Chain Precursor Research Articles

Mining the Cinnabaramide Biosynthetic Pathway to Generate Novel Proteasome Inhibitors

The cinnabaramides and salinosporamides are mixed PKS/NRPS natural products isolated from a terrestrial streptomycete and a marine actinomycete, respectively. They interfere with the proteasome and thus potentially inhibit the growth of cancer cells. The compounds exhibit a γ-lactam-β-lactone bicyclic ring structure attached to a cyclohexenyl unit and a PKS side chain. As a first step towards improving anticancer activity and permitting genetic approaches to novel analogues, we have cloned and characterized the cinnabaramide biosynthetic genes from Streptomyces sp. JS360. In addition to the expected PKS and NRPS genes, the cluster encodes functionalities for the assembly of the hexyl side chain precursor. The corresponding enzymes exhibit relaxed substrate specificities towards a number of synthesized precursors, enabling production of novel chlorinated cinnabaramides. These were isolated and analyzed for activity, revealing that derivatives bearing a chlorine atom in the PKS side chain show higher inhibitory potentials towards the proteasome's proteolytic subunits (especially the trypsin and chymotrypsin units) and higher cytotoxicities towards human tumor cell lines than the parent cinnabaramide A. Although their activities towards the proteasome were weaker than that of salinosporamide A, the cinnabaramides were found to inhibit the growth of various fungi with greater potency.

Profiling of skin anti-aging related proteins in human dermal fibroblasts by decursin in Angelica gigas Nakai

The extract of Angelica gigas Nakai by supercritical CO2 increased the expression of collagen synthesis-related proteins in human dermal fibroblast, including type 1(α-2) collagen chain precursor (pI 9.08, MW 129.7), procollagen C-endopeptidase (pI 7.40, MW 47.97), and prolyl 4-hydroxylase (pI 5.49, MW 60.90). It also increased the expression level of interaction-related proteins, α-actinin (pI 5.47, MW 105.5), integrin-β1 (pI 5.27, MW 88.46). The expression levels of these proteins by pure decursin were similar to those by supercritical fluid extract. By the dose concentration experiment, decursin in A. gigas was found to play the major role in expression level increases. Proteome analysis proved that decursin in A. gigas promoted synthesis of proteins related to skin anti-aging in human dermal fibroblasts.

Synthesis of silver nanoparticles within intercalated clay/polymer nanocomposite via in situ electron transfer reaction

In this study, we present a new process for the synthesis of silver nanoparticles using intercalated clay/polymer nanocomposites via in situ electron transfer reduction process. Clay minerals are first intercalated by amphiphilic urethane precursor chains (UAN) and nano-dispersed at hydrophobic monomer (styrene). AgNO 3 crystals were added and mixed with UAN/styrene mixture to dissociate it not only by forming complex with silicate layers of clay minerals but also by making complex with polyethylene oxide chains of UAN chains. Radicals formed by UV-radiation caused reduction of dissolved Ag + to silver nanoparticles (in situ electron transfer reaction) and copolymerization of clay/UAN/styrene mixture (radical polymerization) simultaneously, resulting in direct conversion of the solution to silver nanoparticle dispersed clay/polymer nanocomposite film without additional reduction process. Size and morphology of silver nanoparticles formed within silicate layers depended on the surface property of clay minerals, which could be confirmed by XRD and TEM measurements.

The Differences Between Products Of Gene Expression In Male, Female And Hermaphrodite Cucumber Floral Buds (Cucumis Sativus L.)

