Induction and regulation of ethylene biosynthesis and ripening by pectic oligomers in tomato pericarp discs

The effect of pectic oligomers and 1-aminocyclopropane carboxylic acid on ethylene biosynthesis and color change was studied in ripening tomato pericarp discs excised from mature-green tomato fruit (Lycopersicon esculentum Mill.). Pectic oligomers induced at least four distinct responses when added to pericarp discs: (a) a short-term, transient increase in ethylene biosynthesis; (b) a long-term, persistent increase in climacteric ethylene in discs excised from mature-green fruit; (c) an advance in ripening processes, as indicated by increased reddening of the disc surfaces; and (d) a darkening of the treated endocarp surface. Pectic oligomers appear to affect the ripening of exocarp and endocarp tissues by different mechanisms. In exocarp tissues, the acceleration of reddening by pectic oligomers might simply be a consequence of induced ethylene biosynthesis. In endocarp tissues, the acceleration of reddening appears to be a direct effect of oligomers on ripening processes. We suggest that the rate of ripening of endocarp tissues may be regulated, in part, by the release of pectic oligomers from the cell walls of adjacent exocarp tissues. Exocarp and endocarp tissues of pericarp discs appear to differ in their sensitivity to ethylene at each maturity stage, and to exhibit independent changes in sensitivity to ethylene as ripening progresses. The tissue-specific pattern of reddening in tomato pericarp may result from this differential sensitivity to endogenous ethylene concentrations.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").     

Effect of pectic oligomers on physiological responses of chilling injury in discs excised from zucchini (Cucurbita pepo L.).

The effect of pectic oligomers (OG) on ethylene biosynthesis, electrolyte leakage (EL), and CO(2) production was studied in discs excised from zucchini fruit (Cucurbita pepo L.) and stored at 20 or 2.5 degrees C. At 20 degrees C, OG enhanced ethylene biosynthesis and had a transient effect on decreasing EL, but showed little effect on respiratory rate; both the amount and size of the oligomer were important in changing both ethylene synthesis and EL. At 2.5 degrees C, OG increased both ethylene biosynthesis and respiratory rate with a maximum effect at 100 microg of oligomer and peaking at 6 h; shorter oligomers demonstrated an even greater effect on ethylene biosynthesis, but differences were smaller in respiratory rate. EL at 2.5 degrees C was affected most by 1 microg of OG and by monomeric galacturonic acid, with transient increases that peaked at 8 h. We suggest a signaling role for OG in the early steps of cold acclimation or chilling injury.     

Dual peptide nucleic acid- and peptide-functionalized shell cross-linked nanoparticles designed to target mRNA toward the diagnosis and treatment of acute lung injury

In this work, multifunctional biosynthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell cross-linked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient, and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the K(d) values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.     

Distribution analyses of chain substituents of lipoteichoic acids by chemical degradation

The lipoteichoic acid from Lactococcus lactis Kiel 48337 was analyzed. It had 61% of its glycerophosphate residues substituted with alpha-D-galactopyranosyl residues. Non-substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage. The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE-Sephadex into 10 pairs of molecular species with n from 1 to 10. The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents. A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid. Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern. The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P = 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.     

4-O-α-d-galactopyranosyl-d-galactose: Efficient synthetic routes from “polygalacturonic acid”

Enzymic hydrolysis of “polygalacturonic acid” gave a mixture of oligomers which was fractionated by ion-exchange chromatography. The resulting di- and tri-saccharides were treated, respectively, with methanol and ethylene oxide, and the resulting esters were reduced with sodium borohydride. Treatment of the products with acetic anhydride and sulfuric acid, followed by deacetylation, produced the title compound.     

A novel mechanism and kinetic model to explain enhanced xylose yields from dilute sulfuric acid compared to hydrothermal pretreatment of corn stover

Pretreatment of corn stover in 0.5% sulfuric acid at 160 °C for 40 min realized a maximum monomeric plus oligomeric xylose yield of 93.1% compared to a maximum of only 71.5% for hydrothermal (no added mineral acid) pretreatment at 180 °C for 30 min. To explain differences in dilute acid and hydrothermal yields, a fast reacting xylan fraction (0.0889) was assumed to be able to directly form monomeric xylose while a slow reacting portion (0.9111) must first form oligomers during hydrothermal pretreatment. Two reactions to oligomers were proposed: reversible from fast reacting xylan and irreversible from slow reacting xylan. A kinetic model and its analytical solution simulated xylan removal data well for dilute acid and hydrothermal pretreatment of corn stover. These results suggested that autocatalytic reactions from xylan to furfural in hydrothermal pretreatment were controlled by oligomeric xylose decomposition, while acid-catalytic reactions in dilute acid pretreatment were controlled by monomeric xylose decomposition.     

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, ¹H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

Microbiological Aspects of Ethylene Oxide Sterilization: III. Effects of Humidity and Water Activity on the Sporicidal Activity of Ethylene Oxide

An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 +/- 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed.     

