with PEG-cholesterol, increased I _Ca,L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I _Ca,L more strongly as compared with cholesterol (MβCD). Poly(ethylene glycol) conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I _Ca,L.     

Effects of Modification of Amino Groups with Poly(Ethylene Glycol) on a Recombinant Uricase from Bacillus fastidiosus

After modification with monomethoxyl-poly(ethylene glycol)-5000, a recombinant intracellular uricase from Bacillus fastidiosus ATCC 29604 showed residual activity of about 65%, a thermo-inactivation half-life >85 h, a circulating half-life about 20 h in rats in vivo, consistent effects of common cations, and consistent optima for reaction temperature and pH. Thus, this uricase can be formulated via modification with monomethoxyl-poly(ethylene glycol).     

Design and Characterization of an Adhesive Matrix Based on a Poly(Ethyl Acrylate, Methyl Methacrylate)

The main issue in the development of transdermal patches made of poly(ethyl acrylate, methyl methacrylate) (Eudragit NE 40D, PMM) is the shrinkage phenomenon during the spreading of the latex onto the release liner. To solve this problem, the latex is usually freeze-dried and then re-dissolved in an organic solvent (method 1). To simplify the production process, we prepared an adhesive matrix by adding to the commercial PMM latex a plasticizer and an additive (anti-shrinkage agent) that avoids the shrinkage of the water dispersion spread onto the release liner (method 2). In some cases the active ingredient itself, such as potassium diclofenac (DK) and nicotine (NT), works as anti-shrinkage agent. In this work, the effects of the preparation method, types and concentrations of the plasticizer (triacetin and tributyl citrate) on the adhesive properties of the transdermal patches were investigated. The adhesive properties of the prepared patch were determined by texture analysis, peel adhesion test and shear adhesion. The PMM/plasticizer interactions were evaluated by ATR-FTIR spectroscopy. Furthermore, the in vitro skin permeation profiles of DK and NT released from the patch were determined by Franz cell method. Generally speaking, the variables that mainly modify the adhesive properties are the concentration and type of the plasticizer. The skin permeation profiles of DK and NT from the patch prepared by method 2 overlapped with those obtained with the commercial products. The results underline that the PMM latex can be used conveniently in the development of transdermal patches.     

Formation of Phosphatidyl 1-(2-Hydroxyethyl)-2,3,5-Trialkyl Pyridinium on Heating Phosphatidyl Ethanolamine with n-Alkanal

From the reaction products of phosphatidyl ethanolamine and n-alkanais such as pro-pionaldehyde or n-heptaldehyde at 60°C for 2 hr, a series of phosphatidyl l-(2-hydroxyethyl)-2,3,5-trialkyl pyridiniums were isolated by silica gel column chromatography and were identified by the elementary analysis, UV, IR and NMR spectral analysis of alkali and acid hydrolyzates of the compounds. The course of formation reaction of pyridinium ring was discussed.     

Characterization of Genes Encoding Poly(A) Polymerases in Plants: Evidence for Duplication and Functional Specialization

Background

Poly(A) polymerase is a key enzyme in the machinery that mediates mRNA 3′ end formation in eukaryotes. In plants, poly(A) polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A) polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied.

Methodology/Principal Findings

Using bioinformatics tools, poly(A) polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A) polymerase gene, other species possessed between two and six possible poly(A) polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A) polymerase genes (but not the C. reinhardtii gene) possessed almost identical intron positions within the poly(A) polymerase coding sequences, suggesting that all plant poly(A) polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A) polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A) polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis.

Conclusions/Significance

Our results indicate that poly(A) polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A) polymerases is a novel cytoplasmic poly(A) polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A) polymerases in mammals.     

环氧基高分子磁性复合微球固定化木瓜蛋白酶的研究

研究以甲基丙烯酸环氧丙酯(GMA)为单体,二甲基丙烯酸乙二醇酯(EDMA)为交联剂,聚乙烯醇(PVA)为分散剂,在Fe3O4磁性纳米粒子存在的条件下,合成了交联度为25%的磁性高分子复合微球(GMAE-DMA).并以这种微球为载体,进行了对木瓜蛋白酶的固定化研究.探讨了最佳的固定化条件如下:温度为25℃,反应时间20h,pH值为8.5,给酶量为160mg/g.同时以酪蛋白为底物,研究了固定化酶的酶学性质,结果表明:固定化酶对不同pH值环境的耐受力、热稳定性和操作稳定性都有较大幅度的提高.实验证明这种高分子磁性复合微球是一种优良的固定化酶载体.     

Immobilization of Lipase from Mucor Miehei Onto Poly (Ethylene) Based Graft Copolymers

Lipase from Mucor miehei was immobilized covalently onto hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-HEMA). This hydrolysis of the copolymer was achieved using 0.1 M NaOH over different periods of time, under controlled conditions. The graft copolymers and their hydrolyzed equivalents were characterized by scanning electron microscopy (SEM) and by differential scanning calorimetry analysis (DSC). Water sorption studies were undertaken to provide a measure of relative hydrophobicity of the samples.

The lipase immobilization reaction was studied in order to assess the effects of controlling various important parameters. These include the nature of the buffering medium, the time over which the immobilization was allowed to occur, the concentration of the activating and coupling agent used (CMC) and the concentration of enzyme employed during attempts at effective immobilization. The immobilized lipase was used in the hydrolysis of triolein (glycerol trioleate). From this study, the apparent K_M, the optimum pH for hydrolysis and the optimum temperature for hydrolysis were revealed.

The suitability of hydrolyzed poly(ethylene)-g.co-HEMA as a support in the immobilization of lipase was assessed by determination of the amount of lipase coupled to the support and by assessment of the retention of activity of the immobilized lipase after its exposure to the immobilization reagents, procedure and conditions.     

Synthesis of 9-[1-(1-Hydroxyethyl)-3-(Phosphonomethoxy)Propyl] Adenine and Prodrug as Possible Antiviral Agents

The appropriately protected C-1′-hydroxyethyl-3-hydroxypropyl-N⁹-adenine nucleoside was prepared from 1-pivaloyloxy-5-tert-butyldiphenylsilyloxy-3-pentanol and adenine through the Mitsunobu reaction. One of the terminal hydroxyls was converted to the phosphonomethoxy derivative and the prodrug.     

Functional reconstitution of influenza A M2(22-62)

Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K(+)] gradient-induced membrane potential and [H(+)] gradient), M2(22-62) showed a transport rate of 78 H(+)/tetramer-s (pH(o) 6.0, pH(i) 8.0, nominal V(m)=-114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K(+) flux of 0.1K(+)/tetramer-s, and also demonstrated that the K(+) permeability, relative to H(+), was 2.8 × 10(-6). Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H(+) selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pK(a) of 6.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Poly[3-(3,4-Dihydroxyphenyl)glyceric Acid]: A New Biologically Active Polymer from Two Comfrey Species Symphytum asperumand S. caucasicum(Boraginaceae)

Two high-molecular water-soluble preparations with high anticomplementary and antioxidant activity were isolated from the roots of Symphytum asperum and S. caucasicum. Their main chemical constituent was found to be poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene] according to IR and NMR spectroscopy.     

Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.