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Sample records for l-lysine cap alpha

  1. [L-lysine-alpha-oxidase activity of some Trichoderma species].

    PubMed

    Smirnova, I P; Khaduev, S Kh

    1984-01-01

    Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.

  2. [Antiherpetic activity of l-lysine-alpha-oxidase in different dosage forms].

    PubMed

    Selishcheva, A A; Alekseev, S B; Smirnova, I P; Podboronov, V M

    2003-01-01

    The antiviral effect of different medicinal forms on base of L-lysin-alpha-oxidase (LO) in treatment of experimental keratits and skin diseases induced by herpes simplex virus type 1 (HSV-1) at the rabbits was examined. Treatment with gel form of LO was more effective in comparison with water solution form of LO. This effect was tested by morphological, histological and immunoblotting methods in dynamics of viral disease.

  3. N alpha- and N epsilon-D-galacturonoyl-L-lysine amides: properties and possible occurrence in plant cell walls.

    PubMed

    Perrone, P; Hewage, C M; Sadler, I H; Fry, S C

    1998-12-01

    Three representatives of a novel class of amide (isopeptide) glycoconjugates have been synthesised: N alpha-D-galacturonoyl-L-lysine and N epsilon-D-galacturonoyl-L-lysine and N epsilon-D-polygalacturonoyl-L-lysine. Galacturonoyl-lysine amide bonds were labile in 2 M trifluoroacetic acid at 120 degrees and in alkali, but relatively stable in cold acid. The amide bonds were resistant to digestion by Driselase, Pronase and trypsin. The polysaccharide backbone of N epsilon-D-polygalacturonoyl-L-lysine was hydrolysed by Driselase to yield two major ninhydrin-positive compounds which were shown by 1H and 13C NMR spectroscopy to be tri- and tetra-alpha-(1-->4)-D-galacturonoyl-L-lysines. To investigate the possible natural occurrence of N-galacturonoyl isopeptide bonds, we fed cell-suspension cultures of spinach and tomato with D-[6-14C]glucuronic acid, which radio-labels pectic polysaccharides. The radioactive cell walls were digested with, sequentially, Driselase, mild acid, and proteinases. On electrophoresis at pH 2.0, several of the radioactive digestion-products were cathodic. Some of the cathodic products yielded [14C]galacturonic acid upon complete acid hydrolysis. The existence of these products is compatible with the presence of novel N-galacturonoyl isopeptide bonds, which could serve as cross-links in plant cell walls.

  4. Enzymic synthesis of L-lysine from DL-alpha-amino-epsilon-caprolactam by new microbial strains.

    PubMed

    Plhácková, K; Vojtísek, V; Plachý, J

    1982-01-01

    The production of L-lysine from DL-alpha-amino-epsilon-caprolactam (DL-ACL) by new strains producing L-alpha-amino-epsilon-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis of L-ACL by Cryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified as Pseudomonas sp. were determined. Synthesis of L-alpha-amino-epsilon-caprolactamase is induced by DL-ACL or L-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10% aqueous DL-ACL to L-lysine by a mixture of native cells in a mass ratio of 1 : 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 degrees C and pH 8.0.

  5. [Effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai ВКМF-4268D on pheochromocytoma PC12 cell line].

    PubMed

    Lukasheva, E V; Ribakova, Yu S; Fedorova, T N; Makletsova, M G; Arinbasarova, A Yu; Medentzev, A G; Berezov, T T

    2015-01-01

    L-Amino acid oxidases (L-ААО, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Н2О2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ВКМF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.

  6. Broadening the antibacterial spectrum of histidine kinase autophosphorylation inhibitors via the use of ε-poly-L-lysine capped mesoporous silica-based nanoparticles.

    PubMed

    Velikova, Nadya; Mas, Nuria; Miguel-Romero, Laura; Polo, Lorena; Stolte, Ellen; Zaccaria, Edoardo; Cao, Rui; Taverne, Nico; Murguía, José Ramón; Martinez-Manez, Ramon; Marina, Alberto; Wells, Jerry

    2017-02-01

    Two-component systems (TCS) regulate diverse processes such as virulence, stress responses, metabolism and antibiotic resistance in bacteria but are absent in humans, making them promising targets for novel antibacterials. By incorporating recently described TCS histidine kinase autophosphorylation inhibitors (HKAIs) into ε-poly-L-lysine capped nanoparticles (NPs) we could overcome the Gram negative (Gr-) permeability barrier for the HKAIs. The observed bactericidal activity against Gr- bacteria was shown to be due to the enhanced delivery and internalization of the HKAIs and not an inhibitory or synergistic effect of the NPs. The NPs had no adverse effects on mammalian cell viability or the immune function of macrophages in vitro and showed no signs of toxicity to zebrafish larvae in vivo. These results show that HKAIs are promising antibacterials for both Gr- and Gr+pathogens and that NPs are a safe drug delivery technology that can enhance the selectivity and efficacy of HKAIs against bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. L-lysine fermentation.

    PubMed

    Anastassiadis, Savas

    2007-01-01

    Amino acids are the basic bioelements of proteins, which are the most important macromolecules for the functions of humans and animals. Out of the 20 L-amino acids, ecumenically found in most of living organisms, L-lysine is one of the 9 amino acids which are essential for human and animal nutrition. L-lysine is useful as medicament, chemical agent, food material (food industry) and feed additive (animal food). Its demand has been steadily increasing in recent years and several hundred thousands tones of L-lysine (about 800,000 tones/year) are annually produced worldwide almost by microbial fermentation. The stereospecificity of amino acids (the L isomer) makes the fermentation advantageous compared with synthetic processes. Mutant auxotrophic or resistant to certain chemicals strains of so-called gram positive coryneform bacteria are generally used, including the genera Brevibacterium and Corynebacterium, united to the genus. The significance of Research and Development increased rapidly since the discovery of fermentative amino acid production in the fifties (S. Kinoshita et al., Proceedings of the International Symposium on Enzyme Chemistry 2:464-468 (1957)), leading to innovative fermentation processes which replaced the classical manufacturing methods of L-lysine like acid hydrolysis. L-Lysine is separated and purified by suitable downstream processes involving classical separation or extraction methods (ultrafiltration or centrifugation, separation or ion exchange extraction, crystallization, drying) and is sold as a powder. Alternatively, spray dried pellets or liquid fermentation broth can be used as animal feed supplement. On behalf of today's strong competition in amino acid industry, Biotechnology companies are continuously aiming in innovative research developments and use complex management concepts and business strategies, towards gaining market leadership in the field of amino acid production.

  8. Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-Helix and beta-sheet conformations

    SciTech Connect

    Grigsby, J.J.; Blanch, H.W.; Prausnitz, J.M.

    2001-10-30

    Because poly-L-lysine (PLL) can exist in the {alpha}-helix or {beta}-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and {alpha}-helix or {beta}-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the {beta}-sheet conformation than for the {alpha}-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.

  9. High activity of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and cytolytic perforin in cloned cell lines is not demonstrable in in-vivo-induced cytotoxic effector cells.

    PubMed Central

    Dennert, G; Anderson, C G; Prochazka, G

    1987-01-01

    Recent observations have suggested striking similarities between complement-mediated and cell-mediated lysis. Both pathways share the terminal insertion of channels into target membranes, and unique esterases have been postulated to participate in the activation of cytolytic effector molecules. Since killer-specific esterases and channel-forming proteins can be demonstrated in in vitro cell lines, it is important to ascertain that the described esterase and channel-forming proteins are also present in killer cells from in vivo sources. Results presented here show that killer-cell-specific N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase is induced in vitro concomitant with the sensitization of cytotoxic effector cells. In contrast, in-vivo-primed cytotoxic T cells or natural killer (NK) cells fail to express high levels of this enzyme. Assay of different cytotoxic effector cells reveals the presence of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase in clones with T killer and NK activity, but enzyme levels do not correlate with cytolytic activity nor does inhibition of esterase activity interfere with granule-mediated cell lysis. A similar result is seen with granule-mediated cytolytic activity. Cloned NK and T killer cell lines possess granules that are able to lyse erythrocyte targets. However, T killer cells sensitized in mixed lymphocyte culture or in vivo have no detectable cytotoxic granules. Cytotoxic granules are also not detected in NK cells isolated from animals. PMID:2955414

  10. Intermediate filaments in. cap alpha. -keratins

    SciTech Connect

    Fraser, R.D.B.; MacRae, T.P.; Parry, D.A.D.; Suzuki, E.

    1986-03-01

    Previous x-ray diffraction studies on the ..cap alpha..-keratins of hair and wool have revealed that the intermediate filaments (IF) have a helical structure rendered imperfect by a precisely defined dislocation. It has also been possible to deduce a surface lattice for the IF and to determine the number of IF molecules associated with each lattice point. In this work this information is combined with data on the ionic interactions between the coiled-coil rope segments of the IF molecules to provide a plausible model for the pattern of interactions that stabilize the framework of the IF in the hard ..cap alpha..-keratins. Similar interaction studies of the proteins from the IF in the so-called soft ..cap alpha..-keratin from the stratum corneum layer of the skin suggest that they are likely to have an essentially similar pattern.

  11. Changes in the amide I FT-IR bands of poly-L-lysine on spray-drying from alpha-helix, beta-sheet or random coil conformations.

    PubMed

    Mauerer, Alexander; Lee, Geoffrey

    2006-02-01

    Poly-L-lysine (PLS) was spray-dried in a laboratory-scale, mini spray-dryer at Tin/Tout =150/90-95 degrees C from three different liquid feeds composed mainly of alpha-helix, beta-sheet or random coil conformations of the homopolypeptide. FT-IR analysis of the liquid feeds, the spray-dried solids, and the re-dissolved solids was performed by considering the deconvoluted and second-derivative amide I spectra, as well as a Gaussian curve fitting procedure. All three initial conformations were transformed by spray-drying to anti-parallel beta-sheet with bands at 1623 and 1690 cm(-1). The beta-sheet liquid feed showed a band at 1616 cm(-1) indicating a denatured, extended chain structure that was also converted to anti-parallel beta-sheet on spray-drying. The shift to beta-sheet cannot therefore be a simple result of forming the conformation with the strongest H-bonds in the dried state. We suggest that steric effects arising from the close approach of the globular polypeptide molecules during drying make the anti-parallel beta-sheet structure energetically favorable in the solid state. This suggestion is supported by the effects of trehalose on the FT-IR amide I bands of the spray-dried PLS. No stabilizing effects were observed on either the initial alpha-helix or beta-sheet (extended chain) conformations. Random coil could be partially stabilized. Again, no direct relation to H-bond strength is evident. The efficacy of the trehalose could be related to the ability of newly-formed trehalose/PLS intermolecular H-bonds to stabilize the intramolecular H-bonds of the secondary structural elements.

  12. Use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-(3-/sup 3/H)Trimethyl-DL-lysine

    SciTech Connect

    Stein, R.; England, S.

    1981-09-01

    6-N-(3-/sup 3/H)Trimethyl-DL-lysine was synthesized from 6-N-acetyl-L-lysine by the following chemical scheme: 6-N-acetyl-L-lysine ..-->.. 2-keto-6-N-acetylcaproic acid ..-->.. 2-(3-/sup 3/H)keto-6-N-acetylcaproic acid ..-->.. 2-(3-/sup 3/H)keto-6-N-acetylcaproic acid oxime ..-->.. 6-N-(3-/sup 3/H)acetyl-DL-lysine ..-->.. DL-(3-/sup 3/H)lysine ..-->.. 2-N-(3-/sup 3/H)formyl-DL-lysine ..-->.. 2-(3-/sup 3/H)formyl-6-N-trimethyl-DL-lysine ..-->.. 6-N-(3-/sup 3/H)trimethyl-DL-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rate kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-L-lysine hydroxylase activity as measured by tritium release from 6-N-(3-/sup 3/H)trimethyl-DL-lysine were: ..cap alpha..-ketoglutarate, ferrous ions, L-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-(3-/sup 3/H)trimethyl-DL-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-L-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.

  13. Biological efficacy of L-lysine preparations containing biomass compared to L-lysine-HCl.

    PubMed

    Schutte, J B; Pack, M

    1994-01-01

    In two growth experiments on broilers of 6 to 27 days of age, the biological efficacy of three L-lysine preparations containing biomass was evaluated in comparison to L-lysine.HCl. L-lysine from the different sources was added in graded levels to a basal diet deficient in lysine, but adequate in all other nutrients. From multi-exponential regression analysis of weight gain and feed conversion responses, biological efficacy of the three L-lysine preparations containing biomass was estimated to be 97%, 103%, and 113%, respectively. None of these estimates was significantly different from 100%. Thus, it is concluded that the efficacy of the L-lysine preparations to deliver lysine is equivalent to L-lysine.HCl.

  14. cap alpha. -2 adrenergic receptor: a radiohistochemical study

    SciTech Connect

    Unnerstall, J.R.

    1984-01-01

    ..cap alpha..-2 adrenergic agents have been shown to influence blood pressure, heart rate and other physiological and behavioral functions through interactions with adrenergic pathways within the central nervous system. Pharmacologically relevant ..cap alpha..-1 adrenergic receptors were biochemically characterized and radiohistochemically analyzed in intact tissue sections of the rat and human central nervous system. The anatomical distribution of the ..cap alpha..-2 receptors, labeled with the agonist (/sup 3/H)para-aminoclonidine, verified the concept that ..cap alpha..-2 receptors are closely associated with adrenergic nerve terminals and that ..cap alpha..-2 agents can influence autonomic and endocrine function through an action in the central nervous system. Since ..cap alpha..-2 agonists can influence sympathetic outflow, ..cap alpha..-2 binding sites were closely analyzed in the intermediolateral cell column of the thoracic spinal cord. The transport of putative presynaptic ..cap alpha..-2 binding sites in the rat sciatic nerve was analyzed by light microscopic radiohistochemical techniques. Finally, in intact tissue section of the rat central nervous system, the biochemical characteristics of (/sup 3/H)rauwolscine binding were analyzed. Data were also shown which indicates that the synthetic ..cap alpha..-2 antagonist (/sup 3/H)RX781094 also binds to ..cap alpha..-2 receptors with high-affinity. Further, the distribution of (/sup 3/H)RX781094 binding sites in the rat central nervous system was identical to the distribution seen when using (/sup 3/H)para-aminoclonidine.

  15. MFTF-. cap alpha. + T shield design

    SciTech Connect

    Gohar, Y.

    1985-01-01

    MFTF-..cap alpha..+T is a DT upgrade option of the Tandem Mirror Fusion Test Facility (MFTF-B) to study better plasma performance, and test tritium breeding blankets in an actual fusion reactor environment. The central cell insert, designated DT axicell, has a 2-MW/m/sup 2/ neutron wall loading at the first wall for blanket testing. This upgrade is completely shielded to protect the reactor components, the workers, and the general public from the radiation environment during operation and after shutdown. The shield design for this upgrade is the subject of this paper including the design criteria and the tradeoff studies to reduce the shield cost.

  16. MFTF-. cap alpha. + T progress report

    SciTech Connect

    Nelson, W.D.

    1985-04-01

    Early in FY 1983, several upgrades of the Mirror Fusion Test Facility (MFTF-B) at Lawrence Livermore National Laboratory (LLNL) were proposed to the fusion community. The one most favorably received was designated MFTF-..cap alpha..+T. The engineering design of this device, guided by LLNL, has been a principal activity of the Fusion Engineering Design Center during FY 1983. This interim progress report represents a snapshot of the device design, which was begun in FY 1983 and will continue for several years. The report is organized as a complete design description. Because it is an interim report, some parts are incomplete; they will be supplied as the design study proceeds. As described in this report, MFTF-..cap alpha..+T uses existing facilities, many MFTF-B components, and a number of innovations to improve on the physics parameters of MFTF-B. It burns deuterium-tritium and has a central-cell Q of 2, a wall loading GAMMA/sub n/ of 2 MW/m/sup 2/ (with a central-cell insert module), and an availability of 10%. The machine is fully shielded, allows hands-on maintenance of components outside the vacuum vessel 24 h after shutdown, and has provisions for repair of all operating components.

  17. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    SciTech Connect

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  18. Anisotropic. cap alpha. -emission of on-line separated isotopes

    SciTech Connect

    Wouters, J.; Vandeplassche, D.; van Walle, E.; Severijns, N.; Van Haverbeke, J.; Vanneste, L.

    1987-12-10

    The technical realization of particle detection at very low temperatures (4K) has made it possible to study for the first time the anisotropic ..cap alpha..-decay of oriented nuclei which have been produced, separated and implanted on line. The measured ..cap alpha..-angular distributions reveal surprising new results on nuclear aspects as well as in solid state physics. The nuclear structure information from these data questions the older ..cap alpha..-decay theoretical interpretation and urges for a reaxamination of the earliest work on anisotropic ..cap alpha..-decay.

  19. Structural Basis for l-Lysine Feedback Inhibition of Homocitrate Synthase

    SciTech Connect

    Bulfer, Stacie L.; Scott, Erin M.; Pillus, Lorraine; Trievel, Raymond C.

    2010-09-02

    The {alpha}-aminoadipate pathway of lysine biosynthesis is modulated at the transcriptional and biochemical levels by feedback inhibition. The first enzyme in the {alpha}-aminoadipate pathway, homocitrate synthase (HCS), is the target of the feedback regulation and is strongly inhibited by L-lysine. Here we report the structure of Schizosaccharomyces pombe HCS (SpHCS) in complex with L-lysine. The structure illustrates that the amino acid directly competes with the substrate 2-oxoglutarate for binding within the active site of HCS. Differential recognition of the substrate and inhibitor is achieved via a switch position within the ({alpha}/{beta}){sub 8} TIM barrel of the enzyme that can distinguish between the C5-carboxylate group of 2-oxoglutarate and the {epsilon}-ammonium group of L-lysine. In vitro and in vivo assays demonstrate that mutations of the switch residues, which interact with the L-lysine {epsilon}-ammonium group, abrogate feedback inhibition, as do substitutions of residues within the C-terminal domain that were identified in a previous study of L-lysine-insensitive HCS mutants in Saccharomyces cerevisiae. Together, these results yield new insights into the mechanism of feedback regulation of an enzyme central to lysine biosynthesis.

  20. Trafficking of. cap alpha. -L-fucosidase in lymphoid cells

    SciTech Connect

    DiCioccio, R.A.; Brown, K.S.

    1987-05-01

    The quantity of ..cap alpha..-L-fucosidase in human serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. To investigate this, lymphoid cell lines derived from individuals with either low, intermediate or high ..cap alpha..-L-fucosidase in serum were established. Steady state levels of extracellular ..cap alpha..-L-fucosidase protein and activity overlapped among the cell lines. Thus, in vivo serum phenotypes of ..cap alpha..-L-fucosidase are not adequately expressed in this system. ..cap alpha..-L-Fucosidase was also metabolically labelled with /sup 35/S-methionine, immunoprecipitated, and examined by SDS-PAGE. Cells pulse-labelled from 0.25-2 h had a major intracellular form of enzyme (Mr = 58,000). Cells pulsed for 1.5 h and chased for 21 h with unlabeled methionine had an intracellular form of Mr = 60,000 and an extracellular form of Mr = 62,000. Cells treated with chloroquine had only the 58,000-form both intra- and extra-cellularly. Moreover, chloroquine did not effect the quantitative distribution of ..cap alpha..-L-fucosidase between cells and medium. In fibroblasts, chloroquine enhanced the secretion of newly made lysosomal enzymes and blocked the processing of intercellular enzyme forms from a higher to a lower molecular mass. Thus, there are trafficking differences between ..cap alpha..-L-fucosidase in lymphoid cells and lysosomal enzymes in fibroblasts. This suggests that alternative targeting mechanisms for lysosomal enzymes exist in these cells.

  1. Systematics of. cap alpha. decay of even--even isotones

    SciTech Connect

    Poplavskii-breve, I.V.

    1987-02-01

    On the basis of an analysis of experimental data we have investigated for the first time the ..cap alpha.. decay of even--even isotones. We have established that the ..cap alpha..-decay energy of isotones depends on the number of protons approximately according to a linear law. We have shown that the Geiger--Nuttall law is valid both for isotopes and isobars, and also for isotones. The deviations from the Geiger--Nuttall law are due to the shell structure of the nucleus. The regularities observed in the ..cap alpha.. decay of isotones have been used to estimate the magnitudes of the ..cap alpha..-decay energies, the kinetic energies of the emitted ..cap alpha.. particles, and the partial half-lives for ..cap alpha.. decay of the known and unknown neutron--deficient nuclei /sup 202//sup ,//sup 204/Ra, /sup 210/Th, /sup 228//sup ,//sup 230/Pu, /sup 234//sup ,//sup 236/Cm, /sup 242//sup ,//sup 244/Fm, /sup 250//sup ,//sup 258/No, and /sup 254//sup ,//sup 256/Ku.

  2. Synthesis and Characterization of L-Lysine Conjugated Silver Nanoparticles Smaller Than 10 nM

    PubMed Central

    Bonor, Jeremy; Reddy, Vandhana; Akkiraju, Hemanth; Dhurjati, Prasad; Nohe, Anja

    2015-01-01

    A rapid and convenient batch method for synthesizing lysine-conjugated silver nanoparticles of approximately 5 nm of size was developed. Nanoparticles of size less than 100 nm exhibit significant medical potential. L-Lysine demonstrates potential for therapeutic applications and silver nanoparticles are an optimal choice for drug delivery because of its intrinsic anti-platelet, anti-bacterial and anti-inflammatory capabilities. Current synthesis protocols for Lysine-capped particles under 10 nm are time consuming and tedious and allow only for the sythesis of small quantities of particles. The synthesis of Lysin-capped silver nanoparticles was based on the reaction in which AgNO3 was reduced by excess NaBH4. L-Lysine, a known essential amino acid, served as the capping agent to minimize initial aggregation. The particles were then separated by size chromatography. Capping occurred through the amide bond on L-Lysine as determined by FT-IR. The conjugation of the particle to the amide bond is important, since this leaves the amino group of Lysine open to further modifications. The particles were further characterized in regards to their shape, size and stability. Finally we demonstrated that the synthesized particles exhibit limited to no toxicity in cells, using HEK 293 cell line as a model system. Our sythesis protocol can be successfully used for scale-up and synthesis of high quantities of nanoparticles. PMID:26478827

  3. Kinetic peculiarities of human tissue kallikrein: 1--substrate activation in the catalyzed hydrolysis of H-D-valyl-L-leucyl-L-arginine 4-nitroanilide and H-D-valyl-L-leucyl-L-lysine 4-nitroanilide; 2--substrate inhibition in the catalyzed hydrolysis of N alpha-p-tosyl-L-arginine methyl ester.

    PubMed

    Sousa, Marinez O; Miranda, Tânia L S; Maia, Caroline N; Bittar, Eustáquio R; Santoro, Marcelo M; Figueiredo, Amintas F S

    2002-04-01

    Hydrolysis of D-valyl-L-leucyl-L-lysine 4-nitroanilide (1), D-valyl-L-leucyl-L-arginine 4-nitroanilide (2), and N alpha-p-tosyl-L-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis-Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis-Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme-substrate complex and a free substrate molecule.

  4. Amiloride interacts with renal. cap alpha. - and. beta. -adrenergic receptors

    SciTech Connect

    Howard, M.J.; Mullen, M.D.; Insel, P.A.

    1987-07-01

    The authors have used radioligand binding techniques to assess whether amiloride and certain analogues of amiloride (ethylisopropyl amiloride and benzamil) can bind to adrenergic receptors in the kidney. They found that amiloride could compete for (/sup 3/H)rauwolscine (..cap alpha../sub 2/-adrenergic receptors), (/sup 3/H)prazosin (..cap alpha../sub 1/-adrenergic receptors), and (/sup 125/I)iodocyanopindolol (..beta..-adrenergic receptors) binding in rat renal cortical membranes with inhibitor constants of 13.6 /plus minus/ 5.7, 24.4 /plus minus/ 7.4, and 8.36 /plus minus/ 13.5 ..mu..M, respectively. Ethylisopropyl amiloride and benzamil were from 2- to 25-fold more potent than amiloride in competing for radioligand binding sites in studies with these membranes. In addition, amiloride and the two analogues competed for (/sup 3/H)prazosin sites on intact Madin-Darby canine kidney cells and amiloride blocked epinephrine-stimulated prostaglandin E/sub 2/ production in these cells. They conclude that amiloride competes for binding to several classes of renal adrenergic receptors with a rank order of potency of ..cap alpha../sub 2/ > ..cap alpha../sub 1/ > ..beta... Binding to, and antagonism of, adrenergic receptors occurs at concentrations of amiloride that are lower than previously observed nonspecific interactions of this agent.

  5. Photoreactivity of. cap alpha. -fluorinated phenyl alkyl ketones

    SciTech Connect

    Wagner, P.J.; Thomas, M.J.; Puchalski, A.E.

    1986-11-26

    The photoreactivities of the mono-, di-, and tri-..cap alpha..-fluorinated acetophenones have been compared to that of acetophenone itself. All four ketones have similar triplet excitation energies; the three fluorinated ketones have reduction potentials 0.5-0.7 eV lower than that of acetophenone. Triplet reactivity toward alkylbenzenes keeps increasing with fluorine substitution, since the rate-determining step becomes charge-transfer complexation as the ketone reduction potential decreases. The primary/tertiary C-H selectivity toward p-cymene increases with the number of fluorines. Triplet reactivity toward cyclopentane also is increased by fluorination but peaks at two fluorines, since the lowest triplet switches from n,..pi..* to ..pi..,..pi..* with two or three fluorines and ..pi..,..pi..* triplets are unreactive in simple hydrogen atom abstraction. In contrast, ..cap alpha..-fluorination of valerophenone does not significantly increase the rate of triplet ..gamma..-hydrogen abstraction. The inductive effect on reactivity apparently is offset by a conformational effect. The ..cap alpha..-fluorinated phenones give predominantly cyclobutanols instead of Norrish type II elimination. ..cap alpha..-Fluoroacetophenone forms predominantly acetophenone and HF when irradiated with 2-propanol, in what appears to be a short chain process involving electron transfer to ketone followed by fluoride ion loss. Finally, the radical coupling products in these reactions are formed in varying yields, depending on solvent and additives.

  6. 5. cap alpha. -reductase activity in rat adipose tissue

    SciTech Connect

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-11-01

    We measured the 5 ..cap alpha..-reductase activity in isolated cell preparations of rat adipose tissue using the formation of (/sup 3/H) dihydrotestosterone from (/sup 3/H) testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5..cap alpha..-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10/sup -8/ M), when added to the medium, caused a 90% decrease in 5..cap alpha..-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5..cap alpha..-reductase activity in each tissue studied.

  7. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

    SciTech Connect

    Wiktorowicz, J.E.; Whitman, J.M.

    1986-05-01

    Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated several more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.

  8. Tritium systems for the mirror upgrade MFTF-. cap alpha. +T

    SciTech Connect

    Finn, P.A.

    1983-01-01

    The tritium systems and the tritium needed for MFTF-..cap alpha..+T were assessed. This mirror reactor produces 20 MW of power, has 10% availability, and uses tritium positive neutral beams with 20 to 30% total gas efficiency as fuelers. The two burn times considered were 10 h and 100 h. A reference processing case was developed including types, sizes, and cost of units.

  9. Effect of. cap alpha. -ketobutyrate on the metabolism of pyruvate and palmitate in isolated rat hepatocytes

    SciTech Connect

    Brass, E.P.

    1986-05-01

    Alpha-ketobutyrate (..cap alpha..KB), an intermediate in the catabolism of threonine and methionine, is decarboxylated to propionyl-CoA. The authors have reported that propionate (PROP) inhibits oxidative metabolism in rate hepatocytes. Based on these observations, the present study examined the effects of ..cap alpha..KB on pyruvate and palmitate metabolism in hepatocytes isolated from fed rats. Similar to PROP, ..cap alpha..KB (10mM) inhibited palmitate oxidation and this inhibition was diminished when 10mM carnitine (CN) was added (35 +/- 6% inhibition without CN, 22 +/- 8% with CN). ..cap alpha..KB inhibited the conversion of 3-/sup 14/C-pyruvate to glucose and CO/sub 2/. Inhibition of pyruvate metabolism by ..cap alpha..KB was concentration-dependent. At equal concentrations, ..cap alpha..KB inhibited pyruvate metabolism to a greater extent than PROP. Addition of CN partially reversed the effects of PROP on pyruvate metabolism, but not those of ..cap alpha..KB despite the generation of propionylcarnitine when ..cap alpha..KB and CN were included in the incubation. These results demonstrate that accumulation of ..cap alpha..KB can impair normal hepatocyte metabolism. While some of the effects of ..cap alpha..KB can be explained on the basis of propionyl-CoA formation, ..cap alpha..KB has effects on pyruvate metabolism not explainable by this mechanism.

  10. cap alpha. -Methyl-p-tyrosine shifts circadian temperature rhythms

    SciTech Connect

    Cahill, A.L.; Ehret, C.F.

    1982-09-01

    ..cap alpha..-Methyl-p-tyrosine shifts the acrophase (time of highest temperature) of the circadian temperature rhythm of the rat to earlier or later times of day depending on the phase of the cicadian cycle at which the drug is administered. When ..cap alpha..-methyl-p-tyrosine methyl ester HCl is injected intraperitoneally at a dose of 100 mg/kg late in the projected 8-h light phase, the acrophase of the intraperitoneal temperature rhythm is delayed by up to 3 h.However, when the same dose of drug is given 9-10 h into the projected 16-h dark phase of the daily cycle, the acrophase of the temperature rhythm occurs about 2 h earlier than expected. The times of ..cap alpha..-methyl-p-tyrosine administration leading to maximal phase delays or advances are correlated with the times of minimal and maximal turnover of norepinephrine in the hypothalamus. These results suggest that changing rates of norepinephrine turnover in the hypothalamus may regulate the circadian temperature rhythm in rats. The results also emphasize the fact that the effects of drugs may vary as a function of the time of administration. This fact must be taken into account in pharmacologic testing.

  11. Human hTM. cap alpha. gene: Expression in muscle and nonmuscle tissue

    SciTech Connect

    MacLeod, A.R.; Gooding, C.

    1988-01-01

    The authors isolated a cDNA clone from a human skeletal muscle library which contains the complete protein-coding sequence of a skeletal muscle ..cap alpha..-tropomyosin. This cDNA sequence defines a fourth human tropomyosin gene, the hTM..cap alpha.. gene, which is distinct from the hTM/sub nm/ gene encoding a closely related isoform of skeletal muscle ..cap alpha..-tropomyosin. In cultured human fibroblasts, the hTM..cap alpha.. gene encodes both skeletal-muscle- and smooth-muscle-type ..cap alpha..-tropomyosins by using an alternative mRNA-splicing mechanism.

  12. Water reuse in the l-lysine fermentation process

    SciTech Connect

    Hsiao, T.Y.; Glatz, C.E.

    1996-02-05

    L-Lysine is produced commercially by fermentation. As is typical for fermentation processes, a large amount of liquid waste is generated. To minimize the waste, which is mostly the broth effluent from the cation exchange column used for l-lysine recovery, the authors investigated a strategy of recycling a large fraction of this broth effluent to the subsequent fermentation. This was done on a lab-scale process with Corynebacterium glutamicum ATCC 21253 as the l-lysine-producing organisms. Broth effluent from a fermentation in a defined medium was able to replace 75% of the water for the subsequent batch; this recycle ratio was maintained for 3 sequential batches without affecting cell mass and l-lysine production. Broth effluent was recycled at 50% recycle ratio in a fermentation in a complex medium containing beet molasses. The first recycle batch had an 8% lower final l-lysine level, but 8% higher maximum cell mass. In addition to reducing the volume of liquid waste, this recycle strategy has the additional advantage of utilizing the ammonium desorbed from the ion-exchange column as a nitrogen source in the recycle fermentation. The major problem of recycling the effluent from the complex medium was in the cation-exchange operation, where column capacity was 17% lower for the recycle batch. The loss of column capacity probably results from the buildup of cations competing with l-lysine for binding.

  13. l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

    PubMed

    Nærdal, Ingemar; Netzer, Roman; Irla, Marta; Krog, Anne; Heggeset, Tonje Marita Bjerkan; Wendisch, Volker F; Brautaset, Trygve

    2017-02-20

    Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysE(MGA3). Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysE(PB1) and lysE2(PB1). The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysE(Cg) from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysE(Cg) while overexpression of lysE(MGA3), lysE(PB1) and lysE2(PB1) had no measurable effect.

  14. Alpha-human atrial natriuretic polypeptide (. cap alpha. -hANP) specific binding sites in bovine adrenal gland

    SciTech Connect

    Higuchi, K.; Nawata, H.; Kato, K.I.; Ibayashi, H.; Matsuo, H.

    1986-06-13

    The effects of synthetic ..cap alpha..-human atrial natriuretic polypeptide (..cap alpha..-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. ..cap alpha..-hANP did not inhibit basal aldosterone secretion. ..cap alpha..-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of aCTH (10/sup -8/M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of (/sup 125/I) ..cap alpha..-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of ..cap alpha..-hANP.

  15. Influence of the. cap alpha. /sub 1/ and. cap alpha. /sub 2/ fractions on the low temperature carbonization of coal tar pitch

    SciTech Connect

    Pashchenkova, L.F.; Rusinovskaya, N.N.; Puzyreva, E.B.

    1980-01-01

    The investigations showed that increasing the ..cap alpha../sub 1/ content of the pitch retards the formation, growth and consolidation of mesophase spherules during low-temperature carbonization. The quinoline-soluble ..cap alpha../sub 2/ fraction accelerates the formation, growth, and consolidation of the spherules.

  16. cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

    SciTech Connect

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-05-15

    cDNA cloning has identified the presence in the human genome of three genes encoding ..cap alpha.. subunits of pertussis toxin substrates, generically called G/sub i/. They are named ..cap alpha../sub i/-1, ..cap alpha../sub i/-2 and ..cap alpha../sub i/-3. However, none of these genes has been functionally identified with any of the ..cap alpha.. subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A/sub 2/, G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K/sup +/ channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver ..cap alpha../sub i/-3 and the partial amino acid sequence of proteolytic fragments of the ..cap alpha.. subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of ..cap alpha../sub i/-3, thus identifying it as ..cap alpha../sub k/. The probable identity of ..cap alpha../sub i/-1 with ..cap alpha../sub p/ and possible roles for ..cap alpha../sub i/-2, as well as additional roles for ..cap alpha../sub i/-1 and ..cap alpha../sub i/-3 (..cap alpha../sub k/) are discussed.

  17. ACTH and. cap alpha. -melanotropin in central temperature control

    SciTech Connect

    Lipton, J.M.; Glyn, J.R.; Zimmer, J.A.

    1981-11-01

    Adrenocorticotropin (ACTH) and ..cap alpha..-melanotropin (..cap alpha..-MSH) occur in brain tissue known to be important to temperature control. These peptides cause hypothermia if they are injected centrally in sufficient doses, but they do not act on the central set point of temperature control. Instead they appear to inhibit central pathways for heat conservation and production. In addition to their hypothermic capability, these peptides are antipyretic when given centrally in doses that have no effect on normal body temperature. ACTH has previously been associated with fever reduction in both clinical and experimental studies, and it may be that endogenous central ACTH is important for limitation of maximal fever. The hypothermic and antipyretic effects of ACTH do not depend on stimulation of the adrenal cortex because they are also observed in adrenalectomized rabbits. Nor is the antipyretic effect limited to the rabbit inasmuch as a comparable effect has been demonstrated in the squirrel monkey. The two peptides may be involved in central mediation of normal thermoregulation and fever, perhaps limiting the febrile response and other rises in body temperature by acting as neurotransmitters or neuromodulators in central thermoregulatory pathways.

  18. cap alpha. -Alkenylphenones - A new class of acid corrosion inhibitors

    SciTech Connect

    Frenier, W.W.; Growcock, F.B.; Lopp, V.R. )

    1988-09-01

    In this paper, a new class of corrosion inhibitors for steel in strong HCI is described. When formulated with small amounts of surfactants, ..cap alpha..-Alkenylphenones provide excellent protection for oilfield steel in contact with strong HCI (up to 28%). The protection levels are similar to, and sometimes superior to, those provided by alkynols. In 15% HCI, benzylallyl alcohol (BAA) appears to be a common intermediate, i.e., in the acid after 24 h when any of the four different phenyl ketones are used. Poly(phenylvinyl ketone) (PPVK), which could form on the surface from reaction of absorbed BAA, was the major component of the thin surface film after corrosion tests with the four ketones.

  19. Probable new type of reaction mechanism: Double. cap alpha. direct transfer process

    SciTech Connect

    Xu Shu-wei; Wu Guo-hua; Miao Rong-zhi; Han Fei

    1983-10-01

    It is assumed that /sup 8/Be consists of two ..cap alpha.. particles which are close to each other in configuration space. A spectroscopic density of /sup 8/Be cluster in the residue nuclei is then obtained, which is proportional to the square of the preformation probability of ..cap alpha.. particle at nuclear surface. Using the improved method of parametrization of EFR-DWBA overlap integral,/sup 1//sup en-dash//sup 2/ we calculate the double differential energy spectra and angular distributions of ..cap alpha.. particles for the reactions /sup 209/Bi (/sup 12/C, ..cap alpha..) /sup 217/Fr and extract the preformation probability of ..cap alpha.. particle at the surface of /sup 217/Fr nuclei from fitting the experimental data. The agreement within the range of calculation error between the preformation probabilities extracted from transfer reactions and ..cap alpha.. decay suggests that the reaction /sup 209/Bi(/sup 12/C, ..cap alpha..) /sup 217/Fr may be explained as a double ..cap alpha.. direct transfer process.

  20. Immunochemical studies on thymosin: radioimmunoassay of thymosin. cap alpha. /sub 1/

    SciTech Connect

    McClure, J.E.; Lameris, N.; Wara, D.W.; Goldstein, A.L.

    1982-01-01

    A sensitive and specific radioimmunoassay (RIA) has been developed to measure thymosin ..cap alpha../sub 1/ in human blood and tissue extracts. Thymosin ..cap alpha../sub 1/ originally isolated from bovine thymosin fraction 5, has an identical primary structure in bovine and human species. Antiserum to synthetic thymosin ..cap alpha../sub 1/ was prepared in rabbits. A synthetic analogue, N-Ac-(Tyr/sup 1/)thymosin ..cap alpha../sub 1/ was radioiodinated by utilizing Na /sup 125/I and soluble lactoperoxidase. The RIA is capable of detecting as small an amount as 40 pg thymosin ..cap alpha../sub 1/ in serum/plasma and does not significatly cross-react with other putative thymic hormones or other purified blood proteins that have been assayed. Proteolytic degradation of thymosin ..cap alpha../sub 1/ does not occur during the collection, storage, or assay of serum or plasma. The concentration of thymosin ..cap alpha../sub 1/ in blood is highest in utero, decreases sharply after birth, and appears to remain fairly constant during the first 15 yr of life. Maternal blood levels of thymosin ..cap alpha../sub 1/ appear to be above normal during pregnancy.

  1. Improved radioimmunoassay for thymosin. cap alpha. 1 recognizes the N-14 amino terminus

    SciTech Connect

    Naylor, P.H.; Goldstein, A.L.

    1986-03-01

    Thymosin ..cap alpha../sub 1/(T..cap alpha../sub 1/) is a biologically active thymic peptide currently undergoing trials as an immunomodulator in cancer patients and patients with immunodeficiencies. Abnormally elevated levels of T..cap alpha../sub 1/ have been found in the serum of individuals with or at risk for AIDS, with T-cell leukemias, and chronic progressive multiple sclerosis. Absorption of the current antibody with a synthetic C-14 fragment of T..cap alpha../sub 1/ results in an antisera specific for the N-14 amino terminus of T..cap alpha../sub 1/, which measures significantly higher levels of T..cap alpha../sub 1/ in serum from normal individuals and significantly increases the sensitivity of the assay. Ongoing studies indicate that this new RIA for T..cap alpha../sub 1/ will be useful in monitoring changes of immunoreactive T..cap alpha../sub 1/ in serum with age and in patients with known or suspected T-cell abnormalities.

  2. Radical ions of alpha,alpha'-bis(diphenylamino)-capped oligothiophenes: a combined spectroelectrochemical and theoretical study.

    PubMed

    Rohde, Dirk; Dunsch, Lothar; Tabet, Ahacine; Hartmann, Horst; Fabian, Jürgen

    2006-04-27

    A new homologous series of alpha,alpha'-bis(diphenylamino)-capped oligothiophenes, prepared by a palladium-catalyzed coupling reaction of stannylated 2-diphenylaminothiophenes with 2-mono- or 2,5-dibromothiophenes and their homologues, was studied by in situ ESR/UV-vis/NIR spectroelectrochemistry. In general, the oxidation potentials of these oligothiophenes were found to be proportional to the inverse number of thiophene units. However, the potential slope of the first oxidation is completely different from that of higher oxidation steps. Trication radicals were identified by electron spin resonance (ESR) for higher thiophene homologues in addition to monocation radicals (polarons). According to the ESR hyperfine structures, the unpaired electron is delocalized in the conjugated system. In contrast to the parent alpha,alpha'-bis(diphenylamino)-capped oligothiophenes, the UV-vis/NIR absorption maxima of the oxidized species strongly depend on the number of thiophene units. Spin-restricted and spin-unrestricted Kohn-Sham density functional calculations were used to explain and to understand these properties. Absorption wavelength and intensities were calculated by the time-dependent density functional response theory. Unrestricted density functional calculations of oligothiophene dications (bipolarons) with five or more thiophene rings result in spin-broken structures which may be considered as two-polaron biradicals (polaron pairs).

  3. Purification and characterization of the human platelet. cap alpha. /sub 2/-adrenergic receptor

    SciTech Connect

    Shreeve, S.M.; Kerlavage, A.R.; Fraser, C.M.; Mariani, A.P.; Venter, J.C.

    1986-05-01

    The ..cap alpha../sub 2/-receptor (..cap alpha../sub 2/-R) from human platelets has been purified to homogeneity using a four step process. An affinity column was prepared by coupling p-aminoclonidine to CH-Sepharose 4B via the p-NH/sub 2/ group. Digitonin solubilized ..cap alpha../sub 2/-R bound to the affinity matrix were eluted with 100 ..mu..M phentolamine and directly applied to a DEAE-Sepharose column. Bound receptors were eluted with a linear gradient of 0-500 mM NaCl, pooled and chromatographed on HPLC size exclusion columns. Three peaks of ..cap alpha../sub 2/-R binding were eluted from HPLC columns (t = 33, 42, 47 min). Radioiodination of HPLC eluates and analysis by SDS-PAGE indicated that ..cap alpha../sub 2/-R binding was associated with a 75-85 kDa protein. These data suggest that the ..cap alpha../sub 2/-R may exist in monomeric and oligomeric forms in the purified state and support previous target size data which indicate that the ..cap alpha../sub 2/-R exists as a dimer in the native membrane. The pure radioiodinated ..cap alpha../sub 2/-R (77-85 kDa) is a glycoprotein with terminal sialic acid or N-acetylglucosamine residues and has a pI of 4.1 on column isoelectric focusing. These data are consistent with those previously reported on the partially purified ..cap alpha../sub 2/-R. Electron micrographs confirm the oligomeric nature and size of the pure ..cap alpha../sub 2/-R.

  4. Enzymic and chemical synthesis of epilson-N-(L-propionyl-2)-L-lysine.

    PubMed

    Fujioka, M; Tanaka, M

    1978-10-01

    Pyruvate was shown to act as an oxo acid substrate in the reverse direction of saccharopine dehydrogenase [epsilon N-(L-glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine-forming)] reaction. The enzymic condensation product of lysine and pyruvate was isolated and identified as epsilon-N-(L-propionyl-2)-L-lysine by comparison with the synthetic compound. A method for the chemical preparation of diastereoisomers of epsilon-N-(propionyl-2)-L-lysine is also described.

  5. Characteristics of binding of human seminal. cap alpha. -inhibin-92 to human pituitary membranes

    SciTech Connect

    Ramasharma, K.; Li, C.H.

    1987-06-01

    The authors investigated the binding of /sup 125/I-labeled ..cap alpha..-inhibin-92 (a 92-residue peptide) to human pituitary membrane preparations. Unlabeled ..cap alpha..-inhibin-92 competed effectively with the labeled peptide for binding to the membranes. Binding was also inhibited by both ..cap alpha..-inhibin-52 and ..cap alpha..-inhibin-31, but less effectively. Scatchard analysis of the ..cap alpha..-inhibin-92 binding data indicated the presence of high-affinity binding sites (1.35 nM/mg of membrane protein) with an apparent K/sub d/ of 0.37 nM. When /sup 125/I-labeled ..cap alpha..-inhibin-92 was covalently crosslinked to the pituitary membrane preparation with disuccinimidyl suberate and the solubilized labeled receptor complex was analyzed by NaDodSO/sub 4//PAGE under either reducing or nonreducing conditions, a single radioactive band at an apparent molecular weight of 90,000 +/- 5000 observed. These data suggest that human pituitary has specific binding sites for ..cap alpha..-inhibins.

  6. Limited proteolysis by macrophage elastase inactivities human. cap alpha. /sub 1/-proteinase inhibitor

    SciTech Connect

    Banda, M.J.; Clark, E.J.; Werb, Z.

    1980-12-01

    Ever since the initial description of ..cap alpha../sub 1/-proteinase inhibitor (..cap alpha../sub 1/PI), the role of this plasma glycoprotein and its allelic polymorphism in disease and in healthy physiology has been the subject of much investigation, ..cap alpha../sub 1/PI inactivates a number of serine proteinases, including granulocyte elastase, and thus affords protection from the connective tissue degradation mediated by this class of proteinases. Because an imbalance in the ratio between ..cap alpha../sub 1/PI and proteinase may contribute to the development of destructive lung diseases, proteinases have been implicated in the pathogenesis of pulmonary emphysema. Both macrophages and polymorphonuclear leukocytes have been implicated in disruption of the ..cap alpha../sub 1/PI-proteinase balance. In this report, a new mechanism for alteration of the ..cap alpha../sub 1/PI-proteinase balance is demonstrated. It was found that the purified form of macrophage elastase catalytically degrades and inactivates ..cap alpha../sub 1/PI so that it no longer inhibits the elastinolytic activity of granulocyte elastase.

  7. Metal-capped alpha-cyclodextrins: squaring the circle.

    PubMed

    Armspach, D; Matt, D

    2001-07-02

    This report describes the four-step synthesis of 6A,6D-diamino-6A,6D-dideoxy-2(A),2(B),2(C),2(D),2(E),2(F),3(A),3(B),3(C),3(D),3(E),3(F), 6(B),6(C),6(E),6(F)-hexadeca-O-methylcyclomaltohexaose (5), a methylated alpha-CD that bears two -CH(2)NH(2) ligands located on diametrically opposed glucopyranose rings. Reaction of 5 with K(2)PtCl(4) afforded the water-soluble chelate complex [PtCl(2)(5)] where the metal center is bonded to cis-arranged nitrogen atoms. A single-crystal X-ray structure of the latter reveals the high distortion imposed on the CD structure by the short metallo-organic cap. The cyclodextrin core adopts an unprecedented elongated, almost rectangular shape, the shortest and longest O(4)(n). O(4)(n+3) distances being respectively 5.44 and 9.98 A. Two opposing glucose rings are no longer in the usual (4)C(1) chair conformation, but adopt an elongated (O)S(2) skew-boat structure. The observed CD-flattening produces a highly preorganized hydrophilic pocket that complexes through multiple hydrogen bonding a single water molecule. Complex [PtCl(2)(5)] and the corresponding synthetic intermediates were characterized by elemental analysis, MS, and IR and NMR spectroscopy.

  8. Membrane transfer of. cap alpha. -tocopherol: influence of soluble. cap alpha. -tocopherol-binding factors from the liver, lung, heart and brain of the rat

    SciTech Connect

    Murphy, D.J.; Mavis, R.D.

    1981-10-25

    The pH of liver supernatant was lowered from 7.4 to 5.1, which removed 23% of the soluble protein and 97% of the lipid-soluble phosphate, increased the total ..cap alpha..-tocopherol transfer activity 1.3-fold and the specific activity of the transfer rate 1.6-fold. This transfer activity was proportional to time up to 4 min and to protein concentrations up to 0.1 mg/ml. Fractionation of the pH 5.1-treated liver supernatant by gel filtration produced a single peak of ..cap alpha..-tocopherol transfer activity of M/sub r/ = 34,000 and a single peak of ..cap alpha..-tocopherol-binding activity which was coincident with the transfer activity. The transfer rate of this peak of activity was 316 pmol/min/mg of protein, a 9-fold purification over the original untreated supernatant. This ..cap alpha..-tocopherol transfer rate was reduced by 83 and 96% following pronase digestion or heat treatment (80/sup 0/C) of the soluble fraction, respectively, while trypsin digestion reduced the transfer rate only 18% and phospholipase C digestion had no effect. Untreated liver supernatant possessed the peak of binding activity of M/sub r/ = 34,000 and a high molecular weight binding fraction that eluted at the void volume. Heart and brain supernatants also possessed an ..cap alpha..-tocopherol-binding fraction that eluted at the void volume, while lung supernatant lacked binding activity.

  9. Distribution of G/sub o. cap alpha. / mRNA and protein in bovine tissues

    SciTech Connect

    Price, S.R.; Tsai, S.C.; Adamik, R.; Angus, C.W.; Van Meurs, K.P.; Czarnecki, S.; Bruckwick, E.C.; Moss, J.; Vaughan, M.

    1987-05-01

    G/sub o..cap alpha../ is a 39 kDa guanyl nucleotide-binding protein similar in structure and function to G/sub s..cap alpha../ and G/sub i..cap alpha../ in the adenylate cyclase complex and transducin (G/sub t..cap alpha../) in the retinal photon receptor system. A bovine retinal cDNA clone, lambdaG09, that encodes the complete amino acid sequence of G/sub o..cap alpha../ has been isolated. Nick-translated lambdaG09 cDNA and a 5' end-labeled oligonucleotide probe complementary to a 24 base sequence unique to G/sub o..cap alpha../ were used as probes for Northern analysis of poly(A)/sup +/ RNA from bovine tissues. A major 4.0 kb mRNA was detected in brain and retina and in lesser amounts in heart. Several smaller mRNAs also hybridized with both probes in these tissues and in liver and lung. G/sub o..cap alpha../ protein was identified using rabbit polyclonal antibodies directed against purified bovine G/sub o..cap alpha../ and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation. Soluble and membrane proteins were incubated with toxin and (/sup 32/P)NAD and then separated by gel electrophoresis before transfer to nitrocellulose for immunoreaction and subsequent autoradiography. A radiolabeled and immunoreactive 39 kDa membrane protein was found principally in retina and brain, and to a lesser extent, in heart. Thus, in the tissues examined, distribution of the 4.0 kb mRNA parallels that of the immunoreactive G/sub o..cap alpha../ with relatively small amounts in heart and larger amounts in brain and retina.