AbstractCucumber is a species in which sex expression has been extensively studied. Sexual differentiation is controlled by genotypic and environmental factors. The main genes responsible for sex determination have been described but the mechanism of their action remains unexplained. In this study we attempted to find cDNA clones which can be connected with sex differentiation and flower development in cucumber.Two pairs of nearly isogenic lines: GY-3 (gynoecious – FFMMGG) and HGY-3 (hermaphrodite - FFmmGG), B10 (monoecious - ffMMGG) and 2gg (gynoecious - FFMMgg) were used to search for differences in gene expression in young (1 – 2mm) cucumber floral buds.In order to obtain differentially expressed cDNA clones the differential screening and the differential subtraction chain (DSC) methods were used. Altogether above 900 cDNA clones were isolated and part of them were randomly chosen and sequenced (tab. 1 and 2). To observe the expression patterns of isolated cDNA clones in developing flowers at different developmental stages, we performed in situ RT-PCR. Here we present the results for two cDNA clones designed as 216GY3 and 35GY3.The expression of 35 Gy3 clone, similar to hypotehetical protein from A. Thaliana, was localised in the stamen primordium and petals of floral buds, whereas there was no expression in male and hermaphrodite floral buds. The 216 Gy3 cDNA clone similar to chaperonine 60 beta chain precursor was strongly expressed in the pistil primordium in the male buds, whereas in female and hermaphrodite buds the oserved signals was very weak.SUMMARY & CONCLUSIONS The most of isolated and identified clones are involved in same part of a light or hormone signaling cascade and they may participate in the “cascade of sex expression” in cucumber.Signals of expression of clone 35GY3 were observed only in female cucumber flower buds and were the strongest in a site where as it seems only the development of stamens should be inhibited.The accumulation of large amounts of the transcripts of 216 GY3 clone in primordia of male flower pistils which will thus not develop is interesting.

Sieve‐based device for MALDI sample preparation. III. Its power for quantitative measurements

The solid sample inhomogeneity is a weak point of traditional MALDI deposition techniques that reflects negatively on quantitative analysis. The recently developed sieve-based device (SBD) sample deposition method, based on the electrospraying of matrix/analyte solutions through a grounded sieve, allows the homogeneous deposition of microcrystals with dimensions smaller than that of the laser spot. In each microcrystal the matrix/analyte molar ratio can be considered constant. Then, by irradiating different portions of the microcrystal distribution an identical response is obtained. This result suggests the employment of SBD in the development of quantitative procedures. For this aim, mixtures of different proteins of known molarity were analyzed, showing a good relationship between molarity and intensity ratios. This behaviour was also observed in the case of proteins with quite different ionic yields. The power of the developed method for quantitative evaluation was also tested by the measurement of the abundance of IGPP[Oxi]GPP[Oxi]GLMGPP (m/z 1219) present in the collagen-α-5(IV) chain precursor, differently expressed in urines from healthy subjects and diabetic-nephropathic patients, confirming its overexpression in the presence of nephropathy. The data obtained indicate that SBD is a particularly effective method for quantitative analysis also in biological fluids of interest.

Efficacy of calcium supplementation for human bone health by mass spectrometry profiling and cathepsin K measurement in plasma samples

Osteoporosis is a common disease among older people, especially postmenopausal women. Calcium supplementation is effective in decreasing the occurrence of osteoporosis. We tested the effect of different calcium sources (i.e., calcium carbonate chew, milk mineral chew, milk drink and placebo chew) by direct mass spectrometry (dMS) profiling and cathepsin K measurement in the serum of subjects. The dMS method is promising for plasma biomarker discovery, and cathepsin K level in the plasma is an indicator for osteoporosis. Our result shows that dMS detected characteristic ion peaks after different calcium supplement interventions; ion peak 4281.0 m/z was commonly inhibited by all three treatments. This ion peak was identified to be a fragment of follistatin-related protein 3 precursor by means of the "Lift" mode of MS/MS. The other differential ion peaks were also successfully identified: 1786.5 m/z (upregulated after calcium carbonate chew) was shown to be one fragment of transcription factor jun-B; the parent protein of 3504.7 m/z (upregulated after milk drink) was a collagen alpha-2 (type I) chain precursor; the ion peak of 3359.6 m/z (downregulated after milk mineral chew) was one fragment of family 31 glucosidase. Cathepsin K is significantly inhibited only by calcium carbonate chew treatment, indicating this form of calcium supplement has some advantage over other sources of supplementation.