Synthesis of polyamide oligomers based on 14-amino-3,6,9, 12-tetraoxatetradecanoic acid

A series of oligomers of polyamides based on 14-amino-3,6,9, 12-tetraoxatetradecanoic acid monomers (ATTAn) was synthesized. These materials were designed as monodisperse analogues of poly(ethylene glycol) for use in biomedical applications where reproducible behavior is important. Synthesis of the monomer was evaluated using two routes. For small-scale preparations, tetraethylene glycol (TEG) was monoprotected with dihydropyran, converted to an alkoxide, and alkylated with ethyl bromoacetate. On larger scales, TEG was alkylated directly by treatment with sodium, followed by ethyl bromoacetate. The amine function was introduced by mesylation followed by treatment with sodium azide. Reduction of the azide to amino groups was performed over Pd/C using either hydrogen or formic acid as proton sources. Assembly of the oligomers was accomplished using standard DCC/NHS chemistry and an iterative dimerization sequence after appropriate deprotection of a pair of monomers. The amino group was protected by retaining the azido group as a latent amine. A series of ATTAn oligomers was prepared (n = 1-8). A lipid conjugate of the octamer, ATTA8-DSPE, was synthesized.     

Photocurable biodegradable liquid copolymers: synthesis of acrylate-end-capped trimethylene carbonate-based prepolymers, photocuring, and hydrolysis

Various photocurable liquid biodegradable trimethylene carbonate (TMC)-based (co)oligomers were prepared by ring-opening (co)polymerization of TMC with or without L-lactide (LL) using low molecular weight poly(ethylene glycol) (PEG) (mol wt 200, 600, or 1000) or trimethylolpropane (TMP) as an initiator. Resultant (co)oligomers were pastes, viscous liquids, or liquids at room temperature, depending on the monomer composition and monomer/initiator ratio. Liquid (co)oligomers were subsequently end-capped with acrylate groups. Upon visible-light irradiation in the presence of camphorquinone as a radical generator, rapid liquid-to-solid transformation occurred to produce photocured solid. The photocuring yield increased with photoirradiation time, photointensity, and camphorquinone concentration. The photocured polymers derived from low molecular weight PEG (PEG200) and TMP exhibited much reduced hydrolysis potential compared with PEG1000-derived polymers in terms of weight loss, water uptake, and swelling depth. Force-distance curve measurements by nanoindentation using atomic force microscopy clearly showed that Young's moduli of the photocured polymer films decreased with increasing hydrolysis time. Their potential biomedical applications are discussed.     

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.     

Fluorescent probe and permeability to cells of isopoly (S-carboxymethyl-L-cysteine) derivative of nucleic acid bases.

Isopoly(S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases were found to form stable complex with oligo-DNA in vitro. Fluorescent probed isopoly(S-carboxymethyl cysteine) derivatives of nucleic acid bases were prepared as antisense oligomers. The transfection of the oligomer into cells was carried out by HVJ-liposome method. Fluorescence was observed from the cells treated with HVJ-liposome including fluorescent probed oligomers.     

Laccase-catalyzed polymerization of two phenolic compounds studied by matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with collision-induced dissociation experiments

Enzymatic oxidation of two phenolic compounds [syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) and 2,6-dimethylphenol] was studied. The products of laccase- and laccase-mediator-catalyzed oxidation reactions were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and further analyzed by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) MS with collision-induced dissociation (CID) experiments. For the oligomers of syringic acid, some variability was observed in MALDI-TOF analysis. However, the origin of this variability could not be resolved on the basis of MALDI-TOF spectra due to the poor resolution of the instrument in use. The strength of ESI-FTICR MS was the high-resolution data provided from oligomers of syringic acid. The CID experiments were extremely useful for structural studies of oligomers and verified that the variability of the products was due to the end groups; the phenolic hydroxyl group was modified during the oxidation.     

Impact of RGD nanopatterns grafted onto titanium on osteoblastic cell adhesion

This work reports on the synthesis of titanium bone implants functionalized with nanoparticles (NPs) containing Arg-Gly-Asp-Cys peptide (RGDC) and shows the adhesion behavior of cells seeded on these materials. RGDC peptides were first conjugated to a norbornenyl-poly(ethylene oxide) macromonomer (Nb-PEO). Then, functional NPs with a size of ～300 nm and constituted of polynorbornene core surrounded by poly(ethylene oxide) shell were prepared by ring-opening metathesis polymerization in dispersed medium. The grafting density of these NPs on the titanium surface is up to 2 NPs·μm(-2) (80 pmol of RGDC per cm(-2) of NP surface). Cell adhesion was evaluated using preosteoblast cells (MC3T3-E1). Results of cells cultured for 24 h showed that materials grafted with NPs functionalized with RGDC peptides enhance specific cell adhesion and can create filopodia-like structures among NP sites by stressing the cells.     

Bouquet-type dendrimerlike poly(ethylene oxide)s with a focal aldehyde and peripheral hydroxyls

Doubly functionalized dendrimerlike poly(ethylene oxide)s (PEOs) carrying 16 hydroxyl groups at their periphery and one aldehyde group at their focal point were synthesized up to the fourth generation through an iterative divergent approach. First, a protected aldehyde dihydroxyl compound, namely, 3,3-diethoxy-1,2-propanediol, was used as initiator for the anionic ring-opening polymerization (AROP) of ethylene oxide after partial deprotonation (30%) in dimethyl sulfoxide. The two hydroxyls carried by the PEO chain ends of the first generation were subsequently derivatized so as to generate four hydroxyls via a two-step reaction (allylation and osmylation). The next generations of such dendrimerlike PEOs were grown upon repeating the same cycle of AROP and chain-end modification. At the completion of these reactions, the acetal group present at the core was deprotected under acidic conditions to afford the targeted dendrimerlike PEO of fourth generation with a central aldehyde group. The reactivity and accessibility of the latter function was demonstrated upon its conjugation with aniline used as a model compound.