  10. Photoinduced reductive dissolution of. cap alpha. -Fe/sub 2/O/sub 3/ by bisulfite

    SciTech Connect

    Faust, B.C.; Hoffmann, M.R.

    1986-09-01

    The kinetics of the photoinduced reductive dissolution of ..cap alpha..-Fe/sub 2/O/sub 3/ by bisulfite in anoxic colloidal suspensions have been investigated. Quantum yields, ..cap alpha../sub Fe(II)/, as a function of wavelength for Fe(II)/sub aq/ production were obtained. Photochemical reactivity has been discussed in terms of the excitation of the O/sup 2 -/ to Fe/sup 3 +/ charge-transfer band of ..cap alpha..-Fe/sub 2/O/sub 3/ and in terms of photoassisted charge-transfer reactions of Fe(III)-S(IV) curficial complexes.

  11. Increased concentration of. cap alpha. - and. gamma. -endorphin in post mortem hypothalamic tissue of schizophrenic patients

    SciTech Connect

    Wiegant, V.M.; Verhoef, C.J.; Burbach, J.P.H.; de Wied, D.

    1988-01-01

    The concentrations of ..cap alpha..-, ..beta..- and ..gamma..-endorphin were determined by radioimmunoassay in HPLC fractionated extracts of post mortem hypothalamic tissue obtained from schizophrenic patients and controls. The hypothalamic concentration of ..cap alpha..- and ..gamma..-endorphin was significantly higher in patients than in controls. No difference was found in the concentration of ..beta..-endorphin, the putative precursor of ..cap alpha..- and ..gamma..-endorphins. These results suggest a deviant metabolism of ..beta..-endorphin in the brain of schizophrenic patients. Whether this phenomenon is related to the psychopathology, or is a consequence of ante mortem farmacotherapy, remains to be established.

  12. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    SciTech Connect

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  13. Neutron diffraction of. cap alpha. ,. beta. and. gamma. cyclodextrins: hydrogen bonding patterns

    SciTech Connect

    Hingerty, B.E.; Klar, B.; Hardgrove, G.; Betzel, C.; Saenger, W.

    1983-01-01

    Cyclodextrins (CD's) are torus-shaped molecules composed of six (..cap alpha..), seven (..beta..) or eight (..gamma..) (1 ..-->.. 4) linked glucoses. ..cap alpha..-CD has been shown to have two different structures with well-defined hydrogen bonds, one tense and the other relaxed. An induced-fit-like mechanism for ..cap alpha..-CD complex formation has been proposed. Circular hydrogen bond networks have also been found for ..cap alpha..-CD due to the energetically favored cooperative effect. ..beta..-CD with a disordered water structure possesses an unusual flip-flop hydrogen bonding system of the type O-H H-O representing an equilibrium between two states; O-H O reversible H-O. ..gamma..-CD with a disordered water structure similar to ..beta..-CD also possesses the flip-flop hydrogen bond. This study demonstrates that hydrogen bonds are operative in disordered systems and display dynamics even in the solid state.

  14. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    SciTech Connect

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  15. Growth and characterization of L-Lysine adipate crystal

    NASA Astrophysics Data System (ADS)

    Ramya, K.; Saraswathi, N. T.; Raja, C. Ramachandra

    2017-05-01

    Nonlinear optical crystals of L-Lysine adipate were grown by liquid diffusion method at room temperature. The lattice parameters were determined by single crystal X-ray diffraction analysis. Fourier Transform Infrared and Raman spectral studies were carried out to confirm functional groups present in the crystal. Optical property of crystal was examined by UV-vis-NIR studies. Molecular structure of grown crystal was established using 1H NMR and 13C NMR studies. Thermal stability and decomposition were analyzed using Thermo Gravimetric and Differential Thermal analysis. Second harmonic generation efficiency of crystal was determined by Kurtz Perry powder technique.

  16. Sugar Substrates for l-Lysine Fermentation by Ustilago maydis

    PubMed Central

    Sánchez-Marroquín, A.; Ledezma, M.; Carreño, R.

    1970-01-01

    The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; KLa in the fermentors, 0.41 mmoles of O2 per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content. PMID:5485081

  17. Sugar substrates for L-lysine fermentation by Ustilago maydis.

    PubMed

    Sánchez-Marroquín, A; Ledezma, M; Carreño, R

    1970-11-01

    The extracellular production of l-lysine in media with cane sugar, blackstrap molasses, or clarified sugar-cane juice by a previously obtained mutant of Ustilago maydis was studied. Enzymatically inverted clarified juice (medium J-3) gave 2.9 g of lysine per liter under the following conditions: inoculum, 5%; pH 5.8; temperature, 30 C; K(La) in the fermentors, 0.41 mmoles of O(2) per liter per min; fermentation time, 72 hr. The concentrate, obtained by direct evaporation and drying of the fermentation broth, could be used as a possible feed supplement because of its amino-acid and vitamin content.

  18. Synthesis and Conformations of Dendronized Poly(L-lysine)

    PubMed Central

    Lee, Cameron C.; Fréchet, Jean M. J.

    2008-01-01

    Dendronized polymers based on a poly(L-lysine) backbone have been synthesized up to the fourth generation. The hydroxyl-terminated polymers are completely water-soluble, which makes them good candidates for drug delivery applications. The dendronized polypeptide backbones are helical at lower generations, but undergo a dramatic conformational change from α-helical to disordered upon increasing the dendron size to the third generation. This conformational change, attributed to steric repulsions between dendrons, is supported by spectroscopic measurements while chain extension upon dendronization is confirmed by scanning force microscopy. PMID:18833337

  19. Antibodies to the. cap alpha. -subunit of insulin receptor from eggs of immunized hens

    SciTech Connect

    Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

    1985-11-01

    Simple methods for the generation, purification, and assay of antibodies to the ..cap alpha..-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the ..cap alpha..-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the ..beta..-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.

  20. Demonstration of. cap alpha. /sub 2/-adrenergic receptors in rat pancreatic islets using radioligand binding

    SciTech Connect

    Cherksey, B.; Mendelsohn, S.; Zadunaisky, J.; Altszuler, N.

    1982-11-01

    The type of the ..cap alpha..-adrenergic receptors on rat pancreatic islet cells was characterized directly using specific radioligands and displacement agonists and antagonists. Scatchard plots for binding of (/sup 3/H)clonidine (..cap alpha../sub 2/-agonist) revealed a dissociation constant, K/sub d/ of 0.542 +/- 0.1 nM and density of binding sites (B/sub max/) of 50.4 +/- 3.6 fmole/mg protein. Similar values were obtained with (/sup 3/H)dihydroergocryptine (antagonist). The various agonists displaced (/sup 3/H)clonidine with the following order of potency: clonidine > epinephrine approx. = norepinephrine > isoproterenol. Yohimbine, the ..cap alpha../sub 2/-antagonist, was very effective in displacing (/sup 3/H)clonidine, whereas the ..cap alpha../sub 1/-antagonist, prazosin, was much less effective. The data indicate that the ..cap alpha..-adrenergic receptors on rat pancreatic islets are of the ..cap alpha../sub 2/ subtype.

  1. New, tritium-release assay for 25-hydroxyvitamin D-1. cap alpha. -hydroxylase

    SciTech Connect

    Brown, A.J.; Perlman, K.; DeLuca, H.F.

    1986-05-01

    A new, rapid assay for 25-hydroxyvitamin D (25-OH-D)-1..cap alpha..-hydroxylase has been developed using 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ as substrate. This compound was prepared by reduction of 1-oxo-25-hydroxycyclovitamin D/sub 3/ with (/sup 3/H)NaBH/sub 4/, separation of the 1..cap alpha..- and 1..beta..-hydroxy products by HPLC, subsequent treatments with methylsulfonylchloride and lithium aluminum hydride, cycloreversion, and saponification. The 1..cap alpha..- and 1..beta..-tritiated substrates were tested in the solubilized and reconstituted chick 1..cap alpha..-hydroxylase system. After incubation, the reaction mixture was passed through a reversed phase silica cartridge to separate (/sup 3/H)H/sub 2/O from the labeled substrate. The cartridges were then washed with methanol to elute all vitamin D metabolites, and the amount of 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ was measured by HPLC. In addition, identical reaction mixtures using 25-OH-(26,27-/sup 3/H)D/sub 3/ as substrate were extracted and analyzed by HPLC for 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/. Reactions with 25-OH-(1..cap alpha..-/sup 3/H)D/sub 3/ produced (/sup 3/H)H/sub 2/O comparable to the amount of 1,25-(OH)/sub 2/(26,27-/sup 3/H)D/sub 3/ and negligible (/sup 3/H) in 1,25-(OH)/sub 2/D/sub 3/. Conversely, reactions with 25-OH-(1..beta..-/sup 3/H)D/sub 3/ produced negligible (/sup 3/H)H/sub 2/O but produced 1,25-(OH)/sub 2/(/sup 3/H)D/sub 3/ comparable to that from reactions with 25-OH-(26,27-/sup 3/H)D/sub 3/. The results indicate that 1..cap alpha..-hydroxylation specifically displaces the 1..cap alpha..-hydrogen of 25-OH-D/sub 3/ and that the release of the 1..cap alpha..-/sup 3/H provides an accurate measure of vitamin D 1..cap alpha..-hydroxylation.

  2. Regulation of the synthesis of barley aleurone. cap alpha. -amylase by gibberellic acid and calcium ions

    SciTech Connect

    Jones, R.L.; Carbonell, J.

    1984-09-01

    The effects of gibberellic acid (GA/sub 3/) and calcium ions on the production of ..cap alpha..-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA/sub 3/ or CA/sup 2 +/ show qualitative and quantitative changes in hydrolase production following incubation in either GA/sub 3/ or CA/sup 2 +/ or both. In cubation in H/sub 2/O or CA/sup 2 +/ results in the production of low levels of ..cap alpha..-amylase or acid phosphatase. The addition of GA/sub 3/ to the incubation medium causes 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of CA/sup 2 +/ at 10 millimolar causes a further 8- to 9-fold increase in ..cap alpha..-amylase release and a 75% increase in phosphatase release. Production of ..cap alpha..-amylase isoenzymes is also modified by the levels of GA/sub 3/ and CA/sup 2 +/ in the incubation medium. ..cap alpha..-amylase 2 is produced under all conditions of incubation, while ..cap alpha..-amylase 1 appears only when layers are incubated in GA/sub 3/ or GA/sub 3/ plus CA/sup 2 +/. The synthesis of ..cap alpha..-amylases 3 and 4 requires the presence of both GA/sub 3/ and CA/sup 2 +/ in the incubation medium. Laurell rocket immunoelectrophoresis shows that two distinct groups of ..cap alpha..-amylase antigens are present in incubation media of aleurone layers incubated with both GA/sub 3/ and CA/sup 2 +/, while only one group of antigens is found in media of layers incubated in GA/sub 3/ alone. Strontium ions can be substituted for CA/sup 2 +/ in increasing hydrolase production, although higher concentrations of Sr/sup 2 +/ are requried for maximal response. We conclude that GA/sub 3/ is required for the production of ..cap alpha..-amylase 1 and that both GA/sub 3/ and either CA/sup 2 +/ or Sr/sup 2 +/ are required for the production of isoenzymes 3 and 4 of barley aleurone ..cap alpha..-amylase. 22 references, 8

  3. Human cDNA clones for an. cap alpha. subunit of G/sub i/ signal-transduction protein

    SciTech Connect

    Bray, P.; Carter, A.; Guo, V.; Puckett, C.; Kamholz, J.; Spiegel, A.; Nirenberg, M.

    1987-08-01

    Two cDNA clones were obtained from a lambdagt11 cDNA human brain library that correspond to ..cap alpha../sub i/ subunits of G signal-transduction proteins (where ..cap alpha../sub i/ subunits refer to the ..cap alpha.. subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain ..cap alpha../sub i/ is highly homologous to that of bovine brain ..cap alpha../sub i/ and the predicted amino acid sequences are identical. However, human and bovine brain ..cap alpha../sub i/ cDNAs differ significantly from ..cap alpha../sub i/ cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain ..cap alpha../sub i/ cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage ..cap alpha../sub i/ cDNAs. These results suggest there are at least two classes of ..cap alpha../sub i/ mRNA

  4. Production of transforming growth factor. cap alpha. in human pancreatic cancer cells: evidence for a superagonist autocrine cycle

    SciTech Connect

    Smith, J.J.; Derynck, R.; Korc, M.

    1987-11-01

    Previous work showed that cultured human pancreatic cancer cells overexpress the epidermal growth factor (EGF) receptor. In the present study, the authors sought to determine whether some of these cell lines produce transforming growth factor ..cap alpha.. (TGF-..cap alpha..). Utilizing a radiolabeled TGF-..cap alpha.. cDNA in hybridization experiments, they determined that ASPC-1, T/sub 3/M/sub 4/, PANC-1, COLO-357, and MIA PaCa-2 cell lines expressed TGF-..cap alpha.. mRNA. Serum-free medium conditioned by T/sub 3/M/sub 4/ and ASPC-1 cells contained significant amounts of TGF-..cap alpha.. protein. Although unlabeled TGF-..cap alpha.. readily competed with /sup 125/I-labeled EGF for binding, each cell line exhibited lower surface binding and internalization of /sup 125/I-labeled TGF-..cap alpha.. as compared to /sup 125/I-labeled EGF. Both TGF-..cap alpha.. and EGF significantly enhanced the anchorage-independent growth of PANC-1, T/sub 3/M/sub 4/, and ASPC-1 cells. However, TGF-..cap alpha.. was 10- to 100-fold more potent than EGF. These findings suggest that the concomitant overexpression of EGF receptors and production of TGF-..cap alpha.. may represent an efficient mechanism for certain cancer cells to obtain a growth advantage.

  5. Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

    SciTech Connect

    Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.; Pessin, J.E.

    1988-05-03

    Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.

  6. cap alpha. -Methylglucoside satisfies only Na/sup +/-dependent transport system of intestinal epithelium

    SciTech Connect

    Kimmich, G.A.; Randles, J.

    1981-01-01

    The unidirectional influx of ..cap alpha..-methylglucoside (..cap alpha..-MG) by isolated chicken intestinal epithelial cells is 98% inhibited by phlorizin. The remaining 2% of the total influx occurs in the absence of Na/sup +/, is not sensitive to phloretin, and is equal to the diffusional entry rate for 2-deoxyglucose. The glucoside is much more strongly accumulated (75-fold) than 3-O-methylglucose (3-OMG) (10-fold). Inhibitors of the serosal sugar carrier (phloretin, cytochalasin B, theophylline, and flavanoids) do not enhance ..cap alpha..-MG accumulation. It is concluded that the glycoside is not a substrate for the intestinal serosal transport system. Steady-state gradients of the sugar can be represented accurately by a concentrative, phlorizin-sensitive system that is opposed by a diffusional efflux process.

  7. Chronotypic induction of tyrosine aminotransferase by. cap alpha. -methyl-p-tyrosine. [Rat liver

    SciTech Connect

    Cahill, A.L.; Ferguson, S.M.; Ehret, C.F.

    1981-04-06

    ..cap alpha..Methyl-p-tyrosine induced hepatic tyrosine aminotransferase activity to different extents depending upon the time of day of administration of the drug. Maximal induction occurred when ..cap alpha..-methyl-p-tyrosine (100 mg/kg) was injected intraperitoneally during the first several hours of the light phase of the daily cycle, but the magnitude of the induction depended on the nutritional state of the animal. Induction was 4- to 5-fold greater in fasting rats. The effect of ..cap alpha..-methyl-ptyrosine on hepatic tyrosine aminotransferase is believed to be mediated by decreases in hypothalamic norepinephrine. This hypothesis was supported by the demonstration that decreasing levels of hypothalamic norepinephrine at times of day when hypothalamic turnover of norepinephrine was greatest resulted in the greatest induction of tyrosine aminotransferase, while lowering hypothalamic norepinephrine at times when turnover was minimal resulted in minimal induction of tyrosine aminotransferase.

  8. Crystal structures of. cap alpha. - and. beta. -CdUO/sub 4/

    SciTech Connect

    Yamashita, T.; Fujino, T.; Masaki, N.; Tagawa, H.

    1981-04-01

    The structural parameters of ..cap alpha..- and ..beta..-CdUO/sub 4/ crystals are determined by x-ray powder diffraction technique. ..cap alpha..-CdUO/sub 4/ is rhombohedral and cell parameters are a = 6.233(3) A and ..cap alpha.. = 36.12(5)/sup 0/. ..beta..-CdUO/sub 4/ crystallizes in a C-centered orthorhombic cell with a = 7.023(4), b = 6.849(3), c = 3.514(2) A. The space groups are R3m for ..cap alpha..-CdUO/sub 4/ and Cmmm for ..beta..-CdUO/sub 4/. ..cap alpha..-CdUO/sub 4/: 1U in (000), 1Cd in (1/2 1/2 1/2), 2O(1) in +-(uuu), 2O(2) in +-(vvv); u = 0.113, v= 0.350, Z = 1. ..beta..-CdUO/sub 4/: 2U in (000; 1/2 1/2 0), 2Cd in (1/2 0 1/2; 0 1/2 1/2), 40(1) in (0, +-y, 0; 1/2, 1/2 +-y, 0), 4O(2) in (+-x, 0, 1/2; 1/2 +-x, 1/2, 1/2); x = 0.159, y = 0.278, Z = 2. ..beta..-CdUO/sub 4/ contains collinear uranyl UO/sub 2//sup 2 +/ groups with a U-O(1) distance of 1.91 A, located either along or parallel to the c axis whereas the U-O(1) bond length in ..cap alpha..-CdUO/sub 4/ is 1.98 A which is longer than the usual uranyl bond length.

  9. Sequence heterogeneity, multiplicity, and genomic organization of. cap alpha. - and. beta. -tubulin genes in Sea Urchins

    SciTech Connect

    Alexandraki, D.; Ruderman, J.V.

    1981-12-01

    The authors analyzed the multiplicity, heterogeneity, and organization of the genes encoding the ..cap alpha.. and ..beta.. tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. ..cap alpha.. and ..beta..-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The ..cap alpha.. cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of ..cap alpha.. tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The ..beta.. cDNA insertion contains the coding sequence for the 100 C-terminal amino acids of ..beta.. tubulin and 83 base pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous ..cap alpha..- and ..beta..-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of ..cap alpha..-tubulin genes with ..beta..-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

  10. Cardiovascular and endocrine response to hemorrhage after. cap alpha. /sub 1/-blockade in lambs and ewes

    SciTech Connect

    Block, S.M.; Rose, J.C.; Ernest, J.M.; Flowe, K.; South, S.; Zimmerman, C.

    1987-02-01

    To evaluate the role of the ..cap alpha../sub 1/-adrenergic system in the response to hemorrhage during development, lambs and adult sheep were chronically catheterized and hemorrhaged after pretreatment with prazosin or vehicle. The adults became markedly more hypotensive after ..cap alpha../sub 1/-blockade and hemorrhage than after vehicle and hemorrhage, whereas the lambs were no more hypotensive when hemorrhaged after prazosin. In the adults and the lambs hemorrhage produced elevations in plasma renin activity and arginine vasopressin measured by radioimmunoassay. However, after prazosin, the adults had a far greater increase in arginine vasopressin levels than after vehicle treatment.

  11. Reactions of. cap alpha. -oxides in the presence of hexamethylenetetramine and glycerine diphenyl ether

    SciTech Connect

    Nikolaev, P.V.; Sveshnikova, N.F.; Ignatov, V.A.

    1987-11-20

    Hexamethylenetetramine (HMTA) is widely used as a catalyst for the condensation and hardening of compositions based on epoxide oligomer. To provide objective information about the reaction scheme and the kinetics of reactions in which epoxide oligomers participate we studied a model reaction system. The model epoxide oligomer selected was phenyl glycidyl ether (PGE) and the ..cap alpha..,..gamma..-diphenyl ether of glycerine (GDPE). The reference substances in the differential thermal analysis were magnesium oxide, aluminum oxide, and GDPE. Monitoring of the progress of the isothermal reaction was effected by determining the ..cap alpha..-oxide group mercurimetrically. The concentration of HMTA was determined iodometrically.

  12. A new perspective on beta-sheet structures using vibrational Raman optical activity: from poly(L-lysine) to the prion protein.

    PubMed

    McColl, Iain H; Blanch, Ewan W; Gill, Andrew C; Rhie, Alexandre G O; Ritchie, Mark A; Hecht, Lutz; Nielsen, Kurt; Barron, Laurence D

    2003-08-20

    The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.

  13. Quantification of Nε-(2-Furoylmethyl)-L-lysine (furosine), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry.

    PubMed

    Troise, Antonio Dario; Fiore, Alberto; Wiltafsky, Markus; Fogliano, Vincenzo

    2015-12-01

    The control of Maillard reaction (MR) is a key point to ensure processed foods quality. Due to the presence of a primary amino group on its side chain, lysine is particularly prone to chemical modifications with the formation of Amadori products (AP), Nε-(Carboxymethyl)-L-lysine (CML), Nε-(Carboxyethyl)-L-lysine (CEL). A new analytical strategy was proposed which allowed to simultaneously quantify lysine, CML, CEL and the Nε-(2-Furoylmethyl)-L-lysine (furosine), the indirect marker of AP. The procedure is based on stable isotope dilution assay followed by liquid chromatography tandem mass spectrometry. It showed high sensitivity and good reproducibility and repeatability in different foods. The limit of detection and the RSD% were lower than 5 ppb and below 8%, respectively. Results obtained with the new procedure not only improved the knowledge about the reliability of thermal treatment markers, but also defined new insights in the relationship between Maillard reaction products and their precursors.

  14. Lauryl-poly-L-lysine: A New Antimicrobial Agent?

    PubMed Central

    Thuault, Véronique; Mangas, Arturo; Thienpont, Anne; Geffard, Michel

    2014-01-01

    The development of multiple antibiotic resistance is a global problem. It is necessary to find new tools whose mechanisms of action differ from those of currently used antibiotics. It is known that fatty acids and cationic polypeptides are able to fight bacteria. Here, we describe the synthesis of fatty acids linked to a polypeptide with antibacterial activity. The linkage of fatty acids to a polypeptide is reported to increase the antibacterial effect of the linked fatty acid in comparison with free fatty acids (FA) or free poly-L-lysine (PLL) or a mixture of both (FA free + PLL free). A number of C6–C18 fatty acids were linked to PLL to obtain new synthetic products. These compounds were assessed in vitro to evaluate their antibacterial activity. Some fatty acid-PLLs showed a good ability to fight bacteria. Their bactericidal activity was evaluated, and, lauryl linked to PLL was found to be the most active product against both Gram-positive and Gram-negative bacteria. This new active component showed a good degree of specificity and reproducibility and its minimum inhibitory concentration (MIC) was comparatively good. The antibacterial activity of the lauryl-PLL compound suggests that it is a new and promising antimicrobial agent. PMID:24660058

  15. A remarkable member of the polyoxometalates: the eight-nickel-capped alpha-keggin polyoxoazonickelate.

    PubMed

    Dong, Lanjun; Huang, Rudan; Wei, Yongge; Chu, Wei

    2009-08-17

    The eight-nickel-capped polyoxoazonickelate, [Ni(20)(OH)(24)(MMT)(12)(SO(4))](NO(3))(2).6H(2)O (1; MMT = 2-mercapto-5-methyl-1,3,4-thiadiazole), has been synthesized, which has an alpha-Keggin structure with eight nickel caps. In this structure, the polyatom is the late transition metal Ni(II); the central heteroatom is S, and the organic terminal ligand becomes the primary part of the Keggin structure. This is a Keplerate-type cluster, which shows a central Ni(II)(12) cuboctahedron formed by the 12 Ni(II) centers of the classical alpha-Keggin core and a Ni(II)(8) hexahedron formed by the eight nickel caps.

  16. Photoaffinity labeling of human platelet and rabbit kidney. cap alpha. -adrenoceptors with (/sup 3/H)SKF 102229

    SciTech Connect

    Regan, J.W.; Raymond, J.R.; Lefkowitz, R.J.; DeMarinis, R.M.

    1986-06-13

    A newly developed ..cap alpha../sub 2/-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of approx. 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, ..cap alpha../sub 2/-adrenoceptors have been covalently labeled following photolysis in the presence of (/sup 3/H)SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of ..cap alpha../sub 2/-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of (/sup 3/H)SKF 102229 into the ..cap alpha../sub 2/-adrenoceptor was very good. Thus, following photolysis up to 90% of the ..cap alpha../sub 2/-adrenoceptors could be irreversibly labeled with (/sup 3/H)SKF 102229.

  17. A novel potentiometric biosensor for determination of L-lysine in commercial pharmaceutical L-lysine tablet and capsule.

    PubMed

    Yarar, Saniye; Karakuş, Emine

    2016-01-01

    The construction of an L-lysine biosensor on ammonium-selective poly(vinylchloride) (PVC) membrane electrode is described in this study. The construction procedure occurs in two stages: (I) the preparation of ammonium-selective poly(vinylchloride) (PVC) membrane electrode and (II) the chemical immobilization of lysine oxidase on this ammonium-selective electrode by using glutaraldehyde. The ammonium ions produced after enzymatic reaction were determined potentiometrically. The sensitivity of the lysine biosensor against ammonium ions and lysine were studied. The response time, linear working range, reproducibility and life time of the biosensor were also determined. The interfering effect of other amino acids on the biosensor performance was also studied and potentiometric selectivity coefficients were calculated. Although the biosensor responded mainly against tyrosine, a lot of amino acids and ascorbic acid that can be present in some real samples did not show any important interference. Additionally, lysine assay in commercial pharmaceutical lysine tablets and capsules was also successfully carried out. The results were in good agreement with previously reported values.

  18. cap alpha. /sub 2/-Adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production

    SciTech Connect

    Jones, S.B.; Toews, M.L.; Turner, J.T.; Bylund, D.B.

    1987-03-01

    Preincubation of HT29 human colonic adenocarcinoma cells with ..cap alpha../sub 2/-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a (/sup 3/H)adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is ..cap alpha../sub 2/-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an ..cap alpha../sub 2/-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (..cap alpha../sub 1/-adrenergic) and sotalol (..beta..-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of ..cap alpha../sub 2/-adrenergic responses that are not mediated by a decrease in cyclic AMP.

  19. Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

    SciTech Connect

    Cox, G.S.; Rimerman, R.A.

    1988-08-23

    The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..cap alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.

  20. Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia

    PubMed Central

    1994-01-01

    A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin- deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes. PMID:8195289

  1. Synthesis and Phase Behavior of Poly(N-isopropylacrylamide)-b-Poly(L-Lysine Hydrochloride) and Poly(N-Isopropylacrylamide-co-Acrylamide)-b-Poly(L-Lysine Hydrochloride)

    PubMed Central

    Spasojević, Milica; Vorenkamp, Joop; Jansen, Mark R. P. A. C. S.; de Vos, Paul; Schouten, Arend Jan

    2014-01-01

    The synthesis of poly(N-isopropylacrylamide)-b-poly(L-lysine) and poly(N-isopropylacrylamide-co-acrylamide)-b-poly(L-lysine) copolymers was accomplished by combining atom transfer radical polymerization (ATRP) and ring opening polymerization (ROP). For this purpose, a di-functional initiator with protected amino group was successfully synthetized. The ATRP of N-isopropylacrylamide yielded narrowly dispersed polymers with consistent high yields (~80%). Lower yields (~50%) were observed when narrowly dispersed random copolymers of N-isopropylacrylamide and acrylamide where synthesized. Amino-terminated poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide-co-acrylamide) were successfully used as macroinitiators for ROP of N6-carbobenzoxy-L-lysine N-carboxyanhydride. The thermal behavior of the homopolymers and copolymers in aqueous solutions was studied by turbidimetry, dynamic light scattering (DLS) and proton nuclear magnetic resonance spectroscopy (1H-NMR). PMID:28788130

  2. Amperometric biosensor based on diamond paste for the enantioanalysis of L-lysine.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Nejem, R'afat Mahmoud; van Staden, Jacobus Frederick; Aboul-Enein, Hassan Y

    2012-05-15

    An amperometric biosensor was proposed for the enantioanalysis of L-lysine. The biosensor is based on the impregnation of L-lysine oxidase in diamond paste. The potential used for the determination of l-lysine was 650 mV. The biosensor exhibited a linear concentration range between 1 and 100 nmol/L with a limit of detection of 4 pmol/L. The selectivity of the biosensor is high over other amino acids, such as L-serine, L-leucine, L-aspartic acid, L-glutamic acid, histamine, glycine. The proposed biosensor can be applied for the determination of L-lysine in serum samples and pharmaceutical compounds. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Interaction between Ca/sup + +/-channel antagonists and. cap alpha. /sub 2/-adrenergic receptors in rabbit ileal cell membrane

    SciTech Connect

    Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

    1986-03-05

    An interaction between Ca/sup + +/-channel antagonists and the ..cap alpha../sub 2/-adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an ..cap alpha../sub 2/-agonist, stimulated NaCl absorption apparently by Ca/sup + +/-channel antagonism since it inhibited /sup 45/Ca/sup + +/ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca/sup + +/-channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of /sup 3/H-yohimbine, a specific ..cap alpha../sub 2/-adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd/sup + +/ inhibited the specific binding of /sup 3/H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca/sup + +/-channels and ..cap alpha../sub 2/-adrenergic receptors in ileal basolateral membranes. Some Ca/sup + +/-channel antagonists alter ..cap alpha../sub 2/-adrenergic binding to the receptor and ..cap alpha../sub 2/-agonist binding leads to changes in Ca/sup + +/ entry. A close spatial relationship between the Ca/sup + +/-channel and the ..cap alpha../sub 2/-receptor could explain the data.

  4. Bacillus seohaeanensis sp. nov., a halotolerant bacterium that contains L-lysine in its cell wall.

    PubMed

    Lee, Jae-Chan; Lim, Jee-Min; Park, Dong-Jin; Jeon, Che Ok; Li, Wen-Jun; Kim, Chang-Jin

    2006-08-01

    A halotolerant, round-endospore-forming, aerobic, Gram-positive bacterium, designated BH724(T), was isolated from a solar saltern at Taean in Korea. Cells of this strain were rod-shaped and found to be non-motile. Strain BH724(T) grew at salinities of 0-10 % (w/v) NaCl with an optimum of 3 % (w/v) NaCl and at temperatures of 15-50 degrees C with an optimum of 40 degrees C. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain BH724(T) belonged to the genus Bacillus and that Bacillus aquimaris TF-12(T), Bacillus marisflavi TF-11(T) and Bacillus vietnamensis JCM 11124(T) were its closest neighbours, sharing 97.3, 97.2 and 97.0 % 16S rRNA gene sequence similarity, respectively. The genomic DNA G+C content was 39 mol% and the predominant menaquinone was MK-7. Its major cellular fatty acids were anteiso-C(15 : 0), iso-C(15 : 0), iso-C(16 : 0) and iso-C(14 : 0). The peptidoglycan type was A1alpha, linked directly through l-lysine. On the basis of morphological, chemotaxonomic, physiological and phylogenetic properties, strain BH724(T) represents a novel species of the genus Bacillus, for which the name Bacillus seohaeanensis sp. nov. is proposed. The type strain is BH724(T) (=KCTC 3913(T)=DSM 16464(T)).

  5. Agonist-promoted desensitization and phosphorylation of. cap alpha. /sub 1/-adrenergic receptors coupled to stimulation of phosphatidylinositol metabolism

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Cotecchia, S.; Caron, M.G.; Lefkowitz, R.J.

    1986-03-05

    In the DDT/sub 1/ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha../sub 1/-adrenergic receptor (..cap alpha../sub 1/-AR) agonist norepinephrine (NE) promotes rapid attenuation of ..cap alpha../sub 1/-AR-mediated phosphatidylinositol (PI) metabolism which is paralleled by rapid phosphorylation of the ..cap alpha../sub 1/-AR. Cells were labeled by incubation with /sup 32/P/sub i/. Coincubation with NE (100 ..mu..M) significantly increases the rate of /sup 32/P-labeling of both PI and phosphatidic acid. Pretreatment of cells with 100 ..mu..M NE (in the presence of 1 ..mu..M propranolol to prevent ..beta..-AR interactions) results in a drastic attenuation of the NE response on PI metabolism. ..cap alpha../sub 1/-AR from labeled cells can be solubilized and purified by affinity chromatography on Affigel-A55414 and wheat germ agglutinin agarose chromatography. SDS-PAGE of purified ..cap alpha../sub 1/-AR shows a NE-promoted increase in phosphorylation of the M/sub r/ 80K ligand binding peptide. Stoichiometry of phosphorylation increases from approx. 1 mol phosphate/mol ..cap alpha../sub 1/-AR in the basal condition to approx. 2.5 after NE treatment. Both desensitization and phosphorylation are rapid being maximal within 10-20 min of agonist exposure. These results together with previous findings that phorbol esters promote rapid ..cap alpha../sub 1/-AR uncoupling and phosphorylation suggest that receptor phosphorylation is an important mechanism of regulation of ..cap alpha../sub 1/-AR receptor responsiveness.

  6. Comparative investigation of the binding characteristics of poly-L-lysine and chitosan on alginate hydrogel.

    PubMed

    Ren, Ying; Xie, Hongguo; Liu, Xiaocen; Bao, Jie; Yu, Weiting; Ma, Xiaojun

    2016-03-01

    The binding properties of poly-L-lysine and chitosan to alginate have been evaluated quantitatively and compared. Poly-L-lysine bound to alginate hydrogel more rapidly than chitosan as poly-L-lysine has a smaller molar hydrodynamic volume. In addition, poly-L-lysine showed a much higher binding capacity (6.14:1) for alginate hydrogel beads than chitosan (2.71:1), and a little higher binding stoichiometry (0.58) to sodium alginate molecules in solution than chitosan (0.49). An exothermic heat of alginate-poly-L-lysine complexes formation of 2.02 kJ/mol was detected. For alginate-chitosan complexes, the binding enthalpy has been seen to be -3.49 kJ/mol. The stability of the polyelectrolyte complexes was related to their binding enthalpy. The alginate-poly-L-lysine complexes could be disintegrated and rebuilt. By contrast, chitosan was bound with alginate in a steady state. These results provide fundamental insights regarding the structure and property relationships of macromolecules, and will be helpful in designing and selecting appropriate polymers.

  7. Self-degradation of tissue adhesive based on oxidized dextran and poly-L-lysine.

    PubMed

    Matsumura, Kazuaki; Nakajima, Naoki; Sugai, Hajime; Hyon, Suong-Hyu

    2014-11-26

    We have developed a low-toxicity bioadhesive based on oxidized dextran and poly-L-lysine. Here, we report that the mechanical properties and degradation of this novel hydrogel bioadhesive can be controlled by changing the extent of oxidation of the dextran and the type or concentration of the anhydride species in the acylated poly-L-lysine. The dynamic moduli of the hydrogels can be controlled from 120 Pa to 20 kPa, suggesting that they would have mechanical compatibility with various tissues, and could have applications as tissue adhesives. Development of the hydrogel color from clear to brown indicates that the reaction between the dextran aldehyde groups and the poly-L-lysine amino groups may be induced by a Maillard reaction via Schiff base formation. Degradation of the aldehyde dextran may begin by reaction of the amino groups in the poly-L-lysine. The gel degradation can be ascribed to degradation of the aldehyde dextran in the hydrogel, although the aldehyde dextran itself is relatively stable in water. The oxidized dextran and poly-L-lysine, and the degraded hydrogel showed low cytotoxicities. These findings indicate that a hydrogel consisting of oxidized dextran and poly-L-lysine has low toxicity and a well-controlled degradation rate, and has potential clinical applications as a bioadhesive.

  8. Stable yeast transformants that secrete functional. cap alpha. -amylase encoded by cloned mouse pancreatic cDNA

    SciTech Connect

    Filho, S.A.; Galembeck, E.V.; Faria, J.B.; Frascino, A.C.S.

    1986-04-01

    Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomyces cerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.

  9. Determination of the half-lives of. cap alpha. disintegration and spontaneous fission of /sup 242/Cm

    SciTech Connect

    Chang, H.Q.; Xu, J.C.; Wen, T.Q.

    1981-04-01

    Followup measurements have been made by using the method of low geometry for ..cap alpha.. disintegration of /sup 242/Cm. It has been obtained that the half-life of ..cap alpha.. disintegration is T/sub ..cap alpha../ = 163.02 +- 0.11 d. This value is in accord with other recent results. Besides, the method of the specific activity has been used to determine the half-life of spontaneous fission of /sup 242/Cm, T/sub f/ = (7.46 +- 0.06) x 10/sup 6/ yr. This value has provided a check for two appreciably different existing results.

  10. Separation of Cf, Es, and Fm by eluative chromatography using ammonium. cap alpha. -hydroxyisobutyrate

    SciTech Connect

    Mikheev, N.B.; Kamenskaya, A.N.; Auerman, L.N.; Kulyukhin, S.A.; Rumer, I.A.; Novichenko, V.L.

    1987-11-01

    The method of separation of californium, einsteinium, fermium, and certain lanthanides, based on the use of ammonium ..cap alpha..-hydroxyisobutyrate as the eluent, has been improved. The use of a 0.10-0.14 M solution of this reagent and columns (9 x 0.5 cm) filled with the resin Aminex SB with particle size 20-25 ..mu.., permitted the production of about 0.5 ..mu..g of einsteinium-253 from irradiated californium-252 with coefficient of purification of einsteinium from californium in two cycles of adsorption and elution of approx. 10/sup 10/. The coefficient of separation of californium and einsteinium is equal to 1.6 and that of einsteinium and fermium 1.9. The behavior of certain lanthanides in the separation of californium, einsteinium, and fermium using a 0.010 M solution of ammonium ..cap alpha..-hydroxyisobutyrate is discussed.

  11. Physiological significance of benzo(. cap alpha. )pyrene adsorbed to carbon blacks: elution studies, AHH determinations

    SciTech Connect

    Buddingh, F.; Bailey, M.J.; Wells, B.; Haesemeyer, J.

    1981-07-01

    Desorption of benzo(..cap alpha..)pyrene from commercial carbon blacks by tissue fluids in vitro is compared to arylhydrocarbon hydroxylase induction in mice at three levels of carbon black exposure. Less than 0.005% of the adsorbed benzo(..cap alpha..)pyrene content determined by soxhlet extraction in toluene is eluted by human plasma, swine serum, swine lung homogenate and swine lung washings. Statistically significant differences in elution efficiency are observed for the various tissue fluids and carbon blacks. There is no detectable increase in AHH level in mouse lung or liver tissues even at the highest carbon black exposure and consumption rate of 1g/g bdywt/year.

  12. Structural studies of. cap alpha. -bungarotoxin. 1. Sequence-specific /sub 1/H NMR resonance assignments

    SciTech Connect

    Basus, V.J.; Billeter, M.; Love, R.A.; Stroud, R.M.; Kuntz, I.D.

    1988-04-19

    The authors report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the /sup 1/H NMR spectrum of ..cap alpha..-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper. Significant differences exist between the solution structure described here and the crystal structure of ..cap alpha..-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure. These differences include a larger ..beta..-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin ..cap alpha..-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper.

  13. Role of DNA polymerase. cap alpha. in chromosomal aberration production by ionizing radiation

    SciTech Connect

    Bender, M.A.

    1983-01-01

    Aphidicolin is a tetracyclic diterpinoid fungal antibiotic which inhibits DNA synthesis in eukaryotic cells by interfering specifically with DNA polymerase ..cap alpha.., apparently by binding to and inactivating the DNA-polymerase ..cap alpha.. complex. We have shown that aphidicolin, like other inhibitors of DNA synthesis, both induces chromosomal aberrations in human peripheral lymphocytes, and, as a post-treatment, interacts synergistically with x rays to produce greatly enhanced aberration yields. The present experiments explore the effects of aphidicolin in human lymphocytes in the post-DNA-synthetic G/sub 2/ phase of the cell cycle. These experiments utilized labeling with tritiated thymidine to positively identify cells in the S phase at the time of treatment, and used serial colcemid collections and fixations to determine aberration yields over as much of the G/sub 2/ phase as feasible. Because DNA polymerase ..cap alpha.. is the only DNA synthetic or repair enzyme known to be affected by aphidicolin, we infer that this enzyme is directly involved in the repair of DNA lesions which can result in visible chromosomal aberrations. (DT)

  14. Study of the /sup 7/Li(/sup 3/He,. cap alpha. )/sup 6/Li* reaction

    SciTech Connect

    Lin, B.S.C.

    1980-01-01

    Differential cross sections for formation of the 5.37 MeV (J'' = 2/sup +/, T = 1) level in /sup 6/Li have been measured via the reaction /sup 7/Li(/sup 3/He,..cap alpha..)/sup 6/Li at incident /sup 3/He energies in the range 0.7 to 2.5 MeV and for angles in the range 20/sup 0/ less than or equal to theta (Lab) less than or equal to 160/sup 0/. Data for the g.s., 2.18 and 3.56 MeV excited states of /sup 6/Li were also obtained. The results for the g.s. and the first two excited states are in good agreement with previous measurements and show no clear resonance structure. The excitation function for the 5.37 MeV state, however, exhibits resonance structure at incident /sup 3/He energies of 1.45 and 2.15 MeV corresponding to formation of states in the compound nucleus, /sup 10/B, at 18.8 and 19.3 MeV excitation respectively. The experimental data were compared with the results of DWBA calculations using the computer code DWUCK4. Coincident events between the first emitted alpha particle and the decay alpha's or proton's were detected using a counter telescope in the reaction plane at several appropriately chosen angle pairs at an incident /sup 3/He energy of 2.15 MeV. Branching ratios for the allowed decay modes /sup 5/He + p and ..cap alpha.. + p + n, as well as the isospin forbidden decay into ..cap alpha.. + d, were deduced from the data.

  15. L-lysine effectively blocks renal uptake of 125I- or 99mTc-labeled anti-Tac disulfide-stabilized Fv fragment.

    PubMed

    Kobayashi, H; Yoo, T M; Kim, I S; Kim, M K; Le, N; Webber, K O; Pastan, I; Paik, C H; Eckelman, W C; Carrasquillo, J A

    1996-08-15

    In this study, we investigated the ability of L-lysine to block renal uptake of 125I- or 99mTc- labeled Fv fragments. Anti-Tac disulfide-stabilized Fv fragment (dsFv) was derived from a murine monoclonal antibody that recognizes the alpha subunit of the interleukin-2 receptor (IL-2R alpha). The 125I- or 99mTc-labeled dsFv was injected i.v. into non-tumor-bearing nude mice or into nude mice bearing SP2/Tac (IL-2R alpha positive) and SP2/0 (IL-2R alpha negative) tumor. We then evaluated the pharmacokinetics of L-[3H]lysine and the effect of L-lysine dose, timing of administration, and route of delivery on catabolism and biodistribution of i.v. dsFv. Peak renal uptake of i.v. or i.p. injected L-[3H]lysine occurred within 5 and 15 min, respectively. The kidney uptake of L-lysine exhibited a dose-response effect. When L-lysine was coinfused or injected shortly before dsFv, renal uptake of dsFv was blocked to < 5% of the control, but longer intervals were less effective. Aminosyn II and Travasol 10% (parenteral amino acid solutions) also blocked renal uptake of radiolabeled dsFv. Administration of L-lysine did not alter the blood kinetics and slightly increased the tumor uptake of dsFv, but it did prevent catabolism in the kidney and resulted in lower amounts of catabolites in the serum and urine. In conclusion, we have shown that a blocking dose of lysine, injected with or immediately before the injection of radiolabeled dsFv, is most effective in blocking the renal uptake of dsFv. This is consistent with the rapid uptake of L-[3H]lysine by the kidney and is further substantiated by the relative ineffectiveness of lysine injected immediately after the radiolabeled dsFv injection.

  16. Stimulation of the synthesis of 15-hydroxyeicosatetraenoic acid (15-HETE) and 6-keto-prostaglandin F/sub 1. cap alpha. / (6-keto-PGF/sub 1. cap alpha. /) by cultured human umbilical veins

    SciTech Connect

    Ibe, B.O.; Johnson, A.R.; Falck, J.R.; Campbell, W.B.

    1986-03-05

    These studies were designed to investigate the synthesis of 6-keto-PGF/sub 1..cap alpha../ and 15-HETE in cultured human endothelial cells. The identification of the 15-HETE in these cells was made by UV absorption and gas chromatography/mass spectrometry. Specific radioimmunoassays were developed to quantify the synthesized 6-keto-PGF/sub 1..cap alpha../ and 15-HETE. The release of 15-HETE and 6-keto-PGF/sub 1..cap alpha../ was stimulated by arachidonic acid, histamine or the calcium ionophore A23187. The release of 15-HETE paralleled the release of 6-keto-PGF/sub 1..cap alpha../ and was both concentration-related and time-dependent. Aspirin, ibuprofen and indomethacin inhibited both the formation of 6-keto-PGF/sub 1..cap alpha../ and 15-HETE in similar concentrations. These data indicate that agents which stimulate PGI/sub 2/ synthesis also stimulate the synthesis of 15-HETE. Also, they implicate the cyclooxygenase pathway in the synthesis of 6-keto PGF/sub 1..cap alpha../ and 15-HETE in human endothelial cells.

  17. Synthesis of complex pyridine bases in the reaction of. cap alpha. ,omega-nitrileacetylenes with acetylene, catalyzed by cobalt complexes

    SciTech Connect

    Dzhemilev, U.M.; Selimov, F.A.; Khafizov, V.R.

    1987-01-20

    It has been shown that ..cap alpha..,omega-nitrileacetylenes under the action of homogeneous cobalt-containing catalysts undergo transformations into pyridine derivatives. In order to expand the scope of this method for synthesis of complex pyridine bases, for investigation of the reactivity of nitrileacetylenes of various structure in the reaction of cooligomerization with acetylene, as well as for the introduction to these reactions of new types of ..cap alpha..,omega-nitrileacetylenes, containing in their molecules an oxygen atom, they studied the homo- and codimerization of ..cap alpha..,omega-nitrileacetylenes with acetylene under the action of a Co(2-ethyl hexanoate)/sub 2/-AIR/sub 3/ catalyst in a toluene solution. Cyclodimerization of acetylene with ..cap alpha..,omega-nitrileacetylenes, catalyzed by a Co(2-ethyl hexanoate)/sub 2/-AlEt/sub 3/ system gives new types of mono- and bicyclic pyridines.

  18. ORIGEN-S (. cap alpha. ,n) neutron source spectra in borosilicate glass containing HLW (high-level waste)

    SciTech Connect

    Hermann, O.W.; Parks, C.V.; Ludwig, S.B.

    1987-01-01

    There is growing interest in the methodology and computational software for evaluating the (..cap alpha..,n) source spectra produced in mixtures of high-level waste (HLW) and borosilicate glass. The need for this development has been seen in previous work involving the analysis of HLW in borosilicate glass. Descriptions and applications of the ORIGEN-S method of computing neutron source spectra by both (..cap alpha..,n) reactions and spontaneous fission of UO/sub 2/ spent fuel have been reported previously. This summary presents a significant expansion of the ORIGEN-S (..cap alpha..,n) model to include ..cap alpha..-interactions with the light elements of borosilicate glass.

  19. Estrogen receptor binding radiopharmaceuticals: II. Tissue distribution of 17. cap alpha. -methylestradiol in normal and tumor-bearing rats

    SciTech Connect

    Feenstra, A.; Vaalburg, W.; Nolten, G.M.J.; Reiffers, S.; Talma, A.G.; Wiegman, T.; van der Molen, H.D.; Woldring, M.G.

    1983-06-01

    Tritiated 17..cap alpha..-methylestradiol was synthesized to investigate the potential of the carbon-11-labeled analog as an estrogen-receptor-binding radiopharmaceutical. In vitro, 17..cap alpha..-methylestradiol is bound with high affinity to the cytoplasmic estrogen receptor from rabbit uterus (K/sub d/ = 1.96 x 10/sup -10/M), and it sediments as an 8S hormone-receptor complex in sucrose gradients. The compound shows specific uptake in the uterus of the adult rat, within 1 h after injection. In female rats bearing DMBA-induced tumors, specific uterine and tumor uptakes were observed, although at 30 min the tumor uptake was only 23 to 30% of the uptake in the uterus. Tritiated 17..cap alpha..-methylestradiol with a specific activity of 6 Ci/mmole showed a similar tissue distribution. Our results indicate that a 17 ..cap alpha..-methylestradiol is promising as an estrogen-receptor-binding radiopharmaceutical.

  20. Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

    PubMed

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Isaka, Yoshitaka; Rakugi, Hiromi

    2014-09-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC.

  1. The Preventive Effect of L-Lysine on Lysozyme Glycation in Type 2 Diabetes.

    PubMed

    Mirmiranpour, Hossein; Khaghani, Shahnaz; Bathaie, S Zahra; Nakhjavani, Manouchehr; Kebriaeezadeh, Abbas; Ebadi, Maryam; Gerayesh-Nejad, Siavash; Zangooei, Mohammad

    2016-01-01

    Lysozyme is a bactericidal enzyme whose structure and functions change in diabetes. Chemical chaperones are small molecules including polyamines (e.g. spermine), amino acids (e.g. L-lysine) and polyols (e.g. glycerol). They can improve protein conformation in several stressful conditions such as glycation. In this study, the authors aimed to observe the effect of L-lysine as a chemical chaperone on structure and function of glycated lysozyme. In this study, in vitro and in vivo effects of L-lysine on lysozyme glycation were investigated. Lysozyme was incubated with glucose and/or L-lysine, followed by an investigation of its structure by electrophoresis, fluorescence spectroscopy, and circular dichroism spectroscopy and also assessment of its bactericidal activity against M. lysodeikticus. In the clinical trial, patients with type 2 diabetes mellitus (T2DM) were randomly divided into two groups of 25 (test and control). All patients received metformin and glibenclamide for a three months period. The test group was supplemented with 3 g/day of L-lysine. The quantity and activity of lysozyme and other parameters were then measured. Among the test group, L-lysine was found to reduce the advanced glycation end products (AGEs) in the sera of patients with T2DM and in vitro condition. This chemical chaperone reversed the alteration in lysozyme structure and function due to glycation and resulted in increased lysozyme activity. Structure and function of glycated lysozyme are significantly improved by l-lysine; therefore it can be considered an effective therapeutic supplementation in T2DM, decreasing the risk of infection in these patients.