Analysis of the urinary peptidome associated withHelicobacter pyloriinfection

To investigate the relationship between urinary peptide changes and Helicobacter pylori (H. pylori) infection using urinary peptidome profiling. The study was performed in volunteers (n=137) who gave informed consent. Urinary peptides were enriched by magnetic beads based weak cation exchange chromatography and spectrums acquired by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). ClinProTools bioinformatics software was used for statistical analysis and the recognition of peptide patterns. The marker peptides were identified by LTQ Obitrap XL tandem MS. Approximately 50 proteins or peptides which loaded onto the magnetic beads were detected by MALDI-TOF MS. By optimizing the parameters of the model, the Genetic Algorithm model had good recognition capability (97%) and positive predictive value (94%). Based on the model, 2 markers with molecular masses of 6788 and 1912 Da were found that differentiated between H. pylori positive and negative volunteers. The m/z 1912 sequence was parsed as SKQFTSSTSYNRGDSTF. The peptide was identified as isoform 1 of the fibrinogen α chain precursor, whose concentration in urine was markedly higher in H. pylori infected volunteers than in H. pylori non-infected ones. The appearance of urinary fibrinogen degradation products is caused by an active H. pylori-induced process.

뫼스바우어 분광법에 의한 양친매성 고분자 망상구조 나노복합체의 자기적 성질 연구

Magnetic nanocomposites contained iron oxide were synthesized by through cross-linking polymerization of dimethylacetamide (DMAc) solution and toluen solution on the amphiphilic polymer networks based on urethan acrylate nonionomer (UAN) precursor chains. For the study on microscopic structures and magnetic properties of the magnetic nanoparticles, FESEM and XRD and Mossbauer spectroscopy were used. The results investigated show that there are magnetic nanoparticles of in samples and the magnetic nanocomposites contained iron oxide in polymer networks of UAN using DMAc solution are more smaller than using toluen solution. All of the Fe ions in the samples present and the magnetic property of samples are paramagnetic by superparamagnetic effect at room temperature.

Synthesis and characterization of cross-linked poly(esterurethane)s for elastomeric bearings

Summary — Cross-linked poly(esterurethane) elastomers (PEURs) have been synthesized in a two-step synthesis. In the first step poly(ethylene adipate) diol was reacted with 4,4’-diphenylmethane diisocyanate, then the obtained prepolymers were chain extended with bifunctional precursor (1,3-propane diol or 1,5-pentane diol) chains and a trifunctional cross-linker (glycerine) at stoichiometric ratios, resulting in a series of cross-linked products with various hard segment (HS) of various structure and content. Their thermal and mechanical properties were determined. Due to phase separation and cross-linked hard segments, propane diol based polyurethane exhibited high mechanical properties. The replacement of propane diol with pentane diol had a negative effect on these mechanical properties. The described cross-linked PEURs possessed strain/shape recovery properties.

Effect of pyrolysis temperature on the microstructure of disordered carbon nanowires

Without any catalyst or pressure, bulk non-graphitizing carbon does not exhibit any change in its atomic order (or graphitize) even at 3000 °C. We propose a route for significant graphitization in polymer-derived disordered carbon materials at moderate temperatures by pre-aligning the precursor polymer chains. Accordingly, we employ a template-based fabrication process, where the capillary forces align the polyfurfuryl alcohol chains in the form of nanowires with diameter from 100–200 nm. The nanowires are pyrolyzed at temperatures ranging from 600 °C to 2000 °C and characterized with transmission electron and Raman microscopy as well as electrical conductivity measurements. The observed significant change in atomic rearrangement is explained by the alignment of precursor chains during the templated synthesis.

Toughness and fracture energy of PDMS bimodal and trimodal networks with widely separated precursor molar masses

End-linked PDMS bimodal and trimodal networks display enhanced mechanical properties in uniaxial extension over those of unimodal networks with similar modulus when the molar masses of their precursor chains are widely separated. These multimodal networks have optimal mechanical properties when the short chains are near their overlap concentration and sustain most of the load, but the volume of the system is still dominated by the ductile long chain component. Such elastomers can be stretched to large elongations before fracture while displaying an upturn in stress at high strain. Improvement in fracture energy of pre-cut bimodal and trimodal networks over that of unimodal networks is much less pronounced and appears to be dictated by the average molar mass of the effective elastic strands in each network.

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra- and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration-based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration-based washing. In the centrifugation-based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin-G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3-4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin-G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood.

Proteomic profiling of plasma microparticles following deep-vein thrombosis

Evaluation of: Ramacciotti E, Hawley A, Wrobleski S et al. Proteomics of microparticles after deep vein thrombosis. Thromb. Res. DOI: 10.1016/j.thromres.2010.01.019 (2010) (Epub ahead of print).Deep-vein thrombosis (DVT) remains a significant health problem, underlining the need for improved understanding of the molecular mechanisms that regulate this condition. Hypercoagulability and DVT are associated with microparticle (MP) release from both blood and vascular cells into the plasma. In order to identify novel effectors and/or biomarkers of DVT, Ramacciotti et al. have compared the proteomic profiles of plasma MPs isolated from six healthy individuals, nine with DVT and nine with leg pain but negative for DVT. Using an isobaric tags for relative and absolute quantitation (iTRAQ)-based approach, galectin-3 binding protein and α-2 macroglobulin were shown to be significantly upregulated (20% and above) in MPs of individuals with DVT. Nine proteins depleted, including fibrinogen β and γB chain precursors. Their methods and significant findings are reviewed within this article.

Association of hydrophobically α,ω‐end‐capped poly(2‐methyl‐2‐oxazoline) in water

AbstractThe α,ω‐end‐capped poly(2‐methyl‐2‐oxazoline) (Cn‐POXZ‐Cn) have been synthesized by a one‐pot process using cationic ring‐opening polymerization with an appropriate initiator and terminating agent. The polymers bearing different alkyl groups C12 and C18 have molecular weight in the range of 2.4 × 103 to 14 × 103 with a small polydispersity index. The solution behavior of the free chains has been analyzed in a nonselective solvent, dichloromethane, by small‐angle neutron scattering and dynamic light scattering. These amphiphilic polymers associate in water to form flower‐like micellar structures. Critical micelle concentrations, investigated by fluorescence technique, are in the range of 0.03–0.5 g L−1 and are dependent on the hydrophilic/lipophilic balance. The structural properties of the aggregates have also been investigated by viscometry. Intrinsic viscosities of these polymers are in the same range as that of the precursors poly(2‐methyl‐2‐oxazoline) (POXZ) and mono‐functionalized polymers. Large viscosity increase corresponding to intermicellar bridging was observed in the vicinity of the micelle overlap concentration. Addition of hydroxypropyl β‐cyclodextrin (HβCD) has dissociated the aggregates and the intrinsic viscosities of the HβCD‐end‐capped chains have become comparable with the ones of POXZ precursor chains. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2477–2485, 2010

Abstract 4585: Verification of prostate cancer biomarker candidates by nanoparticle-capture MRM

Abstract Prostate adenocarcinoma (PCa) is highly diffuse between older men, and biomarkers that allow discrimination of cancerous pathologies from benign diseases, identification of clinical aggressive cancer with high specificity and monitor cancer progression are required. Recently we applied a new workflow for the discovery of candidate biomarkers based on hydrogel nanoparticle capture of LMW serum borne analytes. Comparing serum from pre and post prostatectomy patients we identified 112 candidates biomarkers. Our novel nanoparticle technology provides a rapid and near 100% efficient means to capture and greatly amplify the resting concentration of LMW protein analytes. Since most of these markers do not have well performing specific antibodies commercially available, we utilized MRM to verify their differential expression as first identified by Orbitrap-MS/MS. Hydrogel nanoparticle serum pre-processing was used for both Orbitrap-LTQ based discovery and MRM verification such that the same analyte fractionation workflow was used but different downstream mass spectrometry. MRM was performed on a Thermo Quantum triple quadrupole-MS using the nanoparticle trapped analyte archive as the input for analysis. The following biomarker candidates were selected for MRM verification: Apolipoprotein E, Diacylglycerol kinase alpha, complement 1 subcomponent q B chain precursor, complement component 1 q subcomponent gamma polypeptide, serpin peptidase inhibitor, vitronectin precursor, angiotensinogen preproprotein, H4 histone family member J, fibronectin 1 isoform 2, orosomucoid 1 precursor, alpha-2-HS-glycoprotein, annexin I. Several prostate candidate biomarkers were verified by Nanoparticle capture-MRM, thus providing data supporting the use and continued improvement of this novel biomarker discovery system for the identification of protein biomarker panels for early disease detection and therapeutic monitoring. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4585.