  2. Acetohydroxyacid Synthase, a Novel Target for Improvement of l-Lysine Production by Corynebacterium glutamicum▿ †

    PubMed Central

    Blombach, Bastian; Hans, Stephan; Bathe, Brigitte; Eikmanns, Bernhard J.

    2009-01-01

    The influence of acetohydroxy acid synthase (AHAS) on l-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower Km for the substrate pyruvate and an about fourfold-lower Vmax; (ii) a slightly increased Km for the substrate α-ketobutyrate with an about twofold-lower Vmax; and (iii) insensitivity against the inhibitors l-valine, l-isoleucine, and l-leucine (10 mM each). Introduction of the modified AHAS into the l-lysine producers C. glutamicum DM1729 and DM1933 increased l-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased l-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to l-valine, l-isoleucine, and l-leucine auxotrophy and to further-improved l-lysine production. In batch fermentations, C. glutamicum DM1729 ΔilvB produced about 85% more l-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 ΔilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the l-lysine biosynthetic pathway. PMID:19047397

  3. Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

    PubMed Central

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Rakugi, Hiromi

    2014-01-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. PMID:24652795

  4. Control of. cap alpha. -amylase mRNA accumulation by gibberellic acid and calcium in barley aleurone layers

    SciTech Connect

    Deikman, J.; Jones, R.L.

    1985-01-01

    Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA/sub 3/) with or without 5 millimolar CaCl/sub 2/ shows that ..cap alpha..-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca/sup 2 +/. No difference was observed in ..cap alpha..-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA/sub 3/ with 5 millimolar CaCl/sub 2/ and layers incubated in GA/sub 3/ alone. RNA isolated from layers incubated for 12 hours in GA/sub 3/ with and without CA/sup 2 +/. A cDNA clone for ..cap alpha..-amylase was isolated and used to measure ..cap alpha..-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca/sup 2 +/ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca/sup 2 +/ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for ..cap alpha..-amylase synthesized in Ca/sup 2 +/-deprived aleurone layers was translatable. Ca/sup 2 +/ is required for the synthesis of ..cap alpha..-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.

  5. Limited proteolysis of human leukocyte interferon-. cap alpha. 2 and localization of the monoclonal antibody-binding antigenic determinant

    SciTech Connect

    Kostrov, S.V.; Chernovskaya, T.V.; Khodova, O.M.; Borukhov, S.I.; Ryzhavskaya, A.S.; Izotova, L.S.; Strongin, A.Ya.

    1986-05-20

    Large peptide fragments of human leukocyte interferon-..cap alpha..2 (INF-..cap alpha..2) were produced by limited proteolysis with trypsin, pepsin, thermolysin, and Bacillus amyloliquefaciens serine proteinase, and the ability of the fragments to react with murine monoclonal antibodies NK2, directed toward INF-..cap alpha..2, was studied by the immunoblotting technique. The region of the sequence 110-149 is the most sensitive to proteinase attack and evidently is exposed on the surface of the INF-..cap alpha..2 molecule. The INF-..cap alpha..2 fragments 1-139, 1-147, and 1-149 react with antibodies, whereas the fragments 1-109 and 1-112 do not bind NK2 antibodies. A comparison of the primary structure of the families of human leukocyte and murine leukocyte INF in the region of the sequence 110-139 and an analysis of the ability of human INF differing in amino acid sequence to interact with NK2 antibodies suggested that the antigenic determinant that binds monoclonal antibodies NK2 is the sequence Glu/sub 114/-Asp/sub 115/-Ser/sub 116/-He/sub 117/ of the INF-..cap alpha..2 molecule.

  6. Human transforming growth factor. beta. -. cap alpha. /sub 2/-macroglobulin complex is a latent form of transforming growth factor. beta

    SciTech Connect

    Huang, S.S.; O'Grady, P.; Huang, J.S.

    1987-05-01

    Human platelet-derived transforming growth factor ..beta.. (TGF..beta..) has been shown to be present as a high molecular weight latent form in human serum. Appearance of transforming growth factor activity, along with the change from high molecular weight form to low molecular weight form, was observed following treatment of the latent form of TGF..beta.. with acid or urea, suggesting that the latent form of TGF..beta.. is a complex of TGF..beta.. and a high molecular weight binding protein. Human ..cap alpha../sub 2/-M has been found to be a plasma binding protein for platelet-derived growth factor (PDGF) in serum or plasma. TGF..beta.. and PDGF share similar properties. They, therefore, investigated the interaction between /sup 125/I-TGF..beta.. and ..cap alpha../sub 2/M. /sup 125/I-TGF..beta.. and purified human ..cap alpha../sub 2/M formed a complex as demonstrated by polyacrylamide gel electrophoresis. Most of the /sup 125/I-TGF..beta..-..cap alpha../sub 2/M complex could be dissociated by acid or urea treatment. These results suggest that ..cap alpha../sub 2/M is a binding protein for TGF..beta.. and that TGF..beta..-..cap alpha../sub 2/M complex may be the latent form of TGF..beta.. in serum.

  7. Na/sup +/-dependent transport of /sup 14/C-L-lysine across bullfrog alveolar epithelium

    SciTech Connect

    Kim, K.J.; Crandall, E.D.

    1986-03-01

    Transepithelial transport of the basic amino acid L-lysine has been studied utilizing the isolated intact bullfrog lung mounted in the Ussing chamber. Lungs were excised from doubly pithed bullfrogs and sandwiched between two hemichambers. /sup 14/C-(U)-L-lysine was added to the upstream reservoir of amphibian Ringer solution, while the tissue was short-circuited. Two lungs from the same animal were used simultaneously to determine the two opposite unidirectional fluxes. Downstream and upstream radioactivities were assayed and used to estimate the apparent permeability (P) of the labeled lysine. Results indicate that the apparent P of /sup 14/C-L-lysine measured in the alveolar (M) to the pleural (S) direction is 19.06 (+- 2.84) x 10/sup -7/ cm/s and P in the S to M direction is 3.29 (+- 0.02) x 10/sup -7/ cm/s. When the 100 mM NaCl in the bath was replaced by 110 mM choline chloride, the flux of /sup 14/C-L-lysine from the alveolar to the pleural side decreased to the same value as that in the opposite direction. The flux from the pleural to the alveolar direction in the absence of Na/sup +/ did not change. These results suggest that the alveolar epithelium exhibits Na/sup +/-dependent amino acid (L-lysine) transport in the M->S, but not in the S->M, direction.

  8. Gastroprotective effects of L-lysine salification of ketoprofen in ethanol-injured gastric mucosa.

    PubMed

    Cimini, Annamaria; Brandolini, Laura; Gentile, Roberta; Cristiano, Loredana; Menghini, Paola; Fidoamore, Alessia; Antonosante, Andrea; Benedetti, Elisabetta; Giordano, Antonio; Allegretti, Marcello

    2015-04-01

    Ketoprofen L-lysine salt (KLS), a NSAID, is widely used for its analgesic efficacy and tolerability. L-lysine salification was reported to increase the solubility and the gastric absorption and tolerance of ketoprofen. Since the management of NSAIDs gastrotoxicity still represents a major limitation in prolonged therapies, mainly when gastric lesions are present, this study investigated the gastro-protective activity of L-lysine by using a well-established model of gastric mucosa injury, the ethanol-gastric injury model. Several evidences show that the damaging action of ethanol could be attributed to the increase of ROS, which plays a key role in the increase of lipid peroxidation products, including malonyldialdehyde and 4-hydroxy-2-nonenal. With the aim to unravel the mechanism of L-lysine gastroprotection, cellular MDA levels and 4-HNE protein adducts as markers of lipid peroxidation and a panel of key endogenous gastro-protective proteins were assayed. The data obtained indicate a gastroprotective effect of L-lysine on gastric mucosa integrity.

  9. Oligo(L-lysine)-induced titanium dioxide: Effects of consecutive lysine on precipitation

    NASA Astrophysics Data System (ADS)

    Ahn, Sungjun; Park, Sangwoo; Lee, Sang-Yup

    2011-11-01

    Biomineralization of metal oxide utilizes biomolecular substances, such as peptides and proteins, to induce mineralization of metal precursors in a mild aqueous solution. In this study, we investigated biomineralization of an abiological substance, titanium dioxide (TiO 2), by oligo(L-lysine). Specifically, we systemically studied the influence of the number of consecutive lysine on TiO 2 precipitation. Oligo(L-lysine) was chosen as a homopeptide lysine source whose lysine quantity was adjusted. When oligo(L-lysine) contains more than three consecutive lysine, it induces notably fast precipitation, while single and dilysine do not readily form TiO 2 precipitates. Precipitation of TiO 2 was promoted with the length of oligo(L-lysine). The oligo(L-lysine) was associated with TiO 2 precipitate, which was confirmed by spectroscopic and thermogravitational analyses. The outcomes of this research provide a plausible rationale for explaining precipitation of the Ti precursor that is highly dependent on peptide sequences.

  10. Actin capping protein alpha maintains vestigial-expressing cells within the Drosophila wing disc epithelium.

    PubMed

    Janody, Florence; Treisman, Jessica E

    2006-09-01

    Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing.

  11. Controllable synthesis of functional nanocomposites: Covalently functionalize graphene sheets with biocompatible L-lysine

    NASA Astrophysics Data System (ADS)

    Mo, Zunli; Gou, Hao; He, Jingxian; Yang, Peipei; Feng, Chao; Guo, Ruibin

    2012-09-01

    In this paper a novel method to synthesize functionalize graphene sheets (Gs) by biocompatible L-lysine (Gs/Lys) is reported. The method was composed of two steps: (1) we controllably synthesized self-assembly Gs/Lys-Cu-Lys through the terminal amino of copper L-lysine (Lys-Cu-Lys) attaching to graphite oxide (GO) and then reducing. (2) Obtained the Gs/Lys by eliminating the copper ion. This method could also be used to functionalize other nanomaterials by L-lysine. The Gs/Lys nanocomposites are water-soluble, biocompatible, and above all, it is a chiral material of graphene, which is proposed by us. This novel material will be promising for more applications of graphene. The formation of Gs/Lys nanocomposites were confirmed by scanning electron microscopy (SEM), Fourier-transform infrared spectra (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermal gravimetric (TG) analysis.

  12. Unidirectional growth of pure and L-lysine added ADP crystals from aqueous solution

    NASA Astrophysics Data System (ADS)

    Salarian, Samaneh; Dizaji, Hamid Rezagholipour

    2014-01-01

    Pure and L-lysine added ammonium dihydrogen phosphate (ADP) crystals were grown in the <001> direction by Sankaranarayanan-Ramasamy (S-R) method. The grown crystals were characterized by X-Ray diffractometry (XRD), UV-Vis spectroscopy, Fourier Transform Infrared (FT-IR) and Vicker's Microhardness analysis. XRD spectrum of each of the grown crystals proved its crystallinity. The crystals showed good transparency in the entire visible region. FT-IR spectra of the specimens revealed the presence of functional groups in them. The hardness of the pure and L-lysine added ADP crystals were measured and that of the added one was found higher. Meanwhile, it was found that the ADP crystals (pure and L-lysine added) grown by S-R method had higher hardness compared to ADP crystal grown by conventional method.

  13. A novel amperometric biosensor based on a co-crosslinked L-lysine-α-oxidase/overoxidized polypyrrole bilayer for the highly selective determination of L-lysine.

    PubMed

    Guerrieri, Antonio; Ciriello, Rosanna; Cataldi, Tommaso R I

    2013-09-17

    An amperometric biosensor for the determination of L-lysine based on L-lysine-α-oxidase immobilized by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film is described. The optimization of experimental parameters, such as pH and flow rate, permitted to minimize significantly substrate interferences even using a low specific, commercial enzyme. The relevant biases introduced in the measurement of lysine were just about 1% for L-arginine, L-histidine and L-ornithine, roughly 4% for L-phenylalanine and L-tyrosine. The developed approach allowed linear lysine responses from 0.02 mM up to 2 mM with a sensitivity of 41 nA/(mM × mm(2)) and a detection limit of 4 μM (S/N=3). No appreciable loss in lysine sensitivity was observed up to about 40 days. Allowing polypyrrole layer to remove interference from electroactive compounds, the present method revealed suitable to detect L-lysine in a pharmaceutical and cheese sample, showing a good agreement with the expected values. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Comprehensive assessment of the L-lysine production process from fermentation of sugarcane molasses.

    PubMed

    Anaya-Reza, Omar; Lopez-Arenas, Teresa

    2017-07-01

    L-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of L-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to L-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of L-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the L-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g L-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

  15. Radiation degradation of methyl. cap alpha. -chloroacrylate-methacrylonitrile copolymers. [Gamma rays

    SciTech Connect

    Chen, C.Y.; Pittman, C.U. Jr.; Helbert, J.N.

    1980-01-01

    The radiation degradation behavior of methyl ..cap alpha..-chloroacrylate (MCA) and methacrylonitrile (MCN) copolymers has been investigated as part of a program to develop high-sensitivity polymeric resists for integrated circuit manufacture. High-molecular-weight copolymers were prepared by emulsion techniques. Several different copolymer compositions were prepared varying from 19 to 68 mol % MCA. These copolymers were fractionated and then subjected to ..gamma.. irradiation from a /sup 60/Co source. The G/sub s/ - G/sub x/, G/sub s/ - 4G/sub x/ values were determined from anti M/sub n//sup -1/ and anti M/sub w//sup -1/ versus dose plots, and the G/sub s/ and G/sub x/ values were then calculated. Molecular weights of both unirradiated and irradiated polymers were analyzed by membrane osmometry and gel permeation chromatography. All copolymers exhibited higher degradation susceptibilities than that of poly(methyl methacrylate), which has G/sub s/ = 1.3. The individual G/sub s/ and G/sub x/ values of the copolymers were found to fall between those of the two homopolymers, poly(methyl ..cap alpha..-chloracrylate) (G/sub s/ = 6.0) and polymethacrylonitrile (G/sub s/ = 3.1). The dependence of G/sub s/ and G/sub x/ values on molecular weight was minor. The crosslinking susceptibility of the poly(methyl ..cap alpha..-chloroacrylate) (G/sub x/ = 0.8) was greatly decreased by copolymerization with MCN. Relatively small amounts of MCN caused alarge drop in G/sub x/, i.e., G/sub x/ = 0.15 at 32% MCN and G/sub x/ = 0.03 at 51% MCN. The observation could be attributed to the decreasing probability that crosslinking sites, in the MCA monomer units on adjacent chains, would lie in close proximity. 3 figures, 8 tables.

  16. Radiation-induced cationic polymerization of limonene oxide,. cap alpha. -pinene oxide, and. beta. -pinene oxide

    SciTech Connect

    Aikins, J.A.; Williams, F.

    1984-01-01

    After suitable drying, the subject monomers in the form of neat liquids undergo radiation-induced polymerization with no apparent side reactions and high conversions to precipitatable polymers of low molecular weight. A cationic mechanism is evidenced by the strongly retarding effect of tri-n-propylamine on the polymerization rate. At 25/sup 0/C, limonene oxide gives the highest polymerization rates, an average conversion of 36% per Mrad being obtained in comparison with values of 5.7 and 7.3% per Mrad for the ..cap alpha..-pinene and ..beta..-pinene oxides, respectively. Similarly, the average anti DP/sub n/ decreases from 11.8 for the limonene oxide polymer to 5.6 and 4.0 for the ..cap alpha..-pinene oxide and ..beta..-pinene oxide polymers, respectively. A high frequency of chain transfer to monomer is indicated in each case by the fact that the kinetic chain lengths are estimated to be on the order of a hundred times larger than the anti DP/sub n/ values. Structural characterization of the limonene oxide polymer by /sup 1/H and /sup 13/C NMR spectroscopy provides conclusive evidence that the polymerization proceeds by the opening of the epoxide ring to yield a 1,2-trans polyether. Similar NMR studies on the polymers formed from the ..cap alpha..-pinene and ..beta..-pinene oxides show that in the polymerization of these monomers, the opening of the epoxide ring is generally accompanied by the concomitant ring opening of the cyclobutane ring structure to yield a gem-dimethyl group in the main chain. The detection of isopropenyl end groups in the pinene oxide polymers is also consistent with this mode of propagation being followed by chain (proton) transfer to monomer.

  17. Quasi-relativistic SCF X. cap alpha. study of octahedral 5f/sup 1/ complexes

    SciTech Connect

    Thornton, G.; Roesch, N.; Edelstein, N.

    1980-05-01

    Quasi-relativistic SCF X..cap alpha.. calculations have been carried out for the octahedral 5f/sup 1/ complexes Pa/sup IV/X/sub 6//sup 2 -/, U/sup V/X/sub 6//sup -/(X = F, Cl, Br, I), and Np/sup VI/F/sub 6/. The 5f ..-->.. 5f excitation energies calculated by using the transition-state method agree well with the available absorption spectra. Ionic effects appear to dominate the trends observed in the f-orbital ligand field splitting.

  18. Involvement of prostaglandins F/sub 2. cap alpha. / and E/sub 1/ with rabbit endometrium

    SciTech Connect

    Orlicky, D.J.

    1985-01-01

    Several growth factors and hormones are thought to play a role in the growth control of endometrial cells. The authors have shown that prostaglandin F/sub 2..-->../ (PGF/sub 2..cap alpha../) is a growth factor for primary cultures of rabbit endometrium cultured in chemically-defined serum-free medium and that prostaglandin E/sub 1/ (PGE/sub 1/) antagonizes the PGF/sub 2..-->../ induction of growth. Both (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ bind in a time and temperature dependent, dissociable, saturable and specific manner. The binding of (/sup 3/H)PGF/sub 2..cap alpha../ and (/sup 3/H)PGE/sub 1/ can be both down and up regulated and is enzyme sensitive. PGE /sub 1/ stimulates intracellular cAMP synthesis and accumulation in a time and concentration dependent manner. PGF/sub 2..cap alpha../ probably exerts its effects through an amiloride-sensitive intermediate. Both PGF/sub 2..cap alpha../ and PGE/sub 1/ are constitutively synthesized by these primary cultures, and they have shown this synthesis to be both drug and hormone sensitive. They hypothesize that it is the ratio, rather than the absolute quantities, of PGF/sub 2..cap alpha../ and PGE/sub 1/ which is of more importance in the regulation of endometrial cell growth. Furthermore, they believe this regulation of endometrial growth plays a role in control of proliferation during the decidual response and that a derangement in the ratio of these prostaglandins may lead to either infertility or hyperplasia. The ability of these cultures to synthesize prostaglandins in a hormonally regulatable manner may be of importance in the study of dysmenorrhea and uterine cramping as caused by the myometrial contracting prostaglandin, PGF/sub 2..cap alpha../.

  19. Stimulation of bovine heart pyruvate dehydrogenase kinase by. cap alpha. -ketoisovalerate in the presence of thiamin pyrophosphate

    SciTech Connect

    Robertson, J.G.; Barron, L.L.; Olson, M.S.

    1987-05-01

    Purified bovine heart pyruvate dehydrogenase complex was used to study pyruvate dehydrogenase kinase (PDH kinase) regulation. Previously, they showed that KCl and NH4Cl stimulate PDH kinase, and that thiamin pyrophosphate over the range of 1-80 ..mu..M completely blocks PDH kinase stimulation by 20 mM KCl, whereas thiamin pyrophosphate has very little inhibitory effect in the absence of KCl. Under inhibitory conditions, 100 ..mu..M thiamin pyrophosphate and 20 mM KCl, addition of 1 mM ..cap alpha..-ketoisovalerate stimulated PDH kinase activity 1.5-fold. Addition of 1 mM ..cap alpha..-ketoisovalerate had a similar stimulatory effect in the presence of 1 mM NH4Cl and 100 ..mu..M thiamin pyrophosphate. Half-maximal stimulation occurred at about 200 ..mu..M ..cap alpha..-ketoisovalerate in both cases. Inhibition by thiamin pyrophosphate was uncompetitive, and the effect of ..cap alpha..-ketoisovalerate on thiamin pyrophosphate inhibition was to shift both Vm and Km toward control values without changing the slope of the reciprocal plot. After incubation of pyruvate dehydrogenase complex with ..cap alpha..-ketoisovalerate and gel filtration on Sephadex G-25 to remove ..cap alpha..-ketoisovalerate, PDH kinase activity remained stimulated relative to controls incubated with only buffer. In bovine heart mitochondria solubilized in 0.2% Triton X-100 and incubated with (..gamma..-/sup 32/P)ATP, ..cap alpha..-ketoisovalerate stimulated PDH phosphorylation in the presence of thiamin pyrophosphate, as detected by SDS-PAGE and autoradiography. The results indicate that acylation of pyruvate dehydrogenase complex results in PDH kinase stimulation, but only in the presence of monovalent cation and thiamin pyrophosphate. Furthermore, the results also suggest that this effect may be relevant under more in vivo conditions.

  20. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and. cap alpha. factor pheromones

    SciTech Connect

    Chan, R.K.; Otte, C.A.

    1982-01-01

    Saccharomyces cerevisiae MATa cells carrying mutations in either sst1 or sst2 are supersensitive to the G1 arrest induced by ..cap alpha.. factor pheromone. When sst1 mutants were mixed with normal SST/sup +/ cells, the entire population recovered together from ..cap alpha.. factor arrest, suggesting that SST/sup +/ cells helped sst1 mutants to recover. Complementation tests and linkage analysis showed that sst1 and bar1, a mutation which eliminates the ability of MATa cells to act as a ''barrier'' to the diffusion of ..cap alpha.. factor, were lesions in the same genes. These findings suggest that sst1 mutants are defective in recovery from ..cap alpha.. factor arrest because they are unable to degrade the pheromone. In contrast, recovery of sst2 mutants was not potentiated by the presence of SST/sup +/ cells in mixing experiments. When either normal MATa cells or mutant cells carrying defects in sst1 or sst2 were exposed to ..cap alpha.. factor for 1 h and then washed free of the pheromone, the sst2 cells subsequently remained arrested in the absence of ..cap alpha.. factor for a much longer time than SST/sup +/ or sst1 cells. These observations suggest that the defect in sst2 mutants is intrinsic to the cell and is involved in the mechanism of ..cap alpha.. factor action at some step after the initial interaction of the pheromone with the cell. The presence of an sst2 mutation appears to cause a growth debility, since repeated serial subculture of haploid sst2-1 strains led to the accumulation of faster-growing revertants that were pheromone resistant and were mating defective (''sterile'').

  1. cap alpha. -D-Mannopyranosylmethyl-P-nitrophenyltriazene effects on the degradation and biosynthesis of N-linked oligosaccharide chains on. cap alpha. /sub 1/-acid glycoprotein by liver cells

    SciTech Connect

    Docherty, P.A.; Aronson, N.N. Jr.

    1986-05-01

    The effects of ..cap alpha..-D-mannopyranosylmethyl-p-nitrophenyltriazene (..cap alpha..-ManMNT) on the degradation and processing of oligosaccharide chains on ..cap alpha../sub 1/-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver blocked the complete degradation of endocytosed N-acetyl (/sup 14/C)glucosamine-labeled asialo-AGP and caused the accumulation of Man/sub 2/GlcNAc/sub 1/ fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally-derived (/sup 14/C)GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully-glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the ..cap alpha..-ManMNT-treated hepatocytes remained sensitive to digestion with endoglycosidase H, were resistant to neuraminidase, and consisted of Man/sub 9-7/GlcNAc/sub 2/ structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since ManMNT is likely to irreversibly inactivate ..cap alpha..-D-mannosidases, the return of AGP to secretory forms with complex chains after 24 h probably resulted from synthesis of new processing enzymes.

  2. Presynaptic UNC-31 (CAPS) is required to activate the G alpha(s) pathway of the Caenorhabditis elegans synaptic signaling network.

    PubMed

    Charlie, Nicole K; Schade, Michael A; Thomure, Angela M; Miller, Kenneth G

    2006-02-01

    C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal G alpha(s) pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(s) pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal G alpha(s) pathway near synaptic active zones.

  3. Sensitive radioimmunoassay of. cap alpha. human atrial natriuretic polypeptide using monoclonal antibody recognizing human form ring structure

    SciTech Connect

    Naomi, S.; Umeda, T.; Iwaoka, T.; Miura, F.; Ohno, M.; Sasaki, M.; Oishi, S.; Sato, T.; Takatsu, K.

    1988-01-01

    A monoclonal antibody (C351) against ..cap alpha.. human atrial natriuretic polypeptide (..cap alpha..hANP) recognizing human form ring structure was established and applied to a radioimmunoassay of plasma ..cap alpha..hANP. The minimum detectable amount in terms of 10 % radioligand displacement relative to zero dose were 0.28 fmol/tube, corresponding to 0.7 fmol/ml in plasma after extraction using Sep-Pak C18 cartridges. When the mean plasma levels at recumbent position in fasted morning were compared in 10 young (<30 years) and 10 elderly (greater than or equal to50 years) healthy subjects taking normal sodium diet, it was slightly higher in the latter. After i.v. infusion of hypertonic saline at a rate of 0.24 ml/kg/min for 20 min in 6 normal subjects (26 to 35 years), it was increased from 4.1 +/- 0.4 to 5.9 +/- 0.7 fmol/ml (p<0.01). In 6 patients with essential hypertension (34 to 57 years), it was elevated with high salt intake. From these results, the radioimmunoassay of plasma IR-..cap alpha..hANP using MAb C351 seems to be quite suitable to detect rather small changes at low plasma concentrations and to investigate a physiological importance of ..cap alpha..hANP in man.

  4. Reaction of HO/sub 2//O/sub 2//sup -/ with. cap alpha. -tocopherol in ethanolic solutions

    SciTech Connect

    Arudi, R.L.; Sutherland, M.W.; Bielski, B.H.J.

    1982-01-01

    The HO/sub 2/ perhydroxyl radical reacts with ..cap alpha..-tocopherol in 85% ethanol containing some H/sub 2/SO/sub 4/, EDTA, and O/sub 2/. The resulting transient has a spectral maximum near 390 ..mu... The final product is mostly ..cap alpha..-tocopherylquinone. Best reproducibility for reaction of O/sub 2//sup -/ with ..cap alpha..-tocopherol was obtained in a deoxygenated reaction mixture of 26 +- 3 ..mu..M O/sub 2//sup -/, 0.0565M ..cap alpha..-tocopherol, 5..mu..M EDTA, and 0.005 M KOH in 85% EtOH; the upper limit for the reaction was 6.0 +- 3.0 M/sup -1/ s/sup -1/, indicating that for all practical purposes O/sub 2//sup -/ does not react at all with ..cap alpha..-tocopherol. Preliminary experiments with Trolox, a vitamin E model compound, indicates that it too reacts with HO/sub 2/ but not with O/sub 2//sup -/. Membrane-bound tocopherols in vivo may fulfil a dual antioxidant role. (DLC)

  5. Analysis of a mouse. cap alpha. -globin gene mutation induced by ethylnitrosourea

    SciTech Connect

    Popp, R.A.; Bailiff, E.G.; Skow, L.C.; Johnson, F.M.; Lewis, S.E.

    1983-09-01

    A DBA/2 mouse treated with ethylnitrosourea sired an offspring whose hemoglobin showed an extra band following starch gel electrophoresis. The variant hemoglobin migrated to a more cathodal posititon in starch gel. Isoelectric focusing indicated that chain 5 of the mutant hemoglobin migrated to a more cathodal position than the normal chain 5 from DBA/2 mice and that the other ..cap alpha..-globin, chain 1, was not affected. On focusing gels the phenotype of the mutant allele, Hba/sup y9/, was expressed without dominance to normal chain 5, and Hba/sup y9/ / Hba/sup y9/ homozygotes were fully viable in the laboratory. The molecular basis for the germinal mutation was investigated by analyzing the amino acid sequence of chain 5/sup y9/, the mutant form of ..cap alpha..-chain 5. A single amino acid substitution (His ..-->.. Leu) at position 89 was found in chain 5/sup y9/. The authors propose that ethylnitrosourea induced an A ..-->.. T transversion in the histidine codon at position 89 (CAC ..-->.. CTC). This mutation has apparently not been observed previously in humans, mice or other mammals, and its novel occurrence may be indicative of other unusual mutational events that do not ordinarily occur in the absence of specific mutagen exposure.

  6. Removal of. cap alpha. -tocopherol from blood and its comparison with other lipids

    SciTech Connect

    Gardner, H.K.; Vang, M.J.; Mavis, R.D.

    1986-01-01

    The blood decay curve of ..cap alpha..-tocopherol in rats was compared with those of the two major blood lipids by labeling rat serum in vitro with /sup 3/H-..cap alpha..-tocopherol (AT), /sup 3/H-cholesterol (CHO) or /sup 3/H-trioleoylglycerol (TO) and injecting it into the bloodstream. For the three lipids, loss from blood was biphasic. The half time of the faster decay was 2-4 minutes. The slower curve decayed with half times of 42, 289 and 990 minutes for TO, AT and CHO, respectively. This intermediate rate of AT removal is consistent with its accompanying both of the major blood lipids as they are removed by their respective mechanisms or with a process specific for AT. To investigate the role of liver in the faster curve, animals were hepatectomized. TO and CHO loss remained biphasic after liver removal. However, AT loss became monophasic, with a loss rate intermediate between the non-hepatectomized fast and slow decays. This demonstrates a central role for liver in the metabolism of blood-borne AT and a mode of removal distinct from the other two lipids.

  7. Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    SciTech Connect

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-05-01

    Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/sub z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

  8. Stabilization of collagen with EDC/NHS in the presence of L-lysine: a comprehensive study.

    PubMed

    Usha, R; Sreeram, K J; Rajaram, A

    2012-02-01

    This paper reports the effect of L-lysine on the conformational, rheological, and thermal properties of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross linked collagen and investigates the influence of l-lysine on the self assembly processes of collagen. In the absence of L-lysine, the rheological characterization of collagen cross linked with EDC/NHS showed an increase in shearing stress with shearing speed indicating that the collagen chains become rigid and the molecules are reluctant to flow. On the other hand, the increase in shearing stress with shearing speed is comparatively much less in the presence of L-lysine indicating a greater flexibility of the collagen molecules. The self assembly processes of collagen treated with EDC/NHS in the absence and presence of L-lysine were characterized using powder XRD, FT-IR, polarizing optical microscopy and kinetic studies. XRD studies show an increase in peak intensity and sharpness in the presence of L-lysine indicating the enhancement of crystallinity of collagen nano-fibrils. FT-IR results suggest that the incorporation of L-lysine in the EDC/NHS cross linking favors the molecular stability of collagen. From the present study, it is possible to conclude that the pre-treatment of collagen with L-lysine enhances EDC/NHS cross linking and can be used for biomaterial applications.

  9. Cloning, sequencing, and expression of the gene coding for the human platelet. cap alpha. /sub 2/-adrenergic receptor

    SciTech Connect

    Kobilka, B.K.; Matsui, H.; Kobilka, T.S.; Yang-Feng, T.L.; Francke, U.; Caron, M.G.; Lefkowitz, R.J.; Regan, J.W.

    1987-10-30

    The gene for the human platelet ..cap alpha../sub 2/-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of ..cap alpha../sub 2/-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human ..beta../sub 2/- and ..beta../sub 1/-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional ..cap alpha../sub 2/-adrenergic receptor subtypes.

  10. Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

    SciTech Connect

    Kim, M.H.; Neubig, R.R.

    1986-03-05

    High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonist (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.

  11. Generation of hydrogen using hematite (. cap alpha. -Fe/sub 2/O/sub 3/) electrode

    SciTech Connect

    Bhardwaj, R.C.; Jadhav, C.M.; Khan, M.M.T.

    1983-12-01

    Recent reports by Somorjai produced considerable interest in photocatalytic production of hydrogen from water using P- and ntype polycrystalline iron oxide assembly. The authors report the constant generation of hydrogen using semi-conductor electrode of hematite (..cap alpha..-Fe/sub 2/O/sub 3/) available in nature. The naturally occurring hematite was ground and powdered, the powder then pressed in the form of pellets of 0.8 cm/sup 2/ diameter and connected with a copper lead using silver eposy. The P-type Fe/sub 2/O/sub 3/ electrode was prepared by pressing the ..cap alpha..-Fe/sub 2/O/sub 3/ powder with fine powder of MgO (different weight percentage) and heating them in air at 1350 - 1400/sup 0/C in air. When both electrodes were connected and dipped in 0.1 M Na/sub 2/SO/sub 4/ aqueous solution, hydrogen was evolved at the rate 3 X 10/sup 5/ molecules/hr on irradiation with 450 W xenon lamp. The hydrogen was detected by gas chromatography. It was observed that the production of hydrogen decreases with time and stops completely after sometime. The reactivation by flowing oxygen gas through the reaction cell for an hour, as reported by Somorjai, was not successful. This may have been due to the deterioration of p-type samples by gradual reduction of the iron oxide surfaces by a small fraction of the photoproduced hydrogen. This deterioration was stopped up to a certain extent by deposition of a thin film of indium on the p-type electrode. The progress to improve the efficiency of this system is being accomplished by changing the grain size of polycrystalline iron oxide and by depositing different metal films of different thickness on p-type electrode. It appears from preliminary observations that hematite available in nature contains impurities of SiO/sub 2/ and, on heating at higher temperatures, the hematite (..cap alpha..-Fe/sup 2/O/sub 3/) becomes doped automatically and gives an n-type electrode. The spectral studies show that it absorbs maximum light at

  12. Chemical and physical consequences of. cap alpha. and. beta. /sup -/ decay in the solid state

    SciTech Connect

    Young, J.P.; Haire, R.G.; Peterson, J.R.; Ensor, D.D.

    1984-01-01

    Interesting chemical and structural phenomena can occur when radioactive materials are stored in the solid state. Extensive studies have been made of both the chemical and physical status of progeny species that result from the ..cap alpha.. or ..beta.. /sup -/ day of actinide ions in several different compounds. The samples have been both initially pure actinide compounds - halides, oxides, etc. and actinides incorporated into other non-radioactive host materials, for example lanthanide halides. In general, the oxidation state of the actinide progeny is controlled by the oxidation state of its parent (a result of heredity). The structure of the progeny compound seems to be controlled by its host (a result of environment). These conclusions are drawn from solid state absorption spectral studies, and where possible, from x-ray diffraction studies of multi-microgram sized samples. 13 references, 4 figures, 4 tables.

  13. H/sub. cap alpha. / photon yield in fuelling of tokamaks

    SciTech Connect

    McNeill, D.H.

    1988-08-01

    The number of H/sub ..cap alpha../ photons emitted per entering particle is examined for three types of fueling in tokamaks: wall and limiter efflux, gas puff, and solid pellet injection. These sources correspond to greater involvement of heavy particles in the breakup of entering molecules and to an increasing degree of thermodynamic equilibrium in the radiating zone. The effect of wall absorption and charge exchange on the photon yield in normal tokamak discharges is examined with a molecular influx model. The emission from a perturbing gas puff is discussed qualitatively as an extension to this model. The photon yield from injected pellets corresponds to a limit of extremely high neutral influx and is estimated with a simple hydrodynamical model and measured pellet cloud parameters. 23 refs., 4 figs., 1 tab.

  14. Interaction of poly(L-lysines) with negatively charged membranes: an FT-IR and DSC study.

    PubMed

    Schwieger, Christian; Blume, Alfred

    2007-04-01

    The influence of the binding of poly(L-lysine) (PLL) to negatively charged membranes containing phosphatidylglycerols (PG) was studied by DSC and FT-IR spectroscopy. We found a general increase in the main transition temperature as well as increase in hydrophobic order of the membrane upon PLL binding. Furthermore we observed stronger binding of hydration water to the lipid head groups after PLL binding. The secondary structure of the PLL after binding was studied by FT-IR spectroscopy. We found that PLL binds in an alpha-helical conformation to negatively charged DPPG membranes or membranes with DPPG-rich domains. Moreover we proved that PLL binding induces domain formation in the gel state of mixed DPPC/DPPG or DMPC/DPPG membranes as well as lipid remixing in the liquid-crystalline state. We studied these effects as a function of PLL chain length and found a significant dependence of the secondary structure, phase transition temperature and domain formation capacity on PLL chain length and also a correlation between the peptide secondary structure and the phase transition temperature of the membrane. We present a system in which the membrane phase transition triggers a highly cooperative secondary structure transition of the membrane-bound peptide from alpha-helix to random coil.

  15. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite

    PubMed Central

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-01-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs. PMID:25565321

  16. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite.

    PubMed

    Ganter, Markus; Rizopoulos, Zaira; Schüler, Herwig; Matuschewski, Kai

    2015-04-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.

  17. An overlooked effect of glycine betaine on fermentation: prevents caramelization and increases the L-lysine production.

    PubMed

    Xu, Jianzhong; Xia, Xiuhua; Zhang, Junlan; Guo, Yanfeng; Zhang, Weiguo

    2014-10-01

    This article focuses on the effects of glycine betaine on preventing caramelization, and increasing DCW and L-lysine production. The additional glycine betaine not only decreased the browning intensity (decreased 4 times), and the concentrations of 5-hydroxymethylfurfural (decreased 7.8 times) and furfural (decreased 12 times), but also increased the availability of glucose (increased 17.5%) for L-lysine production. The DCW and L-lysine production were increased by adding no more than 20 mM glycine betaine, whereas the DCW and L-lysine production were decreased with the reduction of pH values, although pH had a better response to prevent caramelization than did glycine betaine. For L-lysine production, the highest increase (40%) was observed on the media with 20 mM glycine betaine. The crucial enzymes in glycolysis and L-lysine biosynthesis pathway were investigated. The results indicated that additional glycine betaine increases the activity of enzymes in glycolysis, in contrast to the effect of pH. All the results indicated that glycine betaine can be used to prevent caramelization and increase the L-lysine production. By applying this strategy, glucose would not be have to be separated from the culture media during autoclaving so that factories can save production costs and shorten the fermentation period.

  18. Enhanced L-lysine production from pretreated beet molasses by engineered Escherichia coli in fed-batch fermentation.

    PubMed

    He, Xun; Chen, Kequan; Li, Yan; Wang, Zhen; Zhang, Hong; Qian, Juan; Ouyang, Pingkai

    2015-08-01

    Faster sugar consumption rate and low-cost nitrogen source are required for the chemical biosynthesis using molasses. Five pretreatment methods were applied to beet molasses prior to fermentation through engineered Escherichia coli, respectively, and corn steep liquid was used as an organic nitrogen source to replace expensive yeast extract. Furthermore, the effects of different feeding strategy in fed-batch fermentation on L-lysine production were investigated. The experimental results showed that combined tricalcium phosphate, sulfuric acid, and activated carbon pretreatment method (TPSA) pretreatment could improve the sugar consumption rate most greatly, and the initial total sugar concentration of 35 g/L from TPSA-pretreated beet molasses gave the best results with respect to L-lysine production, dry cell weight concentration, and L-lysine yield in batch fermentation. Moreover, a mixture of low-cost corn steep liquid and yeast extract containing equal amount of nitrogen could be used as the organic nitrogen source for effective L-lysine fermentation, and constant speed feeding strategy of TPSA-pretreated beet molasses promoted L-lysine production by engineered E. coli. The TPSA-pretreated beet molasses had a sugar consumption rate of 1.75 g/(L h), and a L-lysine yield of 27.81% was achieved, compared with the theoretical yield of 62% by glucose. It was clarified that the pretreatment significantly enhanced the conversion of sugars in beet molasses to L-lysine.

  19. Supramolecular Hydrogels from Self-Assembly of di-Fmoc-L-lysine

    NASA Astrophysics Data System (ADS)

    Hashemnejad, Seyed Meysam; Naas, Kinsey; Kundu, Santanu

    Mechanical properties and nanostructure of a supramolecular hydrogel formed by self-assembly of di-fluorenylmethyloxycarbonyl-lysine (di-Fmoc-L-lysine) are reported here. Hydrogels were prepared by solvent switch technique in which water was added to a solution of di-Fmoc-L-lysine in dimethyl sulfoxide (DMSO). Mechanical properties of the gels were investigated using shear and cavitation rheology. The gels display strain-softening behavior at moderate strain values. Morphological investigations of the samples were conducted using FTIR and CD spectroscopy, electron microscopy, and atomic force microscopy (AFM). Self-assembled fibers with lateral dimensions ranging from 10 to 50 nm were captured in microscopy studies. FTIR results indicate β-sheet-like conformation of the peptides in the hydrogel.

  20. A giant market and a powerful metabolism: L-lysine provided by Corynebacterium glutamicum.

    PubMed

    Eggeling, Lothar; Bott, Michael

    2015-04-01

    L-lysine is made in an exceptional large quantity of currently 2,200,000 tons/year and belongs therefore to one of the leading biotechnological products. Production is done almost exclusively with mutants of Corynebacterium glutamicum. The increasing L-lysine market forces companies to improve the production process fostering also a deeper understanding of the microbial physiology of C. glutamicum. Current major challenges are the identification of ancillary mutations not intuitively related with product increase. This review gives insights on how cellular characteristics enable to push the carbon flux in metabolism towards its theoretical maximum, and this example may also serve as a guide to achieve and increase the formation of other products of interest in microbial biotechnology.

  1. L-lysine as adjunctive treatment in patients with schizophrenia: a single-blinded, randomized, cross-over pilot study

    PubMed Central

    2011-01-01

    Background Accumulating evidence suggests that the brain's nitric oxide (NO) signalling system may be involved in the pathophysiology of schizophrenia and could thus constitute a novel treatment target. The study was designed to investigate the benefit of L-lysine, an amino acid that interferes with NO production, as an add-on treatment for schizophrenia. Methods L-lysine, 6 g/day, was administered to 10 patients with schizophrenia as an adjunctive to their conventional antipsychotic medication. The study was designed as a single-blinded, cross-over study where patients were randomly assigned to initial treatment with either L-lysine or placebo and screened at baseline, after four weeks when treatment was crossed over, and after eight weeks. Results L-lysine treatment caused a significant increase in blood concentration of L-lysine and was well tolerated. A significant decrease in positive symptom severity, measured by the Positive And Negative Syndrome Scale (PANSS), was detected. A certain decrease in score was also observed during placebo treatment and the effects on PANSS could not unequivocally be assigned to the L-lysine treatment. Furthermore, performance on the Wisconsin Card Sorting Test was significantly improved compared to baseline, an effect probably biased by training. Subjective reports from three of the patients indicated decreased symptom severity and enhanced cognitive functioning. Conclusions Four-week L-lysine treatment of 6 g/day caused a significant increase in blood concentration of L-lysine that was well tolerated. Patients showed a significant decrease in positive symptoms as assessed by PANSS in addition to self-reported symptom improvement by three patients. The NO-signalling pathway is an interesting, potentially new treatment target for schizophrenia; however, the effects of L-lysine need further evaluation to decide the amino acid's potentially beneficial effects on symptom severity in schizophrenia. Trial registration NCT00996242 PMID

  2. First Crystal Structure of l-Lysine 6-Dehydrogenase as an NAD-dependent Amine Dehydrogenase*

    PubMed Central

    Yoneda, Kazunari; Fukuda, Junya; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2010-01-01

    A gene encoding an l-lysine dehydrogenase was identified in the hyperthermophilic archaeon Pyrococcus horikoshii. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. The expressed enzyme is the most thermostable l-lysine dehydrogenase yet described, with a half-life of 180 min at 100 °C. The product of the enzyme's catalytic activity is Δ1-piperideine-6-carboxylate, which makes this enzyme an l-lysine 6-dehydrogenase (EC 1.4.1.18) that catalyzes the reductive deamination of the ϵ- amino group and a type of NAD-dependent amine dehydrogenase. The three-dimensional structure of the enzyme was determined using the mercury-based multiple-wavelength anomalous dispersion method at a resolution of 2.44 Å in the presence of NAD and sulfate ion. The asymmetric unit consisted of two subunits, and a crystallographic 2-fold axis generated the functional dimer. Each monomer consisted of a Rossmann fold domain and a C-terminal catalytic domain, and the fold of the catalytic domain showed similarity to that of saccharopine reductase. Notably, the structures of subunits A and B differed significantly. In subunit A, the active site contained a sulfate ion that was not seen in subunit B. Consequently, subunit A adopted a closed conformation, whereas subunit B adopted an open one. In each subunit, one NAD molecule was bound to the active site in an anti-conformation, indicating that the enzyme makes use of pro-R-specific hydride transfer between the two hydrides at C-4 of NADH (type A specificity). This is the first description of the three-dimensional structure of l-lysine 6-dehydrogenase as an NAD-dependent amine dehydrogenase. PMID:20056607

  3. Biomimetic niche for neural stem cell differentiation using poly-L-lysine/hyaluronic acid multilayer films.

    PubMed

    Lee, I-Chi; Wu, Yu-Chieh; Cheng, En-Ming; Yang, Wen-Ting

    2015-05-01

    Polyelectrolyte multilayer films have been suggested as tunable substrates with flexible surface properties that can modulate cell behavior. However, these films' biological effects on neural stem/progenitor cells have rarely been studied. Herein, biomimetic multilayer films composed of hyaluronic acid and poly-L-lysine were chosen to mimic the native extracellular matrix niche of brain tissue and were evaluated for their inductive effects, without the addition of chemical factors. Because neural stem/progenitor cells are sensitive to substrate properties, it is important that this system provides control over the surface charge, and slight stiffness variations are also possible. Both of these factors affect neural stem/progenitor cell differentiation. The results showed that neural stem/progenitor cells were induced to differentiate on the poly-L-lysine/hyaluronic acid multilayer films with 0.5-4 alternating layers. In addition, the neurite outgrowth length was regulated by the surface charge of the terminal layer but did not increase with the layer number. In contrast, the quantity of differentiated neurons was enhanced slightly as the number of layers increased but was not affected by the surface charge of the terminal layer. In sum, material pairs in the form of native poly-L-lysine/hyaluronic acid films achieved important targets for neural regenerative medicine, including enhancement of the neurite outgrowth length, regulation of neuron differentiation, and the formation of a network. These extracellular matrix-mimetic poly-L-lysine/hyaluronic acid multilayer films may provide a versatile platform that could be useful for surface modification for applications in neural engineering. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  4. Effect of poly-L-lysine and neuraminidase on the infectivity of Trypanosoma cruzi in cultured HeLa cells.

    PubMed

    Gamarro, F; Castanys, S; Ruiz-Perez, L M; Adroher, F J; Osuna, A

    1985-01-01

    The percentage of parasitisation and index of adherence of Trypanosoma cruzi has been studied when host HeLa cells or metacyclic forms were pretreated with neuraminidase or with poly-L-lysine. The percentage of parasitisation was significatively reduced (P less than or equal to 0.001) when cells were pretreated with poly-L-lysine while pretreatment with neuraminidase caused no apparent effects. On the other hand, the adherence of the metacyclic forms pretreated with poly-L-lysine or neuraminidase was significantly higher than that of the control group.

  5. A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

    PubMed Central

    Steffen, Victoria; Otten, Julia; Engelmann, Susann; Radek, Andreas; Limberg, Michael; Koenig, Bernd W.; Noack, Stephan; Wiechert, Wolfgang; Pohl, Martina

    2016-01-01

    Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization. PMID:27690044

  6. A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis.

    PubMed

    Steffen, Victoria; Otten, Julia; Engelmann, Susann; Radek, Andreas; Limberg, Michael; Koenig, Bernd W; Noack, Stephan; Wiechert, Wolfgang; Pohl, Martina

    2016-09-28

    Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector(®) microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

  7. Multi-technique characterization of poly-L-lysine dendrigrafts-Cu(II) complexes for biocatalysis.

    PubMed

    Rossi, Jean-Christophe; Maret, Barbara; Vidot, Kevin; Francoia, Jean-Patrick; Cangiotti, Michela; Lucchi, Susanna; Coppola, Concetta; Ottaviani, Maria Francesca

    2015-02-01

    Poly-L-lysine is a biocompatible polymer used for drug or gene delivery, for transport through cellular membranes, and as nanosized magnetic resonance imaging contrast agents. Cu(II)-poly-L-lysine complexes are of particular interest for their role in biocatalysis. In this study, poly-L-lysine dendrigrafts (DGLs) at different generations (G2, G3, and G4) are synthesized and characterized in absence and presence of Cu(II) by means of electron paramagnetic resonance (EPR), UV-Vis, potentiometric titration and circular dichroism (CD). The analysis is performed as a function of the [Cu(II)]/[Lys] (=R) molar ratio, pH and generation by identifying differently flexible complexes in different dendrimer regions. The amine sites in the lateral chains become increasingly involved with the increase of pH. The good agreement and complementarity of the results from the different techniques provide an integrate view of the structural and dynamic properties of Cu(II)-DGL complexes implementing their use as biocatalysts. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Human transforming growth factor type. cap alpha. coding sequence is not a directed-acting oncogene when overexpressed in NIH 3T3 cells

    SciTech Connect

    Finzi, E.; Fleming, T.; Segatto, O.; Pennington, C.Y.; Bringman, T.S.; Derynck, R.; Aaronson, S.A.

    1987-06-01

    A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type ..cap alpha.. (TGF-..cap alpha..). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-..cap alpha.. was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. Binding assays were done using (/sup 125/I)-EGF. When NIH 3T3 clonal sublines were selected by transfection of TGF-..cap alpha.. expression vectors in the presence of a dominant selectable market, they were shown to secrete large amounts of TGF-..cap alpha.. into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-..cap alpha.. monoclonal antibody. These results indicated that secreted TGF-..cap alpha.. interacts with its receptor at a cell surface location. Single cell-derived TGF-..cap alpha..-expressing sublines grew to high saturation density in culture. These and other results imply that TGF-..cap alpha.. exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-..cap alpha.. is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

  9. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and. cap alpha. factor pheromones

    SciTech Connect

    Chan, R.K.; Otte, C.A.

    1982-01-01

    Eight independently isolated mutants which are supersensitive (Sst/sup -/) to the G1 arrest induced by the tridecapeptide pheromone ..cap alpha.. factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by ..cap alpha.. factor. These mutants carries lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to ..cap alpha.. factor, but MAT..cap alpha.. sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT..cap alpha.. cells. Even in the absence of added ..cap alpha.. pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology (''shmoo'' shape) that normally develops only after MATa cells are exposed to ..cap alpha.. factor. This ''self-shmooing'' phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT..cap alpha.. diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT..cap alpha.. sst2-1/sst2-1) were still insensitive to ..cap alpha.. factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked nor centromere distal to MAT on the right arm of chromosome III.

  10. Enhancement of ε-poly-L-lysine production coupled with precursor L-lysine feeding in glucose-glycerol co-fermentation by Streptomyces sp. M-Z18.