Proteomics of microparticles after deep venous thrombosis

Background Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells. Methods To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05. Results Two proteins (Galectin-3 Binding Protein, [Gal3BP], R = 1.76 and Alpha-2 macroglobulin [A2M] R = 1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R = 0.65). Conclusions These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.

Effect of network structure on the stress–strain behaviour of endlinked PDMS elastomers

Endlinked poly(dimethylsiloxane) (PDMS) networks synthesized from telechelic precursor chains of different molar mass were prepared with varying volume fractions of non-reactive chains acting as solvent. Uniaxial extension and compression measurements were performed on these networks to investigate their stress–strain behaviour. The effect of the network structure and of solvent on the stress–strain behaviour is examined by controlling the extent of crosslinks and entanglements during the network synthesis. The master curve of the Rubinstein and Panyukov non-affine slip-tube (NAST) model provide an adequate fit to most of the extension and compression data. Furthermore, the crosslink and entanglement parameters of the NAST model ( G c and G e) are found to be in general in reasonable agreement with the 2 C 1 and 2 C 2 parameters of the Mooney–Rivlin continuum model applied to the extension data. For high molar mass precursor chains, the entanglement contribution to the modulus surpasses the crosslink contribution.

Structural Hierarchy of Regulatory Elements in the Folding and Transport of an Intestinal Multidomain Protein

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHalpha, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHbeta region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHbeta and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydrolase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHalpha. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV.

Effects of Erysiphe pisi on protein profiles and ribulose-1,5-bisphosphate carboxylase content in resistant and susceptible pea (Pisum sativum × Pisum fulvum) plants

The fate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was examined in leaves of both resistant and susceptible plants from a Pisum sativum × Pisum fulvum cross after challenge with Erysiphe pisi. High performance liquid chromatography and 1-dimensional gel electrophoresis revealed a 51 kDa protein that was much more abundant in leaves of resistant versus susceptible plants, and which was identified as the Rubisco large chain precursor by mass spectrometry. Greater quantities of Rubisco in resistant tissue suggested that the protein may serve as an important nutrient source for E. pisi in infected susceptible leaves. Liquid chromatography — tandem mass spectrometry revealed three defense-response proteins only in extracts from resistant tissue, while proteins similar to the antimicrobial peptides viscotoxin and phoratoxin from mistletoes, and bubble protein from the yeast Williopsis mrakii, were found only in susceptible tissue. Greater numbers of DNA synthesis and regulation proteins were also identified in the susceptible vs. resistant tissue, probably resulting from changes in host metabolism induced by E. pisi.

Poly(terthiophene)s from copolymer precursors via solid-state oxidative conversion

Herein, we report the synthesis of conducting poly (terthiophene)s using a side chain precursor polymer approach. Random copolymers were prepared by ring opening metathesis polymerization of two norbornylene monomers, one containing a pendant terthiophene group and the other containing a pendant acetate group. Solid-state oxidative conversion of the terthiophene units was used to produce conductive polymers. Oxidative solid-state conversion was successful for copolymers containing as little as 1 mol % of terthiophene comonomer. The electrical and optical properties of CPs were studied as a function of the amount of electroactive moiety, terthiophene (3T), present in the copolymer. The CPs were found to have conductivity varying between 10−1 and 10−4 S/cm depending on the precursor copolymer compositions. The CPs obtained from all precursors had no significant difference in their energy gaps and showed blue to orange color transitions when switching from the oxidized to the neutral states, respectively. The absorbance intensity at 426 nm for poly(3T) from the precursors fits the Beer–Lambert law corresponding to the range of initial 3T content in the precursor copolymer composition (from 1 to 100 mol %). © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 756–763, 2010