    PubMed

    Chen, Xu-Sheng; Ren, Xi-Dong; Zeng, Xin; Zhao, Fu-Lin; Tang, Lei; Zhang, Hong-Jian; Zhang, Jian-Hua; Mao, Zhong-Gui

    2013-12-01

    ε-Poly-L-lysine (ε-PL), one of the only two homo-poly amino acids known in nature, is used as a preservative. In this study, strategies of feeding precursor L-lysine into 5 L laboratory scale fermenters, including optimization of L-lysine concentration and time, was investigated to optimize the production of ε-PL by Streptomyces sp. M-Z18. The optimized strategy was then used in ε-PL fed-batch fermentation in which glucose and glycerol served as mixed carbon sources. In this way, a novel ε-PL production strategy involving precursor L-lysine coupled with glucose-glycerol co-fermentation was developed. Under optimal conditions, ε-PL production reached 37.6 g/l, which was 6.2 % greater than in a previous study in which glucose and glycerol co-fermentation was performed without added L-lysine (35.14 g/l). To the best of our knowledge, this is the first report of the enhancement of ε-PL production through L-lysine feeding to evaluate the use of fermenters. Meanwhile, the role of L-lysine in the promotion of ε-PL production, participating ε-PL synthesis as a whole, was first determined using the L-[U-(13)C] lysine labeling method. It has been suggested that the bottleneck of ε-PL synthesis in Streptomyces sp. M-Z18 is in the biosynthesis of precursor L-lysine. The information obtained in the present work may facilitate strain improvement and efficient large-scale ε-PL production.

  11. Enhancing L-Lysine Production of Beet Molasses by Engineered Escherichia coli Using an In Situ Pretreatment Method.

    PubMed

    He, Xun; Qi, Yanbin; Chen, Kequan; Li, Yan; Ouyang, Pingkai

    2016-07-01

    Reducing the viscosity of molasses environmentally and selectively removing the harmful ingredients for microbes are the keys to promoting the bioavailability of molasses. A simple and environmental in situ pretreatment method integrating surfactants and alkali was developed to reduce the viscosity of molasses prior to L-lysine production using Escherichia coli ZY0217. Adding activated carbon and modified orange peel based on the in situ pretreatment process effectively removed pigments and excessive zinc in the molasses and also significantly increased the cell growth and L-lysine yield from E. coli ZY0217. The experimental results showed that a mixture of secondary alkane sulfonate, an anionic surfactant, and HodagCB-6, a non-ionic surfactant, effectively reduced the viscosity of the molasses more so than any single surfactant. When the surfactant mixture was added at a concentration of 0.04 g/L to the molasses, the ω value was 0.4, and when ammonia was added at 0.6 %, the lowest viscosity of 705 mPa · s was obtained. Further, 91.5 % of the color and 86.68 % of the original levels of zinc were removed using an activated carbon and modified orange peel treatment on the molasses with the lowest viscosity, which further promoted cell growth and L-lysine production. In the fed-batch cultivation process, the L-lysine concentration achieved using a constant-speed feeding strategy was 45.89 g/L, with an L-lysine yield of 27.18 %, whereas the L-lysine yield from untreated molasses was only 10.13 %. The increase in L-lysine yield was related to the reduced viscosity and the detoxification of the molasses. Lastly, the pretreatment was found to significantly enhance the conversion of sugars in the molasses to L-lysine.

  12. Characterization of a pyridoxal-5'-phosphate-dependent l-lysine decarboxylase/oxidase from Burkholderia sp. AIU 395.

    PubMed

    Sugawara, Asami; Matsui, Daisuke; Takahashi, Narumi; Yamada, Miwa; Asano, Yasuhisa; Isobe, Kimiyasu

    2014-11-01

    A novel enzyme, which catalyzed decarboxylation of l-lysine into cadaverine with release of carbon dioxide and oxidative deamination of l-lysine into l-2-aminoadipic 5-semialdehyde with release of ammonia and hydrogen peroxide, was found from a newly isolated Burkholderia sp. AIU 395. The enzyme was specific to l-lysine and did not exhibit enzyme activities for other l-amino acids, l-lysine derivatives, d-amino acids, and amines. The apparent Km values for l-lysine in the oxidation and decarboxylation reactions were estimated to be 0.44 mM and 0.84 mM, respectively. The molecular mass was estimated to be 150 kDa, which was composed of two identical subunits with molecular mass of 76.5 kDa. The enzyme contained one mol of pyridoxal 5'-phosphate per subunit as a prosthetic group. The enzyme exhibiting decarboxylase and oxidase activities for l-lysine was first reported here, while the deduced amino acid sequence was homologous to that of putative lysine decarboxylases from the genus Burkholderia.

  13. cap alpha. -chain locus of the T-cell antigen receptor is involved in the t(10; 14) chromosome translocation of T-cell acute lymphocytic leukemia

    SciTech Connect

    Kagan, J.; Finan, J.; Letofsky, J.; Besa, E.C.; Nowell, P.C.; Croce, C.M.

    1987-07-01

    Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C/sub ..cap alpha../ (the constant region of TCRA (the ..cap alpha..-chain locus of the T-cell antigen receptor gene)), but not for V/sub ..cap alpha../ (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V/sub ..cap alpha../genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V/sub ..cap alpha../ and the C/sub ..cap alpha../ genes. These results suggest that the translocation of the C/sub ..cap alpha../ locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which the authors propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.

  14. Trypsin pretreatment dissociates the effect of GTP and Na/sup +/ on ligand binding to human platelet. cap alpha. /sub 2/-adrenoreceptors

    SciTech Connect

    Periyasamy, S.M.; Somani, P.

    1986-03-01

    The authors recently showed that maximal concentration of trypsin (TR) inactivated only 70-80% of specific (/sup 3/H)yohimbine (/sup 3/H-Y) binding to human platelet ..cap alpha../sub 2/-adrenoreceptors (..cap alpha../sub 2/-AR). The effect of TR on the interaction between the agonist, epinephrine, and GTP or Na/sup +/ on /sup 3/H-Y binding to ..cap alpha../sub 2/-AR of human platelets was investigated in the present study. Partially purified ..cap alpha../sub 2/-AR protein was pretreated with 500 ..mu..g TR for 3 min at 35/sup 0/C, the proteolytic action was terminated by specific TR-inhibitor, and the membranes were washed twice prior to specific ligand binding studies. Under these conditions, TR produced a 40-50% reduction of specific /sup 3/H-Y binding. Further characterization of the specific ligand binding by Scatchard plots showed that TR did not modify the affinity of the ..cap alpha../sub 2/-AR to the agonist or the antagonist or the antagonists. However, in the TR-pretreated membranes, the ability of GTP to reduce the receptor affinity for epinephrine was lost. The effect of Na/sup +/, on the other hand, remained intact under these conditions. Thus, TR can selectively destroy the GTP-..cap alpha../sub 2/-AR but not Na/sup +/-..cap alpha../sub 2/-AR interaction suggesting that the GTP- and Na/sup +/-binding proteins are distinct.

  15. cap alpha. -Naphthylisothiocyanate (ANIT) stimulates the release of superoxide by rat neutrophils in vitro

    SciTech Connect

    Roth, R.A.; Hewett, J.

    1986-03-01

    ..cap alpha..-Naphthylisothiocyanate (ANIT) is an hepatotoxicant that produces cholestasis and hyperbilirubinemia in rats. Its mechanism of action is unknown. The observation that polymorphonuclear leukocytes (PMNs) accumulate in the bile ductular region of the liver following ANIT administration prompted us to examine the ability of ANIT to stimulate these cells. PMNs elicited from rat peritoneum were treated with ANIT in vitro to test for the release of superoxide anion (O/sub 2//sup -/). ANIT stimulated O/sub 2//sup -/ release from PMNs in a concentration-dependent manner. Maximal O/sub 2//sup -/ release was achieved by an ANIT concentration of 110 ..mu.. M. O/sub 2//sup -/ release was rapid after the first few minutes of ANIT addition and ceased entirely between 10 and 15 minutes. An increase in the extracellular activity of lactate dehydrogenase also occurred after a 5-10 minute lag phase following ANIT addition. PMNs exposed to ANIT also failed to exclude trypan blue dye, either in the presence or in the absence of superoxide dismutase and catalase, suggesting a direct, oxygen radical-independent, cytotoxic effect of ANIT on PMNs. Release of the lysosomal enzyme, ..beta..-glucuronidase, also occurred within 5 min following exposure of PMNs to ANIT. These results indicate that ANIT stimulates the release of cytotoxic agents from rat PMNs in vitro and suggests that the direct stimulation of PMNs in vivo may contribute to ANIT-induced hepatotoxicity in rats.

  16. Removal of. cap alpha. -tocopherol from blood and its comparison with other lipids: studies of inhibition

    SciTech Connect

    Gardner, H.K.; Mavis, R.D.

    1986-05-01

    To investigate the mechanism of ..cap alpha..-tocopherol (AT) uptake into tissues, loss of /sup 3/H-AT from blood was characterized and compared with the losses of two major blood lipids: /sup 3/H-cholesterol (CHO) and /sup 3/H-trioleoylglycerol (TO). Male Long-Evans rats (200-325 gm) were injected with serum labelled with one lipid, and bled from the tail from 1-240 min. In one group heparin (HEP), an inhibitor of lipoprotein lipase which mediates uptake of TO into tissues, was intravenously injected prior to serum and following corn oil gavage. The control group was only gavaged (GAV). A third group was injected with labelled serum which had been incubated with 1,2-cyclohexanedione (CHD), a reagent which modifies the receptors responsible for removal of CHO-rich low density lipoproteins from blood. Labelled serum incubated only with borate buffer (BO) was injected into the fourth group. HEP slowed TO loss from 2-220 min, but left CHO loss unchanged. AT loss was slowed by HEP from 100 min on. That AT responded to HEP but over a time span different from that of TO suggests that AT may be removed by a mechanism distinct from that of TO but sensitive to HEP. CHD slowed CHO loss from 40-240 min while TO and AT loss were uninhibited. This argues against a mechanism of removal common to both AT and CHO.

  17. Characterization of D-enzyme (4-. cap alpha. -glucanotransferase) in Arabidopsis leaf

    SciTech Connect

    Lin, T.P.; Preiss, J.

    1988-01-01

    Two major forms of D-enzyme (4-..cap alpha..-glucanotransferase, EC 2.4.1.25) were successfully separated from most of the amylase activity using FPLC-Mono Q column chromatography. Transfer of a maltosyl group was observed upon the incubation of D-enzyme with maltotriose and D-(U-/sup 14/C)glucose. About 4.5% of the radioactivity was transferred to maltotriose in 2 hours. End product analysis showed the accumulation of glucose and maltopentaose from maltotriose within the first 10 minutes of the reaction. Several other maltodextrins were also observed with longer incubation times, although maltose was never produced. A quantitative measurement of maltodextrin production from the reaction of (/sup 14/C)maltotriose with D-enzyme showed that the quantity of maltotriose decreased from 100% to 31% after 3 hours incubation, while glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, and higher maltodextrins increased in amount. Glucose is the major product throughout the course of the reaction of D-enzyme with maltotriose. Maltotriose, in addition to glucose, are the major products in the reaction of D-enzyme with maltodextrins with a chain length greater than maltotriose. This study confirms the existence of a transglycosylase that disproportionates amaltotriose and higher maltodextrins by transferring maltosyl or maltodextrinyl groups between maltodextrins resulting in the production of glucose and different maltodextrins, but not maltose.

  18. Radiation-induced cationic polymerization of limonene oxide,. cap alpha. -pinene oxide, and. beta. -pinene oxide

    SciTech Connect

    Aikins, J.A.; Williams, F.

    1985-01-01

    After suitable drying, the subject monomers in the form of neat liquids undergo radiation-induced polymerization with no apparent side reactions and high conversions to precipitatable polymers of low molecular weights. A high frequency of chain (proton) transfer to monomer is indicated by the fact that the kinetic chain lengths are estimated to be several hundred times larger than the range of DP/sub n/ values (12-4). Structural characterization of the limonene oxide polymer by /sup 1/H and /sup 13/C NMR spectroscopy provides conclusive evidence that the polymerization proceeds by the opening of the epoxide ring to yield a 1,2-trans polyether. Similar NMR studies on the polymers formed from the ..cap alpha..-pinene and ..beta..-pinene oxides show that the opening of the epoxide ring for these monomers is generally accompanied by the concomitant ring opening of the cyclobutane ring structure to yield a gem-di-methyl group in the main chain.

  19. Neutron-multiplicity distributions for (. cap alpha. ,xn. gamma. ) reactions with E/sub. cap alpha. / = 50--120 MeV and the pre-equilibrium neutron deexcitation processes

    SciTech Connect

    Maeda, K.; Shibata, T.; Ejiri, H.; Sakai, H.

    1983-08-01

    Pre-equilibrium and equilibrium deexcitation processes for (..cap alpha..,xn ..gamma..) reactions induced by 50--120 MeV ..cap alpha.. particles were studied. Reaction channels were identified by measuring rotational ..gamma.. rays characteristic of the reaction residues. The branching of the reaction channels gave neutron multiplicity distributions. Features characteristic of the pre-equilibrium process were seen in the reaction channels with small neutron multiplicity x. An exciton model calculation code was developed so as to incorporate both multiparticle emission at the fast pre-equilibrium stage and multiparticle p evaporation at the slow equilibrium stage. The calculation reproduced the neutron multiplicity distributions in the whole range of E/sub ..cap alpha../ = 50--120 MeV. The pre-equilibrium fractions and the entry lines from the pre-equilibrium stage to the equilibrium one were deduced. The pre-equilibrium fractions were found to be approximately 40--60 %, being rather independent of the individual reaction channel. The entry lines slowly increase from 25 to 35 MeV with increasing projectile energy.

  20. Kinetics of ozonation. 4. Reactions of ozone with. cap alpha. -tocopherol and oleate and linoleate esters in carbon tetrachloride and in aqueous micellar solvents

    SciTech Connect

    Giamalva, D.H.; Church, D.F.; Pryor, W.A.

    1986-10-15

    Vitamin E (..cap alpha..-tocopherol; ..cap alpha..-T) is known to protect animals against the deleterious effects of ozone in polluted air; one such effect is the ozone-initiated autooxidation of polyunsaturated fatty acids (PUFA) that occur in membranes. In order to assess the possibility of a direct reaction of ozone with ..cap alpha..-T competing with the very fast ozone-PUFA reaction, we have measured the rates of reaction of ozone with ..cap alpha..-T, oleic acid, and linoleic acid. I CCl/sub 4/ as solvent, ..cap alpha..-T reacts with ozone with a rate constant of about 5500 M/sup -1/ s/sup -1/; methyl oleate and methyl linoleate react 2 orders of magnitude faster. In aqueous micellar solutions the rate constants for ..cap alpha..-T and the fatty acids are more similar. The k for the ozone/..cap alpha..-T reaction is about 1 x 10/sup 6/ M/sup -1/ s/sup -1/ at pH 7, but decreases as the solution becomes more acidic; the k's for oleic acid and linoleic acid are ca. 1 x 10/sup 6/ M/sup -1/ s/sup -1/ and exhibit no significant pH dependence. Since the ratio of fatty acids to ..cap alpha..-T in membranes is typically at least 100-1000 to 1, we conclude that the direct reaction of ozone with ..cap alpha..-T is unlikely. Thus, the protection that vitamin E provides to animals breathing ozone-containing air must result from vitamin E acting as a free radical scavenger. We have also detected the ..cap alpha..-tocopheroxyl radical as an intermediate from the reaction of ozone with ..cap alpha..-T both in CCl/sub 4/ and aqueous micelles using electron spin resonance spectroscopy. The authors suggest that the observation of this intermediate is consistent with an initial electron transfer from ..cap alpha..-T to ozone.

  1. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

    PubMed

    Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

    2012-03-01

    Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

  2. Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake.

    PubMed

    Leonetti, J P; Degols, G; Lebleu, B

    1990-01-01

    We have previously shown that antisense oligomers linked to poly(L-lysine) (PLL) exhibit antiviral properties against vesicular stomatitis virus (VSV) at concentrations lower than 1 microM. The conjugation to PLL provides an interesting alternative to natural or neutral oligomers to increase the biological effects of antisense oligomers. The internalization pathway of oligomer-PLL conjugates as compared to unconjugated oligomers has been studied in L929 cells. In parallel to their enhanced antiviral activity, PLL increases greatly the uptake of fluorescently tagged oligomers. This internalization follows a classical endocytic pathway and the oligomer has to be cleaved from PLL in the cell to exhibit an antiviral effect.

  3. Bladder carcinogenesis in rats subjected to ureterosigmoidostomy and treated with L-lysine.

    PubMed

    Dornelas, Conceição Aparecida; Santos, Alessandra Marques Dos; Correia, Antonio Lucas Oliveira; Juanes, Camila de Carvalho; Coelho, João Paulo Ferreira; Cunha, Bianca Lopes; Maciel, André Vinicius Vieira; Jamacaru, Francisco Vagnaldo Fechine

    2016-01-01

    to evaluate the effect of L-lysine in the bladder and intestinal epithelia in rats submitted to vesicosigmoidostomy. we divided forty Wistar rats into four groups: group I - control group (Sham); group II - submitted to vesicosigmoidostomy and treated with L-lysine 150mg/kg; group III - submitted only to vesicosigmoidostomy; and group IV - received L-lysine 150mg/kg. After eight weeks the animals were sacrificed. in the bladders of all operated animals we observed simple, papillary and nodular hyperplasia of transitional cells, transitional cell papillomas and squamous metaplasia. As for the occurrence of aberrant crypt foci in the colons of operated animals, we did not observe statistically significant differences in any of the distal, proximal and medium fragments, or in all fragments together (p=1.0000). Although statistically there was no promotion of carcinogenesis in the epithelia of rats treated with L-lysine in the observed time, it was clear the histogenesis of bladder carcinogenesis in its initial phase in all operated rats, this being probably associated with chronic infection and tiny bladder stones. o objetivo deste trabalho é avaliar o efeito da L-lisina nos epitélios vesical e intestinal de ratas submetidas à vesicossigmoidostomia. quarenta ratas Wistar, foram divididas em quatro grupos: grupo I- grupo controle (Sham); grupo II- submetido à vesicossigmoidostomia e tratado com L-lisina 150mg/kg; grupo III- submetido apenas à vesicossigmoidostomia; e grupo IV- recebeu L-lisina 150mg/kg. Após oito semanas os animais foram sacrificados. na bexiga de todos os animais operados observou-se hiperplasia simples, papilar e nodular de células transicionais, papiloma de células transicionais e metaplasia escamosa. Quanto à ocorrência de focos de criptas aberrantes nos colos dos animais operados, não foi evidenciado diferença estatística significante em nenhum dos fragmentos distal, proximal e médio, e todos juntos (P=1,0000). apesar de

  4. Influence of poly(L-lysine) on the structure of dipalmitoylphosphatidylglycerol/water dispersions studied by X-ray scattering.

    PubMed

    Förster, G; Schwieger, C; Faber, F; Weber, T; Blume, A

    2007-04-01

    The interaction between the negatively charged phospholipid DPPG and positively charged poly(L: -lysine) (PLL) of different lengths was studied by X-ray scattering in the SAXS and WAXS region. As a reference pure DPPG (Na salt) was investigated over a wide temperature range (-30 to 70 degrees C). The phase behavior of DPPG in aqueous and in buffer/salt dispersions showed a metastable subgel phase at low temperatures and a recrystallization upon heating before reaching the liquid-crystalline phase. The presence of additional salt stabilizes the bilayer structure and decreases the recrystallization temperature. Large changes in the SAXS region are not connected with changes in chain packing. In DPPG/PLL samples, the PLL is inserted between adjacent headgroup layers and liberates counterions which give rise to a freezing point depression. In the complex with DPPG PLL form an alpha-helical secondary structure at pH 7 and temperatures below the gel to liquid-crystalline phase transition. This prevents DPPG from recrystallization and strongly increases the stacking order. The lamellar repeat distance is decreased and fixed by the helix conformation of PLL in the gel phase. PLL with n = 14 is too short to form helices and is squeezed out reversibly from the interbilayer space upon cooling by freezing of trapped water. In dispersions with longer PLLs (n > 400) at -20 degrees C a 1D crystallization of PLL alpha-helices in the aqueous layer between the headgroups takes place. A structural model is presented for the lateral periodic complex, which is similar to the known cationic lipid/DNA complex.

  5. Assay and properties of rat yolk sac 25-hydroxyvitamin D/sub 3/ 1. cap alpha. -hydroxylase

    SciTech Connect

    Paulson, S.K.; Phelps, M.; DeLuca, H.F.

    1986-11-04

    An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D/sub 3/ 1..cap alpha..-hydroxylase (1..cap alpha..-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D/sub 3/ produced from incubations of yolk sac homogenates was extracted, purified by Sephadex LH-20 chromatography and straight- and reverse-phase high-performance liquid chromatography (HPLC), and measured by a competitive binding assay using chick intestinal receptor. The reaction is linear with time for up to 45 min at a substrate concentration of 80 ..mu..M and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 x 10/sup -3/ M) was found to inhibit 1-hydroxylation, but a 90% carbon monoxide-10% oxygen atmosphere did not, leaving open the question of involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, inhibited 1..cap alpha..-hydroxylation.

  6. Selective inhibition by chloramphenicol of pregnenolone-16. cap alpha. -carbonitrile-inducible rat liver cytochrome P-450 isozymes

    SciTech Connect

    Graves, P.E.; Kaminsky, L.S.; Halpert, J.

    1986-03-01

    Pregnenolone-16 ..cap alpha..-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effect of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of /sup 14/C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver.

  7. Clearance of. cap alpha. -aminoisobutyric acid during in-situ perfusion of the guinea pig placenta

    SciTech Connect

    Kelman, B.J.; Sikov, M.R.

    1983-05-01

    Extensive investigation of the transport of ..cap alpha..-aminoisobutyric acid (AIB; a nonmetabolized amino acid) has shown that AIB is actively transported from mother to fetus across the hemochorial placenta of the guinea pig. As a step towards clarifying the relative rolls of active and passive movements of amino acids across the placenta, it would be useful to obtain concurrent measurements of transplacental movements of a substance which crosses the placenta rapidly by simple diffusion (water) and of a substance which is actively transported across the placenta (AIB). In our study, placentas from guinea pigs between 59 and 61 days of gestation were perfused in situ through cannulated umbilical vessels with the maternal circulation left intact. Tritiated water and /sup 14/C-AIB were injected into a maternal jugular vein and maternal blood samples were obtained at 1 to 10 minute intervals; perfusate samples were collected sequentially after one pass through the placenta. Clearance of /sup 14/C-AIB from mother to fetus (AIB/sub MF/) and AIB concentrations in placental tissue, maternal plasma, and perfusate were consistent in magnitude with data obtained by other invetigators who have clearly shown an active transport of AIB in the placenta. On the other hand, in this study AIB/sub MF/ ranged from approximately 50% to 96% of the clearance of /sup 3/H-labeled water from mother to fetus (T/sub MF/) and that changes in AIB/sub MF/ correlated closely with changes in T/sub MF/ in all perfusions. Thus, it appears that AIB/sub MF/ closely paralleled T/sub MF/ and these data suggest that a relatively large component of AIB/sub MF/ is of passive origin in the in situ placenta.

  8. Transport of. cap alpha. -aminoisobutyric acid by Streptococcus pyogenes and its derived L-form

    SciTech Connect

    Reizer, J.; Panos, C.

    1982-01-01

    We studied the uptake of ..cap alpha..-aminoisobutyric acid (AIB) in Streptococcus pyogenes and its physiologically isotonic L-form. S. pyogenes cells starved for glucose or treated with carbonyl cyanide-m-chlorophenyl hydrazone accumulated limited amounts of AIB. A high apparent K/sub m/ value characterized the glucose-independent transport of AIB. The rate and extent of AIB accumulation significantly increased in the presence of glucose. Two saturable transport components with distinct apparent K/sub m/values characterized glycolysis-coupled transport of AIB. A biphasic Lineweaver-Burk plot was also obtained for L-alanine transport by glycolyzing S. pyogenes cells. AIB seems to share a common transport system(s) with glycine, L- and D-anine, L-serine, and L-valine. This was shown by the competitive exchange efflux of accumulated AIB. About 30% of the AIB uptake was not inhibited by a saturating amount of L-valine, indicating the existence of more than one system for AIB transport, p-Chloromercuribenzoate markedly inhibited the accumulation of AIB by both glycolyzing and glucose-starved cells. In contrast, carbonyl cyanide-m-chlorophenyl hydrazone affected only metabolism-dependent uptake of AIB, which was also sensitive to dinitrophenol, N-ethylmaleimide, iodoacetate, fluoride (NaF), arsenate, and N,N'-dicyclohexylcarbodiimide. These results are interpreted according to the chemiosmotic theory of Mitchell, whereby a proton motive force constitutes the driving force for AIB accumulation. AIB was not accumulated by the L-form. However, a temporary accumulation of AIB by a counterflow mechanism and a saturable system with a low apparent affinity were demonstrated for AIB transport by this organism. We suggest that a deficiency in the coupling of energy to AIB transport is responsible for the apparent lack of active AIB accumulation by the L-form.

  9. Isolation and characterization of a novel ε-poly-L-lysine producing strain: Streptomyces griseofuscus.

    PubMed

    Li, Shu; Tang, Lei; Chen, Xusheng; Liao, Lijuan; Li, Feng; Mao, Zhonggui

    2011-04-01

    ε-poly-L-lysine (ε-PL) is a homo-poly-amino acid of L-lysine which is used as a safe food preservative. The productivity of ε-PL in currently reported wild type strains is low. This study was aimed at finding novel ε-PL producing strains with higher productivity and new fermentative characters. An improved detection method was employed using methylene blue as an ε-PL secretion indicator. 137 strains forming transparent circles were isolated. The best one was identified as Streptomyces griseofuscus according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. The fermentative behavior of S. griseofuscus was investigated, and the ε-PL production was enhanced to 2.3 g/L in 5-L bioreactor by a pH control strategy. The yield of ε-PL reached 7.5 g/L in the fed-batch process. Compared with the reported wild type strains, S. griseofuscus produced relatively higher amounts of ε-PL, and might be a promising Streptomyces for ε-PL production.

  10. Low cytotoxic tissue adhesive based on oxidized dextran and epsilon-poly-L-lysine.

    PubMed

    Hyon, Suong-Hyu; Nakajima, Naoki; Sugai, Hajime; Matsumura, Kazuaki

    2014-08-01

    A novel adhesive hydrogel consisting of dextran and epsilon-poly(L-lysine) (dextran-PL) with multiple biomedical applications was developed. Periodate oxidation in aqueous media almost stoichiometrically introduces aldehyde groups in dextran molecules, and aldehyde dextran can react with the primary amino groups in epsilon-PL (ɛ-PL) at neutral pH to form a hydrogel. The gelation time of the hydrogel can be easily controlled by the extent of oxidation in dextran and of the acylation in ɛ-PL by anhydrides. The shear adhesion strength of dextran-PL was 10 times higher than that of fibrin glue, when wet collagen sheets were selected as test specimens. The cytotoxicity of aldehyde dextran and ɛ-PL were 1000 times lower than that of glutaraldehyde and poly(allylamine). The considerably low cytotoxicity of aldehyde dextran could be ascribed to its low reactivity with amine species when compared with glutaraldehyde. In contrast, a high reactivity of amino groups in ɛ-PL was observed when compared with glycine, L-lysine, and gelatin, which could be explained by their poor dissociation at neutral pH, thus leading to low cytotoxicity.

  11. Separation of lacquer polysaccharides and interaction with poly-L-lysine.

    PubMed

    Bai, Yuting; Yoshida, Takashi

    2013-10-15

    A naturally occurring acidic lacquer polysaccharide with glucuronic acid at the terminals of the complex branches has specific biological activities including promotion of blood coagulation and antitumor activities. The polysaccharide has two molecular weight fractions M¯n=10×10(4) and M¯n=3.0×10(4). In the present work, two pure fractions were isolated for the first time by Sephadex G-100 column chromatography. Then, each fraction was treated with diluted alkaline solution to decrease the molecular weights to M¯n=3.0×10(4) and M¯n=1.4×10(4), respectively. The NMR and IR spectra and specific rotations of the fractionated and original lacquer polysaccharides were almost identical, suggesting that the lacquer polysaccharides are an associated structure with several low molecular weight polysaccharides of M¯n=1.4×10(4). Interactions between each lacquer polysaccharide and poly-L-lysine, a model compound of proteins and peptides with positively-charged amino groups, were investigated by surface plasmon resonance (SPR) to elucidate the biological mechanism. The apparent dissociation-rate (kd), association-rate (ka), and dissociation constant (KD) obtained by SPR indicate that the lacquer polysaccharides had weaker interactions with poly-L-lysine than sulfated polysaccharides and that the interaction depended on the molecular weight. These SPR results suggest that the specific biological activities of lacquer polysaccharides originate from electrostatic interaction.

  12. Engineering of Corynebacterium glutamicum with an NADPH-Generating Glycolytic Pathway for l-Lysine Production ▿

    PubMed Central

    Takeno, Seiki; Murata, Ryosuke; Kobayashi, Ryosuke; Mitsuhashi, Satoshi; Ikeda, Masato

    2010-01-01

    A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP+ to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis. PMID:20851994

  13. L-lysine-L-tartaric acid: New molecular complex with nonlinear optical properties. Structure, vibrational spectra and phase transitions

    SciTech Connect

    Debrus, S.; Marchewka, M.K. . E-mail: mkm@int.pan.wroc.pl; Baran, J.; Drozd, M.; Czopnik, R.; Pietraszko, A.; Ratajczak, H.

    2005-09-15

    The first X-ray diffraction and vibrational spectroscopic analysis of a novel complex between L-lysine and L-tartaric acid is reported. The structure was solved in two temperatures (320 and 260 K) showing incommensurate phase between them. Room-temperature powder infrared and Raman measurements for the L-lysine-L-tartaric acid molecular complex (1:1) were carried out. DSC measurements on powder samples indicate two phase transitions points at about 295, 300 and 293, 300 K, for heating and cooling, respectively, with noticeable temperature interval between them. Second harmonic generation efficiency d {sub eff}=0.35 d {sub eff} (KDP)

  14. Adhesion of plant roots to poly-L-lysine coated polypropylene substrates.

    PubMed

    Towler, Melissa J; Weathers, Pamela J

    2003-03-06

    The ability to immobilize plant tissue in a bioreactor is an important process tool. We have shown that roots of several species rapidly attach to poly-L-lysine coated polypropylene mesh in a liquid environment. Using transformed roots of Artemisia annua as a model, the attachment process was found to be enhanced by sheep serum, but not BSA and inhibited by excess Mn(2+), but unaffected by Ca(2+) or Mg(2+). Attempts to characterize the molecule(s) responsible for binding using lectins and antibodies showed that the binding site does not appear to be glycosylated or vitronectin-like. This method of rapid attachment should prove useful for controlled immobilization of roots in bioreactors. Copyright 2002 Elsevier Science B.V.

  15. Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(L-lysine)–DNA Polyplexes

    PubMed Central

    Ketola, Tiia-Maaria; Hanzlíková, Martina; Leppänen, Linda; Raviña, Manuela; Bishop, Corey J.; Green, Jordan J.; Urtti, Arto; Lemmetyinen, Helge; Yliperttula, Marjo; Vuorimaa-Laukkanen, Elina

    2013-01-01

    The mechanism of polyethylenimine–DNA and poly(L-lysine)–DNA complex formation at pH 5.2 and 7.4 was studied by a time-resolved spectroscopic method. The formation of a polyplex core was observed to be complete at approximately N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. The data were analyzed further both by an independent binding model and by a cooperative model for multivalent ligand binding to multisubunit substrate. At pH 5.2, the polyplex formation was cooperative at all N/P ratios, whereas for pH 7.4 at N/P < 0.6 the polyplex formation followed independent binding changing to cooperative binding at higher N/Ps. PMID:23941196

  16. Polyethylene glycol-grafted poly-L-lysine as polymeric gene carrier.

    PubMed

    Choi, Y H; Liu, F; Kim, J S; Choi, Y K; Park, J S; Kim, S W

    1998-06-01

    A new series of gene carriers, polyethylene glycol (PEG)-grafted poly-L-lysine (PLL, mol. wt. = 25000) with three different PEG-grafted ratios (5, 10 and 25 mole%, which means 5, 10 and 25% of epsilon-amino group of PLL was modified by PEG), was synthesized. These new gene carriers, named comb-shaped PEG-g-PLL copolymer, showed a 5- to 30-fold increase in transfection efficiency compared to PLL alone on a human carcinoma cell line. It is likely that Hep G2 cells were transfected by plasmid DNA/PEG-g-PLL complexes through an endocytosis mechanism due to the fact that chloroquine increased transfection efficiency. Although Lipofectin, a cationic lipid formulation, showed slightly higher transfection efficiency than PEG-g-PLL in Hep G2 cells, our designed PEG-g-PLL demonstrated lower cytotoxicity, early gene expression and maintenance of gene expression for up to 96 h.

  17. Antimicrobial effect of medical adhesive composed of aldehyded dextran and ε-Poly(L-Lysine).

    PubMed

    Lee, Jeong-Hyun; Kim, Hye-Lee; Lee, Mi Hee; Taguchi, Hideaki; Hyon, Suong-Hyu; Park, Jong-Chul

    2011-11-01

    Infection of surgical wounds is a severe problem. Conventional tissue reattachment methods have limits of incomplete sealing and high susceptibility to infection. Medical adhesives have several advantages over traditional tissue reattachment techniques, but still have drawbacks, such as the probability of infection, low adhesive strength, and high cytotoxicity. Recently, a new medical adhesive (new-adhesive) with high adhesive strength and low cytotoxicity, composed of aldehyded dextran and ε-poly(L-lysine), was developed. The antimicrobial activity of the new-adhesive was assayed using agar media and porcine skin. In the agar diffusion method, inoculated microorganisms that contacted the new-adhesive were inactivated, but this was not dependent on the amount of new-adhesive. Similar to the agar media results, the topical antimicrobial effect of new-adhesive was confirmed using a porcine skin antimicrobial assay, and the effect was not due to physical blocking based on comparison with the group whose wounds were wrapped.

  18. A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-L-lysine.

    PubMed

    Zhou, Chuncai; Li, Peng; Qi, Xiaobao; Sharif, Abdul Rahim Mohamed; Poon, Yin Fun; Cao, Ye; Chang, Matthew W; Leong, Susanna Su Jan; Chan-Park, Mary B

    2011-04-01

    Hydrogels made from epsilon-poly-l-lysine-graft-methacrylamide (EPL-MA) have been found to have impressive wide spectrum antimicrobial activity against both bacteria (specifically Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus) and fungi (specifically Candida albicans and Fusarium solani). The EPL-MA hydrogel also possesses in vitro biocompatibility and EPL-MA solution is relatively non-hemolytic: the concentration needed for onset of human red blood cell (hRBC) hemolysis is 12,500 μg/mL so that the selectivity for the pathogenic microorganisms over hRBCs is 230-1560. Further, EPL-MA hydrogel can be conveniently ultraviolet-immobilized onto plasma-treated plastic surfaces to form thin highly adherent antimicrobial hydrogel coatings for medical devices and implants.

  19. Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.

    PubMed

    Babic, Michal; Horák, Daniel; Trchová, Miroslava; Jendelová, Pavla; Glogarová, Katerina; Lesný, Petr; Herynek, Vít; Hájek, Milan; Syková, Eva

    2008-03-01

    New surface-modified iron oxide nanoparticles were developed by precipitation of Fe(II) and Fe(III) salts with ammonium hydroxide and oxidation of the resulting magnetite with sodium hypochlorite, followed by the addition of poly( L-lysine) (PLL) solution. PLL of several molecular weights ranging from 146 ( L-lysine) to 579 000 was tested as a coating to boost the intracellular uptake of the nanoparticles. The nanoparticles were characterized by TEM, dynamic light scattering, FTIR, and ultrasonic spectrometry. TEM revealed that the particles were ca. 6 nm in diameter, while FTIR showed that their surfaces were well-coated with PLL. The interaction of PLL-modified iron oxide nanoparticles with DMEM culture medium was verified by UV-vis spectroscopy. Rat bone marrow stromal cells (rMSCs) and human mesenchymal stem cells (hMSC) were labeled with PLL-modified iron oxide nanoparticles or with Endorem (control). Optical microscopy and TEM confirmed the presence of PLL-modified iron oxide nanoparticles inside the cells. Cellular uptake was very high (more than 92%) for PLL-modified nanoparticles that were coated with PLL (molecular weight 388 00) at a concentration of 0.02 mg PLL per milliliter of colloid. The cellular uptake of PLL-modified iron oxide was facilitated by its interaction with the negatively charged cell surface and subsequent endosomolytic uptake. The relaxivity of rMSCs labeled with PLL-modified iron oxide and the amount of iron in the cells were determined. PLL-modified iron oxide-labeled rMSCs were imaged in vitro and in vivo after intracerebral grafting into the contralateral hemisphere of the adult rat brain. The implanted cells were visible on magnetic resonance (MR) images as a hypointense area at the injection site and in the lesion. In comparison with Endorem, nanoparticles modified with PLL of an optimum molecular weight demonstrated a higher efficiency of intracellular uptake by MSC cells.

  20. Non-Invasive Evaluation of Alginate/Poly-L-lysine/Alginate Microcapsules by Magnetic Resonance Microscopy

    PubMed Central

    Constantinidis, Ioannis; Grant, Samuel C.; Celper, Susanne; Gauffin-Holmberg, Isabel; Agering, Kristina; Oca-Cossio, Jose A.; Bui, Jonathan D.; Flint, Jeremy; Hamaty, Christine; Simpson, Nicholas E.; Blackband, Stephen J.

    2007-01-01

    In this report, we present data to demonstrate the utility of 1H MR microscopy to noninvasively examine alginate/poly-L-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-L-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6±6.2 μm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 minutes). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T2 relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T2 over the duration of the experiment, while ADC was unaffected. This decrease in T2 is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T2 was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T2 or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions. PMID:17239948

  1. Uptake of L-lysine by a double mutant of Saccharomyces cerevisiae.

    PubMed

    García, J C; Kotyk, A

    1988-01-01

    A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport. Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system. The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol. The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate). The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine. The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule. The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre. It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached. Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.

  2. Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the. cap alpha. subunits

    SciTech Connect

    Takahashi, M.; Catterall, W.A.

    1987-08-25

    Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against ..cap alpha.. subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, ..beta.. subunits (M/sub r/ 50,000) and lambda subunits (M/sub r/ 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with (/sup 3/H)isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-(methoxycarbonyl)pyridine-3-carboxylate ((/sup 3/H)PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the ..cap alpha../sub 2/ subunits of the skeletal muscle channel. The calcium channels were radiolabeled with /sup 32/P and /sup 125/I. It is concluded that these two calcium channels have a homologous, but distinct, ..cap alpha.. subunit as a major polypeptide component.

  3. Reaction mechanism of the /sup 12/C(/sup 3/He,. cap alpha. )/sup 11/C reaction

    SciTech Connect

    Kudo, Y.

    1982-11-01

    The mechanism of the reactions /sup 12/C(/sup 3/He, ..cap alpha..)/sup 11/C leading to the (3/2)/sup -/ ground state and (5/2)/sup -/ second excited state of /sup 11/C is studied using finite-range distorted-wave Born approximation with all one-step direct and exchange processes. By taking into account in detail the cluster structure of /sup 11/C and /sup 12/C, the transition to the (5/2)/sup -/ state which is a j forbidden direct process can be explained well by the exchange process.

  4. Acute CNS syndrome from /sup 10/B(n,. cap alpha. )/sup 7/Li irradiation of mouse brain

    SciTech Connect

    Slatkin, D.N.; Stoner, R.D.; Rosander, K.M.; Kalef-Ezra, J.K.; Laissue, J.A.

    1986-01-01

    The purpose of this study is to compare absorbed doses of /sup 10/B-neutron capture radiation and x radiation in the brains of head-exposed, body-shielded mice, at LD/sub 50/ for acute (<4 days post exposure) mortality from the central nervous system (CNS) radiation syndrome. This comparison leads to an estimate of the in vivo relative biological effectiveness (RBE) of heavy particle radiation (helium and lithium ions) from the /sup 10/B(n,..cap alpha..)/sup 7/Li nuclear reaction.

  5. Catalytic versatility of Bacillus pumilus. beta. -xylosidase: glycosyl transfer and hydrolysis promoted with. cap alpha. - and. beta. -D-xylosyl fluoride

    SciTech Connect

    Kasumi, T.; Tsumuraya, Y.; Brewer, C.F.; Kersters-Hilderson, H.; Claeyssens, M.; Hehre, E.J.

    1987-06-02

    Bacillus pumilus ..beta..-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of ..beta..-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with ..cap alpha..- and ..beta..-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of ..beta..-D-xylopyranosyl fluoride at a high rate, V = 6.25 ..mu..mol min/sup -1/ mg/sup -1/ at 0/sup 0/C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon ..cap alpha..-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, /sup 1/H NMR spectra of a digest of ..cap alpha..-D-xylosyl fluoride showed the substrate to be specifically converted to ..cap alpha..-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this inverting enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from ..cap alpha..-D-xylosyl fluoride to form a ..beta..-D-xylosidic product, followed by hydrolysis of the latter to produce ..cap alpha..-D-xylose. The transient intermediate product formed enzymically from ..cap alpha..-D-xylosyl fluoride in the presence of (/sup 14/C)xylose was isolated and shown by its specific radioactivity and /sup 1/H NMR spectrum as well as by methylatino and enzymic analyses to be 4-O-..beta..-D-xylopyranosyl-D-xylopyranose containing one (/sup 14/C)xylose residue.

  6. Human interleukin 1. beta. (IL-1. beta. ), a more powerful inducer of bone demineralization than interleukin 1. cap alpha. IL-1. cap alpha. ), parathyroid hormone (PTH) or prostaglandin E/sub 2/ (PGE/sub 2/) in vitro

    SciTech Connect

    Chin, R.C.; Hodges, Y.C.; Allison, A.C.

    1986-03-01

    Effects of human IL-1..cap alpha.. and IL-1..beta.., prepared by recombinant DNA technology on cultures of rat fetal long bones, prelabelled with /sup 45/Ca were studied. IL-1..beta.. was found to be the most powerful inducer of bone calcium loss so far known. Maximal activity (2.5 times the control rate of calcium loss) was induced by IL-1..beta.. at concentrations between 1 x 10/sup -10/ M to 6 x 10/sup -12/ M. With IL-1..cap alpha.. maximal activity (1.5 times the control rate of calcium loss) was obtained at 6 x 10/sup -10/ M. With bovine PTH (1-34) maximal activity (1.8 times the control rate of calcium loss) was obtained at 1 x 10/sup -8/ M. With PGE/sub 2/ maximal activity (1.6 times the control rate of calcium loss) was obtained at 1 x 10/sup -7/ M. The calcium loss induced by IL-1..beta.. was inhibited in the presence of 1 x 10/sup -7/ M indomethacin, 5 x 10/sup -5/ M naproxen or ketorolac, or 5 x 10/sup -6/ M cyclohexamide. These findings suggest that protein synthesis and prostaglandin formation are required to mediate bone demineralization induced by IL-1..beta...

  7. Immunochemical detection of a primase activity related subunit of DNA polymerase. cap alpha. from human and mouse cells using the monoclonal antibody

    SciTech Connect

    Yagura, T.; Kozu, T.; Seno, T.; Tanaka, S.

    1987-12-01

    A hybrid cell line (HDR-854-Er) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase ..cap alpha.. was established by immunizing mice with DNA replicase complex (DNA polymerase ..cap alpha..-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralized the primase activity as assessed either by the direct primase assay (incorporation of (..cap alpha..-/sup 32/P)AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase ..cap alpha.. activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase ..cap alpha... The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDA) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3 S DNA polymerase ..cap alpha.. which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase ..cap alpha... Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDA polypeptide is associated with DNA polymerase ..cap alpha.., and not with the primase. These results indicate that the 77-kDa polypeptide detected with the E4 antibody is not the primase but is a subunit firmly bound to DNA polymerase ..cap alpha.. catalytic polypeptide and yet influences the activity of the associated DNA primase.

  8. Influence of baking conditions and precursor supplementation on the amounts of the antioxidant pronyl-L-lysine in bakery products.

    PubMed

    Lindenmeier, Michael; Hofmann, Thomas

    2004-01-28

    The influence of baking conditions and dough supplements on the amounts of the antioxidant and Phase II-Enzyme modulating, protein-bound 2,4-dihydroxy-2,5-dimethyl-1-(5-acetamino-5-methoxycarbonyl-pentyl)-3-oxo-2H-pyrrol (pronyl-L-lysine) in bakery products was investigated in quantitative studies. These studies revealed high amounts of the antioxidant in bread crust, only low amounts in the crumb, and the absence of this compound in untreated flour. The amounts of pronyl-L-lysine were found to be strongly influenced by the intensity of the thermal treatment. For example, increasing the baking time from 70 to 210 min or increasing the baking temperature from 220 to 260 degrees C led to a 5- or 3-fold increase in the concentrations of this antioxidant in the crust, respectively. In addition, modifications in the recipe showed to have a major impact on pronyl-L-lysine formation. For example, substituting 5% of the flour with the lysine-rich protein casein or with 10% of glucose increased the amounts of the antioxidant by more than 200%. Quantitative analyses of commercial bread samples collected from German bakeries revealed the highest amount of 43 mg/kg for a full grain bread, followed by a rye/wheat bread, both of which have been sourdough fermented. A mixed-grain bread as well as pale wheat bread, both prepared without sourdough fermentation, contained significantly lower amounts of pronyl-L-lysine, and German pretzels, which are treated with a dilute sodium hydroxide solution prior to baking, contained only trace amounts of pronyl-L-lysine (e.g., less than 5 mg/kg were detectable in pretzels). Systematic studies revealed that the decrease of the pH value induced by microbial acid formation during sourdough fermentation is the clue for producing high amounts of pronyl-L-lysine in baking products. These data clearly demonstrate for the first time that the amounts of the antioxidant and chemopreventive compound pronyl-L-lysine in bakery products is strongly

  9. Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

    PubMed

    Jakobsen, Oyvind M; Brautaset, Trygve; Degnes, Kristin F; Heggeset, Tonje M B; Balzer, Simone; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

    2009-02-01

    Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

  10. Genomic clone encoding the. cap alpha. chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules

    SciTech Connect

    Cosgrove, L.J.; Sandrin, M.S.; Rajasekariah, P.; McKenzie, I.F.C.

    1986-02-01

    LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa ..beta.. subunit but are distinguished by their ..cap alpha.. chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. The authors report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in lambda phase Charon 4A and subsequent rescue of the lambda phage. Transfection with the purified recombinant lambda DNA yielded a transfectant that expressed the three human ..cap alpha.. chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine ..beta.. chain.

  11. Inhibition of reticulocyte lysate protein synthesis by heavy metal ions involves eIF-2. cap alpha. phosphorylation

    SciTech Connect

    Matts, R.; Hurst, R.; Schatz, J.

    1987-05-01

    Heavy metal ions inhibit protein synthesis and disaggregate polyribosomes in several tissues and cell types. They have initiated studies to determine the mechanism by which this inhibition occurs utilizing the hemin-supplemented rabbit reticulocyte lysate as a model system. The inhibition of protein synthesis observed in the presence of heavy metal ions occurs with biphasic kinetics in a concentration dependent manner, which correlates with the biological toxicity of the ion. Highly toxic ions (AsO/sub 2//sup -/, Cd/sup + +/, Hg/sup + +/, Pb/sup + +/) inhibit protein synthesis by 50% at concentrations of 5-10 ..mu..M. In comparison, Cu/sup + +/, Fe/sup + +/, Zn/sup + +/ and GSSG inhibited protein synthesis by 50% at concentrations of 60, 250, 300 and 350 ..mu..M, respectively. The inhibition of protein synthesis was accompanied by the phosphorylation of eIF-2..cap alpha.., and was reversed or prevented by the addition of 1 mM DTT, 10 mM cAMP, 2 mM MgGTP, eIF-2 or the reversing factor (RF). Glucose-6-phosphate was found to have no effect. The data indicate that the inhibition of protein synthesis observed with the addition of heavy metal ions to reticulocyte lysates is due to the phosphorylation of eIF-2..cap alpha...

  12. Jet physics in e/sup +/e/sup -/ annihilation: evidence for the running of. cap alpha. /sub s/

    SciTech Connect

    Bethke, S.

    1988-03-01

    The energy dependence of the relative production rate of 3-jet events is studied in hadronic e/sup +/e/sup -/ annihilation events at centre of mass energies between 22 and 56 GeV, using the data of the JADE, MARK-II and AMY collaborations at PETRA, PEP and TRISTAN. Three jet events are defined by a jet finding algorithm which is closely related to the definition of resolvable jets used in O(..cap alpha../sub s//sup 2/) perturbative QCD calculations, where the relative production rate of 3-jet events is roughly proportional to the strong coupling strength, ..cap alpha../sub s/. The observed production rates of 3-jet events decreases significantly with increasing centre of mass energy. The results, which are independent of fragmentation model calculations, can be directly compared to theoretically calculated jet production rates and are in good agreement with the QCD expectations of a running coupling strength, while the hypothesis of an energy independent coupling constant can be excluded with a significance of 5 standard deviations. Based on these results, the presented jet analysis also provides the possibility to detect first signs of the production of new and heavy particles in the early stage of data taking at SLC and LEP.

  13. Radiative corrections to e/sup +/e/sup -/ reactions to all orders in. cap alpha. using the renormalization group

    SciTech Connect

    Tsai, Y.S.

    1983-01-01

    Renormalization group technique is used to improve the accuracy of the lowest order radiative corrections in QED. The exponentiation of infrared terms comes automatically. It also leads to exponentiation of the vertex functions. It predicts the existence of conversion of photons into pairs and the result agrees with the Kroll-Wada relation. Kinoshita-Lee-Nauenberg cancellation of mass singularities occurs to all order in ..cap alpha.. in leading log approximation in the final state if we sum over all the final states. Higher order corrections to the order ..cap alpha../sup 3/ asymmetry is shown to be small. The results are used to derive useful formulas for the radiative corrections to processes such as e/sup +/e/sup -/ ..-->.. ..mu../sup +/..mu../sup -/, e/sup +/e/sup -/ ..-->.. ..mu../sup +/..mu../sup -/..gamma.., e/sup +/e/sup -/ ..-->.. hadron continuum, e/sup +/e/sup -/ ..-->.. very narrow resonance such as phi, and e/sup +/e/sup -/ ..-->.. not very narrow resonance such as Z/sup 0/.

  14. Search for P-odd asymmetry in the. cap alpha. emission in the capture of polarized thermal neutrons by /sup 6/Li and /sup 10/B nuclei

    SciTech Connect

    Vesna, V.A.; Egorov, A.I.; Kolomenskii, A.; Kornyushkin, A.F.; Lobashev, V.M.; Okunev, I.S.; Peskov, B.G.; Pirozhkov, A.N.; Smotritskii, L.M.; Titov, N.A.

    1983-09-10

    Measurements by an integral method in a geometry ruling out an effect of a P-even left-right asymmetry have yielded limitations on the magnitude of the P-odd asymmetry in several reactions: Vertical Bara/sub p/Vertical Bar<1.4 x 10/sup -6/ for the reaction /sup 6/Li(n,..cap alpha..)/sup 3/H, Vertical Bara/sub p/Vertical Bar<8 x 10/sup -6/ for the reaction /sup 10/B(n,..cap alpha../sub 0/)/sup 7/Li, and Vertical Bara/sub p/Vertical Bar<1.5 x 10/sup -6/ for the reaction /sup 10/B(n,..cap alpha../sub 1/)/sup 7/Li* (90% confidence level).

  15. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    SciTech Connect

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-10-01

    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  16. Antifreeze effect of carboxylated ε-poly-L-lysine on the growth kinetics of ice crystals.

    PubMed

    Vorontsov, Dmitry A; Sazaki, Gen; Hyon, Suong-Hyu; Matsumura, Kazuaki; Furukawa, Yoshinori

    2014-08-28

    Some biological substances control the nucleation and growth of inorganic crystals. Antifreeze proteins, which prohibit ice crystal growth in living organisms, promise are also important as biological antifreezes for medical applications and in the frozen food industries. In this work, we investigated the crystallization of ice in the presence of a new cryoprotector, carboxylated ε-poly-L-lysine (COOH-PLL). In order to reveal the characteristics and the mechanism of its antifreeze effect, free-growth experiments of ice crystals were carried out in solutions with various COOH-PLL concentrations and degrees of supercooling, and the depression of the freezing point and growth rates of the tips of ice dendrites were obtained using optical microscopy. Hysteresis of growth rates and depression of the freezing point was revealed in the presence of COOH-PLL. The growth-inhibition effect of COOH-PLL molecules could be explained on the basis of the Gibbs-Thomson law and the use of Langmuir's adsorption isotherm. Theoretical kinetic curves for hysteresis calculated on the basis of Punin-Artamonova's model were in good agreement with experimental data. We conclude that adsorption of large biological molecules in the case of ice crystallization has a non-steady-state character and occurs more slowly than the process of embedding of crystal growth units.

  17. Poly-L-lysine Prevents Senescence and Augments Growth in Culturing Mesenchymal Stem Cells Ex Vivo

    PubMed Central

    Heo, June Seok; Kim, Hyun Ok; Song, Seung Yong; Lew, Dae Hyun; Choi, Youjeong

    2016-01-01

    Mesenchymal stem cells (MSCs) possess great therapeutic potential. Efficient in vitro expansion of MSCs is however necessary for their clinical application. The extracellular matrix (ECM) provides structural and biochemical support to the surrounding cells, and it has been used as a coating substrate for cell culture. In this study, we have aimed to improve the functionality and stemness of MSCs during culture using poly-L-lysine (PLL). Functionality of MSCs was analysed by cell cycle analysis, differentiation assay, β-galactosidase staining, and RT-PCR. Furthermore, we assessed the global gene expression profile of MSCs on uncoated and PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth rate with increased S-phase and upregulated expression of the stemness markers. In addition, their osteogenic differentiation potential was increased, and genes involved in cell adhesion, FGF-2 signalling, cell cycle, stemness, cell differentiation, and cell proliferation were upregulated, compared to that of the MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoated plates expressed higher β-galactosidase than the MSCs on PLL-coated plates. We demonstrate that PLL provides favourable microenvironment for MSC culture by reversing the replicative senescence. This method will significantly contribute to effective preparation of MSCs for cellular therapy. PMID:27403437

  18. Metabolomic analysis of antimicrobial mechanisms of ε-poly-L-lysine on Saccharomyces cerevisiae.

    PubMed

    Bo, Tao; Liu, Miao; Zhong, Cheng; Zhang, Qian; Su, Qin-Zhi; Tan, Zhi-Lei; Han, Pei-Pei; Jia, Shi-Ru

    2014-05-14

    ε-Poly-L-lysine (ε-PL), a naturally occurring amino acid homopolymer, has been widely used as a food preservative. However, its antimicrobial mechanism has not been fully understood. This study investigated the antimicrobial mode of action of ε-PL on a yeast, Saccharomyces cerevisiae. When treated with ε-PL at the concentration of 500 μg/mL, cell mortality was close to 100% and the phospholipid bilayer curvature, pores, and micelles on the surface of S. cerevisiae were clearly observed by scanning electron microscopy (SEM). At the level of 200 μg/mL, ε-PL significantly inhibited the cell growth of S. cerevisiae. When treated with 50 μg/mL ε-PL, the yeast cell was able to grow but the cell cycle was prolonged. A significant increase in cell membrane permeability was induced by ε-PL at higher concentrations. Metabolomics analysis revealed that the ε-PL stress led to the inhibition of primary metabolic pathways through the suppression of the tricarboxylic acid cycle and glycolysis. It is therefore proposed that the microbiostatic effect of ε-PL at lower levels on S. cerevisiae is achieved by inducing intracellular metabolic imbalance via disruption of cell membrane functions. Moreover, the results suggested that the antimicrobial mechanism of ε-PL on S. cerevisiae can in fact change from microbiostatic to microbicidal when the concentration of ε-PL increased, and the mechanisms of these two modes of action were completely different.

  19. Application of biodegradable dendrigraft poly-l-lysine to a small interfering RNA delivery system.

    PubMed

    Kodama, Yukinobu; Kuramoto, Haruka; Mieda, Yukari; Muro, Takahiro; Nakagawa, Hiroo; Kurosaki, Tomoaki; Sakaguchi, Miako; Nakamura, Tadahiro; Kitahara, Takashi; Sasaki, Hitoshi

    2017-01-01

    Dendrigraft poly-l-lysine (DGL), including its central core, consists entirely of lysine, hence it is completely biodegradable. We applied DGL in a small interfering RNA (siRNA) delivery system. Binary complexes with siRNA and DGL had particle sizes of 23-73 nm and ζ-potentials of 34-42 mV. The siRNA-DGL complexes showed significant silencing effects in a mouse colon carcinoma cell line expressing luciferase (Colon26/Luc cells). The siRNA-DGL complexes induced slight cytotoxicity and hematological toxicity at a high charge ratio of DGL to siRNA, probably because of their cationic charges. Therefore, we recharged the siRNA-DGL complexes with γ-polyglutamic acid (γ-PGA), a biodegradable anionic compound, which was reported to reduce the cytotoxicity of cationic complexes. The ternary complexes showed particle sizes of 35-47 nm at a charge ratio of greater than 14 to siRNA with negative charges. Strong silencing effects of the ternary complexes were observed in Colon26/Luc cells without cytotoxicity or hematological toxicity. The cellular uptake and degradation of the binary and ternary complexes were confirmed by fluorescence microscopy. The ternary complexes suppressed luciferase activity in the tumor after direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, a potentially important siRNA delivery system was constructed using biodegradable DGL.

  20. Mixed poly(dopamine)/poly(L-lysine) (composite) coatings: from assembly to interaction with endothelial cells.

    PubMed

    Zhang, Yan; Lynge, Martin E; Teo, Boon M; Ogaki, Ryosuke; Städler, Brigitte

    2015-08-01

    Engineered polymer films are of significant importance in the field of biomedicine. Poly(dopamine) (PDA) is becoming more and more a key player in this context. Herein, we deposited mixed films consisting of PDA and poly(L-lysine) (PLL) of different molecular weights. The coatings were characterized by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and X-ray photoelectron spectroscopy. The protein adsorption to the mixed films was found to decrease with increasing amounts of PLL. PDA/PLL capsules were also successfully assembled. Higher PLL content in the membranes reduced their thickness while the ζ-potential increased. Further, endothelial cell adhesion and proliferation over 96 h were found to be independent of the type of coating. Using PDA/PLL in liposome-containing composite coatings showed that sequential deposition of the layers yielded higher liposome trapping compared to one-step adsorption except for negatively charged liposomes. Association/uptake of fluorescent cargo by adherent endothelial cells was found to be different for PDA and PDA/PLL films. Taken together, our findings illustrate the potential of PDA/PLL mixed films as coatings for biomedical applications.

  1. Crystal structure, spectroscopic investigation and thermal properties of L-lysine p-toluenesulfonate

    NASA Astrophysics Data System (ADS)

    Wang, L.; Wang, D. H.; Zhang, G. H.; Xu, D.; Deng, W. X.

    2016-03-01

    A novel organic crystal was prepared from L-lysine (Lly) and p-toluenesulfonic acid (pTS), which was grown from an aqueous solution by slow cooling method. The crystal system and the lattice parameters have been confirmed by single crystal X-ray diffraction studies. The FT-IR, FT-Raman, 1H-NMR and 13C-NMR spectral of the crystal have been recorded and analyzed. The spectral analyses confirmed the presence of various functional groups and the molecular configurations in LLTS crystal. The UV-Vis-NIR transmittance spectrum has been carried out which shows the cutoff wavelength around 280 nm. The thermal properties of crystal have been evaluated from thermogravimetric (TG) and differential thermal analysis (DTA). The melting point of grown crystal is fairly high, at around 259 °C. The nonlinear optical (NLO) properties of LLTS crystal were demonstrated by powder SHG experiment and also by quantum chemical calculations. The powder SHG efficiency of LLTS crystal is relatively low and very different from theoretical calculation results.

  2. Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

    PubMed

    Liu, Shuai; Yang, Jixiang; Ren, Hongqi; O'Keeffe-Ahern, Jonathan; Zhou, Dezhong; Zhou, Hao; Chen, Jiatong; Guo, Tianying

    2016-03-01

    Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their cationic properties is imperative. Herein, two uncharged functional diblock oligomers P(DMAEL-b-NIPAM) and P(DMAEL-b-Vlm) were prepared from a lactose derivative, N-iso-propyl acrylamide (NIPAM) as well as 1-vinylimidazole (Vlm) and further functionalized with four small ligands folate, glutathione, cysteine and arginine, respectively, aiming to enhance the interactions of complexes with cells, which were quantified utilizing a quartz crystal microbalance (QCM) biosensor, circumventing the tedious material screening process of cell transfection. Upon incorporation with PLL and DNA, the multifunctional oligomers endow the formulated ternary complexes with great properties suitable for transfection, such as anti-aggregation in serum, destabilized endosome membrane, numerous functional sites for promoted endocytosis and therefore robust transfection activity. Furthermore, different from the conventional strategy of decreasing cytotoxicity by reducing the charge density, the multifunctional oligomer incorporation strategy maintains the highly positive charge density, which is essential for efficient cellular uptake. This system develops a new platform to modify natural polycations towards clinical gene therapy.

  3. [Effect of oxygen-vectors on the production of ε-poly-L-lysine].

    PubMed

    Bo, Fangfang; Xu, Zhaoxian; Sun, Zhuzhen; Cao, Changhong; Xia, Jun; Xui, Hong; Feng, Xiaohai

    2015-03-01

    To enhance the production of ε-poly-L-lysine (ε-PL) by improving dissolved oxygen level of the fermentation system, different oxygen-vectors were added to broth and n-dodecane was screened as the best oxygen-vector. The best amount of n-dodecane was 0.5% (V/V) and the best time was at start of the fermentation. In a fed-batch fermentation in a 5 L bioreactor, ε-PL concentration reached a maximum of (30.8 ± 0.46) g/L and the dry cell weight obtained was (33.8 ± 0.29) g/L, increasing by 31.6% and 20.7% compared with the control group, respectively. This improvement can be related to 0.5% n-dodecane could maintain dissolved oxygen concentration > 32% of air concentration compared with 23.8% in ε-PL production phase, and the production of a main by-product, poly-L-diaminopropionic acid, fell by 31%. These results indicated that the dissolved oxygen level in the broth was improved by adding n-dodecane, which can inhibit the by-product production and improve the biosynthesis of ε-PL.

  4. Insulin/poly(ethylene glycol)-block-poly(L-lysine) Complexes: Physicochemical Properties and Protein Encapsulation.

    PubMed

    Pippa, Natassa; Kalinova, Radostina; Dimitrov, Ivaylo; Pispas, Stergios; Demetzos, Costas

    2015-06-04

    Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides.

  5. Impact of Ion Binding on Poly-L-Lysine (Un)folding Energy Landscape and Kinetics

    PubMed Central

    Xiong, Kan; Asher, Sanford A.

    2012-01-01

    We utilize T-jump UV resonance Raman spectroscopy to study the impact of ion binding on the equilibrium energy landscape and on (un)folding kinetics of poly-L-lysine (PLL). We observe that the relaxation rates of the folded conformations (including π-helix (bulge), pure α-helix and turns) of PLL are slower than those of short alanine based peptides. The PLL pure α-helix folding time is similar to that of short alanine based peptides. We, for the first time have directly observed that turn conformations are α-helix and π-helix (bulge) unfolding intermediates. ClO4− binding to the lys side chain –NH3+ groups and the peptide backbone slows the α-helix unfolding rate compared to that in pure water, but little impacts the folding rate, resulting in an increased α-helix stability. ClO4− binding significantly increases the PLL unfolding activation barrier but little impacts the folding barrier. Thus, the PLL folding coordinate differs from the unfolding coordinate. The π-helix (bulge) unfolding and folding coordinates do not directly go through the α-helix energy well. Our results clearly demonstrate that PLL (un)folding is not a two-state process. PMID:22612556

  6. Influence of the sulfation degree of glycosaminoglycans on their multilayer assembly with poly-l-lysine.

    PubMed

    Teixeira, Raquel; Reis, Rui L; Pashkuleva, Iva

    2016-09-01

    We report on the build-up and the intrinsic properties of polyelectrolyte multilayer films from poly-l-lysine and glycosaminoglycans (GAGs) with different sulfation degree, i.e. different charge. We used three complementary techniques, namely electrokinetic analysis (EKA), quartz-crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR), to characterize the assembly process and to assess the properties of the obtained films. EKA elucidated the contribution of the polymers charged groups to the net surface charge of the films and suggested that the assembly process is not solely driven by electrostatic interactions. The combined analysis of QCM-D and SPR data demonstrated that the mechanical properties of the films are dependent on the polymer charge: sulfated GAGs (heparin and chondroitin sulfate) form elastic films while hyaluronan (no sulfation) assembles into multilayer constructs with viscous behavior. The contribution of the water content to these distinct regimes is also discussed. Finally, we show that rather complete characterization of the film properties is possible by SPR employing the two-wavelength and two-media approach: thickness, adsorbed mass, refractive index, and interaction kinetics of the assembly process can be studied by SPR alone.

  7. Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth

    PubMed Central

    Al-Jamal, Khuloud T.; Al-Jamal, Wafa’ T.; Akerman, Simon; Podesta, Jennifer E.; Yilmazer, Açelya; Turton, John A.; Bianco, Alberto; Vargesson, Neil; Kanthou, Chryso; Florence, Alexander T.; Tozer, Gillian M.; Kostarelos, Kostas

    2010-01-01

    This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G6) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems. PMID:20150514

  8. Heterologous Production of Hyaluronic Acid in an ε-Poly-l-Lysine Producer, Streptomyces albulus

    PubMed Central

    Yoshimura, Tomohiro; Shibata, Nobuyuki; Hamano, Yoshimitsu

    2015-01-01

    Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo. PMID:25795665

  9. Quantification of l-lysine in cheese by a novel amperometric biosensor.

    PubMed

    Ciriello, Rosanna; Cataldi, Tommaso R I; Crispo, Fabiana; Guerrieri, Antonio

    2015-02-15

    Lysine quantification in cheese by a novel, highly selective amperometric biosensor is reported. Based on l-lysine-α-oxidase immobilized by co-crosslinking onto Platinum (Pt) electrodes modified by overoxidized polypyrrole, the sensor proved almost specific to lysine, sensitive and stable. The pure enzymatic nature of current signals was confirmed by a control electrode modified without enzyme. The precision of the method showed relative standard deviations of 4.7% and 9.2% respectively for Parmigiano Reggiano and Grana Padano cheese (n=5). The recovery data on various cheese, spiked with lysine at 50-100% of the measured content, ranged from 85% to 99%. Different types of cheese were analysed showing lysine concentrations related to the ripening time and the manufacture technology, in agreement with literature data. Within dairy products, no appreciable lysine was detected in yogurts. The method adopted revealed suited to satisfy the demands for precise and sensitive detection of lysine with minimal sample preparation and clean-up. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Shell and core cross-linked poly(L-lysine)/poly(acrylic acid) complex micelles.

    PubMed

    Hsieh, Yi-Hsuan; Hsiao, Yung-Tse; Jan, Jeng-Shiung

    2014-12-21

    We report the versatility of polyion complex (PIC) micelles for the preparation of shell and core cross-linked (SCL and CCL) micelles with their surface properties determined by the constituent polymer composition and cross-linking agent. The negatively and positively charged PIC micelles with their molecular structure and properties depending on the mixing weight percentage and polymer molecular weight were first prepared by mixing the negatively and positively charged polyions, poly(acrylic acid) (PAA) and poly(L-lysine) (PLL). The feasibility of preparing SCL micelles was demonstrated by cross-linking the shell of the negatively and positively charged micelles using cystamine and genipin, respectively. The core of the micelles can be cross-linked by silica deposition to stabilize the assemblies. The shell and/or core cross-linked micelles exhibited excellent colloid stability upon changing solution pH. The drug release from the drug-loaded SCL micelles revealed that the controllable permeability of the SCL micelles can be achieved by tuning the cross-linking degree and the SCL micelles exhibited noticeable pH-responsive behavior with accelerated release under acidic conditions. With the versatility of cross-linking strategies, it is possible to prepare a variety of SCL and CCL micelles from PIC micelles.

  11. Low-Density Neuronal Networks Cultured using Patterned Poly-L-Lysine on Microelectrode Arrays

    PubMed Central

    Jun, Sang Beom; Hynd, Matthew R.; Dowell-Mesfin, Natalie; Smith, Karen L.; Turner, James N.; Shain, William; Kim, Sung June

    2009-01-01

    Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-L-lysine (PLL) using microcontact printing (µCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 µm-wide lines for directing process growth and 20 µm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10–14 days in networks using neuron densities as low as 200 cells/mm2. Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K+ (10–12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors. PMID:17049614

  12. Physical, mechanical and antimicrobial properties of starch films incorporated with ε-poly-L-lysine.

    PubMed

    Zhang, Liming; Li, Ruichao; Dong, Feng; Tian, Aiying; Li, Zhengjun; Dai, Yujie

    2015-01-01

    Starch/ε-poly-L-lysine (ε-PL) composite films were prepared by combining 4% (w/v) gelatinized cornstarch and varying the level of ε-PL. The physical, mechanical and antimicrobial properties of these films were investigated. Fourier-transform infrared spectra (FT-IR) showed that the carbonyl group stretching vibration band of the ε-PL molecule shifted from 1646 cm(-1) to 1673 cm(-1) in the composite films. Differential scanning calorimetry (DSC) results indicated that there were sharp endothermal peaks at 215-230 °C for the composite films. These results indicated that there was an intense interaction between the two components. The films incorporated with ε-PL showed a higher tensile strength (TS) and elongation-at-break (E) than those of the starch film alone. These composite films exhibited effective inhibition against Escherichia coli and Bacillus subtilis, films containing 2% (w/w) ε-PL effectively suppressed the growth of the tested microbes (P<0.05). The starch/ε-PL films showed a low inhibitory effect on Aspergillus niger. This antimicrobial trend of the composite films was in agreement with the results of free ε-PL. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Centromeric Alpha-Satellite DNA Adopts Dimeric i-Motif Structures Capped by AT Hoogsteen Base Pairs.

    PubMed

    Garavís, Miguel; Escaja, Núria; Gabelica, Valérie; Villasante, Alfredo; González, Carlos

    2015-06-26

    Human centromeric alpha-satellite DNA is composed of tandem arrays of two types of 171 bp monomers; type A and type B. The differences between these types are concentrated in a 17 bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C-rich strand of the two main variants of the human alpha-satellite A box. We show that, under acidic conditions, the C-rich strands of two A boxes self-recognize and form a head-to-tail dimeric i-motif stabilized by four intercalated hemi-protonated C:C(+) base pairs. Interestingly, the stack of C:C(+) base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three-dimensional structure, although the residues involved in the formation of the i-motif core are different in each case. Together with previous studies showing that the B box (known as the CENP-B box) also forms dimeric i-motif structures, our finding of this non-canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i-motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Electronic structure of the bcc transition metals: Thermoreflectance studies of bulk V, Nb, Ta, and. cap alpha. TaH/sub x/

    SciTech Connect

    Rosei, R.; Colavita, E.; Franciosi, A.; Weaver, J.H.; Peterson, D.T.

    1980-04-15

    Thermoreflectance studies of bulk samples of V, Nb, Ta, and ..cap alpha..-phase TaH/sub x/ are reported and the results interpreted in terms of recent band calculations. The first interband transition is identified as a transition involving the ..sigma.. band at E/sub F/. An M/sub 3/ critical-point transition is attributed to states at N. Significant changes induced by interstitial hydrogen in solid solution ..cap alpha..-TaH/sub x/ are observed and interpreted as due to hybridization and lowering of the N/sup prime//sub 1/ eigenenergy.

  15. Amphiphilic cationic [dendritic poly(L-lysine)]-block-poly(L-lactide)-block-[dendritic poly(L-lysine)]s in aqueous solution: self-aggregation and interaction with DNA as gene delivery carriers.

    PubMed

    Zhu, Yingdan; Sheng, Ruilong; Luo, Ting; Li, Hui; Sun, Wenyan; Li, Yang; Cao, Amin

    2011-02-11

    A new series of triblock [dendritic poly(L-lysine)]-block-PLLA-block-[dendritic poly(L-lysine)]s (DL(2) -PLLA-DL(2) ) with PLLA block lengths of 11.5-26.5 and double 2-generation PLL dendrons DL(2) as model cationic amphiphiles were synthesized and characterized. Their CAC, self-aggregation and plasmid DNA binding affinities in pure water and PBS were studied. The PLLA block length dependence of particle size, morphology and ξ potential for organized pDNA/amphiphile polyplex aggregates were examined. Finally, toxicities of these DL(2) -PLLA-DL(2) amphiphiles and their polyplexes were assayed by MTT with HeLa, SMMC-7721 and COS-7 cells, and COS-7 cell luciferase and eGFP gene transfection efficacies with these amphiphiles as the delivery carriers were investigated.

  16. Dendritic poly(L-lysine)-b-Poly(L-lactide)-b-dendritic poly(L-lysine) amphiphilic gene delivery vectors: roles of PLL dendritic generation and enhanced transgene efficacies via termini modification.

    PubMed

    Li, Yang; Zhu, Yingdan; Xia, Kejia; Sheng, Ruilong; Jia, Lin; Hou, Xiaodong; Xu, Yuhong; Cao, Amin

    2009-08-10

    As an effort to prepare new efficient gene delivery vectors, we have recently developed and reported an amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) D(2)-PLLA-D(2) with two-generation PLL dendrons and a PLLA block. In this work, we continued to explore the roles of dendritic PLL generation in DNA binding and intracellular delivery of gene, and a new series of amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine)s D(n)-PLLA-D(n) (n = 3-5) were synthesized and were structurally characterized. Furthermore, plasmid DNA binding affinity for these cationic amphiphiles was examined by agarose gel electrophoresis and fluorescence titration assay in pure water and PBS buffer solution containing 150 mM NaCl (pH = 7.4), respectively. By dynamic light scattering (DLS) and transmission electronic microscopy (TEM), the interaction and complexation in between were investigated, concerning the DNA/vector polyplex particle morphologies and zeta potentials. Utilizing a human hepatocellular carcinoma cell-line SMMC-7721, cell toxicity, and gene transfection in vitro were explored. To further improve transgene efficiency for these synthetic cationic amphiphiles as gene delivery vectors, new structural DE(n)-PLLA-DE(n) (n = 2-3) were prepared through an amino termini modification of the D(n)-PLLA-D(n) (n = 2-3) with less toxic 4,7,10,13-tetraazatridecanoic acids, and gene transfection with these DE(n)-PLLA-DE(n) (n = 2-3) was examined with an alternative human gastric carcinoma cell-line HGC-27. As a result, the high plasmid DNA binding affinity, low cytotoxicity, and much enhanced transgene efficacy suggest a new possible clue to design effective synthetic gene delivery vectors with amphiphilic skeleton and less toxic polyamine building blocks.

  17. Drosophila casein kinase I alpha regulates homolog pairing and genome organization by modulating condensin II subunit Cap-H2 levels.

    PubMed

    Nguyen, Huy Q; Nye, Jonathan; Buster, Daniel W; Klebba, Joseph E; Rogers, Gregory C; Bosco, Giovanni

    2015-01-01

    The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

  18. Wet-chemical green synthesis of L-lysine amino acid stabilized biocompatible iron-oxide magnetic nanoparticles.

    PubMed

    Krishna, Rahul; Titus, Elby; Krishna, Rohit; Bardhan, Neelkanth; Bahadur, Dhirendra; Gracio, José

    2012-08-01

    In this paper, we report a novel method for the synthesis of L-Lysine (lys) amino acid coated maghemite (gamma-Fe2O3) magnetic nanoparticles (MNPs). The facile and cost effective method permitted preparation of the high-quality superparamagnetic gamma-Fe2O3 MNPs with hydrophilic and biocompatible nature. For this work, first we synthesized magnetite phase Fe3O4/lys by wet chemical method and oxidized to y-Fe2O3 in controlled oxidizing environment, as evidenced by XRD and VSM magnetometry. The crystallite size and magnetization of gamma-Fe2O3/lys MNPs was found to be 14.5 nm, 40.6 emu/gm respectively. The surface functionalization by L-lysine amino acid and metal-ligand bonding was also confirmed by FTIR spectroscopy. The hydrodynamic diameter, colloidal stability and surface charge on MNPs were characterized by DLS and zeta potential analyser.

  19. Overexpression of transport proteins improves the production of 5-aminovalerate from l-lysine in Escherichia coli

    PubMed Central

    Li, Zhong; Xu, Jing; Jiang, Tongtong; Ge, Yongsheng; Liu, Pan; Zhang, Manman; Su, Zhiguo; Gao, Chao; Ma, Cuiqing; Xu, Ping

    2016-01-01

    Bacterial transporters mediate the exchanges between intracellular and extracellular environments. Modification of transport route could be applied to speed up the metabolic reactions and promote the production of aimed compounds. Herein, lysine 2-monooxygenase (DavB) and δ-aminovaleramidase (DavA) were co-expressed in Escherichia coli BL21(DE3) to produce nylon-5 monomer 5-aminovalerate from l-lysine. Then, PP2911 (4-aminobutyrate transporter in Pseudomonas putida) and LysP (the lysine specific permease in E. coli) were overexpressed to promote 5-aminovalerate production using whole cells of recombinant E. coli. The constructed E. coli strain overexpressing transport proteins exhibited good 5-aminovalerate production performance and might serve as a promising biocatalyst for 5-aminovalerate production from l-lysine. This strategy not only shows an efficient process for the production of nylon monomers but also might be used in production of other chemicals. PMID:27510748

  20. Poly(L-proline) II helix propensities in poly(L-lysine) dendrigraft generations from vibrational Raman optical activity.

    PubMed

    Johannessen, Christian; Kapitán, Josef; Collet, Hélène; Commeyras, Auguste; Hecht, Lutz; Barron, Laurence D

    2009-06-08

    Vibrational Raman optical activity (ROA), measured as small circularly polarized components in Raman scattering from chiral molecules, was applied to study the backbone conformations of the first five generations of poly(L-lysine) dendrigrafts (DGLs) in water. Generation 1 was found to support predominantly the poly(L-proline) II (PPII) conformation, the amount of which steadily decreased with increasing generation, with a concomitant increase in other backbone conformations. This behavior may be due to increasing crowding of the lysine side chains, together with suppression of backbone hydration, with increasing branching. In contrast, the ROA spectra of a series of linear poly(L-lysine)s in water show little change with increasing molecular weight. Our results may have implications for the nonimmunogenic properties of DGLs.

  1. Couette-Taylor crystallizer: Effective control of crystal size distribution and recovery of L-lysine in cooling crystallization

    NASA Astrophysics Data System (ADS)

    Nguyen, Anh-Tuan; Yu, Taekyung; Kim, Woo-Sik

    2017-07-01

    A Couette-Taylor crystallizer is developed to enhance the L-Lysine crystal size distribution and recovery in the case of continuous cooling crystallization. When using the proposed Couette-Taylor (CT) crystallizer, the size distribution and crystal product recovery were much narrower and higher, respectively, than those from a conventional stirred tank (ST) crystallizer. Here, the coefficient of the size distribution for the crystal product from the CT crystallizer was only 0.45, while it was 0.78 in the case of the conventional ST crystallizer at an agitation speed of 700 rpm, mean residence time of 20 min, and feed concentration of 900 (g/L). Furthermore, when using the CT crystallizer, the crystal product recovery was remarkably enhanced up to 100%wt with a mean residence time of only 20 min, while it required a mean residence time of at least 60 min when using the conventional ST crystallizer. This result indicates that the CT crystallizer was much more effective than the conventional ST crystallizer in terms of controlling a narrower size distribution and achieving a 100%wt L-lysine crystal product recovery from continuous cooling crystallization. The advantage of the CT crystallizer over the conventional ST crystallizer was explained based on the higher energy dissipation of the Taylor vortex flow and larger surface area for heat transfer of the CT crystallizer. Here, the energy dissipation of the Taylor vortex flow in the CT crystallizer was 13.6 times higher than that of the random fluid motion in the conventional ST crystallizer, while the surface area per unit volume for heat transfer of the CT crystallizer was 8.0 times higher than that of the conventional ST crystallizer. As a result, the mixing condition and heat transfer of the CT crystallizer were much more effective than those of the conventional ST crystallizer for the cooling crystallization of L-lysine, thereby enhancing the L-lysine crystal size distribution and product recovery.

  2. 47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE).

    PubMed

    Fujikawa, T; Kubota, C; Ando, T; Imamura, S; Tokumaru, M; Yamakuchi, H; Gen, Y; Hyon, S-H

    2016-01-01

    Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P=0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48h and hatched rate until 72h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v

  3. Optimal Poly(l-lysine) Grafting Density in Hydrogels for Promoting Neural Progenitor Cell Functions

    PubMed Central

    Cai, Lei; Lu, Jie; Sheen, Volney; Wang, Shanfeng

    2012-01-01

    Recently we have developed a photo-polymerizable poly(l-lysine) (PLL) that can be covalently incorporated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels to improve their bioactivity by providing positive charges. To explore the potential of these PLL-grafted PEGDA hydrogels as a cell delivery vehicle and luminal filler in nerve guidance conduits for peripheral and central nerve regeneration, we varied the amount of pendent PLL chains in the hydrogels by photo-crosslinking PEGDA with weight compositions of PLL (ϕPLL) of 0, 1%, 2%, 3%, and 5%. We further investigated the effect of PLL grafting density on E14 mouse neural progenitor cell (NPC) behavior including cell viability, attachment, proliferation, differentiation, and gene expression. The amount of actually grafted PLL and charge densities were characterized, showing a proportional increase with the feed composition ϕPLL. NPC viability in 3D hydrogels was significantly improved in a PLL grafting density-dependent manner at days 7 and 14 post-encapsulation. Similarly, NPC attachment and proliferation were promoted on the PLL-grafted hydrogels with increasing ϕPLL up to 2%. More intriguingly, NPC lineage commitment was dramatically altered by the amount of grafted PLL chains in the hydrogels. NPC differentiation demonstrated a parabolic or non-monotonic dependence on ϕPLL, resulting in cells mostly differentiated toward mature neurons with extensive neurite formation and astrocytes rather than oligodendrocytes on the PLL-grafted hydrogels with ϕPLL of 2%, whereas the neutral hydrogels and PLL-grafted hydrogels with higher ϕPLL of 5% support NPC differentiation less. Gene expression of lineage markers further illustrated this trend, indicating that PLL-grafted hydrogels with an optimal ϕPLL of 2% could be a promising cell carrier that promoted NPC functions for treatment of nerve injuries. PMID:22533450

  4. Effects of poly(L-lysine) substrates on attached Escherichia coli bacteria.

    PubMed

    Colville, Keegan; Tompkins, Nicolas; Rutenberg, Andrew D; Jericho, Manfred H

    2010-02-16

    Poly(L-lysine) (PLL) is a cationic polymer that is often used for attaching and immobilizing cells to glass substrates for further investigation by, e.g., AFM techniques. Because of their small size, bacterial attachment is most easily done using thick air-dried PLL coatings--though thinner PLL coatings are also used and are commercially available. Nevertheless, the antimicrobial activity of PLL is well-established. Accordingly, we have investigated the physiological effects of suspended PLL and of PLL coatings on individual Escherichia coli bacteria through the pole-to-pole oscillations of cytoplasmic MinD-GFP fusion proteins. For planktonic bacteria, suspended PLL concentrations at the micromolar level quenched MinD-GFP oscillations and inhibited bacterial growth. On coverslips with PLL coatings prepared by short exposures of the slides to PLL solutions, followed by rinsing, only a fraction of available bacteria attached after hours of settling time. Min oscillations in the attached bacteria, however, were strong and only moderately slowed. On thick PLL coatings, prepared by drying drops on the slides followed by a brief rinse with deionized water, cells attached well within 15 min. With thick coatings, average oscillation periods for bacteria increased significantly, and considerable cell-to-cell variability was also observed; subsequent replacement of buffer with distilled water led to much larger period increases and/or fading of fluorescence intensity. We demonstrate that Min oscillations are a useful metric for bacteria attached to adhesion layers. We suggest that thick PLL coatings should probably be avoided for bacterial attachment, and that even thin PLL coatings can have significant effects on bacterial physiology.

  5. The antimicrobial mechanism of action of epsilon-poly-l-lysine.

    PubMed

    Hyldgaard, Morten; Mygind, Tina; Vad, Brian S; Stenvang, Marcel; Otzen, Daniel E; Meyer, Rikke L

    2014-12-01

    Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles.

  6. Genome shuffling enhanced ε-poly-L-lysine production by improving glucose tolerance of Streptomyces graminearus.

    PubMed

    Li, Shu; Li, Feng; Chen, Xu-Sheng; Wang, Liang; Xu, Jian; Tang, Lei; Mao, Zhong-Gui

    2012-01-01

    The productivity of ε-poly-L: -lysine (ε-PL) in currently reported wild-type strains is low. Here we improved glucose tolerance of a Streptomyces graminearus strain LS-B1 by genome shuffling while simultaneously enhancing the ε-PL productivity. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for recursive protoplast fusion. The positive colonies from library, created by fusing the inactivated protoplasts were screened on agar plates containing different concentrations of glucose. Characterization of all recombinants and wild-type strain in shake-flask fermentation indicated the compatibility of two phenotypes of glucose tolerance and ε-PL yield enhancement. The best performing recombinant, F3-4, was isolated after three rounds of genome shuffling, whose ε-PL production was about 88% higher than that of the parent strain. In batch fermentation test, the ε-PL concentration was obtained as 2.4 g/L by F3-4 compared with 1.6 g/L of wild type. Fed-batch fermentation by F3-4 was carried out and the ε-PL production accumulated to 13.5 g/L when initial glucose concentration was improved from 50 to 85 g/L. Enzyme activities of hexokinase, pyruvate kinase, and citrate synthase revealed that the glycolytic pathway and tricarboxylic acid circle way in F3-4 were more active than those in wild type, which was a possible reason for enhanced ε-PL production.

  7. The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

    PubMed Central

    Hyldgaard, Morten; Mygind, Tina; Vad, Brian S.; Stenvang, Marcel; Otzen, Daniel E.

    2014-01-01

    Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. PMID:25304506

  8. Comb-type prepolymers consisting of a polyacrylamide backbone and poly(L-lysine) graft chains for multivalent ligands.

    PubMed

    Asayama, S; Maruyama, A; Akaike, T

    1999-01-01

    The comb-type copolymers consisting of a polyacrylamide (PAAm) backbone and poly(L-lysine) (PLL) graft chains have been prepared as the "prepolymer" for designing multivalent ligands. To regulate the length and density of the clusters of primary amino groups, the Nalpha-carboxyanhydride of Nepsilon-carbobenzoxy (CBZ)-L-lysine was first polymerized using p-vinylbenzylamine as an initiator. The resulting poly(CBZ-L-lysine) macromonomer was then radically copolymerized with AAm, followed by the deprotection of amino groups. For the model study, the reactive clusters of primary amino groups were completely converted into anion clusters by the reaction with succinic anhydride. The model multivalent ligands having the biotin label on the PAAm backbone were prepared by the terpolymerization of the macromonomer, AAm, and the biotin derivative having a vinyl group. The enzyme-linked immunosorbent assay showed that the biotin with no spacer on the PAAm backbone was recognized by the avidin-peroxidase conjugate specifically. Therefore, the highly sensitive detection of the interaction between cells and various model multivalent ligands was possible. The selective labeling onto the PAAm backbone revealed that the converted anion clusters of graft chains interacted exclusively with the cell and that the backbone was inert to the interaction with the cell. These results indicate that the various PAAm-graft-PLL comb-type copolymers with the defined length and density of the PLL-grafts are the potential prepolymers to investigate and to optimize the affinity of the multivalent ligands for receptors.

  9. Investigating antimicrobial activity in Rheinheimera sp. due to hydrogen peroxide generated by l-lysine oxidase activity.

    PubMed

    Chen, Wen Ming; Lin, Chang Yi; Sheu, Shih Yi

    2010-05-05

    A greenish yellow pigmented bacterial strain, designated GR5, was recently isolated from a freshwater culture pond for a soft-shell turtle. Phylogenetic analyses based on 16S rRNA gene sequences indicate that strain GR5 belongs to the genus Rheinheimera and its only closest neighbor is the type strain of Rheinheimera texasensis (98.2%). Based on the antibiogram assay, strain GR5 possesses a broad spectrum of antimicrobial activity including Gram-positive and Gram-negative bacteria, yeast, algae, and strain GR5 itself. Strain GR5 can synthesize a macromolecule with antimicrobial activity due to the generation of hydrogen peroxide and this antimicrobial effect can be inhibited by catalase. This antimicrobial activity is active only in complex culture media or chemically defined culture media containing l-lysine. This antimicrobial macromolecule in strain GR5 is shown to be a monomeric protein with a molecular mass of 71kDa and isoelectric point of approximately 3.68. Liquid chromatography-tandem mass spectrometry analyses reveal close similarity of a 19-amino acid fragment derived from this protein to the antibacterial protein, AlpP from the marine bacterium Pseudoalteromonas tunicata D2, and to the antibacterial protein, marinocine, from the marine bacterium Marinomonas mediterranea. This study explores the nature of antimicrobial macromolecule such as l-lysine oxidase. This is the first report on a freshwater bacterium producing antimicrobial activity by generating hydrogen peroxide through its enzymatic activity of l-lysine oxidase.

  10. Oral supplementation with L-lysine did not prevent upper respiratory infection in a shelter population of cats.

    PubMed

    Rees, Tina M; Lubinski, Janet L

    2008-10-01

    Cats in animal shelters are highly susceptible to infection by feline herpesvirus (FHV) by virtue of their stress and close proximity to other cats. Animal shelters take several different approaches to prevent FHV-related upper respiratory infections (URIs), including empirically treating all cats with L-lysine, a supplement believed to prevent the replication of FHV and, therefore, manifestations of herpesvirus infections. In this study we tested oral supplementation of L-lysine as a means to prevent URIs. One hundred and forty-four cats were treated with L-lysine in a small amount of canned food once daily. A 'no treatment' group of 147 cats received no lysine during the course of the study. The development of conjunctivitis or URI was tracked between the two groups. In all measures, there was no effect between the two groups, suggesting that lysine was not able to prevent URI or conjunctivitis in our shelter situation. Cats entering shelters encounter stressors that may make them more susceptible to FHV reactivation or infection. Infection control and control of fomite transmission are also key to keeping cats healthy in a group housing situation. The finding that lysine did not prevent URI in this animal shelter suggests that shelters may better use their resources by finding ways to decrease stress among their feline population, focusing on proper infection control measures, and limiting fomite transmission of disease.

  11. Theoretical investigation of the stereochemistry of the polymerization of. cap alpha. -olefins and dienes with the participation of Ziegler-Natta catalysts

    SciTech Connect

    Minsker, K.S.; Karpasas, M.M.

    1986-09-01

    The processes involved in the formation of the active polymerization sites in heterogeneous Ziegler-Natta catalysts have been investigated with consideration of the real structure of the components by the atom-atom potential method, the Monte-Carlo method, a modified diatomics-in-molecules method, and the CNDO/2 method with the aid of the available experimental facts. It has been shown that three types of bimetallic active sites (AS), which differ with respect to the spatial configuration of the coordination sphere, viz., AS-1, AS-2, and AS-3, form, depending on the electronic structure of the homogeneous component R /SUB n/ M, the ionic radius of M (M is a metal from groups I-III), and the unit-cell parameters of the heterogeneous component MeX /SUB m/ (Me is a transition metal from groups IV-VIII). Only the AS-1 sites are stereospecific in the polymerization of ..cap alpha..-olefins and 1,3-dienes (isotactic polyolefins and 1,4-trans-polydienes form); the AS-2 sites are nonstereospecific in the polymerization of ..cap alpha..-olefins, but they form stereoregular 1,4-cis-polydienes; the AS-3 sites are nonstereospecific in the polymerization of both ..cap alpha..-olefins and 1,3-dienes. The phenomenon of stereoregularization in the polymerization of ..cap alpha..-olefins and 1,3-dienes is determined by the steric and electrostatic factors.

  12. Development of a radioiodinated ligand for characterising. cap alpha. /sub 1/-adrenoceptors. [Pentolamine and 2 BETA-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone

    SciTech Connect

    Adams, A.; Jarrott, B.

    1982-03-15

    Two ..cap alpha..-adrenoceptor antagonists, phentolamine and 2-(..beta..-(4-hydroxyphenyl)-ethylaminomethyl)-tetralone (BE 2254) which are phenolic derivatives were radioiodinated after chloramine-T oxidation of Na/sup 125/I and the labelled material isolated by chromatography. /sup 125/I-Phentolamine does not bind selectively to ..cap alpha..-adrenoceptors in guinea pig brain whereas the /sup 125/I-BE 2254 derivative binds rapidly, reversibly and with high affinity to these receptors with a K/sub d/ of 230 pM. At low concentrations of /sup 125/I-BE 2254 (< 100 pM) approx. 90% of the bound radioligand is specifically bound and under these conditions drug displacement studies show that the ligand binds predominantly to the ..cap alpha../sub 1/ subclass of adrenoceptors. Binding measurements to kidney and smooth muscle membrane preparations indicate that /sup 125/I-BE 2254 may also be a useful tool in the study of ..cap alpha..-adrenoceptors in peripheral tissues. The high specific activity of /sup 125/I-BE 2254 permits the use of minimal quantities of membrane material for receptor assay and ligand displacement measurements, e.g. 250 ..mu..g per assay tube, and this provides a significant advantage over the use of existing radioligands such as /sup 3/H-prazosin which requires approx. 40 times as much tissue.

  13. Cyclic AMP regulation of the human glycoprotein hormone. cap alpha. -subunit gene is mediated by an 18-base-pair element

    SciTech Connect

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-04-01

    cAMP regulates transcription of the gene encoding the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the ..cap alpha..-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the ..cap alpha..-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the ..cap alpha..-subunit gene.

  14. Antibacterial activity and mechanism of action of ε-poly-L-lysine

    SciTech Connect

    Ye, Ruosong; Xu, Hengyi; Wan, Cuixiang; Peng, Shanshan; Wang, Lijun; Xu, Hong; Aguilar, Zoraida P.; Xiong, Yonghua; Zeng, Zheling; Wei, Hua

    2013-09-13

    Highlights: •Antibacterial activity and mechanism of ε-PL against E. coli O157:H7 was investigated. •Critical inhibitory factors toward the growth of E. coli O157:H7 by ε-PL was analyzed. •Cell membrane integrity and cell morphology of E. coli O157:H7 was affected by ε-PL. •A positive correlation between reactive oxygen species levels and ε-PL concentration in E. coli O157:H7 cells. •ε-PL induced the expression of different genes related to oxidative/redox stress, SOS response, virulence. -- Abstract: ε-Poly-L-lysine (ε-PL) is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of

  15. Effect of poly-L-lysine coating on macrophage activation by alginate-based microcapsules: assessment using a new in vitro method.

    PubMed

    Juste, Sandy; Lessard, Mélanie; Henley, Nathalie; Ménard, Martin; Hallé, Jean-Pierre

    2005-03-15

    The characteristics of the microcapsule surface, which interacts directly with the host macrophages, may have a role in the biocompatibility of alginate-poly-L-lysine (PLL)-alginate (APA) microcapsule. The objectives of the study were: 1) to develop and validate a simple, rapid, and sensitive in vitro method for assessing microcapsule biocompatibility, based on microcapsule coincubation with macrophages and measurement, by reverse transcriptase-polymerase chain reaction, of cytokine mRNA expression, and 2) to evaluate the effect of alginate purification and PLL coating on macrophage activation. The mRNA expression of tumor necrosis factor-alpha and interleukin-1beta was significantly higher when macrophages were coincubated with beads made with nonpurified compared with purified alginate (p<0.01, p<0.05, respectively) and negative control (p<0.001) or with APA microcapsules compared with non-PLL-coated alginate beads and negative control (p<0.001). The mRNA expression of interleukin-6 differed significantly only when APA microcapsules were compared with a negative control (p<0.05). These results confirm that alginate purification improves microcapsule biocompatibility, and suggest that PLL is not completely covered and/or neutralized by the second alginate incubation and thus has a role in the host macrophage activation. The assay is sensitive to both alginate contaminants and microcapsule surface characteristics and may be a useful tool for the development of biocompatible microcapsules. Copyright (c) 2005 Wiley Periodicals, Inc.

  16. Crystalline perfection, optical and third harmonic generation analyses of non-linear optical single crystal of L-lysine acetate

    NASA Astrophysics Data System (ADS)

    Rani, Neelam; Vijayan, N.; Thukral, Kanika; Maurya, K. K.; Haranath, D.; Bhagavannarayana, G.; Verma, S.; Wahab, M. A.

    2013-03-01

    The potential organic non-linear optical single crystal of L-lysine acetate has been grown by slow evaporation solution growth technique (SEST) at room temperature. It crystallizes in the monoclinic system with space group of P21. The crystalline perfection of the grown single crystal has been examined by high resolution X-ray diffraction analysis (HRXRD). The functional groups of the synthesized compound have been identified by 13C NMR, 1H NMR and FTIR analyses. The optical absorption studies show that the crystal is transparent in the entire visible region with a cut-off wavelength of 236 nm. The optical band gap is found to be 5.29 eV. The steady-state PL spectra was recorded for pure L-lysine acetate crystal at room temperature. The third harmonic generation efficiency of the crystal has been evaluated by Z-scan technique and its non-linear optical coefficient has been calculated. Birefringence measurement has been carried out in order to see the optical homogeneity of the grown specimen. Its electrical properties has been assessed by dielectric measurement at different temperatures. The calculated optical band gap is 5.29 eV. Its thermal parameters like thermal diffusivity (α), thermal effusivity (e), thermal conductivity (k) and heat capacity (Cp) have been determined by photopyroelectric technique. Vickers micro hardness studies were carried out using a Vickers hardness tester equipped with a diamond square indenter. The piezoelectric measurement for L-lysine acetate has been also been carried at ambient condition.

  17. L-lysine uptake in giant vesicles from cardiac ventricular sarcolemma: two components of cationic amino acid transport.

    PubMed

    Lu, Xiaodong; Zheng, Ruifang; Gonzalez, Jorge; Gaspers, Lawrence; Kuzhikandathil, Eldo; Peluffo, R Daniel

    2009-08-01

    Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[(14)C]lysine uptake experiments were conducted within a 10(3)-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na(+)-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (K(m)=222+/-71 microM) and low transport capacity (V(max)=121+/-36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (K(m)=16+/-4 mM) and high capacity (V(max)=4.0+/-0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y(+) family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to beta-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its K(m).

  18. Molecular Identification of Hydroxylysine Kinase and of Ammoniophospholyases Acting on 5-Phosphohydroxy-l-lysine and Phosphoethanolamine*

    PubMed Central

    Veiga-da-Cunha, Maria; Hadi, Farah; Balligand, Thomas; Stroobant, Vincent; Van Schaftingen, Emile

    2012-01-01

    The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40–50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-l-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-l-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-l-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines. PMID:22241472

  19. Molecular identification of hydroxylysine kinase and of ammoniophospholyases acting on 5-phosphohydroxy-L-lysine and phosphoethanolamine.

    PubMed

    Veiga-da-Cunha, Maria; Hadi, Farah; Balligand, Thomas; Stroobant, Vincent; Van Schaftingen, Emile

    2012-03-02

    The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40-50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines.

  20. Characterization of the transport of. cap alpha. -methylaminoisobutyric acid by a human intestinal cell line (HT-29)

    SciTech Connect

    Bergin, L.; Dantzig, A.H.

    1986-03-01

    Under certain growth conditions, the human colon adenocarcinoma cell line HT-29 exhibits intestinal enterocyte-like properties. The differentiated cells possess a brush border with the enzyme markers (aminopeptidase and sucrase) normally associated with the intestine. To aid in the characterization of the transport properties of these cells, the uptake of a non-metabolizable amino acid analog, /sup 14/C-..cap alpha..-methylaminoisobutyric acid (MeAIB) as examined in the HT-29-Al subclone which possesses a brush border. The cells exhibited a time-dependent uptake of MeAIB which was concentrative and sodium-dependent. The pH optimum for uptake was about 7.8. Uptake was inhibited by low temperature, 1 mM ouabain, or 0.5 mM dinitrophenol. A 1 hr-preincubation of the cells in an isotonic KCl solution resulted in a decreased uptake rate, suggesting that a negative membrane potential is important for MeAIB uptake. The rate of 0.5 mM MeABIB uptake was inhibited by 40 to 90% by 5 mM of certain small neutral amino acids such as Ala, Ser, Pro, Gly, met but not by acidic or basic amino acids such as Asp, Glu, Arg or Lys. The uptake of MeAIB appears to be mediated by an amino acid transport carrier similar to the A-system described previously for Chinese hamster ovary cells.

  1. Enhancer and promoter elements directing activation and glucocorticoid repression of the. cap alpha. /sub 1/-fetoprotein gene in hepatocytes

    SciTech Connect

    Guertin, M.; La Rue, H.; Bernier, D.; Wrange, O.; Chevrette, M.; Gingras, M.C.; Belanger, L.

    1988-04-01

    Mutations were introduced in 7 kilobases of 5'-flanking rat ..cap alpha../sub 1/-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.

  2. Coherent neutron scattering study of the vibrations of interstitial deuterium in. cap alpha. -VD/sub 0. 7/

    SciTech Connect

    Rush, J.J.; Rowe, J.M.; Glinka, C.J.; Vagelatos, N.; Flotow, H.E.

    1980-06-15

    The lattice dynamics of a single crystal of ..cap alpha..(bcc)VD/sub 0.7/ have been investigated by coherent inelastic neutron scattering measurements at 295 K. Since the scattering cross section of vanadium is almost entirely incoherent, this study offers a unique opportunity to directly measure the ''band'' modes associated with vibrational displacements of the light atom interstitials as a function of wave vector (xi-bar) and ..omega... Transverse and longitudinal acoustic-phonon groups were observed in the three symmetry directions at energies up to 27 meV. These are generally at higher energies than corresponding modes measured in vanadium by x-ray scattering. The vibrational ''density of states'' for V and VD/sub 0.7/ are also shown to be grossly different. Careful measurement of the ''band-mode'' intensities for several different phonon branches as a function of xi-bar and ..omega.. indicate that the deuterium motions are strongly coupled to the metal lattice. Peaks due to optical vibrations are also observed, which indicate little dispersion of the optic branches in the crystal.

  3. Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

    PubMed Central

    Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

    2015-01-01

    The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

  4. Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase.

    PubMed

    Chen, Dawei; Tanem, Justinn; Frey, Perry A

    2007-02-01

    l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.

  5. Isophotes of a field in the Cygnus loop photographed in the (O III) and (N II)+H. cap alpha. lines

    SciTech Connect

    Sitnik, T.G.; Toropova, M.S.

    1982-11-01

    From interference-filter image-tube photographs of a 9' field in the western part of the Cygnus Loop supernova remnant, taken in the lambda5007 (O III) and lambdalambda 6584, 6563 (N II) + H..cap alpha.. lines, sets of isophotes are derived by an equidensitometry technique based on the Sabattier effect. The emission regions in these lines exhibit a relative displacement, interpreted as evidence for radiative cooling of the gas behind the shock generated in the supernova outburst. An explanation is offered for the differing morphology of the nebular filaments in the (O III) and (N II) + H..cap alpha.. lines. The anomalously high I/sub Otsi/II/I/sub H/..beta.. intensity ratio may reflect a spatial separation of the corresponding emission zones.

  6. Measuring chlorophyll. cap alpha. and /sup 14/C-labeled photosynthate in aquatic angiosperms by the use of a tissue solubilizer. [/sup 14/C-labelled photosynthate

    SciTech Connect

    Beer, S.; Stewart, A.J.; Wetzel, R.G.

    1982-01-01

    A compound that quantitatively correlated with chlorophyll ..cap alpha.. could be measured fluorometrically in the extracts of leaves of three aquatic angiosperms (Myriophyllum heterophyllum Michx., Potamogeton crispus L., Elodea canadensis Michx.) treated with the tissue solubilizer BTS-450. Fluorescent characteristics of the solubilized plant tissues were stable for several weeks in the dark at temperatures up to 60/sup 0/C but rapidly degraded in sunlight or when acidified. /sup 14/C-Labeled photosynthate, which had been fixed by leaf discs during 1- to 10-hour exposure to H/sup 14/CO/sub 3/, was also readily extracted by the tissue solubilizer. Solubilizer extraction can, therefore, be used to determine both chlorophyll ..cap alpha.. content and /sup 14/C incorporation rates in the same leaf sample. The method is practical, because no grinding is required, the fluorescent characteristics of the extracts are stable, and analyses can be performed with very little plant material (about 3 milligrams).

  7. cap alpha. /sub 2/-Adrenergic receptors in platelet membranes of depressed patients: no change in number of /sup 3/H-yohimbine affinity

    SciTech Connect

    Daiguji, M.I.; Meltzer, H.Y.; Tong, C.; U'Pritchard, D.C.; Young, M.; Kravitz, H.

    1981-11-16

    The ..cap alpha../sub 2/-adrenergic receptor density in platelet membranes from normal controls and depressed patients was studied using /sup 3/H-yohimbine, a potent ..cap alpha../sub 2/-adrenergic antagonist, as a radioligand. The KD values of /sup 3/H-yohimbine in control and depressed patient samples were 0.92 +/- 0.07 nM and 0.97 +/- 0.06 nM, respectively. The Bmax values of controls and depressed patients were 240 +/- 19 fmoles/mg protein (125 +/- 13 receptor/platelet, R/PL) and 204 +/- 20 fmoles/mg protein (130 +/- 14 R/PL), respectively. There were no significant differences between the KD and Bmax values of the two groups.

  8. Rocket borne solar eclipse experiment to measure the temperature structure of the solar corona via lyman-. cap alpha. line profile observations

    SciTech Connect

    Argo, H.V.

    1981-01-01

    A rocket borne experiment to measure the temperature structure of the inner solar corona via the doppler broadening of the resonance hydrogen Lyman-..cap alpha.. (lambda1216A) radiation scattered by ambient neutral hydrogen atoms was attempted during the 16 Feb 1980 solar eclipse. Two Nike-Black Brant V sounding rockets carrying instrumented payloads were launched into the path of the advancing eclipse umbra from the San Marco satellite launch platform 3 miles off the east coast of Kenya.

  9. Chiral effects on the /sup 13/C resonances of. cap alpha. -tocopherol and related compounds. A novel illustration of Newman's rule of six

    SciTech Connect

    Brownstein, S.; Burton, G.W.; Hughes, L.; Ingold, K.U.

    1989-02-03

    The 100-MHz /sup 13/C NMR spectrum of (2R,4'R,8'R)-..cap alpha..-tocopherol (natural vitamin E) has been completely assigned with the aid of a number of selectively deuteriated (2R,4'R,8'R)-..cap alpha..-tocopherols. The /sup 13/C NMR spectrum of (2RS,4'RS,8'RS)-..cap alpha..-tocopherol (all-racemic, synthetic vitamin E) has also been measured. Many of the individual carbons in this all-racemic mixture of eight ..cap alpha..-tocopherol stereoisomers give more than one resonance with eight of the carbons (2-CH/sub 3/, 2',3',4',4'-CH/sub 3/, 5', 8', and 9') giving the maximum number of four resonances from each of the four enantiomeric pairs; these resonances have also been assigned. The structurally related 5'-hydroxy-2-(4',8',12'-trimethyltridecyl)-2,4,6,7-tetramethyl-2,3,-dihydrobenzofuran (HTDBF) has been synthesized for the first time in the 2R,4'R,8'R and 2S,4'R,8'R configurations and their /sup 13/C resonances have been assigned. In its all-racemic form this compound also shows up to four resonances from a single carbon. Related observations have been made with phytol and isophytol. A careful examination of these chirally induced chemical shift differences for the individual carbon atoms, ..delta.., reveals a bond-alternation effect with maxima at a separation of one, three, and five bonds from the closest chiral center and with the maximum at a five-bond separation being greater than that at a three-bond separation. 32 references, 2 figures, 4 tables.

  10. Temporary anion states of. pi. -ligand transition-metal carbonyls studied by means of electron transmission spectroscopy and x. cap alpha. calculations

    SciTech Connect

    Modelli, A.; Distefano, G.; Guerra, M.; Jones, D.

    1987-07-22

    The resonances observed in the electron transmission spectra of (benzene)chromium tricarbonyl, (cyclopentadienyl)manganese tricarbonyl, (1,3-butadiene)iron tricarbonyl, and (cyclopentadienyl)cobalt dicarbonyl have been assigned with the aid of MS X..cap alpha.. calculations. In contrast with previous theoretical results, the present calculations on the neutral states show a large net electronic charge transfer from the ..pi.. ligand to the metal.

  11. The suppression of prion propagation using poly-L-lysine by targeting plasminogen that stimulates prion protein conversion

    PubMed Central

    Ryou, Chongsuk; Titlow, William B.; Mays, Charles E.; Bae, Younsoo; Kim, Sehun

    2011-01-01

    Poly-L-lysine (PLL), a homopolymer of amino acid L-lysine (LL), has been frequently used for drug delivery. Here, we report that PLL is an effective agent to inhibit propagation of prions that cause fatal and incurable neurologic disorders in humans and animals, termed prion diseases. In our recent investigation on prion propagation facilitated by conversion of the cellular prion protein (PrP) to the misfolded, disease-associated PrP (PrPSc), we demonstrated that plasminogen stimulates PrP conversion as a cellular cofactor. In the current study, we targeted plasminogen using PLL and assessed its anti-prion efficacy. The results showed that PLL strongly inhibited PrPSc propagation in the cell-free, cell culture, and mouse models of prion disease. These results confirm the role of plasminogen in PrPSc propagation, validates plasminogen as a therapeutic target to combat prion disease, and suggests PLL as a potential anti-prion agent. Therefore, our study represents a proof-of-concept that targeting plasminogen, a cofactor for PrP conversion, using PLL results in suppression of prion propagation, which represents a successful translation of our understanding on details of prion propagation into a potential therapeutic strategy for prion diseases. PMID:21288569

  12. Preparation, characterization, and biocompatibility evaluation of poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid interpenetrating network hydrogels.

    PubMed

    Cui, Ning; Qian, Junmin; Xu, Weijun; Xu, Minghui; Zhao, Na; Liu, Ting; Wang, Hongjie

    2016-01-20

    In the present study, poly(Nɛ-acryloyl-L-lysine)/hyaluronic acid (pLysAAm/HA) interpenetrating network (IPN) hydrogels were successfully fabricated through the combination of hydrazone bond crosslinking and photo-crosslinking reactions. The HA hydrogel network was first synthesized from 3,3'-dithiodipropionate hydrazide-modified HA and polyethylene glycol dilevulinate by hydrazone bond crosslinking. The pLysAAm hydrogel network was prepared from Nɛ-acryloyl-L-lysine and N,N'-bis(acryloyl)-(L)-cystine by photo-crosslinking. The resultant pLysAAm/HA hydrogels had a good shape recovery property after loading and unloading for 1.5 cycles (up to 90%) and displayed a highly porous microstructure. Their compressive moduli were at least 5 times higher than that of HA hydrogels. The pLysAAm/HA hydrogels had an equilibrium swelling ratio of up to 37.9 and displayed a glutathione-responsive degradation behavior. The results from in vitro biocompatibility evaluation with pre-osteoblasts MC3T3-E1 cells revealed that the pLysAAm/HA hydrogels could support cell viability and proliferation. Hematoxylin and eosin staining indicated that the pLysAAm/HA hydrogels allowed cell and tissue infiltration, confirming their good in vivo biocompatibility. Therefore, the novel pLysAAm/HA IPN hydrogels have great potential for bone tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Enzymatic properties and anticancer activity of L-lysine α-oxidase from Trichoderma cf. aureoviride Rifai BKMF-4268D.

    PubMed

    Pokrovsky, Vadim S; Treshalina, Helen M; Lukasheva, Elena V; Sedakova, Ludmila A; Medentzev, Alexander G; Arinbasarova, Anna Yu; Berezov, Temirbolat T

    2013-09-01

    L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (Km for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115-116 kDa; the active dimer consists of two identical 57-58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC50) ranging from 3.0×10 to 7.8×10 U/ml (3.2×10 to 8.2×10 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.

  14. Genome Shuffling and Gentamicin-Resistance to Improve ε-Poly-L-Lysine Productivity of Streptomyces albulus W-156.

    PubMed

    Wang, Liang; Chen, Xusheng; Wu, Guangyao; Zeng, Xin; Ren, Xidong; Li, Shu; Tang, Lei; Mao, Zhonggui

    2016-12-01

    Genome shuffling has been a recently effective method for screening the desirable phenotypes of industrial strains. Here, we combined genome shuffling and gentamicin resistance to improve the production of ε-poly-L-lysine in Streptomyces albulus W-156. Five starting mutants with higher ε-poly-L-lysine (ε-PL) productivities were firstly obtained by atmospheric and room temperature plasma (ARTP) mutagenesis. After three rounds of genome shuffling with increasing concentration of gentamicin for selection, S. albulus AG3-28, was finally got with a production of 3.43 g/L in shaking flask. In a 5-L fermenter, AG3-28 exhibited a higher ε-PL productivity (56.5 g/L) than the initial strain W-156 (37.5 g/L). Key enzyme activities in primary and secondary metabolic pathways were analyzed, and the transcription levels of hrdD and pls were determined by quantitative real time-polymerase chain reaction (qRT-PCR). Increase of key enzyme activities and the upregulation of the gene transcriptional levels demonstrated that ε-PL synthetic pathway in AG3-28 was obviously strengthened, which might be responsible for the high productivity. Moreover, hyper-yield strain AG3-28 was found to produce a slightly lower ε-PL polymerization degree than the parent strain. Amplified fragment length polymorphism (AFLP) analysis reflects the genetic diversity among the derivates after genome shuffling.

  15. Human immunodeficiency virus contains an epitope immunoreactive with thymosin. cap alpha. /sub 1/ and the 30-amino acid synthetic p17 group-specific antigen peptide HGP-30

    SciTech Connect

    Naylor, P.H.; Naylor, C.W.; Badamchian, M.; Wada, S.; Goldstein, A.L.; Wang, S.S.; Sun, D.K.; Thornton, A.H.; Sarin, P.S.

    1987-05-01

    The authors have reported that an antiserum prepared against thymosin ..cap alpha../sub 1/ (which shares a region of homology with the p17 protein of the acquired immunodeficiency syndrome (AIDS)-associated human immunodeficiency virus) effectively neutralized the AIDs virus and prevented its replication in H9 cells. Using HPLC and immunoblot analysis, they have identified from a clone B, type III human T-lymphotropic virus (HTLV-IIIB) extracts a protein with a molecular weight of 17,000 that is immunoreactive with thymosin ..cap alpha../sub 1/. In contrast, no immunoreactivity was found in retroviral extracts from a number of nonhuman species including feline, bovine, simian, gibbon, and murine retroviruses. Heterologous antiserum prepared against a 30-amino acid synthetic peptide analogue (HGP-30) does not cross-react with thymosin ..cap alpha../sub 1/ but does react specifically with the p17 protein of the AIDS virus in a manner identical to that seen with an HTLV-IIIB p17-specific monoclonal antibody. The demonstration that this synthetic analogue is immunogenic and that antibodies to HGP-30 cross-react not only with synthetic peptide but also with the HTLV-IIIB p17 viral protein provides an additional, and potentially more specific, candidate for development of a synthetic peptide vaccine for AIDS. In addition, the p17 synthetic peptide (HGP-3) may prove to be useful in a diagnostic assay for the detection of AIDS virus infection in seronegative individuals.

  16. Effects of kinase inhibitors and potassium phosphate (KPi) on site-specific phosphorylation of branched chain. cap alpha. -ketoacid dehydrogenase (BCKDH)

    SciTech Connect

    Kuntz, M.J.; Shimomura, Y.; Ozawa, T.; Harris, R.A.

    1987-05-01

    BCKDH is phosphorylated by a copurifying kinase at two serine residues on the El..cap alpha.. subunit. Phosphorylation of both sites occurs at about the same rate initially, but inactivation is believed associated only with site 1 phosphorylation. The effects of KPi and known inhibitors of BCKDH kinase, ..cap alpha..-chloroisocaproate (CIC) and branched chain ..cap alpha..-ketoacids (BCKA), on the phosphorylation of purified rat liver BCKDH were studied. Site-specific phosphorylation was quantitated by thin-layer electrophoresis of tryptic peptides followed by densitometric scanning of autoradiograms. Addition of 5 mM KPi was found necessary to stabilize the BCKDH activity at 37/sup 0/C. Increasing the KPi to 50 mM dramatically increased the CIC and BCKA inhibition of site 1 and site 2 phosphorylation. The finding of enhanced sensitivity of inhibitors with 50 mM KPi may facilitate identification of physiologically important kinase effectors. Regardless of the KPi concentration, CIC and the BCKA showed much more effective inhibition of site 2 than site 1 phosphorylation. Although site 1 is the primary inactivating site, predominant inhibition of site 2 phosphorylation may provide a means of modulating kinase/phosphatase control of BCKDH activity under steady state conditions.

  17. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    SciTech Connect

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  18. Association of elongation factor 1 alpha and ribosomal protein L3 with the proline-rich region of yeast adenylyl cyclase-associated protein CAP.

    PubMed

    Yanagihara, C; Shinkai, M; Kariya, K; Yamawaki-Kataoka, Y; Hu, C D; Masuda, T; Kataoka, T

    1997-03-17

    CAP is a multifunctional protein; the N-terminal region binds adenylyl cyclase and controls its response to Ras while the C-terminal region is involved in cytoskeletal regulation. In between the two regions, CAP possesses two proline-rich segments, P1 and P2, resembling a consensus sequence for binding SH3 domains. We have identified two yeast proteins with molecular sizes of 48 and 46 kDa associated specifically with P2. Determination of partial protein sequences demonstrated that the 48-kDa and 46-kDa proteins correspond to EF1 alpha and rL3, respectively, neither of which contains any SH3-domain-like sequence. Deletion of P2 from CAP resulted in loss of the activity to bind the two proteins either in vivo or in vitro. Yeast cells whose chromosomal CAP was replaced by the P2-deletion mutant displayed an abnormal phenotype represented by dissociated localizations of CAP and F-actin, which were colocalized in wild-type cells. These results suggest that these associations may have functional significance.

  19. Synthesis of Nepsilon-protected-L-lysine and gamma-benzyl-L-glutamate N-carboxyanhydrides (NCA) by carbamoylation and nitrosation.

    PubMed

    Vayaboury, W; Giani, O; Collet, H; Commeyras, A; Schué, F

    2004-10-01

    This paper reports on an original process to synthesize N-carboxyanhydrides, which consists of nitrosating N-carbamoylamino acids with a NO/O(2) gas mixture in acetonitrile. The synthesis of several N-carbamoylamino acids of L-lysine was described using potassium cyanate in water. The latter were then nitrosated to yield the corresponding NCA with more or less efficiency. Indeed, the NCA carrying an acid-sensitive protecting group led to a partial deprotection to give the L-lysine NCA salt. The NCA of N(epsilon)-trifluoroacetyl-L-lysine, N(epsilon)-benzyloxycarbonyl-L-lysine and gamma-benzyl-L-glutamate were successfully synthesized with satisfactory yields. Their polymerizability was compared to that of the N(epsilon)-trifluoroacetyl-L-lysine NCA initiated by n-hexylamine in N,N-dimethylformamide. It also showed that this new process of NCA synthesis could be applied to the synthesis of polypeptides and more generally to the protein chemistry.

  20. Internode length in Pisum. Gene na may block gibberellin synthesis between ent-7. cap alpha. -hydroxykaurenoic acid and biggerellin A/sub 12/-aldehyde. [Pisum sativum

    SciTech Connect

    Ingram, T.J.; Reid, J.B.

    1987-04-01

    The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA/sub 12/-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7..cap alpha..-hydroxykaurenoic acid and GA/sub 12/-aldehyde in the na mutant. Feeds of ent(/sup 3/H)kaurenoic acid and (/sup 2/H)GA/sub 12/-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize(/sup 2/H)GA/sub 12/-aldehyde to a number of (/sup 2/H)C/sub 19/-GAs including GA/sub 1/. However, there was no indication in na genotypes for the metabolism of ent-(/sup 3/H)kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolized ent-(/sup 3/H)kaurenoic acid to radioactive compounds that co-chromatographed with GA/sub 1/, GA/sub 8/, GA/sub 20/, and GA/sub 29/. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-(/sup 3/H)kaurenoic acid were similar for both Na and na plants and contained ent-16..cap alpha..,17-dihydroxykaurenoic acid and ent-6..cap alpha..,7..cap alpha..,16..beta..,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7..cap alpha..-hydroxylation.

  1. Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane.

    PubMed

    Reuter, Marcel; Schwieger, Christian; Meister, Annette; Karlsson, Göran; Blume, Alfred

    2009-09-01

    Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L(alpha) or the gel phase L(beta) lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (>or=44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R(P)=0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the

  2. Attenuating l-lysine production by deletion of ddh and lysE and their effect on l-threonine and l-isoleucine production in Corynebacterium glutamicum.

    PubMed

    Dong, Xunyan; Zhao, Yue; Hu, Jinyu; Li, Ye; Wang, Xiaoyuan

    2016-11-01

    The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis.

    PubMed

    Park, Si Jae; Oh, Young Hoon; Noh, Won; Kim, Hye Young; Shin, Jae Ho; Lee, Eun Gyo; Lee, Seungwoon; David, Yokimiko; Baylon, Mary Grace; Song, Bong Keun; Jegal, Jonggeon; Lee, Sang Yup; Lee, Seung Hwan

    2014-10-01

    L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Monitoring of nanoparticle reactivity in solution: interaction of l-lysine and Ru nanoparticles probed by chemical shift perturbation parallels regioselective H/D exchange.

    PubMed

    Martínez-Prieto, Luis M; Baquero, Edwin A; Pieters, Grégory; Flores, Juan C; de Jesús, Ernesto; Nayral, Céline; Delpech, Fabien; van Leeuwen, Piet W N M; Lippens, Guy; Chaudret, Bruno

    2017-05-30

    Thanks to new water-soluble Ru nanoparticles (NPs) stabilized by sulfonated NHC ligands, we demonstrate that it is possible to monitor the catalyst/substrate interaction using NMR chemical shift perturbations (CSPs), under conditions that closely resemble those applied during the enantiospecific C-H deuteration of l-lysine. Correlating the pH dependence of the interaction of l-lysine with the surface of the RuNPs and its subsequent deuteration, our study underscores the importance of oriented binding to the surface as a critical factor for H/D exchange.

  5. Growth, spectral, thermal, optical, mechanical and etching studies of L-lysine semi-maleate (L-LSM) single crystals

    NASA Astrophysics Data System (ADS)

    Vasudevan, V.; Renuka, N.; Ramesh Babu, R.; Ramamurthi, K.

    2015-02-01

    Organic nonlinear optical material, L-lysine semi-maleate (L-LSM) single crystals were grown by slow cooling solution growth technique. The crystal system of grown L-LSM was confirmed by single crystal and powder X-ray diffraction analyzes. Functional groups of the grown crystal have been identified by Fourier Transform Infrared spectral analysis. The proton and carbon NMR spectral studies confirm the presence of hydrogen and carbon in the grown L-LSM. The melting and thermal decomposition temperatures of the crystal were determined using thermogravimetric (TG) and differential scanning calorimetry (DSC) analyses. Optical transparency, second harmonic generation efficiency, micro hardness, dielectric constant and loss, refractive index and birefringence have also been measured. Further, the growth patterns and dislocations present in the grown crystal are studied.

  6. Poly-L-Lysine-Poly[HPMA] Block Copolymers Obtained by RAFT Polymerization as Polyplex-Transfection Reagents with Minimal Toxicity.

    PubMed

    Tappertzhofen, Kristof; Weiser, Franziska; Montermann, Evelyn; Reske-Kunz, Angelika; Bros, Matthias; Zentel, Rudolf

    2015-08-01

    Herein we describe the synthesis of poly-L-lysine-b-poly[N-(2-hydroxypropyl)-metha-crylamide)] (poly[HPMA]) block copolymers by combination of solid phase peptide synthesis or polymerization of α-amino acid-N-carboxy-anhydrides (NCA-polymerization) with the reversible addition-fragmentation chain transfer polymerization (RAFT). In the presence of p-DNA, these polymers form polyplex micelles with a size of 100-200 nm in diameter (monitored by SDS-PAGE and FCS). Primary in vitro studies with HEK-293T cells reveal their cellular uptake (FACS studies and CLSM) and proof successful transfection with efficiencies depending on the length of polylysine. Moreover, these polyplexes display minimal toxicity (MTT-assay and FACS-measurements) featuring a p[HPMA] corona for efficient extracellular shielding and the potential ligation with antibodies.

  7. Carbon- and nitrogen-centered radicals produced from L-lysine by radiation-induced oxidation: A pulse radiolysis study

    NASA Astrophysics Data System (ADS)

    Ito, Takeo; Morimoto, Shota; Fujita, Shin-ichi; Kobayashi, Kazuo; Tagawa, Seiichi; Nishimoto, Sei-ichi

    2008-09-01

    Radical species generated from the reactions of a basic amino acid, L-lysine (Lys), with hydroxyl radicals ( rad OH) and sulfate radical anion ( SO4-rad ) have been detected by the method of pulse radiolysis. On the basis of electron transfer reactivities toward tetranitromethane (TNM), it was demonstrated that reducing carbon-centered radicals are generated as a result of hydrogen abstraction from CH 2 of Lys with a G-value of 1.9 × 10 -7 mol J -1. On the other hand, direct oxidation of L-Lys by SO4-rad formed a transient species with different spectroscopic properties, most likely, the ɛ-N-centered Lys radical.

  8. In vitro transfection mediated by dendrigraft poly(L-lysines): the effect of structure and molecule size.

    PubMed

    Hofman, Jakub; Buncek, Martin; Haluza, Radovan; Streinz, Ludvik; Ledvina, Miroslav; Cigler, Petr

    2013-02-01

    Dendritic poly(L-lysines) (DGL) constitute promising nanomaterials applicable as a nonviral gene-delivery vector. In this study, we evaluate the transfection abilities of four DGL generations with special emphasis on the systematic description of the relationship of how generation (i.e., molecule size) affects the transfection efficacy. Using Hep2 cells, we demonstrated that the capability of unmodified DGL to deliver plasmid is of a magnitude lower than that of jetPEI. On the other hand, employing the Hep2 cell line stably transduced with eGFP, we observed that DGL G5 delivers the siRNA oligonucleotide with the same efficiency as Lipofectamine 2000. In further experiments, it was shown that DGL affords excellent ability to bind DNA, protect it against DNase I attack, and internalize it into cells. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Effects of hydration on the acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Chan-Huot, Monique; Limbach, Hans-Heinrich

    2010-09-21

    Using high resolution solid state (15)N and (13)C NMR spectroscopy we have studied the effects of successive hydration on the (15)N labeled side chain amino groups of solid poly-L-lysine (PLL) in the presence of acids. Generally, hydration leads to the formation of local "ionic fluid" phases composed by flexible side chain ammonium groups, acid anions and small amounts of water. The associated local dynamics reduces the widths of the inhomogeneously broadened (15)N amino signals found for the dry states. The hydration of free base PLL--which consists of mixtures of alpha-helices and beta-pleated sheets--is monitored by a small low-field shift of the amino group signal arising from hydrogen bonding with water, reaching eventually the value of PLL in water at pH 13. No difference for the two conformations is observed. PLL x HF adopts a similar secondary structure with isolated NHF hydrogen bonds; hydration leads only to small low-field shifts which are nevertheless compatible with the formation of ammonium groups in aqueous solution. PLL doped with small amounts of HCl contains ammonium groups which are internally solvated by neighboring free amino groups. Both nitrogen environments are characterized by different chemical shifts. Hydration with less than one water molecule per amino group leads already to a chemical shift averaging arising from fast proton motions along NHN-hydrogen bonds and fast side chain and anion motions.By contrast, the hydration of fully doped PLL x HBr and PLL x HCl is more complex. These systems exist only in beta-pleated sheet conformations forming alkyl ammonium salt structures. Separate (15)N signal components are observed for (i) the dry states, for (ii) wet beta-pleated sheets and for (iii) wet alpha-helices which are successively formed upon hydration. Exchange between these environments is slow, but water motions lead to averaged amino group signals within each of the two wet environments. These results indicate that the different

  10. Terminal modification on mPEG-dendritic poly-(l)-lysine cationic diblock copolymer for efficient gene delivery.

    PubMed

    Sheng, Ruilong; Xia, Kejia; Chen, Jian; Xu, Yuhong; Cao, Amin

    2013-01-01

    The development of new non-viral gene vectors with the advantages of low cytotoxicity and high gene transfection efficiency is a recent trend in gene therapy. In this work, we developed a series of termini-modified mPEG-dendritic poly-(l)-lysine cationic diblock copolymers (mPEG5k-DPL4-CG) by coupling various cationic groups to the dendritic skeleton. Their molecular structures were characterized by (1)H NMR, and the buffering capacities were measured by acid titration. The plasmid DNA (pDNA) binding affinities of the mPEG5k-DPL4-CG copolymers were investigated by EB displacement and agarose gel retardation assay, and the average particle size and surface charge of the polyplexes were analyzed by dynamic light scattering. Cytotoxicity and in vitro gene transfection were evaluated in several cell lines in the presence and absence of serum by the luciferase expression assay. The results indicated that the low molecular weight polyethylenimine (PEI800) termini-modified copolymer, mPEG5k-DPL4-PEI800, possessed high pDNA binding affinity, low cytotoxicity, and high gene transfection capability which were maintained in the presence of serum (10% FBS). It is worth noting that the gene delivery efficiency of the dendritic poly-(l)-lysine gene vector was enhanced by termini modification of suitable cationic blocks. The low cytotoxicity and serum-resistance properties of mPEG5k-DPL4-PEI800 make it a potential long-circulating gene vector in gene therapy applications.

  11. Size and medium conductivity dependence on dielectrophoretic behaviors of gas core poly-L-lysine shell nanoparticles.

    PubMed

    Yang, Chungja; Wu, Chun-Jen; Ostafin, Agnes E; Thibaudeau, Giselle; Minerick, Adrienne R

    2015-04-01

    Dynamic (dis)assembly of biocompatible nanoparticles into 3D, packed structures would benefit drug delivery, films, and diagnostics. Dielectrophoretic (DEP) microdevices can rapidly assemble and manipulate polarizable particles within nonuniform electric fields. DEP has primarily discerned micrometer particles since nanoparticles experience smaller forces. This work examines conductivity and size DEP dependencies of previously unexplored spherical core-shell nanoparticle (CSnp) into 3D particle assemblies. Poly-L-lysine shell material was custom synthesized around a gas core to form CSnps. DEP frequencies from 1 kHz to 80 MHz at fixed 5 volts peak-to-peak and medium conductivities of 10(-5) and 10(-3) S/m were tested. DEP responses of ∼220 and ∼400 nm poly-L-lysine CSnps were quantified via video intensity densitometry at the microdevice's quadrapole electrode center for negative DEP (nDEP) and adjacent to electrodes for positive DEP. Intensity densitometry was then translated into a relative DEP response curve. An unusual nDEP peak occurred at ∼57 MHz with 25-80 times greater apparent nDEP force. All electrical circuit components were then impedance matched, which changed the observed response to weak positive DEP at low frequencies and consistently weak nDEP from ∼100 kHz to 80 MHz. This impedance-matched behavior agrees with conventional Clausius-Mossotti DEP signatures taking into account the gas core's contributions to the polarization mechanisms. This work describes a potential pitfall when conducting DEP at higher frequencies in microdevices and concurrently demonstrates nDEP behavior for a chemically and structurally distinct particle system. This work provides insight into organic shell material properties in nanostructures and strategies to facilitate dynamic nanoparticle assemblies.

  12. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    SciTech Connect

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  13. Distinct structural features of the. cap alpha. and. beta. subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

    SciTech Connect

    Wang, S.Z.; Chen, J.S.; Johnson, J.L.

    1988-04-19

    Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding ..cap alpha.. and ..beta.. subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated ..beta.. subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (M/sub r/ 50,115) whose amino-terminal region is about 50 resides shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp ..cap alpha.. subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the ..cap alpha.. and ..beta.. subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine resides, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.

  14. Role of exchange effects in elastic scattering of. cap alpha. particles and /sup 3/He ions by /sup 6/Li nuclei

    SciTech Connect

    Bragin, V.N.; Burtebaev, N.T.; Dui-brevesebaev, A.D.; Ivanov, G.N.; Sakuta, S.B.; Chuev, V.I.; Chulkov, L.V.

    1986-08-01

    Measurements in the entire angular range are carried out for elastic scattering of ..cap alpha.. particles with energies 36.6 and 50.5 MeV and /sup 3/He ions with energies 34, 50, 60, and 72 MeV by /sup 6/Li nuclei. The experimental data obtained have been analyzed within the framework of the optical model. The contribution to the scattering of the mechanism of elastic transfer of clusters was calculated by the distorted-wave method with a finite interaction radius.

  15. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect L-Lysine Production in Corynebacterium glutamicum.

    PubMed

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-03-09

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in L-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport--NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885--were also expressed at significantly higher levels in the L-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, L-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.

  16. Interference effects between /sup 17/O states populated in the /sup 13/C(/sup 6/Li,d)/sup 17/O*. --> cap alpha. +/sup 13/C reaction

    SciTech Connect

    Cardella, G.; Cunsolo, A.; Foti, A.; Imme, G.; Pappalardo, G.; Raciti, G.; Rizzo, F.; Alamanos, N.; Berthier, B.; Saunier, N.

    1987-12-01

    An analysis of the /sup 13/C(/sup 6/Li,d..cap alpha..)/sup 13/C reaction in the collinear (theta/sub d/ = 0/sup 0/) and noncollinear (theta/sub d/ = 10/sup 0/,8/sup 0/) geometry is made for two peaks observed in the deuteron energy spectrum and corresponding to excitation energies of 16.1 and 13.6 MeV in the /sup 17/O nucleus. It is shown that the reaction proceeds via a direct alpha-transfer process which populates doublets of interfering /sup 17/O levels. Spins, weights, and parities of these levels are obtained by means of a least square procedure.

  17. Tellurium-123m-labeled 23-(isopropyl telluro)-24-nor-5. cap alpha. -cholan-3. beta. -ol: a new potential adrenal imaging agent

    SciTech Connect

    Knapp, F.F. Jr.; Ambrose, K.R.; Callahan, A.P.

    1980-03-01

    Tellurium-123m-labeled 23-(isopropyl telluro)-24-nor-5..cap alpha..-cholan-3..beta..-ol (24-telluracholestanol, or 23-ITC) has been prepared as a potential adrenal-imaging agent. The new agent was synthesized by the coupling of 3..beta..-acetoxy-23-bromo-24-nor-5..cap alpha..-cholan with Te-123m-labeled sodium isopropyl tellurol. Tissue distribution experiments in both male and female rats indicate a high adrenal concentration of radioactivity following administration of this agent. In female rats the adrenal glands accumulated 4.5% of the injected radioactivity only 1 day after administration of Te-123m-23-ITC. The adrenal-to-liver ratio was 42 after 1 day, and this increased to 100 after 3 days. Chromatographic analyses of lipid extracts from adrenal, ovary, liver, and lungs suggest that this agent is metabolized by these tissues. Examination of the rats' excretory products has indicated that approximately 50% of the administered radioactivity is excreted in the feces with 5 days after injection of Te-123m-23-ITC. Moreover, the adrenals and ovaries of rats have been clearly imaged with this agent, both with a rectilinear scanner and with an RC type of proportional-counter camera.

  18. /sup 45/Ca efflux for myometrial cells: comparison of the effects of prostaglandin F/sub 2/. cap alpha. (PGF/sub 2/), oxytocin (OT) and arachidonate (A)

    SciTech Connect

    Katona, G.; Molnar, M.; Toth, M.; Hertelendy, F.

    1986-03-01

    The aim of this study was to measure PGF/sub 2..cap alpha../-induced Ca/sup 2 +/ release from uterine cells and to compare this to the actions of OT and A. Smooth muscle cells isolated from the uterus (shell gland) of laying hens were cultured for 7 days in M199 plus 10% fetal calf serum. The cells were treated with digitonin (20..mu..M) and preloaded with /sup 45/Ca for 40 min. Addition of PGF/sub 2..cap alpha../ caused a biphasic /sup 45/Ca-efflux. There was a small but significant /sup 45/Ca-release within 30 sec (rapid phase) followed by a larger one within 7 min (slow phase). In comparison, both OT and A stimulated /sup 45/Ca efflux during a single, slow phase. The maximal effect of A was observed at < 7 min, whereas that of OT was slower, peaking after 7 min. Mepacrin, an inhibitor of A release, attenuated the action of OT without having any effect on A promoted /sup 45/Ca-efflux. Indomethacin, an inhibitor of PG synthase, failed to suppress the Ca-releasing effect of A suggesting the A itself or a lipoxygenase product may have been responsible for the observed effects. Moreover, these results provide suggestive evidence that A release is an important step in the action of various uterotonic agents converging on the mobilization of intracellular Ca.

  19. Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on L-lysine formation and analysis of systemic consequences.

    PubMed

    Hochheim, Jennifer; Kranz, Angela; Krumbach, Karin; Sokolowsky, Sascha; Eggeling, Lothar; Noack, Stephan; Bocola, Marco; Bott, Michael; Marienhagen, Jan

    2017-02-01

    To explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular L-lysine accumulation by this bacterium. The analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing L-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance L-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM L-lysine with a yield of 0.17 g/g (L-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high L-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower L-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on L-lysine formation. MurE is an attractive target to achieve high L-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated L-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers.

  20. Nitroxyl radicals-modified dendritic poly(l-lysine) as a contrast agent for Overhauser-enhanced MRI.

    PubMed

    Niidome, Takuro; Gokuden, Risa; Watanabe, Kazuto; Mori, Takeshi; Naganuma, Tatsuya; Utsumi, Hideo; Ichikawa, Kazuhiro; Katayama, Yoshiki

    2014-01-01

    Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distribution in animals by enhancing the water proton signal intensity by the overhauser effect. In this study, we constructed a contrast agent by combining PROXYL groups that have nitroxyl radicals with PEG-modified dendritic poly(l-lysine) that accumulates in the tumor by enhanced permeability and retention (EPR) effect. Addition of the PROXYL groups at the PEG chains' termini on KG6 was advantageous in OMRI, because the ESR signal of the nitroxyl radical was maintained without decay caused by mobility restriction, even if the PROXYL groups were attached at 25 mol% on one molecule. After intramuscular injection of the molecule modified at 25 mol%, that is, PR25-PEG-KG6, a significant OMRI signal was observed at the injected site. However, no signal was detected in the tumor after intravenous injection of PR25-PEG-KG6 to a tumor-bearing mouse, although PR25-PEG-KG6 itself accumulated in the tumor. The reason was that the nitroxyl radicals were immediately reduced in the blood after the injection, suggesting that use of stable nitroxyl radicals will enable detection of tumors by OMRI after intravenous injection.

  1. Electrochemical Characterization of Poly-L-Lysine Coating on Indium Tin Oxide Electrode for Enhancing Cell Adhesion.

    PubMed

    Choi, Yonghyun; Yagati, Ajay Kumar; Cho, Sungbo

    2015-10-01

    Nano or microelectrode-based cell chip for stimulating or recording neuronal signals requires better cell adhesion procedures in order to achieve efficient cell based assays for effective cellular diagnosis and for high throughput screening of drug candidates. The cells can be adhered on protein pre-coated sensing electrodes, but the electrochemical characteristics of cells are highly influenced by the electrical charge of the underlying protein interface. Thus, in this study, we report on experimental and theoretical aspects of poly-L-lysine (PLL) adsorption on transparent indium tin oxide (ITO) electrodes and the interaction between PLL and human embryonic kidney 293/GFP cells. PLL coated ITO electrodes showed a lower transfer resistance compared to bare or bovine serum albumin coated ITO electrodes. In addition, they exhibited more positive potential and higher magnitude of redox peak currents with increased immersion time of PLL solution. Finally, results of the impedance analysis showed that adhesion of cells was enhanced by PLL coating on ITO electrodes compared to bare ITO electrodes.

  2. Improvement of ε-poly-L-lysine production through seed stage development based on in situ pH monitoring.

    PubMed

    Sun, Qi-Xing; Chen, Xu-Sheng; Ren, Xi-Dong; Mao, Zhong-Gui

    2015-01-01

    Nissin, natamycin, and ε-poly-L-lysine (ε-PL) are three safe, microbial-produced food preservatives used today in the food industry. However, current industrial production of ε-PL is only performed in several countries. In order to realize large-scale ε-PL production by fermentation, the effects of seed stage on cell growth and ε-PL production were investigated by monitoring of pH in situ in a 5-L laboratory-scale fermenter. A significant increase in ε-PL production in fed-batch fermentation by Streptomyces sp. M-Z18 was achieved, at 48.9 g/L, through the optimization of several factors associated with seed stage, including spore pretreatment, inoculum age, and inoculum level. Compared with conventional fermentation approaches using 24-h-old shake-flask seed broth as inoculum, the maximum ε-PL concentration and productivity were enhanced by 32.3 and 36.6 %, respectively. The effect of optimized inoculum conditions on ε-PL production on a large scale was evaluated using a 50-L pilot-scale fermenter, attaining a maximum ε-PL production of 36.22 g/L in fed-batch fermentation, constituting the first report of ε-PL production at pilot scale. These results will be helpful for efficient ε-PL production by Streptomyces at pilot and plant scales.

  3. Highly sensitive electrochemical detection of palmatine using a biocompatible multiwalled carbon nanotube/poly-l-lysine composite.

    PubMed

    Thirumalraj, Balamurugan; Kubendhiran, Subbiramaniyan; Chen, Shen-Ming; Lin, Kuan-Yu

    2017-03-10

    To date, the natural alkaloids are mostly used in the field of pharmacological applications and the active substance of palmatine was extensively used in cancer therapy and other biomedical applications. Hence, in this study we report a simple preparation of poly-l-lysine (PLL) electro-polymerized on the surface of functionalized multiwalled carbon nanotubes (f-MWCNT) for electrochemical detection of palmatine content in human serum and urine samples. The active amino group of PLL plays a vital role towards the oxidation palmatine and exhibits superior electrocatalytic activity. Under optimum conditions, the prepared f-MWCNT/PLL composite shows a wide linear response range over the palmatine concentration ranging from 0.5µM to 425µM, and a detection limit (LOD) of 0.12µM based on S/N =3 (signal to noise ratio). The real time monitoring of palmatine content in serum and urine samples displays an appropriate recoveries and excellent performance for the practical analysis. The advantage of this developed system was simple, higher electrocatalytic activity, long-term stability and low cost. We hope that the prepared composite opens a new way for the fabrication of different biosensors in the field of biomedical application.

  4. Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL).

    PubMed

    Katakura, Hiromichi; Harada, Atsushi; Kataoka, Kazunori; Furusho, Miki; Tanaka, Fumihiro; Wada, Hiromi; Ikenaka, Kazuhiro

    2004-04-01

    Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity. Copyright 2004 John Wiley & Sons, Ltd.

  5. Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(l-Lysine): Experimental Study and Modeling Approach

    PubMed Central

    Vasiliu, Tudor; Cojocaru, Corneliu; Rotaru, Alexandru; Pricope, Gabriela; Pinteala, Mariana; Clima, Lilia

    2017-01-01

    The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(l-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4. PMID:28629130

  6. Evaluation of Cell-Laden Polyelectrolyte Hydrogels Incorporating Poly(L-Lysine) for Applications in Cartilage Tissue Engineering

    PubMed Central

    Lam, Johnny; Clark, Elisa C.; Fong, Eliza L.S.; Lee, Esther J.; Lu, Steven; Tabata, Yasuhiko; Mikos, Antonios G.

    2016-01-01

    To address the lack of reliable long-term solutions for cartilage injuries, strategies in tissue engineering are beginning to leverage developmental processes to spur tissue regeneration. This study focuses on the use of poly(L-lysine) (PLL), previously shown to up-regulate mesenchymal condensation during developmental skeletogenesis in vitro, as an early chondrogenic stimulant of mesenchymal stem cells (MSCs). We characterized the effect of PLL incorporation on the swelling and degradation of oligo(poly(ethylene) glycol) fumarate) (OPF)-based hydrogels as functions of PLL molecular weight and dosage. Furthermore, we investigated the effect of PLL incorporation on the chondrogenic gene expression of hydrogel-encapsulated MSCs. The incorporation of PLL resulted in early enhancements of type II collagen and aggrecan gene expression and type II/type I collagen expression ratios when compared to blank controls. The presentation of PLL to MSCs encapsulated in OPF hydrogels also enhanced N-cadherin gene expression under certain culture conditions, suggesting that PLL may induce the expression of condensation markers in synthetic hydrogel systems. In summary, PLL can function as an inductive factor that primes the cellular microenvironment for early chondrogenic gene expression but may require additional biochemical factors for the generation of fully functional chondrocytes. PMID:26799859

  7. Polyethylene glycol–poly(ε-benzyloxycarbonyl-l-lysine)-conjugated VEGF siRNA for antiangiogenic gene therapy in hepatocellular carcinoma

    PubMed Central

    Wang, Gangmin; Gao, XiaoLong; Gu, GuoJun; Shao, ZhiHong; Li, MingHua; Wang, PeiJun; Yang, JianRong; Cai, XiaoJun; Li, YongYong

    2017-01-01

    A polyethylene glycol–poly(ε-benzyloxycarbonyl-l-lysine) (PEG-SS-PLL) block copolymer based on a disulfide-linked, novel biodegradable catiomer bearing a PEG-sheddable shell was developed to avoid “PEG dilemma” in nanoparticle intracellular tracking of PEG-PLL where PEG was nondegradable. However, PEG-SS-PLL catiomers have not been used to deliver small interfering VEGF RNA (siVEGF) in antiangiogenesis gene therapy. In this study, we aimed to investigate whether this novel biodegradable catiomer can deliver siVEGF into cancer cells and at the same time have an antitumor effect in a xenograft mouse model. It was found that PEG-SS-PLL efficiently delivered siVEGF with negligible cytotoxicity, and significantly decreased the expression of VEGF at both the messenger-RNA and protein levels both in vitro and in vivo, and thus tumor growth was inhibited. Our findings demonstrated that PEG-SS-PLL/siVEGF could potentially be applied to antiangiogenesis gene therapy for hepatocellular carcinoma. PMID:28533682

  8. Simultaneous Detection of Dopamine and Uric Acid Using a Poly(l-lysine)/Graphene Oxide Modified Electrode

    PubMed Central

    Zhang, Yuehua; Lei, Wu; Xu, Yujuan; Xia, Xifeng; Hao, Qingli

    2016-01-01

    A novel, simple and selective electrochemical method was investigated for the simultaneous detection of dopamine (DA) and uric acid (UA) on a poly(l-lysine)/graphene oxide (GO) modified glassy carbon electrode (PLL/GO/GCE) by differential pulse voltammetry (DPV). The electrochemically prepared PLL/GO sensory platform toward the oxidation of UA and DA exhibited several advantages, including high effective surface area, more active sites and enhanced electrochemical activity. Compared to the PLL-modified GCE (PLL/GCE), GO-modified GCE and bare GCE, the PLL/GO/GCE exhibited an increase in the anodic potential difference and a remarkable enhancement in the current responses for both UA and DA. For the simultaneous detection of DA and UA, the detection limits of 0.021 and 0.074 μM were obtained, while 0.031 and 0.018 μM were obtained as the detection limits for the selective detection of UA and DA, using DPV in the linear concentration ranges of 0.5 to 20.0 and 0.5 to 35 μM, respectively. In addition, the PLL/GO/GCE demonstrated good reproducibility, long-term stability, excellent selectivity and negligible interference of ascorbic acid (AA). The proposed modified electrode was successfully implemented in the simultaneous detection of DA and UA in human blood serum, urine and dopamine hydrochloride injection with satisfactory results. PMID:28335305

  9. Molecular dynamics simulation of spin-lattice NMR relaxation in poly-L-lysine dendrimers: manifestation of the semiflexibility effect.

    PubMed

    Markelov, Denis A; Falkovich, Stanislav G; Neelov, Igor M; Ilyash, Maxim Yu; Matveev, Vladimir V; Lähderanta, Erkki; Ingman, Petri; Darinskii, Anatolii A

    2015-02-07

    NMR relaxation experiments are widely used to investigate the local orientation mobility in dendrimers. In particular, the NMR method allows one to measure the spin-lattice relaxation rate, 1/T1, which is connected with the orientational autocorrelation function (ACF) of NMR active groups. We calculate the temperature (Θ) and frequency (ω) dependences of the spin-lattice NMR relaxation rates for segments and NMR active CH2 groups in poly-L-lysine (PLL) dendrimers in water, on the basis of full-atomic molecular dynamics simulations. It is shown that the position of the maximum of 1/T1(ω) depends on the location of the segments inside the dendrimer. This dependence of the maximum is explained by the restricted flexibility of the dendrimer. Such behavior has been predicted recently by the analytical theory based on the semiflexible viscoelastic model. The simulated temperature dependences of 1/T1 for terminal and inner groups in PLL dendrimers of n = 2 and n = 4 generations dissolved in water are in good agreement with the NMR experimental data, which have been obtained for these systems previously by us. It is shown that in the case of PLL dendrimers, the traditional procedure of the interpretation of NMR experimental data - when smaller values of 1/T1 correspond to higher orientation mobility - is applicable to the whole accessible frequency interval only for the terminal groups. For the inner groups, this procedure is valid only at low frequencies.

  10. Bacteriocin protein BacL1 of Enterococcus faecalis is a peptidoglycan D-isoglutamyl-L-lysine endopeptidase.

    PubMed

    Kurushima, Jun; Hayashi, Ikue; Sugai, Motoyuki; Tomita, Haruyoshi

    2013-12-27

    Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL1 and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL1 and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL1 and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL1 and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL1 nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL1 alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL1 has a peptidoglycan D-isoglutamyl-L-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL1 serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.

  11. Synthesis, growth, crystal structure and characterization of a new organic NLO crystal: L-Lysine 4-nitrophenolate monohydrate (LLPNP)

    NASA Astrophysics Data System (ADS)

    Mahadevan, M.; Magesh, M.; Ramachandran, K.; Anandan, P.; Arivanandhan, M.; Hayakawa, Y.

    2014-09-01

    L-Lysine 4-nitrophenolate monohydrate (LLPNP) has been synthesized and grown by solution growth method at room temperature using deionised water as a solvent. The crystal structure of the materials was solved by single crystal X-ray diffraction analysis and it was found that the material has orthorhombic system. The crystallinity of the grown crystals was studied by the powder X-ray diffraction analysis. Molecular structure of the grown crystal was investigated by 1H NMR spectroscopy. The various functional groups of the sample were identified by Fourier transform infrared and Fourier transform-Raman spectroscopic analyses. Thermal stability of the grown crystal has been studied by Thermogravimetric and Differential thermal (TG&DTA) analysis. The optical absorption of the grown crystals has been ascertained by UV-Vis-NIR absorption studies. Second harmonic generation (SHG) efficiency of the material has been determined by Kurtz and Perry technique and the efficiency was found to be 4.45 and 1.4 times greater than that of standard KDP and urea samples, respectively.

  12. Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide].

    PubMed

    Subr, Vladimír; Konák, Cestmír; Laga, Richard; Ulbrich, Karel

    2006-01-01

    N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.

  13. Fabrication of oriented poly-L-lysine/bacteriorhodopsin-embedded purple membrane multilayer structure for enhanced photoelectric response.

    PubMed

    Li, Rui; Cui, Xiaoqiang; Hu, Weihua; Lu, Zhisong; Li, Chang Ming

    2010-04-01

    A poly-L-lysine (PLL)/bacteriorhodopsin-embedded purple membrane (bR-PM) multilayer film has been successfully constructed by a layer-by-layer (LbL) assembly process to enhance the photoelectric response of bR. The assembly conditions were investigated and optimized. The PLL/bR-PM adsorption process was in situ studied by surface plasmon resonance and the growth of multilayer was further characterized by UV-vis absorption spectroscopy. The results indicate that the amount of adsorbed bR-PM vs. the assembled layer number exhibits linear relationship. The atomic force microscopy images of sequentially assembled PLL/bR-PM bilayers show that the patch structure of bR-PM in the structure is well preserved and the roughness increases with increase of the bilayer number. The peak photocurrent generated from PLL/bR-PM film increases with increase of the PLL/bR-PM bilayers until achieving a maximum value. The photocurrent of bR-PM from the film through PLL assembler is higher than those assembled by other polycations, thus rendering a new platform to effectively enhance the bR photoelectric responses.

  14. Characterization of viability and proliferation of alginate-poly-L-lysine-alginate encapsulated myoblasts using flow cytometry.

    PubMed

    Thakur, Ajit; Sengupta, Ruchira; Matsui, Hideto; Lillicrap, David; Jones, Kim; Hortelano, Gonzalo

    2010-08-01

    Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

  15. Highly sensitive determination of methotrexate at poly (l-lysine) modified electrode in the presence of sodium dodecyl benzene sulfonate.

    PubMed

    Wei, Youli; Luo, Liqiang; Ding, Yaping; Si, Xiaojing; Ning, Yanqun

    2014-08-01

    A simple and sensitive electrochemical sensor based on poly (l-lysine) modified glassy carbon electrode (PLL/GCE) was developed to sensitively detect methotrexate (MTX) in the presence of sodium dodecyl benzene sulfonate (SDBS). Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry were carried out to characterize PLL film which exhibits excellent electrocatalytic activity toward the oxidation of MTX in 0.1M phosphate buffer solution. In addition, the oxidation peak of MTX remained stable at PLL/GCE in the presence of SDBS and its current increased 8 times compared with that at bare GCE without SDBS. Experimental parameters were optimized with regard to pH, electro-polymerization segment, accumulation time and concentration of SDBS. Under optimum conditions, the square wave voltammograms exhibited that the oxidation peak current was linearly proportional to the concentration of MTX in the range of 5nM - 0.2μM with detection limit of 1.7 (±0.06) nM (S/N=3). Moreover, this method was applied to detect MTX in medicinal tablets with satisfying results.

  16. Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite

    NASA Astrophysics Data System (ADS)

    Kaçar, Ceren; Erden, Pınar Esra; Kılıç, Esma

    2017-10-01

    A novel matrix, carboxylated multiwalled carbon nanotubes-tin oxide nanoparticles-graphene-chitosan (c-MWCNTs-SnO2-GR-CS) composite, was prepared for biosensor construction. Lysine oxidase (LOx) enzyme was immobilized covalently on the surface of c-MWCNTs-GR-SnO2-CS composite modified glassy carbon electrode (GCE) using N-ethyl-N‧-(3-dimethyaminopropyl) carbodiimide (EDC) and N-hydroxyl succinimide (NHS). Effects of electrode composition and buffer pH on biosensor response were investigated to optimize the working conditions. The biosensor exhibited wide linear range (9.9 × 10-7 M-1.6 × 10-4 M), low detection limit (1.5 × 10-7 M), high sensitivity (55.20 μA mM-1 cm-2) and fast amperometric response (<25 s) at +0.70 V vs. Ag/AgCl. With good repeatability and long-term stability, the c-MWCNTs-SnO2-GR-CS based biosensor offered an alternative for L-lysine biosensing. The practical applicability of the biosensor in two dietary supplements has also been addressed.

  17. Growth, structural, optical, piezoelectric and etching analysis of L-lysine p-nitrophenolate monohydrate single crystals

    NASA Astrophysics Data System (ADS)

    Alexandar, A.; Lakshmanan, A.; Sakthy Priya, S.; Surendran, P.; Rameshkumar, P.

    2017-09-01

    Nonlinear optical single crystals of L-lysine p-nitrophenolate monohydrate (LLPNP) were grown in aqueous solution by the slow evaporation solution technique (SEST). The grown crystals were subjected to powder X-ray diffraction analysis, (PXRD) and it was found that the title compound was crystallized in the orthorhombic crystal system with noncentrosymmetric space group of P212121. The vibrational frequencies of various functional groups present in the crystal were analyzed using the FTIR spectrum with a wavenumber range between 450 cm‑1 and 4000 cm‑1. The microhardness analysis of the sample revealed that the crystal belongs to the soft material category. Piezoelectric analysis was performed to measure the value of the piezoelectric (d33) coefficient. Blue light emission of the material was confirmed using the photoluminescence spectrum. Thermal stability of the grown crystal was analyzed using a melting point apparatus and it was found that the LLPNP is stable upto 175∘C. Etching analysis was performed at various durations, in order to identify the surface properties of the LLPNP crystal.

  18. An innovative method for immobilizing sucrose isomerase on ε-poly-L-lysine modified mesoporous TiO2.

    PubMed

    Wu, Lingtian; Liu, Yi; Chi, Bo; Xu, Zheng; Feng, Xiaohai; Li, Sha; Xu, Hong

    2015-11-15

    Sucrose isomerase (SIase) is the key enzyme in the enzymatic synthesis of isomaltulose. Mesoporous titanium dioxide (M-TiO2) and ε-poly-L-lysine-functionalized M-TiO2 (EPL-M-TiO2) were prepared as carriers for immobilizing SIase. SIase was effectively immobilized on EPL-M-TiO2 (SI-EPL-M-TiO2) with an enzyme activity of 39.41 U/g, and the enzymatic activity recovery rate up to 93.26%. The optimal pH and temperature of immobilized SIase were 6.0 and 30° C, respectively. SI-EPL-M-TiO2 was more stable in pH and thermal tests than SIase immobilized on M-TiO2 and free SIase. K(m) of SI-EPL-M-TiO2 was 204.92 mmol/L, and vmax was 45.7 μmol/L/s. Batch catalysis reaction of sucrose by SI-EPL-M-TiO2 was performed under the optimal conditions. The half-life period of SI-EPL-M-TiO2 under continuous reaction was 114 h, and the conversion rate of sucrose after 16 batches consistently remained at around 95%, which indicates that SI-EPL-M-TiO2 has good operational stability. Thus, SI-EPL-M-TiO2 can be used as a biocatalyst in food industries.

  19. Investigation of the mechanism(s) involved in decreasing increased fibrinogen activity in hyperglycemic conditions using L-lysine supplementation.

    PubMed

    Mirmiranpour, Hossein; Bathaie, S Zahra; Khaghani, Shahnaz; Nakhjavani, Manouchehr; Kebriaeezadeh, Abbas

    2012-09-01

    Fibrinogen is a plasma glycoprotein that participates in the hemostasis system. Its malfunction has been reported as a consequence of diabetic complications. In this study, the inhibitory effect of L-Lysine (Lys) on the nonenzymatic glycation of fibrinogen was investigated in both in vitro and in vivo conditions. Fibrinogen was incubated with glucose in the presence or absence of Lys. Then, its structure was studied by fluorescence spectroscopy, circular dichroism, and electrophoresis. The Clauss method was used to determine fibrinogen activity. In addition, one of the two groups of type 2 diabetic patients receiving ordinary treatment was additionally treated with Lys for 3 months. Fibrinogen activity and some other parameters were evaluated in their plasma. The results indicated increases in the activity of glycated fibrinogen in both of the in vivo and in vitro experiments. Advanced glycation end products were increased by time, as shown using fluorometry in both the plasma of the diabetic patients and the incubation medium of protein with glucose. The circular dichroism spectra showed some changes in the fibrinogen secondary and tertiary structures after glycation. The electrophoretic mobility of the glycated fibrinogen changed and the cross-link formation between the fibrinogen subunits due to glycation was observed. Lys inhibited all of the mentioned fibrinogen changes both in the in vitro experiments and after its administration to the diabetic patients. Lys, as an inhibitor of protein glycation, improved fibrinogen's structure and function, both in vitro and in vivo. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Influence of crystal habit on the surface free energy and interparticulate bonding of L-lysine monohydrochloride dihydrate.

    PubMed

    Bandyopadhyay, R; Grant, D J

    2000-01-01

    The objective of the present study was to apply a technique to measure the surface energy of crystalline powders without changing the surface properties by compaction, and to relate such measurements to crystal habit and orientation. The surface free energy of uncompacted L-lysine monohydrochloride dihydrate (LH), determined using a modified sessile-drop method, reflected a combined value for the various faces, and was influenced by the relative size of the faces and the orientation of the crystals. The surface free energy values obtained from contact angle measurements were within the possible range calculated from the crystal structure. Discrepancies between the theoretical estimates of interparticulate cohesive strengths and those measured from the tensile strength of powder compacts were used to estimate the flaw sizes (or gaps between the particles) that act as stress concentrators and reduce the tensile strength of the compacts. The flaw sizes indicate packing and compressibility of the various crystal habits. In the absence of compressive load, compacts made out of the equidimensional crystals have the larger flaw sizes (wider cracks or wider gaps between the particles). At higher compaction pressures, the compacts from long rod-shaped crystals have longer crack lengths. The weakness of the compacts made from the long rods at the higher compaction pressures may be because of the longer crack length along the interparticulate boundary, which may result in a higher stress intensity at the crack tip and increased fracture propensity.

  1. Poly-L-lysine brushes on magnetic nanoparticles for ultrasensitive detection of Escherichia coli O157: H7.

    PubMed

    Wu, Jing; Chen, Yan; Wang, Yu; Yin, Hong; Zhao, Zhitao; Liu, Nan; Xie, Mengxia; Chen, Yiping

    2017-09-01

    We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19μg/mg and 16μg/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816μg/mg, protein μg per mg of MPLLBs) and Ab (387μg/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8cfu/mL) and shortened the analysis time (within 2h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Preparation of poly-L-lysine functionalized magnetic nanoparticles and their influence on viability of cancer cells

    NASA Astrophysics Data System (ADS)

    Khmara, I.; Koneracka, M.; Kubovcikova, M.; Zavisova, V.; Antal, I.; Csach, K.; Kopcansky, P.; Vidlickova, I.; Csaderova, L.; Pastorekova, S.; Zatovicova, M.

    2017-04-01

    This study was aimed at development of biocompatible amino-functionalized magnetic nanoparticles as carriers of specific antibodies able to detect and/or target cancer cells. Poly-L-lysine (PLL)-modified magnetic nanoparticle samples with different PLL/Fe3O4 content were prepared and tested to define the optimal PLL/Fe3O4 weight ratio. The samples were characterized for particle size and morphology (SEM, TEM and DLS), and surface properties (zeta potential measurements). The optimal PLL/Fe3O4 weight ratio of 1.0 based on both zeta potential and DLS measurements was in agreement with the UV/VIS measurements. Magnetic nanoparticles with the optimal PLL content were conjugated with antibody specific for the cancer biomarker carbonic anhydrase IX (CA IX), which is induced by hypoxia, a physiologic stress present in solid tumors and linked with aggressive tumor behavior. CA IX is localized on the cell surface with the antibody-binding epitope facing the extracellular space and is therefore suitable for antibody-based targeting of tumor cells. Here we showed that PLL/Fe3O4 magnetic nanoparticles exhibit cytotoxic activities in a cell type-dependent manner and bind to cells expressing CA IX when conjugated with the CA IX-specific antibody. These data support further investigations of the CA IX antibody-conjugated, magnetic field-guided/activated nanoparticles as tools in anticancer strategies.

  3. Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis

    PubMed Central

    Sagong, Hye-Young; Kim, Kyung-Jin

    2017-01-01

    Diaminopimelate epimerase (DapF) is one of the crucial enzymes involved in l-lysine biosynthesis, where it converts l,l-diaminopimelate (l,l-DAP) into d,l-DAP. DapF is also considered as an attractive target for the development of antibacterial drugs. Here, we report the crystal structure of DapF from Corynebacterium glutamicum (CgDapF). Structures of CgDapF obtained under both oxidized and reduced conditions reveal that the function of CgDapF is regulated by redox-switch modulation via reversible disulfide bond formation between two catalytic cysteine residues. Under oxidized condition, two catalytic cysteine residues form a disulfide bond; these same cysteine residues exist in reduced form under reduced condition. Disulfide bond formation also induces a subsequent structural change in the dynamic catalytic loop at the active site, which results in open/closed conformational change at the active site. We also determined the crystal structure of CgDapF in complex with its product d,l-DAP, and elucidated how the enzyme recognizes its substrate l,l-DAP as a substrate. Moreover, the structure in complex with the d,l-DAP product reveals that CgDapF undergoes a large open/closed domain movement upon substrate binding, resulting in a completely buried active site with the substrate bound. PMID:28176858

  4. Development of Microtiter Plate Culture Method for Rapid Screening of ε-Poly-L-Lysine-Producing Strains.

    PubMed

    Liu, Yong-Juan; Chen, Xu-Sheng; Zhao, Jun-Jie; Pan, Long; Mao, Zhong-Gui

    2017-05-24

    ε-Poly-L-lysine (ε-PL) produced by Streptomyces albulus possesses a broad spectrum of antimicrobial activity and is widely used as a food preservative. To extensively screen ε-PL-overproducing strain, we developed an integrated high-throughput screening assay using ribosome engineering technology. The production protocol was scaled down to 24- and 48-deep-well microtiter plates (MTPs). The microplate reader assay was used to monitor ε-PL production. A good correlation was observed between the fermentation results obtained in both 24-(48)-deep-well MTPs and conventional Erlenmeyer flasks. Using this protocol, the production of ε-PL in an entire MTP was determined in <5 min without compromising on accuracy. The high-yielding strain selected through this protocol was also tested in Erlenmeyer flasks. The result showed that the ε-PL production of the high-yielding mutants was nearly 45% higher than that of the parent stain. Thus, development of this protocol is expected to accelerate the selection of ε-PL-overproducing strains.

  5. Acid-base interactions and secondary structures of poly-L-lysine probed by 15N and 13C solid state NMR and Ab initio model calculations.

    PubMed

    Dos, Alexandra; Schimming, Volkmar; Tosoni, Sergio; Limbach, Hans-Heinrich

    2008-12-11

    The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

  6. The influence of poly-(L-lysine) and porin on the domain structure of mixed vesicles composed of lipopolysaccharide and phospholipid: an infrared spectroscopic study.

    PubMed Central

    Lasch, P; Schultz, C P; Naumann, D

    1998-01-01

    Fourier transform infrared (FTIR) spectroscopy has been used to study the thermotropic phase behavior of binary lipid mixtures composed of deuterated phospholipids (PLs) and lipopolysaccharides (LPSs). Furthermore, the influence of an extrinsic high-molecular, polycationic polypeptide (poly-(L-lysine), PLL(500)) and an intrinsic membrane protein (outer membrane protein F, OmpF) on these binary mixtures was investigated by FTIR spectroscopy. "Deep rough" mutant LPS (ReLPS), isolated from Salmonella minnesota R595, and perdeuterated 1,2-dimyristoylphosphatidylethanolamine (DMPEd54) were used as model lipids. Deuteration of one of the lipids permitted the detection of lipid protein interaction with each lipid component separately. For this purpose, the symmetric >CH2 and >CD2 stretching bands were utilized as specific monitors to scrutinize the state of order of the membranes. From the individual phase transition temperatures Tm and the shape of the phase transition profiles, it is established that ReLPS and DMPEd54 are molecularly immiscible. In addition to the two domains of the pure lipid components, a third, domain-like structure is detected that may coexist with these pure domains. This domain-like structure undergoes a gel to liquid-crystalline L1 (beta <--> alpha) phase transition at temperatures distinctly different from that of the respective pure lipid domains. The nature of this type of domain is discussed in terms of a "border region" model that adequately explains the experimentally observed complex phase transition profiles. It is further demonstrated that the extrinsic polycationic polypeptide PLL(500) and the intrinsic, pore-forming protein OmpF isolated from Escherichia coli interact preferentially and highly specifically with the negatively charged ReLPS. Both the synthetic polypeptide and the pore-forming protein increased the tendency of ReLPS and DMPEd54 to segregate into distinct, well-separated domains. Whereas the transition profiles of the

  7. Microstructure characterization and thermal analysis of hybrid block copolymer α-methoxy-poly(ethylene glycol)- block-poly[ ɛ-(benzyloxycarbonyl)- L-lysine] for biomedical applications

    NASA Astrophysics Data System (ADS)

    Izunobi, Josephat U.; Higginbotham, Clement L.

    2010-08-01

    Hybrid block copolymers, which combine the economy and processibility of synthetic polymers with the functionality and highly ordered structures of polypeptides, can enhance control over structure formation at the nanoscale, as well as afford interesting materials that interface with Nature for a diversity of biomedical applications, such as drug delivery, tissue engineering and bioimaging. Hybrid block copolymer, α-methoxy-poly(ethylene glycol)- b-poly[ ɛ-(benzyloxycarbonyl)- L-lysine], MPEG- b-PLL(Z), was synthesized by anionic ring-opening polymerization in excellent yield, and fully characterized using IR, 1H, 13C and 2-D NMR, GPC, TGA, DTGA, DSC, MDSC and polarimetry. Its precursor NCA, ɛ-(benzyloxycarbonyl)- L-lysine N-carboxyanhydride, L-Lys(Z)-NCA, was prepared in 97% yield; and a double doublet observed in the 1850-1750 cm -1 absorption region of its infrared spectrum, for the characteristic NCA carbonyl absorption bands, is discussed.

  8. Immobilization of heparin/poly-l-lysine microspheres on medical grade high nitrogen nickel-free austenitic stainless steel surface to improve the biocompatibility and suppress thrombosis.

    PubMed

    Li, Menghua; Wu, Haide; Wang, Yi; Yin, Tieying; Gregersen, Hans; Zhang, Xiaojuan; Liao, Xiaoling; Wang, Guixue

    2017-04-01

    Thrombosis formation, restenosis, and delayed endothelium regeneration continue to be a challenge for coronary artery stent therapy. To improve the hemocompatibility of cardiovascular implants and to selectively direct vascular cell behavior, a novel heparin/poly-l-lysine microsphere was developed and immobilized on a dopamine-coated surface. We chose medical grade high nitrogen nickel-free austenitic stainless steel as the stent material since it has better biocompatibility. The stability and structural characteristics of the microspheres changed with the heparin: poly-l-lysine concentration ratio. Antithrombin III binding was significantly enhanced. Furthermore, for plasma coagulation tests, the activated partial thromboplastin time and thrombin time were prolonged and depended on the heparinfunction. The modified exhibited excellent stability and anticoagulant activity, and efficiently accelerated endothelialization and anticoagulation. This work has potential application for the design of coronary artery stent surfaces tailored for vascular cell behavior. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. L-Lysine: exploiting powder X-ray diffraction to complete the set of crystal structures of the 20 directly encoded proteinogenic amino acids.

    PubMed

    Williams, P Andrew; Hughes, Colan E; Harris, Kenneth D M

    2015-03-23

    During the last 75 years, crystal structures have been reported for 19 of the 20 directly encoded proteinogenic amino acids in their natural (enantiomerically pure) form. The crystal structure is now reported for the final member of this set: L-lysine. As crystalline L-lysine has a strong propensity to incorporate water under ambient atmospheric conditions to form a hydrate phase, the pure (non-hydrate) crystalline phase can be obtained only by dehydration under rigorously anhydrous conditions, resulting in a microcrystalline powder sample. For this reason, modern powder X-ray diffraction methods have been exploited to determine the crystal structure in this final, elusive case. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Interactions of C-reactive protein with the complement system. II. C- reactive protein-mediated consumption of complement by poly-L-lysine polymers and other polycations

    PubMed Central

    1975-01-01

    Cationic homopolymers of poly-L-lysine were found to activate complement (C) via C-reactive protein (CRP) and deplete C3 and C5 as well as early-acting C components. Maximum C consumption was obtained with polymers of 2,000-8,000 daltons; polymers of 1,700, 11,000, and 23,000 daltons were intermediate in reactivity, while L-lysine, lysyl-L- lysine, tetra-L-lysine, and polymers of 70,000-400,000 daltons lacked significant C-consuming activity. Naturally occurring polycations which consumed C in the presence of CRP included myelin basic proteins, cationic proteins of rabbit leukocytes, and both lysine- and arginine- rich histones; poly-L-arginine polymers of 17,000 but not 65,000 daltons also were C-consuming. Polycations without such reactivity included poly-L-orithine (5,000 and 165,000 daltons), egg white and human lysozymes, and Polybrene. The polycations which failed to induce C consumption via CRP, inhibited its consumption by both active polycations and by C-polysaccharide (CPS). The relative inhibitory capacity of phosphorylcholine and polycations in CPS- and polycations- CRP systems was consistent with the concept that phosphate esters and polycations react at the same or an overlapping combining site. The ability of certain polycations to activate C via CRP increases the potential for initiation of host reactions via C. The capacity of other polycations to inhibit C activation via CRP introduces a potential for physiologic or pharmacologic manipulation. These considerations would seem to expand the potential role of CRP in the initiation and modulation of the inflammatory response. PMID:809531

  11. Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    PubMed Central

    Kim, Hong-Il; Kim, Jong-Hyeon; Park, Young-Jin

    2016-01-01

    Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production. PMID:27005618

  12. Characterization of a targeted gene carrier, lactose-polyethylene glycol-grafted poly-L-lysine and its complex with plasmid DNA.

    PubMed

    Choi, Y H; Liu, F; Choi, J S; Kim, S W; Park, J S

    1999-11-01

    The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.

  13. Amperometric L-lysine enzyme electrodes based on carbon nanotube/redox polymer and graphene/carbon nanotube/redox polymer composites.

    PubMed

    Kaçar, Ceren; Erden, Pınar Esra; Kılıç, Esma

    2017-04-01

    Highly sensitive L-lysine enzyme electrodes were constructed by using poly(vinylferrocene)-multiwalled carbon nanotubes-gelatine (PVF/MWCNTs-GEL) and poly(vinylferrocene)-multiwalled carbon nanotubes-gelatine-graphene (PVF/MWCNTs-GEL/GR) composites as sensing interfaces and their performances were evaluated. Lysine oxidase (LO) was immobilized onto the composite modified glassy carbon electrodes (GCE) by crosslinking using glutaraldehyde and bovine serum albumin. Effects of pH value, enzyme loading, applied potential, electrode composition, and interfering substances on the amperometric response of the enzyme electrodes were discussed. The analytical characteristics of the enzyme electrodes were also investigated. The linear range, detection limit, and sensitivity of the LO/PVF/MWCNTs-GEL/GCE were 9.9 × 10(-7)-7.0 × 10(-4) M, 1.8 × 10(-7) M (S/N = 3), and 13.51 μA mM(-1) cm(-2), respectively. PVF/MWCNTs-GEL/GR-based L-lysine enzyme electrode showed a short response time (<5 s) and a linear detection range from 9.9 × 10(-7) to 7.0 × 10(-4) M with good sensitivity of 17.8 μA mM(-1) cm(-2) and a low detection limit of 9.2 × 10(-8) M. The PVF/MWCNTs-GEL/GR composite-based L-lysine enzyme electrode exhibited about 1.3-fold higher sensitivity than its MWCNTs-based counterpart and its detection limit was superior to the MWCNTs-based one. In addition, enzyme electrodes were successfully applied to determine L-lysine in pharmaceutical sample and cheese.

  14. The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA

    PubMed Central

    Stearns, Nancy A.; Zhou, Shuxia; Petri, Michelle; Binder, Steven R.; Pisetsky, David S.

    2016-01-01

    Antibodies to nuclear antigens (antinuclear antibodies or ANAs) are the serological hallmark of systemic lupus erythematosus (SLE). These antibodies bind diverse nuclear antigens that include DNA, histones and non-histone proteins as well as complexes of proteins with DNA and RNA. Because of the frequency of ANA expression in SLE, testing is an important component of clinical evaluation as well as determination of eligibility for clinical trials or utilization of certain therapies. Immunofluorescence assays have been commonly used for this purpose although this approach can be limited by issues of throughput, variability and difficulty in determining positivity. ELISA and multiplex assays are also useful approaches although these assays may give an incomplete picture of antibodies present. To develop a sensitive and quantitative ANA assay, we have explored an ELISA platform in which plates are pre-coated with a positively charged nucleic acid binding polymer (NABP) to increase adherence of antigens containing DNA or RNA. As a source of antigens, we have used supernatants of Jurkat cells undergoing apoptosis in vitro. As results presented show, a poly-L-lysine (PLL) pre-coat significantly enhances detection of antibodies to DNA as well as antigens such as histones, SSA, SSB and RNP. Comparison of the ELISA assay with the PLL pre-coat with a multiplex assay using the BioPlex® 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a pre-coat with a positively charged polymer can increase the sensitivity of an ANA ELISA using as antigens molecules released from dead and dying cells. This assay platform may facilitate ANA testing by providing an ensemble of antigens more similar in composition and structure with antigens present in vivo, with a NABP promoting adherence via charge-charge interactions. PMID:27611194

  15. Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine.

    PubMed

    Matano, Christian; Uhde, Andreas; Youn, Jung-Won; Maeda, Tomoya; Clermont, Lina; Marin, Kay; Krämer, Reinhard; Wendisch, Volker F; Seibold, Gerd M

    2014-06-01

    Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid L-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed.

  16. The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA.

    PubMed

    Stearns, Nancy A; Zhou, Shuxia; Petri, Michelle; Binder, Steven R; Pisetsky, David S

    2016-01-01

    Antibodies to nuclear antigens (antinuclear antibodies or ANAs) are the serological hallmark of systemic lupus erythematosus (SLE). These antibodies bind diverse nuclear antigens that include DNA, histones and non-histone proteins as well as complexes of proteins with DNA and RNA. Because of the frequency of ANA expression in SLE, testing is an important component of clinical evaluation as well as determination of eligibility for clinical trials or utilization of certain therapies. Immunofluorescence assays have been commonly used for this purpose although this approach can be limited by issues of throughput, variability and difficulty in determining positivity. ELISA and multiplex assays are also useful approaches although these assays may give an incomplete picture of antibodies present. To develop a sensitive and quantitative ANA assay, we have explored an ELISA platform in which plates are pre-coated with a positively charged nucleic acid binding polymer (NABP) to increase adherence of antigens containing DNA or RNA. As a source of antigens, we have used supernatants of Jurkat cells undergoing apoptosis in vitro. As results presented show, a poly-L-lysine (PLL) pre-coat significantly enhances detection of antibodies to DNA as well as antigens such as histones, SSA, SSB and RNP. Comparison of the ELISA assay with the PLL pre-coat with a multiplex assay using the BioPlex® 2200 system indicated good agreement in results for a panel of lupus sera. Together, these studies indicate that a pre-coat with a positively charged polymer can increase the sensitivity of an ANA ELISA using as antigens molecules released from dead and dying cells. This assay platform may facilitate ANA testing by providing an ensemble of antigens more similar in composition and structure with antigens present in vivo, with a NABP promoting adherence via charge-charge interactions.

  17. Cell encapsulation and cryostorage in PVA-gelatin cryogels: incorporation of carboxylated ε-poly-L-lysine as cryoprotectant.

    PubMed

    Vrana, Nihal E; Matsumura, Kazuaki; Hyon, Suong-Hyu; Geever, Luke M; Kennedy, James E; Lyons, John G; Higginbotham, Clement L; Cahill, Paul A; McGuinness, Garrett B

    2012-04-01

    It is desirable to produce cryopreservable cell-laden tissue-engineering scaffolds whose final properties can be adjusted during the thawing process immediately prior to use. Polyvinyl alcohol (PVA)-based solutions provide platforms in which cryoprotected cell suspensions can be turned into a ready-to-use, cell-laden scaffold by a process of cryogelation. In this study, such a PVA system, with DMSO as the cryoprotectant, was successfully developed. Vascular smooth muscle cell (vSMC)-encapsulated cryogels were investigated under conditions of cyclic strain and in co-culture with vascular endothelial cells to mimic the environment these cells experience in vivo in a vascular tissue-engineering setting. In view of the cytotoxicity DMSO imposes with respect to the production procedure, carboxylated poly-L-lysine (COOH-PLL) was substituted as a non-cytotoxic cryoprotectant to allow longer, slower thawing periods to generate more stable cryogels. Encapsulated vSMC with DMSO as a cryoprotectant responded to 10% cyclic strain with increased alignment and proliferation. Cells were stored frozen for 1 month without loss of viability compared to immediate thawing. SMC-encapsulated cryogels also successfully supported functional endothelial cell co-culture. Substitution of COOH-PLL in place of DMSO resulted in a significant increase in cell viability in encapsulated cryogels for a range of thawing periods. We conclude that incorporation of COOH-PLL during cryogelation preserved cell functionality while retaining fundamental cryogel physical properties, thereby making it a promising platform for tissue-engineering scaffolds, particularly for vascular tissue engineering, or cell preservation within microgels.

  18. Mucolytic treatment with N-acetylcysteine L-lysinate metered dose inhaler in dogs: airway epithelial function changes.

    PubMed

    Tomkiewicz, R P; App, E M; Coffiner, M; Fossion, J; Maes, P; King, M

    1994-01-01

    N-acetylcysteine L-lysinate Nacystelyn (L-NAC) is a newly synthesized mucolytic agent, of which the action in vivo has not been well defined. In six healthy mongrel dogs, the rheological properties of mucus, its mucociliary and cough clearability, and the transepithelial potential difference (PD) of the tracheobronchial epithelium were evaluated after placebo and L-NAC metered dose inhaler (MDI) aerosols. The principal index of mucus rigidity, log G*, decreased at all airway sites with L-NAC administration, i.e. the mucus became less rigid and more deformable (the overall change in G* was 0.29 log units, i.e. ca. twofold decrease). The viscoelasticity-derived mucus transportability parameters, mucociliary (MCI) and cough (CCI) clearability indices, increased with L-NAC MDI, particularly CCI, which predicts the effect of mucus rheology on cough clearability. PD increased significantly with L-NAC administration at all measurement sites, which appears to be a novel effect for a direct acting mucolytic agent. Tracheal mucus linear velocity (TMV) increased after L-NAC compared with placebo, as did the normalized frog palate transport rate (NFPTR). The increase in NFPTR was greater than that predicted from the mucus rheological properties alone, suggesting that L-NAC still resident in the collected mucus stimulated the frog palate cilia. The index of mucus flux, the collection rate in mg.min-1, was higher with L-NAC compared with placebo. From our results, we conclude that L-NAC shows potential benefit in terms of improving mucus rheological properties and clearability. It may act, in part, by stimulating the fresh secretion of mucus of lower viscoelasticity. The stimulation of mucociliary clearance could be related to ion flux changes, as indicated by the increase in PD.

  19. Enhanced ε-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus.

    PubMed

    Wang, Liang; Chen, Xusheng; Wu, Guangyao; Li, Shu; Zeng, Xin; Ren, Xidong; Tang, Lei; Mao, Zhonggui

    2017-02-01

    ε-Poly-L-lysine (ε-PL), as a food additive, has been widely used in many countries. However, its production still needs to be improved. We successfully enhanced ε-PL production of Streptomyces albulus FEEL-1 by introducing mutations related to antibiotics, such as streptomycin, gentamicin, and rifampin. Single- and double-resistant mutants (S-88 and SG-31) were finally screened with the improved ε-PL productions of 2.81 and 3.83 g/L, 1.75- to 2.39-fold compared with that of initial strain FEEL-1. Then, the performances of mutants S-88 and SG-31 were compared with the parent strain FEEL-1 in a 5-L bioreactor under the optimal condition for ε-PL production. After 174-h fed-batch fermentation, the ε-PL production and productivity of hyper-strain SG-31 reached the maximum of 59.50 g/L and 8.21 g/L/day, respectively, which was 138 and 105% higher than that of FEEL-1. Analysis of streptomycin-resistant mutants demonstrated that a point mutation occurred in rpsL gene (encoding the ribosomal protein S12). These single and double mutants displayed remarkable increases of the activities and transcriptional levels of key enzymes in ε-PL biosynthesis pathway, which may be responsible for the enhanced mycelia viability, respiratory activity, and ε-PL productions of SG-31. These results showed that the new breeding method, called ribosome engineering, could be a novel and effective breeding strategy for the evolution of ε-PL-producing strains.

  20. Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent, Carboxylated ε-poly-L-lysine

    PubMed Central

    Watanabe, Hitomi; Kohaya, Natsuki; Kamoshita, Maki; Fujiwara, Katsuyoshi; Matsumura, Kazuaki; Hyon, Suong-Hyu; Ito, Junya; Kashiwazaki, Naomi

    2013-01-01

    In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ε-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes. PMID:24376724

  1. Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

    PubMed

    Olivieri, Aldo; O'Sullivan, Jeff; Fortuny, Luis Raimon Alvarez; Vives, Itziar Larrauri; Tipton, Keith F

    2010-04-01

    The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified. Copyright 2010 Elsevier B.V. All rights reserved.

  2. A tyrosinase biosensor based on ordered mesoporous carbon-Au/L-lysine/Au nanoparticles for simultaneous determination of hydroquinone and catechol.

    PubMed

    Tang, Lin; Zhou, Yaoyu; Zeng, Guangming; Li, Zhen; Liu, Yuanyuan; Zhang, Yi; Chen, Guiqiu; Yang, Guide; Lei, Xiaoxia; Wu, Mengshi

    2013-06-21

    A novel biosensor was developed based on tyrosinase immobilization with ordered mesoporous carbon-Au (OMC-Au), L-lysine membrane and Au nanoparticles on a glassy carbon electrode (GCE). It was applied for the simultaneous determination of dihydroxybenzene isomers using differential pulse voltammetry (DPV). The tyrosinase/OMC-Au/L-lysine/Au film was characterized by scanning electron microscopy (SEM) and impedance spectra. Under optimized conditions, the DPV study results for two isomers, hydroquinone (HQ, 1,4-dihydroxybenzene) and catechol (CC, 1,2-dihydroxybenzene) showed low peak potentials, and the peak-to-peak difference was about 135.85 mV, which ensured the anti-interference ability of the biosensor and made simultaneous detection of dihydroxybenzene isomers possible in real samples. DPV peak currents increased linearly with concentration over the range of 4.0 × 10(-7) to 8.0 × 10(-5) M, and the detection limits of hydroquinone and catechol were 5 × 10(-8) M and 2.5 × 10(-8) M (S/N = 3), respectively. The tyrosinase biosensor exhibited good repeatability and stability. In addition, the response mechanism of enzyme catalysed redox on the OMC-Au/L-lysine/Au film modified electrode based on electrochemical study was discussed. The proposed method could be extended for the development of other enzyme-based biosensors.

  3. Development and Validation of New HPLC Method for Simultaneous Estimation of L-Lysine Hydrochloride and L-Carnitine-L-Tartrate in Pharmaceutical Dosage Form

    PubMed Central

    Qadir, M. A.; Ahmed, M.; Hussain, W. A.; Tahir, M. S.

    2015-01-01

    A new analytical method is developed and validates for simultaneous estimation of L-lysine hydrochloride and L-carnitine-L-tartrate in single pharmaceutical dosage form by means of high resolution HPLC. The chromatographic procedure was carried out using C18-5 μm (4.6 mm × 250 mm), column. Optimally suited mobile phase was made to run in an isocratic elution mode whose composition was 10 mM of potassium dihydrogen phosphate adjusted with triethylamine to a pH of 7.5 and pumped at a flow rate of 0.50 ml/min through chromatographic system. The detector's wavelength was 214 nm. The accuracy of the analytical method was determined keeping into account the recovery of analytes, which in this case remains well within the accepted standards that is 95-105%. Detection limit for L-lysine hydrochloride and L-carnitine-L-tartrate are 1.47 μg/ml and 0.85 μg/ml while quantitation limit for L-lysine hydrochloride and L-carnitine-L-tartrate is 4.41 and 2.55 μg/ml, respectively. All the statistical results were validated performing precision, accuracy, linearity, specificity studies for analytes concentration from 70-130%. The outcome of results confirmed that the method was in consonance with the acceptance criteria. PMID:26664059

  4. A reanalysis of the observed interplanetary hydrogen L. cap alpha. emission profiles and the derived local interstellar gas temperature and velocity

    SciTech Connect

    Wu, F.M.; Judge, D.L.

    1980-07-01

    An improved model of the interplanetary emission profile is presented. In this model we utilize a new version of the modified Danby-Camm velocity distribution function to calculate emission profiles, and it appears to be very efficient. Using this model, the hydrogen L..cap alpha.. profiles are determined for various viewing directions, and at a solar distance of 1 AU. It is found that the linewidth of the emission profiles and their Doppler shifts depend strongly on the viewing direction, as well as the assumed interstellar temperature. Previous determinations of the interstellar temperature T/sub 0/ and the inflow velocity V/sub b/, using approximate theoretical hydrogen profiles, are found to be inadequate. These values are recalculated and are determined to be T/sub 0/=7000 +- 1200 K and V/sub b/=19 +- 3 km s/sup -1/.

  5. Amide proton exchange in the. cap alpha. -amylase polypeptide inhibitor tendamistat studied by two-dimensional /sup 1/H nuclear magnetic resonance

    SciTech Connect

    Wang, O.; Kline, A.D.; Wuethrich, K.

    1987-10-06

    The individual amide proton exchange rates in Tendamistat at pH 3.0 and 50/sup 0/C were measured by using two-dimensional ..cap alpha..H nuclear magnetic resonance. Overall, it was found that the distribution of exchange rates along the sequence is dominated by the interstrand hydrogen bonds of the ..beta..-sheet structures. The slowly exchanging protons in the core of the two ..beta..-sheets were shown to exchange via an EX2 mechanism. Further analysis of the data indicates that different large-scale structure fluctuations are responsible for the exchange from the two ..beta..-sheets, even though the three-dimensional structure of Tendamistat appears to consist of a single structural domain.

  6. Diagnosis and screening of small hepatocellular carcinomas: comparison of radionuclide imaging, ultrasound, computed tomography, hepatic angiography, and. cap alpha. /sub 1/-fetoprotein assay

    SciTech Connect

    Takashima, T.; Matsui, O.; Suzuki, M.; Ida, M.

    1982-12-01

    Twenty-nine small (<5 cm) hepatocellular carcinomas in 18 patients were examined by radionuclide imaging (RN), ultrasound (US), computed tomography (CT), hepatic angiography, and serum ..cap alpha../sub 1/-fetoprotein (AFP) assay. Sensitivity was 39% with RN, 50% with US, 56% with CT, and 94% with angiography, including infusion hepatic angiography (IHA). Lesions larger than 3 cm could be detected by all of these methods; those between 2 and 3 cm were generally shown by US and CT but not RN. IHA was essential for diagnosis of lesions less than 2 cm, which were otherwise difficult or impossible to detect except with angiography. As a screening method, AFP was best, followed by US and CT. The authors recommend using AFP and US to minimize expense and radiation exposure. In questionable cases, IHA should be performed.

  7. Effect of the filler on the electrophysical properties of the composite system (Pb-PbS)-copolymer of styrene with. cap alpha. -methylstyrene

    SciTech Connect

    Khlystunova, E.V.; Ovchinnikov, A.A.; Saushkina, E.A.; Mardashev, Yu.S.

    1988-08-10

    The results of studies of the effect of a two-phase contact filler (Pb-PbS) on the electrophysical properties of a system based on a copolymer of styrene with ..cap alpha..-methylstyrene (SAM) are reported in this article. The x-ray phase analysis (XPA) was conducted on a DRON-2 diffractometer. The surface of the filler particles was studied by x-ray photoelectron spectroscopy. The electric resistance of the samples was measured in a thermostated cell. The complex character of the concentration and temperature dependence of the electric conductivity of the (Pb-PbS)-SAM composite system related to the composition of the filler was thus elucidated and it was shown that materials with different electrophysical properties can be prepared from the same composite system by using different amounts of the starting components.

  8. (I-125) 17. cap alpha. -Iodovinyl 11. beta. -methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical

    SciTech Connect

    Jagoda, E.M.; Gibson, R.E.; Goodgold, H.; Ferreira, N.; Francis, B.E.; Reba, R.C.; Rzeszotarski, W.J.; Eckelman, W.C.

    1984-04-01

    17 ..cap alpha..-(/sup 125/I)Iodovinyl 11 ..beta..-methoxyestradiol ((I-125)MIVE/sub 2/) has been prepared with high specific activity (155-2000 Ci/mmol) and a high affinity for the estrogen receptor. In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The radioactive labeling of MIVE/sub 2/ is sufficiently rapid so that (I-123)MIVE/sub 2/ has been synthesized and is currently in clinical trials. These results suggest that MIVE/sub 2/ would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

  9. Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

    SciTech Connect

    Cornett, L.E.; Norris, J.S.

    1987-11-01

    In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

  10. Effect of. cap alpha. /sub 1/-adrenergic stimulation on phosphoinositide metabolism and protein kinase C (PK-C) in rat cardiomyocytes

    SciTech Connect

    Kaku, T.; Lakatta, E.; Filburn, C.R.

    1986-03-01

    Alpha/sub 1/-adrenergic stimulation is known to enhance membrane phospholipid metabolism resulting in increases in inositol phosphates (IP's) and diacylglycerol (DAG). Cardiomyocytes prelabeled with /sup 3/H-myo-inositol were treated with norepinephrine (NE) for 1-15 min, acid extracted, and IP's separated by ion exchange chromatography. Addition of NE (10/sup -5/M) in the presence of propranolol (10/sup -5/ M) and LiCl (9 mM) enhanced the accumulation of IP's, linearly with time up to 15 min, and reached 7.3, and 1.5-fold at 15 min for IP/sub 1/, IP/sub 2/, and IP/sub 3/, respectively. KCl at 30 mM had no effect on accumulation of IP's, but augmented the effect of NE. PK-C activity was measured in both cytosol (S) and particulate (P) fractions of treated cells. NE alone had a negligible effect on membrane PK-C, while 30 mM KCl caused a small increase. However, pretreatment with KCl followed by NE produced a significant increase above that seen with KCl alone. Dioctanoylglycerol also stimulated membrane association of PK-C in these cells. These data suggest that ..cap alpha../sub 1/-adrenergic stimulation of membrane association of myocardial PK-C is mediated by DAG but may be dependent on membrane potential and/or the extent of Ca/sup 2 +/ loading.

  11. Antimicrobial, antioxidant, and antitumor activity of epsilon-poly-L-lysine and citral, alone or in combination

    PubMed Central

    Shi, Ce; Zhao, Xingchen; Liu, Zonghui; Meng, Rizeng; Chen, Xiangrong; Guo, Na

    2016-01-01

    Background Food safety is an important worldwide public health concern, and microbial contamination in foods not only leads to food deterioration and shelf life reduction but also results in economic losses and disease. Objective The main aim of the present study was to evaluate the effect of epsilon-poly-L-lysine (ε-PL) and citral combination against Escherichia coli O157:H7 (E. coli O157:H7) strains. The preliminary antioxidant and antitumor activities were also studied. Design Synergism is a positive interaction created when two compounds combine and exert an inhibitory effect that is greater than the sum of their individual effects. The synergistic antimicrobial effect of ε-PL and citral was studied using the checkerboard method against E. coli O157:H7. The minimal inhibitory concentration, time-kill, and scanning electron microscope assays were used to determine the antimicrobial activity of ε-PL and citral alone or in combination; 2,2-diphenyl-1-picrylhydrazyl-scavenging assay and western blotting were used in antioxidant activity assays; cell viability assay was carried out to finish preliminary antitumor test. Results Minimal inhibitory concentrations of ε-PL and citral resisted to the five E. coli O157:H7 strains were 2–4 µg/mL and 0.5–1 µg/mL, and the fractional inhibitory concentration indices were 0.25–0.375. The results of time-kill assay revealed that a stronger bactericidal effect in a laboratory medium might be exerted in the combination against E. coli O157:H7 than that in a food model. The compounds alone or in combination exhibited a potential 2,2-diphenyl-1-picrylhydrazyl radical–scavenging activity, and the expression of superoxide dismutase 1 and glutathione peroxidase 1 protein increased. The preliminary antitumor activity effect of the combination was better than ε-PL or citral alone. Conclusions These findings indicated that the combination of ε-PL and citral could not only be used as a promising naturally sourced food

  12. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  13. A L-lysine transporter of high stereoselectivity of the amino acid-polyamine-organocation (APC) superfamily: production, functional characterization, and structure modeling.

    PubMed

    Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

    2014-01-17

    Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of L-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of L-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of L-arginine, L-ornithine, L-2,4-diaminobutyric acid, and L-alanine. D-lysine is bound 45 times more weakly than its L-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010-1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for L-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

  14. [Antimycoplasmic Activity of Fermentation Broth of Trichoderma harzianum Rifai F-180, an Organism Producing L-Lysine-α-Oxidase, an Antitumor and Antiviral Enzyme].

    PubMed

    Smirnova, I P; Rakovskaya, I V

    2014-01-01

    A concentrate of the fermentation broth of Trichoderma harzianum Rifai F-180, an organism producing L-lysine-α-oxidase, an antitumor and antiviral enzyme, with the activity in the fermentation broth of 0.54-0.56 U/mI was recovered. The effect of the concentrate on the mycoplasmas growth was investigated for the first time. Two representatives of Mycoplasmafaceae, i.e. Mycoplasma hominis and Mycoplasma fermentans and one representative of Aholeplasmataceae. i. e. Aholeplasma laidlawii were used. It was shown that the fermentation broth inhibited the growth of Mycoplasma hominis after the preliminary exposure. The inhibition rate depended on the mycoplasma inoculation dose and the fermentation broth concentration.

  15. Application of MCR-ALS to reveal intermediate conformations in the thermally induced α-β transition of poly-L-lysine monitored by FT-IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Alcaráz, Mirta R.; Schwaighofer, Andreas; Goicoechea, Héctor; Lendl, Bernhard

    2017-10-01

    Temperature-induced conformational transitions of poly-L-lysine were monitored with Fourier-transform infrared (FT-IR) spectroscopy between 10 °C and 70 °C. Chemometric analysis of dynamic IR spectra was performed by multivariate curve analysis-alternating least squares (MCR-ALS) of the amide I‧ and amide II‧ spectral region. With this approach, the pure spectral and concentration profiles of the conformational transition were obtained. Beside the initial α-helical, the intermediate random coil/extended helices and the final β-sheet structure, an additional intermediate PLL conformation was identified and attributed to a transient β-sheet structure.

  16. Alpha 1-antitrypsin activates lung cancer cell survival by acting on cap-dependent protein translation, vesicle-mediated transport, and metastasis.

    PubMed

    Chang, Seung-Hee; Cho, Kyung-Cho; Yu, Kyeong-Nam; Hong, Seong-Ho; Park, Sungjin; Lee, Ah Young; Kim, Sanghwa; Lee, Somin; Kang, Jeong Won; Chae, Chanhee; Park, Jongsun; Kim, Kwang Pyo; Cho, Myung-Haing

    2016-07-19

    Lung cancer remains the leading cause of cancer-related deaths worldwide. Although elevated expression levels of alpha 1-antitrypsin (AAT) have been reported in lung cancer patients, the precise role of AAT in lung cancer progression and prevention has not yet been fully elucidated. We have explored the mechanisms by which AAT stimulates in lung cancer progression. Here, we used proteomic analyses to compare protein levels following AAT overexpression in normal lung L132 cells containing fundamentally low level of AAT. Overexpression of AAT increased levels of proteins involved in transcription and translation, such as signal transducer and activator of transcription 5B (STAT5B) and eukaryotic translation elongation factor 1-alpha 2 (EEF1A2). Furthermore, dual luciferase activity for cap-dependent protein translation increased a 53% at 24 h and 45% at 48 h in AAT-overexpressing cells compared with control. Overexpression of AAT also increased levels of the vesicular transport protein, GOPC, which inhibited the expression of the autophagy protein, BECN1, thereby possibly increasing cell survival. In addition, overexpression of AAT promoted angiogenesis and cell adhesion through increasing expression of the metastatic protein, thrombospondin 1 (THBS1). In contrast, down-regulation of AAT by short hairpin RNA (shRNA) suppressed cell proliferation, metastasis, and adhesion in human lung adenocarcinoma A549 cells and in the lung tissue of K-rasLA1 lung cancer model mice. These findings strongly suggest that AAT regulation shows promise as an alternative avenue for lung cancer treatment and prevention.

  17. Use of antibodies specific to defined regions of scorpion. cap alpha. -toxin to study its interaction with its receptor site on the sodium channel

    SciTech Connect

    Ayeb, M.E.; Bahraoui, E.M.; Granier, C.; Rochat, H.

    1986-10-21

    Five antibody populations selected by immunoaffinity chromatography for the specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the /sup 125/I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to the antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only ..cap alpha..-helix region (residues 23-32) and a ..beta..-turn structure (residues 32-35) are accessible to the respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.

  18. Solvent effects on the synthesis of ion-exchange membranes by radiation-induced graft polymerization of methyl. cap alpha. ,. beta. ,. beta. -trifluoroacrylate. [Freon 113

    SciTech Connect

    Omichi, H.; Okamoto, J.

    1982-06-01

    Methyl ..cap alpha..,..beta..,..beta..-trifluoroacrylate (MTFA) was grafted onto polyethylene (PE) film and fluorine-containing films to make ion-exchange membranes. In the case of PE the grafting yield was not influenced by the presence of trifluorotrichloroethane (Freon 113) in the reaction mixture, while the presence of methanol decreased the grafting yield. The transversal distribution of graft chains in the film observed by electron-probe x-ray microanalysis showed that when the grafting was carried out in the presence of Freon the amount of graft chains in the central part of PE film was much larger than that at the film surface and that the grafts obtained in the absence of Freon were located mainly at the film surface. The electric resistance of the graft PE film obtained in the presence of Freon decreased more than that of the one obtained in the absence of Freon. The weight loss of the graft films in H/sub 2/O/sub 2/ solution was negligibly small.

  19. Remarks concerning the O(Z. cap alpha. /sup 2/) corrections to Fermi decays, conserved-vector-current predictions, and universality

    SciTech Connect

    Sirlin, A.

    1987-06-01

    Finite-nuclear-size contributions to the O(Z..cap alpha../sup 2/) corrections to Fermi decays are studied for realistic nuclear-charge distributions. In conjunction with the results of Koslowsky et al. and recent papers by the author and Zucchini and by Jaus and Rasche, these refinements lead to an average value scrFt = 3070.6 +- 1.6 s for the accurately measured superallowed Fermi transitions. Correspondingly, V/sub u//sub d/ = 0.9744 +- 0.0010 and V/sub u//sub d/ /sup 2/+V/sub us/ /sup 2/+V/sub ub/ /sup 2/ = 0.9979 +- 0.0021 in good agreement with the three-generation standard model at the level of its quantum corrections. The agreement with conserved-vector-current predictions is very good, with each of the eight transitions differing from the average by <1sigma. The consequences of using two other calculations of the nuclear mismatch correction delta/sub c/, Wilkinson's microscopic analysis and the recent results of Ormand and Brown, are briefly discussed. A useful upper bound on scrFt, independent of the delta/sub c/ calculation, is given.

  20. Mutations in a gene encoding the. cap alpha. subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling

    SciTech Connect

    Jahng, K.Y.; Ferguson, J.; Reed, S.I.

    1988-06-01

    Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identically to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to gene encoding the ..cap alpha.. subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G/sub 1/, deposition of mating-specific cell surface aggultinins, and induction of pheromone-specific mRNa, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

  1. Studies on the effect of polymer coating on solution grown hygroscopic non-linear optical single crystal of L-lysine monohydrochloride.

    PubMed

    Rani, Neelam; Vijayan, N; Maurya, K K; Haranath, D; Saini, Parveen; Rathi, Brijesh; Wahab, M A; Bhagavanarayana, G

    2012-11-01

    Nonlinear optical single crystals are getting attention because of its enormous applications in the area of fiber optic communication and optical signal processing. In this article, we are reporting the single crystal growth of l-lysine monohydrochloride by slow evaporation solution growth technique, by using double distilled water as the solvent. We found that the grown single crystal is bulk in size and fairly transparent. But after a period of time, due to its hygroscopic nature, the transparency is completely vanished and became opaque. Then we have attempted to coat the poly methyl methacrylate (PMMA) polymer on the surface of l-lysine monohydrochloride (l-LMHCL) single crystal by dip coating method. This polymer coating is giving resistance to hygroscopic nature and also acting as thin protective covering layer without affecting the other properties. Then we have systematically studied the different properties of bare, polymer coated and hygroscopic l-LMCHL single crystals. Its crystalline perfection was examined by high resolution X-ray diffractometer and found major differences in crystalline quality. Its structural and optical behavior was assessed by powder X-ray diffraction, UV-vis and luminescence analyses.

  2. Reagentless electrochemiluminescent detection of protein biomarker using graphene-based magnetic nanoprobes and poly-L-lysine as co-reactant.

    PubMed

    Liao, Ni; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Han, Jing; Yuan, Ruo

    2013-07-15

    This work described the construction of a reagentless and ultrasensitive electrochemiluminescence (ECL) immunosensor using poly-L-lysine as a co-reactant with Ru(bpy)3(2+) for signal amplification and magnetic Fe3O4 loaded graphene nanosheet as nanoprobes, which can achieve an impressive detection limit of 0.03 pg/mL human total 3,3',5-triiodothyronine (T3), a kind of diagnostic markers of thyroid disease. The bionanoprobes were prepared based on the coimmobilization of Ru(bpy)3(2+) and T3 detection antibody on the Fe3O4 loaded graphene nanosheet and the sensing interface was achieved by assembling T3 capture antibody on the gold nanoparticles (AuNPs) loaded electro-deposited L-lysine film modified bare glass carbon electrode (GCE). ECL responses were generated from the modified electrodes described above by just immersing them in phosphate buffer solutions (PBS) based on the sandwich-type immunoreactions. T3 was measured quantitatively in the range from 0.1 pg/mL to 10 ng/mL, which exhibits sufficiently high sensitivity and stability. The reagentless ECL immunoassay is a promising approach for the detection of a wide range of molecular analytes.

  3. Transport of amino acids (L-valine, L-lysine, L-glutamic acid) and sucrose into plasma membrane vesicles isolated from cotyledons of developing pea seeds.

    PubMed

    de Jong, A; Borstlap, A C

    2000-10-01

    Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H(+)-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of DeltapH. The imposition of Deltapsi stimulated the influx of valine by 20%, both in the absence and in the presence of DeltapH. Uptake of L-lysine was more strongly stimulated by Deltapsi than by DeltapH, and its DeltapH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of Deltapsi. In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by DeltapH or Deltapsi. Although the transport of sucrose was very low, a stimulating effect of DeltapH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H(+)-symporters for the active uptake of sucrose and amino acids.

  4. Avian serum. cap alpha. /sub 1/-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (. beta. -glycoprotein) counter parts

    SciTech Connect

    Goldfarb, V.; Trimble, R.B.; Falco, M.D.; Liem, H.H.; Metcalfe, S.A.; Wellner, D.; Muller-Eberhard, U.

    1986-10-21

    The physicochemical characteristics of chicken hemopexin, which can be isolated by heme-agarose affinity chromatography, is compared with representative mammalian hemopexins of rat, rabbit, and human. The avian polypeptide chain appears to be slightly longer (52 kDa) than the human, rat, or rabbit forms (49 kDa), and also the glycoprotein differs from the mammalian hemopexins in being an ..cap alpha../sub 1/-glycoprotein instead of a ..beta../sub 1/-glycoprotein. The distinct electrophoretic mobility probably arises from significant differences in the amino acid composition of the chicken form, which, although lower in serine and particularly in lysine, has a much higher glutamine/glutamate and agrinine content, and also a higher proline, glycine, and histidine content, than the mammalian hemopexins. Compositional analyses and /sup 125/I concanavalin A and /sup 125/I wheat germ agglutinin binding suggest that chicken hemopexin has a mixture of three fucose-free N-linked bi- and triantennary oligosaccharides. In contrast, human hemopexin has give N-linked oligosaccharides and an additional O-linked glycan blocking the N-terminal threonine residue, while the rabbit form has four N-linked oligosaccharides. In keeping with the finding of a simpler carbohydrate structure, the avian hemopexin shows only a single band on polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions, whereas the hemopexins of the three mammalian species tested show several bands. In contrast, the isoelectric focusing pattern of chicken hemopexin is very complex, revealing at least nine bands between pH 4.0 and pH band 5.0, while the other hemopexins show a broad smear of multiple ill-defined bands in the same region.Results indicate the hemopexin of avians differs substantially from the hemopexins of mammals, which show a notable similarity with regard to carbohydrate structure and amino acid composition.

  5. Effect of. cap alpha. -tocopherol, butylated-hydroxytoluene and hydroxy-anisole on the activation and binding of aflatoxin B/sub 1/ to macromolecules

    SciTech Connect

    Ch'ih, J.J.; Biedrzycka, D.; Devlin, T.M.

    1987-05-01

    The anti-oxidants, ..cap alpha..-tocopherol(TPA), butylated-hydroxy-toluene(BHT) and hydroxyanisole(BHA) inhibit the carcinogenic and toxic effects of a variety of chemical compounds, their effect on aflatoxin B/sub 1/ (AFB/sub 1/) activation and binding was examined utilizing rat liver microsomes and cells. With a NADPH generating system, oxygen, microsomes, (/sup 3/H)-AFB/sub 1/, 2.2 pmoles/h/mg protein was activated and bound to macromolecules. In hepatocytes, 3.4 and 1.4 pmoles of AFB/sub 1/ per 10/sup 6/ cells were taken up and bound to macromolecules, whereas the nucleic acid fraction contained 0.19 pmoles of bound AFB/sub 1/. Moderate decreases of AFB/sub 1/ activation and binding were observed when TPA was present in both cell-free and hepatocytes systems. Only in hepatocytes, BHT inhibited the AFB/sub 1/ uptake and binding to nucleic acids. BHA, however, inhibited microsomal activation of AFB/sub 1/ by 73%; maximum inhibition was reached at 1 mM. AFB/sub 1/ uptake, and binding to nucleic acids were inhibited by 65% and 79% by BHA. GSH-transferase activity of cells treated with these agents was not altered. The effect of BHA at various concentrations on AFB activation was compared with cytochrome P-450 inhibitors; the ED/sub 50/ of SKF 525A, BHA and metyrapone was 9 uM, 80 uM and 380 uM respectively. The data suggest that TPA, BHA and BHT exert their effect by different mechanisms.

  6. Hydrogen fluoride saccharification of wood: lignin fluoride content, isolation of. cap alpha. -D-glucopyranosyl fluoride and posthydrolysis of reversion products

    SciTech Connect

    Hardt, H.; Lamport, D.J.A.

    1982-04-01

    Wood chips from bigtooth aspen (Populus grandidentata Michx.) were saccharified by reaction with hydrogen fluoride either anhydrous or containing up to 10% v/v water. The reaction products were separated into a solid lignin fraction and a water-soluble saccharide fraction. The fluoride content of the lignin (determined after alkaline fusion) was initially about 1 mg/g wood, but was lowered to 0.1 mg/g wood by grinding and washing. Thus little or no chemical binding of fluoride to lignin occurred during hydrogen fluoride (HF) solvolysis. Analysis of the water-soluble fraction by gel filtration on Biogel P2 columns showed a range of low-molecular-weight oligosaccharides an only 10-20% sugar monomers. Thus considerable reversion occurred during HF evacuation. Posthydrolysis conditions were optimized for these reversion products by varying temperature and acid concentration. Optimal conditions at 1 h were 140/sup 0/C with 100mN sulfuric acid or 225mN hydrofluoric acid resulting in monomer yields of > 90% for 0.5% sugar solutions and > 80% for 10% sugar solutions. After reaction of pure cellulose (filter paper) with hydrogen fluoride in the absence of water, and terminating the reaction with calcium carbonate, the reaction intermediate ..cap alpha..-D-glucopyranosylfluride was isolated with a maximal yield of 0.2 g/g paper. Upon purification via paper chromatography glucosyfluoride was identified by its specific rotation and also by gas chromotography-mass spectrometer of its tetra-O-trimethylsilyl derivative.

  7. An additional fluorenylmethoxycarbonyl (Fmoc) moiety in di-Fmoc-functionalized L-lysine induces pH-controlled ambidextrous gelation with significant advantages.

    PubMed

    Reddy, Samala Murali Mohan; Shanmugam, Ganesh; Duraipandy, Natarajan; Kiran, Manikantan Syamala; Mandal, Asit Baran

    2015-11-07

    In recent years, several fluorenylmethoxycarbonyl (Fmoc)-functionalized amino acids and peptides have been used to construct hydrogels, which find a wide range of applications. Although several hydrogels have been prepared from mono Fmoc-functionalized amino acids, herein, we demonstrate the importance of an additional Fmoc-moiety in the hydrogelation of double Fmoc-functionalized L-lysine [Fmoc(Nα)-L-lysine(NεFmoc)-OH, (Fmoc-K(Fmoc))] as a low molecular weight gelator (LMWG). Unlike other Fmoc-functionalized amino acid gelators, Fmoc-K(Fmoc) exhibits pH-controlled ambidextrous gelation (hydrogelation at different pH values as well as organogelation), which is significant among the gelators. Distinct fibrous morphologies were observed for Fmoc-K(Fmoc) hydrogels formed at different pH values, which are different from organogels in which Fmoc-K(Fmoc) showed bundles of long fibers. In both hydrogels and organogels, the self-assembly of Fmoc-K(Fmoc) was driven by aromatic π-π stacking and hydrogen bonding interactions, as evidenced from spectroscopic analyses. Characterization of Fmoc-K(Fmoc) gels using several biophysical methods indicates that Fmoc-K(Fmoc) has several advantages and significant importance as a LMWG. The advantages of Fmoc-K(Fmoc) include pH-controlled ambidextrous gelation, pH stimulus response, high thermal stability (∼100 °C) even at low minimum hydrogelation concentration (0.1 wt%), thixotropic property, high kinetic and mechanical stability, dye removal properties, cell viability to the selected cell type, and as a drug carrier. While single Fmoc-functionalized L-lysine amino acids failed to exhibit gelation under similar experimental conditions, the pH-controlled ambidextrous gelation of Fmoc-K(Fmoc) demonstrates the benefit of a second Fmoc moiety in inducing gelation in a LMWG. We thus strongly believe that the current findings provide a lead to construct or design various new synthetic Fmoc-based LMW organic gelators for several

  8. Cervical Cap

    MedlinePlus

    ... and remove the cap. How Much Does It Cost? A cervical cap costs about $70 and should be replaced every year. In addition, there is also the cost of the doctor's visit. Many health insurance plans ...

  9. Investigation of interactions between poly-L-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation.

    PubMed

    Ciofani, G; Ricotti, L; Danti, S; Moscato, S; Nesti, C; D'Alessandro, D; Dinucci, D; Chiellini, F; Pietrabissa, A; Petrini, M; Menciassi, A

    2010-04-15

    Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription - polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

  10. Contribution of the net charge to the regulatory effects of amino acids and epsilon-poly(L-lysine) on the gelatinization behavior of potato starch granules.

    PubMed

    Ito, Azusa; Hattori, Makoto; Yoshida, Tadashi; Takahashi, Koji

    2006-01-01

    The effects of lysine (Lys), monosodium glutamate (GluNa), glycine, alanine and epsilon-poly(L-lysine) (PL) with different degrees of polymerization on the gelatinization behavior of potato starch granules were investigated by DSC, viscosity and swelling measurements, microscopic observation, and measurement of the retained amino acid amount to clarify the contribution of the net charge to their regulatory effects on the gelatinization behavior. The amino acids and PL each contributed to an increase in the gelatinization temperature, and a decrease in the peak viscosity and swelling. These effects strongly depended on the absolute value of their net charge. The disappearance of a negative or positive net charge by adjusting the pH value weakened the contribution. The swelling index and size of the potato starch granules changed according to replacement of the swelling medium. The amino acids and PL were easily retained by the swollen potato starch granules according to replacement of the outer solution of the starch granules.

  11. Antiviral activity and possible mechanisms of action of oligonucleotides-poly(L-lysine) conjugates targeted to vesicular stomatitis virus mRNA and genomic RNA.

    PubMed Central

    Degols, G; Leonetti, J P; Gagnor, C; Lemaitre, M; Lebleu, B

    1989-01-01

    Synthetic oligonucleotides (oligomers) complementary to vesicular stomatitis virus (VSV) N protein mRNA have specific antiviral properties at concentrations lower than 1 microM when they are covalently linked to poly(L-lysine) (PLL). Since it is generally postulated that antisense oligomers act at the translational level, oligomers with potential targets on VSV viral mRNA and/or genomic RNA have been tested here. In vitro translation experiments in reticulocyte lysates, in vitro transcription experiments with permeabilized viruses, measurement of viral RNA transcription and accumulation in VSV infected cells, and antiviral experiments demonstrate in our model that antisense oligomers probably also act at other levels. Difficulties in the choice of the most effective antisense oligomer targets are also discussed. Images PMID:2479915

  12. The Natural Antimicrobial Peptide Subtilosin Acts Synergistically with Glycerol Monolaurate, Lauric Arginate, and ε-Poly-l-Lysine against Bacterial Vaginosis-Associated Pathogens but Not Human Lactobacilli

    PubMed Central

    Sutyak Noll, Katia; Prichard, Mark N.; Khaykin, Arkady; Sinko, Patrick J.

    2012-01-01

    Subtilosin is a cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens that has antimicrobial activity against the bacterial vaginosis-associated human pathogen Gardnerella vaginalis. The ability of subtilosin to inhibit G. vaginalis alone and in combination with the natural antimicrobial agents glycerol monolaurate (Lauricidin), lauric arginate, and ε-poly-l-lysine was tested using a checkerboard approach. Subtilosin was found to act synergistically with all of the chosen antimicrobials. These promising results indicate that lower concentrations of subtilosin in combination with other compounds could effectively be used to inhibit growth of the pathogen, thereby decreasing the risk of developed antimicrobial resistance. This is the first report on the effects of subtilosin combined with other natural antimicrobials against G. vaginalis. PMID:22252803

  13. The natural antimicrobial peptide subtilosin acts synergistically with glycerol monolaurate, lauric arginate, and ε-poly-L-lysine against bacterial vaginosis-associated pathogens but not human lactobacilli.

    PubMed

    Noll, Katia Sutyak; Prichard, Mark N; Khaykin, Arkady; Sinko, Patrick J; Chikindas, Michael L

    2012-04-01

    Subtilosin is a cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens that has antimicrobial activity against the bacterial vaginosis-associated human pathogen Gardnerella vaginalis. The ability of subtilosin to inhibit G. vaginalis alone and in combination with the natural antimicrobial agents glycerol monolaurate (Lauricidin), lauric arginate, and ε-poly-L-lysine was tested using a checkerboard approach. Subtilosin was found to act synergistically with all of the chosen antimicrobials. These promising results indicate that lower concentrations of subtilosin in combination with other compounds could effectively be used to inhibit growth of the pathogen, thereby decreasing the risk of developed antimicrobial resistance. This is the first report on the effects of subtilosin combined with other natural antimicrobials against G. vaginalis.

  14. NMR studies of protonation and hydrogen bond states of internal aldimines of pyridoxal 5'-phosphate acid-base in alanine racemase, aspartate aminotransferase, and poly-L-lysine.

    PubMed

    Chan-Huot, Monique; Dos, Alexandra; Zander, Reinhard; Sharif, Shasad; Tolstoy, Peter M; Compton, Shara; Fogle, Emily; Toney, Michael D; Shenderovich, Ilya; Denisov, Gleb S; Limbach, Hans-Heinrich

    2013-12-04

    Using (15)N solid-state NMR, we have studied protonation and H-bonded states of the cofactor pyridoxal 5'-phosphate (PLP) linked as an internal aldimine in alanine racemase (AlaR), aspartate aminotransferase (AspAT), and poly-L-lysine. Protonation of the pyridine nitrogen of PLP and the coupled proton transfer from the phenolic oxygen (enolimine form) to the aldimine nitrogen (ketoenamine form) is often considered to be a prerequisite to the initial step (transimination) of the enzyme-catalyzed reaction. Indeed, using (15)N NMR and H-bond correlations in AspAT, we observe a strong aspartate-pyridine nitrogen H-bond with H located on nitrogen. After hydration, this hydrogen bond is maintained. By contrast, in the case of solid lyophilized AlaR, we find that the pyridine nitrogen is neither protonated nor hydrogen bonded to the proximal arginine side chain. However, hydration establishes a weak hydrogen bond to pyridine. To clarify how AlaR is activated, we performed (13)C and (15)N solid-state NMR experiments on isotopically labeled PLP aldimines formed by lyophilization with poly-L-lysine. In the dry solid, only the enolimine tautomer is observed. However, a fast reversible proton transfer involving the ketoenamine tautomer is observed after treatment with either gaseous water or gaseous dry HCl. Hydrolysis requires the action of both water and HCl. The formation of an external aldimine with aspartic acid at pH 9 also produces the ketoenamine form stabilized by interaction with a second aspartic acid, probably via a H-bond to the phenolic oxygen. We postulate that O-protonation is an effectual mechanism for the activation of PLP, as is N-protonation, and that enzymes that are incapable of N-protonation employ this mechanism.

  15. The Sugar Model: Catalytic Flow Reactor Dynamics of Pyruvaldehyde Synthesis from Triose Catalyzed by Poly-L-Lysine Contained in a Dialyzer

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.; DeVincenzi, Donald (Technical Monitor)

    2000-01-01

    The formation of pyruvaldehyde from triose sugars was catalyzed by poly-L-lysine contained in a small dialyzer (100 MWCO) suspended in a much larger triose substrate reservoir. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. A 400 mM solution of poly-L-lysine contained in a 0.35 ml dialyzer placed in a 120 ml solution of triose substrate (pH 5.5, 40 C) generated pyruvaldehyde 11 -times faster than an a control reaction without the catalytic dialyzer. However, since the catalytic dialyzer's volume was 343-times smaller than the control reaction, the synthetic intensity (rate/volume) of pyruvaldehyde synthesis within the catalytic dialyzer was 3400-times greater than that of the control reaction and substrate solution. A similar result was obtained using a dialyzer with a 500 MWCO value. Acting as a catalytic flow reactor the polylysine catalytic dialyzer synthesized about 3.5 molecules of pyruvaldehyde per lysine residue in 7 days -- an amount of triose equal to twice the weight of the catalyst. At 7 days the catalytic activity of polylysine was 16% of its initial value, a result indicating catalyst-poisoning caused by reaction of pyruvaldehyde with the e-amino groups of polylysine. The dialyzer method of catalyst containment was selected it provides a simple, flexible, and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.

  16. Reduction of the Inflammatory Responses against Alginate-Poly-L-Lysine Microcapsules by Anti-Biofouling Surfaces of PEG-b-PLL Diblock Copolymers

    PubMed Central

    Spasojevic, Milica; Paredes-Juarez, Genaro A.; Vorenkamp, Joop; de Haan, Bart J.; Schouten, Arend Jan; de Vos, Paul

    2014-01-01

    Large-scale application of alginate-poly-L-lysine (alginate-PLL) capsules used for microencapsulation of living cells is hampered by varying degrees of success, caused by tissue responses against the capsules in the host. A major cause is proinflammatory PLL which is applied at the surface to provide semipermeable properties and immunoprotection. In this study, we investigated whether application of poly(ethylene glycol)-block-poly(L-lysine hydrochloride) diblock copolymers (PEG-b-PLL) can reduce the responses against PLL on alginate-matrices. The application of PEG-b-PLL was studied in two manners: (i) as a substitute for PLL or (ii) as an anti-biofouling layer on top of a proinflammatory, but immunoprotective, semipermeable alginate-PLL100 membrane. Transmission FTIR was applied to monitor the binding of PEG-b-PLL. When applied as a substitute for PLL, strong host responses in mice were observed. These responses were caused by insufficient binding of the PLL block of the diblock copolymers confirmed by FTIR. When PEG-b-PLL was applied as an anti-biofouling layer on top of PLL100 the responses in mice were severely reduced. Building an effective anti-biofouling layer required 50 hours as confirmed by FTIR, immunocytochemistry and XPS. Our study provides new insight in the binding requirements of polyamino acids necessary to provide an immunoprotective membrane. Furthermore, we present a relatively simple method to mask proinflammatory components on the surface of microcapsules to reduce host responses. Finally, but most importantly, our study illustrates the importance of combining physicochemical and biological methods to understand the complex interactions at the capsules' surface that determine the success or failure of microcapsules applicable for cell-encapsulation. PMID:25347191

  17. Reduction of the inflammatory responses against alginate-poly-L-lysine microcapsules by anti-biofouling surfaces of PEG-b-PLL diblock copolymers.

    PubMed

    Spasojevic, Milica; Paredes-Juarez, Genaro A; Vorenkamp, Joop; de Haan, Bart J; Schouten, Arend Jan; de Vos, Paul

    2014-01-01

    Large-scale application of alginate-poly-L-lysine (alginate-PLL) capsules used for microencapsulation of living cells is hampered by varying degrees of success, caused by tissue responses against the capsules in the host. A major cause is proinflammatory PLL which is applied at the surface to provide semipermeable properties and immunoprotection. In this study, we investigated whether application of poly(ethylene glycol)-block-poly(L-lysine hydrochloride) diblock copolymers (PEG-b-PLL) can reduce the responses against PLL on alginate-matrices. The application of PEG-b-PLL was studied in two manners: (i) as a substitute for PLL or (ii) as an anti-biofouling layer on top of a proinflammatory, but immunoprotective, semipermeable alginate-PLL100 membrane. Transmission FTIR was applied to monitor the binding of PEG-b-PLL. When applied as a substitute for PLL, strong host responses in mice were observed. These responses were caused by insufficient binding of the PLL block of the diblock copolymers confirmed by FTIR. When PEG-b-PLL was applied as an anti-biofouling layer on top of PLL100 the responses in mice were severely reduced. Building an effective anti-biofouling layer required 50 hours as confirmed by FTIR, immunocytochemistry and XPS. Our study provides new insight in the binding requirements of polyamino acids necessary to provide an immunoprotective membrane. Furthermore, we present a relatively simple method to mask proinflammatory components on the surface of microcapsules to reduce host responses. Finally, but most importantly, our study illustrates the importance of combining physicochemical and biological methods to understand the complex interactions at the capsules' surface that determine the success or failure of microcapsules applicable for cell-encapsulation.

  18. Characterization of myo-inositol utilization by Corynebacterium glutamicum: the stimulon, identification of transporters, and influence on L-lysine formation.

    PubMed

    Krings, Eva; Krumbach, Karin; Bathe, Brigitte; Kelle, Ralf; Wendisch, Volker F; Sahm, Hermann; Eggeling, Lothar

    2006-12-01

    Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.

  19. Genome sequence of thermotolerant Bacillus methanolicus: features and regulation related to methylotrophy and production of L-lysine and L-glutamate from methanol.

    PubMed

    Heggeset, Tonje M B; Krog, Anne; Balzer, Simone; Wentzel, Alexander; Ellingsen, Trond E; Brautaset, Trygve

    2012-08-01

    Bacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into L-lysine and L-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their L-glutamate production levels (406 mmol liter(-1) and 11 mmol liter(-1), respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for L-lysine and L-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol.

  20. Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l-Lysine and l-Glutamate from Methanol

    PubMed Central

    Heggeset, Tonje M. B.; Krog, Anne; Balzer, Simone; Wentzel, Alexander; Ellingsen, Trond E.

    2012-01-01

    Bacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into l-lysine and l-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their l-glutamate production levels (406 mmol liter−1 and 11 mmol liter−1, respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for l-lysine and l-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol. PMID:22610424

  1. Enhancement of Airway Gene Transfer by DNA Nanoparticles Using a pH-Responsive Block Copolymer of Polyethylene Glycol and Poly-L-lysine

    PubMed Central

    Boylan, Nicholas J.; Kim, Anthony J.; Suk, Jung Soo; Adstamongkonkul, Pichet; Simons, Brian W.; Lai, Samuel K.; Cooper, Mark J.; Hanes, Justin

    2011-01-01

    Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of polyethylene glycol and poly-L-lysine (PEG-CK30), have shown considerable promise in human gene therapy clinical trials in the nares, but may be less capable of transfecting cells that lack surface nucleolin. To address this potential shortcoming, we formulated pH-responsive DNA nanoparticles that mediate gene transfer via a nucleolin-independent pathway. Poly-L-histidine was inserted between PEG and poly-L-lysine to form a triblock copolymer system, PEG-CH12K18. Inclusion of poly-L-histidine increased the buffering capacity of PEG-CH12K18 to levels comparable with branched polyethyleneimine. PEG-CH12K18 compacted DNA into rod-shaped DNA nanoparticles with similar morphology and colloidal stability as PEG-CK30 DNA nanoparticles. PEG-CH12K18 DNA nanoparticles entered human bronchial epithelial cells (BEAS-2B) that lack surface nucleolin by a clathrin-dependent endocytic mechanism followed by endo-lysosomal processing. Despite trafficking through the degradative endo-lysosomal pathway, PEG-CH12K18 DNA nanoparticles improved the in vitro gene transfer by ~ 20-fold over PEG-CK30 DNA nanoparticles, and in vivo gene transfer to lung airways in BALB/c mice by ~ 3-fold, while maintaining a favorable toxicity profile. These results represent an important step toward the rational development of an efficient gene delivery platform for the lungs based on highly compacted DNA nanoparticles. PMID:22182747

  2. Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

    PubMed

    Gavilan, Maria P; Arjona, Marina; Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R; Bornens, Michel; Rios, Rosa M

    2015-03-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

  3. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    PubMed Central

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  4. Electrodeposited reduced graphene oxide incorporating polymerization of l-lysine on electrode surface and its application in simultaneous electrochemical determination of ascorbic acid, dopamine and uric acid.

    PubMed

    Zhang, Dongdong; Li, Lingzhi; Ma, Weina; Chen, Xia; Zhang, Yanmin

    2017-01-01

    This paper demonstrates a novel strategy for the construction of a graphene hybrid composites film, which was fabricated by electrodeposited reduced graphene oxide (ERGO) incorporating polymerization of l-lysine (PLL) onto glassy carbon electrode (GCE). Here we show that graphene films can be prepared on electrodes directly from GO dispersions by one-step electrodeposition technique based on electropolymerized PLL as a positively charged polymer interface to adsorb negatively charged GO nanosheets through electrostatic attraction. The thickness of graphene film can be easily controlled by using the electrodeposition technique, a distinct advantage over previously developed methods. The electrochemically reduced process of GO and electropolymerization of l-lysine were investigated by cyclic voltammetry with a wide potential range. The surface morphology of the modified electrode was characterized by scanning electron microscopy. The ERGO/PLL/GCE shows conducive to electron transfer kinetics for Fe(CN)6(3-)/Fe(CN)6(4-) redox probes, compared with bare GCE, PLL/GCE and ERGO/GCE. The electrochemical behaviors of ascorbic acid (AA), dopamine (DA) and uric acid (UA) at ERGO/PLL/GCE were investigated by cyclic voltammetry, and the results suggest that the modified electrode exhibits enhanced electrocatalytic activity toward these important molecules. Under physiological condition and in the co-existence system of AA, DA and UA, the ERGO/PLL/GCE showed linear voltammetric responses in the concentration of 100μM-1200μM for AA, 2.0μM-60μM for DA and 20μM-200μM for UA, and with the detection limits (S/N=3) of 2.0μM, 0.10μM and 0.15μM for AA, DA and UA, respectively. The developed method has been applied to simultaneous determination of AA, DA and UA in human urine with satisfactory recoveries of 104.2%, 95.4% and 99.9%, respectively. This work demonstrates that the attractive features of ERGO/PLL provide promising applications in simultaneous determination of AA, DA

  5. Crystal Structure of Ll-Diaminopimelate Aminotransferase From 'Arabidopsis Thaliana': a Recently-Discovered Enzyme in the Biosynthesis of L-Lysine By Plants And 'Chlamydia'

    SciTech Connect

    Watanabe, N.; Cherney, M.M.; van Belkum, M.J.; Marcus, S.L.; Flegel, M.D.; Clay, M.D.; Deyholos, M.K.; Vederas, J.C.; James, M.N.G.

    2007-07-13

    The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 Angstroms resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modeled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.

  6. Crystal structure of LL-diaminopimelate aminotransferase from Arabidopsis thaliana: a recently discovered enzyme in the biosynthesis of L-lysine by plants and Chlamydia.

    PubMed

    Watanabe, Nobuhiko; Cherney, Maia M; van Belkum, Marco J; Marcus, Sandra L; Flegel, Mitchel D; Clay, Matthew D; Deyholos, Michael K; Vederas, John C; James, Michael N G

    2007-08-17

    The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5'-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the design of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 A resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modelled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.

  7. Chain-length dependence of alpha-helix to beta-sheet transition in polylysine: model of protein aggregation studied by temperature-tuned FTIR spectroscopy.

    PubMed

    Dzwolak, Wojciech; Muraki, Takeshi; Kato, Minoru; Taniguchi, Yoshihiro

    2004-03-01

    The chain-length dependence of the alpha-helix to beta-sheet transition in poly(L-lysine) is studied by temperature-tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L-lysine), comprising polypeptide chains with various lengths, undergo the alpha-beta transition at an intermediate temperature compared to homogeneous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel beta-sheet structure. The tendency is that the longer chain is, the lower the alpha-beta transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or beta sheets in elongating chains of poly(L-lysine). As such helical structures are apparently conducive to the alpha-beta transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid-formation pathways. The antiparallel architecture of the beta sheet is likely to reflect the pretransition interhelical interactions in poly(L-lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel pre-assembly of dipolar alpha helices. Copyright 2004 Wiley Periodicals, Inc.

  8. Identification and characterization of L-lysine decarboxylase from Huperzia serrata and its role in the metabolic pathway of lycopodium alkaloid.

    PubMed

    Xu, Baofu; Lei, Lei; Zhu, Xiaocen; Zhou, Yiqing; Xiao, Youli

    2017-04-01

    Lysine decarboxylation is the first biosynthetic step of Huperzine A (HupA). Six cDNAs encoding lysine decarboxylases (LDCs) were cloned from Huperzia serrata by degenerate PCR and rapid amplification of cDNA ends (RACE). One HsLDC isoform was functionally characterized as lysine decarboxylase. The HsLDC exhibited greatest catalytic efficiency (kcat/Km, 2.11 s(-1) mM(-1)) toward L-lysine in vitro among all reported plant-LDCs. Moreover, transient expression of the HsLDC in tobacco leaves specifically increased cadaverine content from zero to 0.75 mg per gram of dry mass. Additionally, a convenient and reliable method used to detect the two catalytic products was developed. With the novel method, the enzymatic products of HsLDC and HsCAO, namely cadaverine and 5-aminopentanal, respectively, were detected simultaneously both in assay with purified enzymes and in transgenic tobacco leaves. This work not only provides direct evidence of the first two-step in biosynthetic pathway of HupA in Huperzia serrata and paves the way for further elucidation of the pathway, but also enables engineering heterologous production of HupA.

  9. A multi-walled carbon nanotubes-poly(L-lysine) modified enantioselective immunosensor for ofloxacin by using multi-enzyme-labeled gold nanoflower as signal enhancer.

    PubMed

    He, Zuyu; Zang, Shuai; Liu, Yingju; He, Yuan; Lei, Hongtao

    2015-11-15

    The enantioselective detection of trace amounts of ofloxacin is very important in many fields. In this work, an enantioselective and sensitive electrochemical immunosensor was constructed for the detection of chiral antibiotic ofloxacin based on a dual amplification strategy using multiwall carbon nanotubes-poly(L-lysine) as a matrix to immobilize the antigen and multi-enzyme-antibody functionalized gold nanoflowers as an electrochemical detection label. The fabrication process of the dual-amplified immunosensor was characterized by scanning electron microscopy, cyclic voltammogram and electrochemical impedance spectroscopy, respectively. After the optimization of the experimental conditions, a competitive immunoassay, i.e., the association ability with the corresponding antibody between the captured antigen and free S-OFL or R-OFL in the solution, showed that the immunosensor exhibited a sensitive response to S-OFL in the range from 0.26 to 25.6 ng/mL with a detection limit of 0.15 ng/mL as well as a sensitive response to R-OFL in the range from 0.37 to 12.8 ng/mL with a detection limit of 0.30 ng/mL. Along with the acceptable sensitivity and stability, the S-OFL or R-OFL immunosensor showed selective ability to its corresponding enantiomer, suggesting this amplification strategy may hold a potential application in the detection of OFL in food or environment. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. The sugar model: catalytic flow reactor dynamics of pyruvaldehyde synthesis from triose catalyzed by poly-l-lysine contained in a dialyzer

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    2001-01-01

    The formation of pyruvaldehyde from triose sugars was catalyzed by poly-l-lysine contained in a small dialyzer with a 100 molecular weight cut off (100 MWCO) suspended in a much larger triose substrate reservoir at pH 5.5 and 40 degrees C. The polylysine confined in the dialyzer functioned as a catalytic flow reactor that constantly brought in triose from the substrate reservoir by diffusion to offset the drop in triose concentration within the reactor caused by its conversion to pyruvaldehyde. The catalytic polylysine solution (400 mM, 0.35 mL) within the dialyzer generated pyruvaldehyde with a synthetic intensity (rate/volume) that was 3400 times greater than that of the triose substrate solution (12 mM, 120 mL) outside the dialyzer. Under the given conditions the final yield of pyruvaldehyde was greater than twice the weight of the polylysine catalyst. During the reaction the polylysine catalyst was poisoned presumably by reaction of its amino groups with aldehyde reactants and products. Similar results were obtained using a dialyzer with a 500 MWCO. The dialyzer method of catalyst containment was selected because it provides a simple and easily manipulated experimental system for studying the dynamics and evolutionary development of confined autocatalytic processes related to the origin of life under anaerobic conditions.

  11. Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue.

    PubMed

    Ardjomandi, N; Klein, C; Kohler, K; Maurer, A; Kalbacher, H; Niederländer, J; Reinert, S; Alexander, D

    2012-08-01

    The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.

  12. Pore Diameter Dependence and Segmental Dynamics of Poly-Z-L-lysine and Poly-L-alanine Confined in 1D Nanocylindrical Geometry

    NASA Astrophysics Data System (ADS)

    Tuncel, Eylul; Suzuki, Yasuhito; Iossifidis, Agathaggelos; Steinhart, Martin; Butt, Hans-Jurgen; Floudas, George; Duran, Hatice

    Structure formation, thermodynamic stability, phase and dynamic behaviors of polypeptides are strongly affected by confinement. Since understanding the changes in these behaviors will allow their rational design as functional devices with tunable properties, herein we investigated Poly-Z-L-lysine (PZLL) and Poly-L-alanine (PAla) homopolypeptides confined in nanoporous alumina containing aligned cylindrical nanopores as a function of pore size by differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy, Solid-state NMR, X-ray diffraction, Dielectric spectroscopy(DS). Bulk PZLL exhibits a glass transition temperature (Tg) at about 301K while PZLL nanorods showed slightly lower Tg (294K). The dynamic investigation by DS also revealed a decrease (4K) in Tg between bulk and PZLL nanorods. DS is a very sensitive probe of the local and global secondary structure relaxation through the large dipole to study effect of confinement. The results revealed that the local segmental dynamics, associated with broken hydrogen bonds, and segmental dynamics speed-up on confinement.

  13. Ultrasound-induced gelation of fluorenyl-9-methoxycarbonyl-l-lysine(fluorenyl-9-methoxycarbonyl)-OH and its dipeptide derivatives showing very low minimum gelation concentrations.

    PubMed

    Geng, Huimin; Ye, Lin; Zhang, Ai-Ying; Shao, Ziqiang; Feng, Zeng-Guo

    2017-03-15

    Four l-Lysine(Lys)-l-glutamic acid(Glu) dipeptide derivatives (1-4) and their precursor-a single fluorenyl-9-methoxycarbonyl(Fmoc)-l-Lys(Fmoc)-OH amino acid (5) were demonstrated as gelators to gelate a variety of alcohols and aromatic solvents under the sonication conditions. Compared to the routine heating-cooling protocol, the ultrasound substantially brought down the minimum gelation concentrations (MGCs) of the resulting organogels. The Fourier transform infrared spectroscopy (FT-IR) and fluorescence studies revealed that the π-π stacking and hydrogen bonding act as major driving forces for the self-assembly of these lysine-based gelators into supramolecular fibrous three dimensional (3D) network, where the more the Fmoc protecting groups, the gelators are more responsive to ultrasound-stimulus and more conducive to an ordered molecular arrangement reinforcing the intermolecular forces. Moreover, the ultrasound-triggered organogels of 5 exhibited the thixotropic property. Upon imposing a mechanical shear, its gels with the fibrous 3D network structure were unraveled into sols. However, after standing quiescently over time, these sols returned to the gels showing a more ordered lamella-like packing structure as evidenced by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Economical production of poly(ε-l-lysine) and poly(l-diaminopropionic acid) using cane molasses and hydrolysate of streptomyces cells by Streptomyces albulus PD-1.

    PubMed

    Xia, Jun; Xu, Zhaoxian; Xu, Hong; Liang, Jinfeng; Li, Sha; Feng, Xiaohai

    2014-07-01

    Poly(ε-L-lysine) (ε-PL) and poly(L-diaminopropionic acid) (PDAP) co-production by Streptomyces albulus PD-1 from cane molasses and hydrolysate of strepyomyces cells (HSC) was investigated for the first time in this study. The optimal initial total sugar concentration of the cane molasses pretreated with sulfuric acid was determined to be 20 g L(-1), and HSC could substitute for yeast extract for ε-PL and PDAP co-production. When fed-batch fermentation was performed in 1t fermentor with pretreated cane molasses and HSC, 20.6 ± 0.5 g L(-1) of ε-PL and 5.2 ± 0.6 g L(-1) of PDAP were obtained. The amount of strepyomyces cells obtained in one fed-batch fermentation is sufficient to prepare the HSC to satisfy the demand of subsequent fermentations, thus the self-cycling of organic nitrogen source becomes available. These results suggest that the low-cost cane molasses and HSC can be used for the economical production of ε-PL and PDAP by S. albulus PD-1.

  15. Influence of pH and Surface Chemistry on Poly(L-lysine) Adsorption onto Solid Supports Investigated by Quartz Crystal Microbalance with Dissipation Monitoring.

    PubMed

    Choi, Jae-Hyeok; Kim, Seong-Oh; Linardy, Eric; Dreaden, Erik C; Zhdanov, Vladimir P; Hammond, Paula T; Cho, Nam-Joon

    2015-08-20

    Poly(L-lysine) (PLL) adsorption onto various materials has been widely applied as a surface modification strategy and layer-by-layer fabrication method. Considering the role of electrostatic charges, a detailed understanding of the influence of solution pH on PLL adsorption process is important for optimization of PLL coating protocols. Herein, PLL adsorption onto different polar and hydrophilic substrates—silica, an amine-terminated self-assembled monolayer (SAM) on gold, and a carboxyl-terminated SAM on gold—across a range of pH conditions was investigated using the quartz crystal microbalance with dissipation. The adsorption kinetics consisted of an initial rapid phase, followed by a second phase where adsorption rate gradually decelerated. These features were interpreted by applying a mean-field kinetic model implying diffusion-limited adsorption in the first phase and reconfiguration of adsorbed PLL molecules in the second phase. The adsorption kinetics and uptake were found to be sensitive to the pH condition, surface chemistry, and flow rate. The strongest PLL adsorption occurred at pH 11 on all three surfaces while weak PLL adsorption generally occurred under acidic conditions. The surface morphology and roughness of adsorbed PLL layers were investigated using atomic force microscopy, and strong PLL adsorption is found to produce a uniform and smooth adlayer while weak adsorption formed a nonuniform and rough adlayer.

  16. Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

    NASA Astrophysics Data System (ADS)

    Hashimoto, Tomoko; Kawazu, Takeshi; Nagasaki, Takeshi; Murakami, Akira; Yamaoka, Tetsuji

    2012-02-01

    Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

  17. Economic process to co-produce poly(ε-l-lysine) and poly(l-diaminopropionic acid) by a pH and dissolved oxygen control strategy.

    PubMed

    Xu, Zhaoxian; Feng, Xiaohai; Sun, Zhuzhen; Cao, Changhong; Li, Sha; Xu, Zheng; Xu, Zongqi; Bo, Fangfang; Xu, Hong

    2015-01-01

    This study tended to apply biorefinery of indigenous microbes to the fermentation of target-product generation through a novel control strategy. A novel strategy for co-producing two valuable homopoly(amino acid)s, poly(ε-l-lysine) (ε-PL) and poly(l-diaminopropionic acid) (PDAP), was developed by controlling pH and dissolved oxygen concentrations in Streptomyces albulus PD-1 fermentation. The production of ε-PL and PDAP got 29.4 and 9.6gL(-1), respectively, via fed-batch cultivation in a 5L bioreactor. What is more, the highest production yield (21.8%) of similar production systems was achieved by using this novel strategy. To consider the economic-feasibility, large-scale production in a 1t fermentor was also implemented, which would increase the gross profit of 54,243.5USD from one fed-batch bioprocess. This type of fermentation, which produces multiple commercial products from a unified process is attractive, because it will improve the utilization rate of raw materials, enhance production value and enrich product variety. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Grafting of poly-L-lysine dendrigrafts onto polypropylene surface using plasma activation for ATP immobilization - Nanomaterial for potential applications in biotechnology.

    PubMed

    Couturaud, Benoit; Molero Bondia, Andrea; Faye, Clément; Garrelly, Laurent; Mas, André; Robin, Jean Jacques

    2013-10-15

    The present work describes a new environmental friendly strategy for the development of surfaces with high amine density via the grafting of native or modified poly-L-lysine dendrigraft (DGL G3) onto plasma activated polypropylene (PP), polystyrene (PS), polyimide, and polytetrafluoroethylene (PTFE) surface. Modified DGL G3 was prepared by replacement of few peripheral amines by various functionalities. Grafting efficiency was determined by wettability measurements, IRTF, XPS, AFM, and by colorimetry using optimized Coomassie Brilliant Blue method tailored for surface analysis. It was shown that a 4-7nm DGL G3 monolayer with 4×10(14)aminecm(-)(2) was covalently grafted onto various surfaces. Immobilization of adenosine triphosphate on the DGL-g-PP material from dilute solution was studied by bioluminescence and proved the ability of the material to interact with polyanionic biological compounds: 1 ATP complex with 5 amine groups. So, this material has a potential use in diagnostic and more widely for biotechnology due to its high capacity for biomolecule immobilization. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. In vitro labeling of neural stem cells with poly-L-lysine coated super paramagnetic nanoparticles for green fluorescent protein transfection.

    PubMed

    Albukhaty, Salim; Naderi-Manesh, Hossein; Tiraihi, Taki

    2013-04-01

    The magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell (NSC) using poly-L-lysine hydrobromide (PLL)-coated super paramagnetic iron oxide nanoparticles (SPION). The SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 µg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 µg/ml). Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated (SPION) and coated (SPION-PLL) complexes were evaluated by a transmission electron microscope and the zeta potential. PLL at 0.75 μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency of the transfected cells showed green fluorescence. Transfection with SPION is an efficient, non-viral gene transfere method.

  20. A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine: airway epithelial function and mucus changes in dog.

    PubMed

    Tomkiewicz, R P; App, E M; De Sanctis, G T; Coffiner, M; Maes, P; Rubin, B K; King, M

    1995-12-01

    A newly synthesized mucolytic agent, N-acetylcysteine L-lysinate (Nacystelyn) was studied. Tracheal mucus velocity (TMV), transepithelial potential difference (PD), rheological properties, and ion content of collected airway secretions were evaluated in six healthy mongrel dogs after placebo, Nacystelyn (NAL) and acetylcysteine (NAC) metered dose inhaler (MDI) aerosols. Although TMV was increased and viscoelasticity decreased after both treatments, the treatment effect with NAL was significantly greater. Furthermore, NAL increased the negative PD and CI- content of secretions in the trachea, an effect not observed after NAC. Both compounds increased ciliary beat frequency (CBF) on the frog palate at a concentration range similar to that approximated in dog airways. The increased mucociliary clearance could be partially explained by favourable rheological changes combined with stimulation of CBF. Since both compounds break disulfide bonds in mucus polymers, the greater change in mucus rheology and clearance rate after NAL, without change in water content, could be explained by the increase in CI- content. Nacystelyn appears to combine different modes of action which synergistically cause an increase in the clearance rate of airway secretions.

  1. L-lysine pro-prodrug containing trans-ferulic acid for 5-amino salicylic acid colon delivery: synthesis, characterization and in vitro antioxidant activity evaluation.

    PubMed

    Cassano, Roberta; Trombino, Sonia; Cilea, Alessia; Ferrarelli, Teresa; Muzzalupo, Rita; Picci, Nevio

    2010-01-01

    In the present work, we report the synthesis of a new 5-amino salicylic acid (5-ASA) pro-prodrug, useful in Crohn disease treatment, and the evaluation of its antioxidant activity. Using as pharmacological carrier L-lysine amino acid and taking advantage of its intrinsic chemical reactivity, due to the presence of two amino groups, placed on the chiral center and in epsilon-position, we inserted trans-ferulic acid in epsilon-position, through amidation reaction, esterified with methanol the carboxylic group and, finally, submitted the free amino group to diazotation with 5-ASA, principal drug for inflammatory bowel diseases (IBD) care. All intermediates of synthesis and the final product (derivative A) were characterized with usual spectroscopic techniques, as FT-IR, GC/MS and (1)H-MNR. Finally, the derivative A antioxidant activity in inhibiting the lipid peroxidation, in rat-liver microsomal membranes, induced in vitro by two different sources of free radicals, 2,2'-azobis (2-amidinopropane) (AAPH) and tert-butyl hydroperoxide (tert-BOOH), was evaluated. Our pro-prodrug could be successfully applied in pharmaceutical field both as prodrug of 5-ASA than as carrier of trans-ferulic acid.

  2. Effect of Ethyl Ester L-Lysine Triisocyanate addition to produce reactive PLA/PCL bio-polyester blends for biomedical applications.

    PubMed

    Visco, Annamaria; Nocita, Davide; Giamporcaro, Alberto; Ronca, Sara; Forte, Giuseppe; Pistone, Alessandro; Espro, Claudia

    2017-04-01

    We report in this paper the effects of Ethyl Ester L-Lysine Triisocyanate (LTI) on the physical-mechanical properties of Poly(lactide)/Poly(ε-caprolactone) (PLA/PCL) polyesters blends. The PLA/PCL ratios considered were 20/80, 50/50 and 80/20 (wt/wt %) and LTI was added in amounts of 0.0-0.5-1.0 phr. PLA and PCL reacted with LTI during processing in a Brabender twin screw internal mixer to produce block copolymers in-situ. The resulting blends have been characterized by torque measurements, uniaxial tensile tests, Differential Scanning Calorimeter, contact angle measurements with a Phosphate Buffered Saline (PBS) solution, ATR analysis and morphological SEM observations. Experimental results highlighted how LTI enhanced interaction and dispersion of the two components, resulting into a synergic effect in mechanical properties. Mechanical and physical properties can be tailored by changing the blend composition. The most noticeable trend was an increase in ductility of the mixed polymers. Besides, LTI decreased blend's wet ability in PBS and lowered the starting of crystalline phase formation for both polymers, confirming an interaction among them. These reactive blends could find use as biomedical materials, e.g. absorbable suture threads or scaffolds for cellular growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Dietary L-Lysine Suppresses Autophagic Proteolysis and Stimulates Akt/mTOR Signaling in the Skeletal Muscle of Rats Fed a Low-Protein Diet.

    PubMed

    Sato, Tomonori; Ito, Yoshiaki; Nagasawa, Takashi

    2015-09-23

    Amino acids, especially L-leucine, regulate protein turnover in skeletal muscle and have attracted attention as a means of increasing muscle mass in people suffering from malnutrition, aging (sarcopenia), or a bedridden state. We previously showed that oral administration of L-lysine (Lys) by gavage suppressed proteolysis in skeletal muscles of fasted rats. However, the intake of Lys in the absence of other dietary components is unlikely in a non-experimental setting, and other dietary components may interfere with the suppressive effect of Lys on proteolysis. We supplemented Lys to a 10% casein diet and investigated the effect of Lys on proteolysis and autophagy, a major proteolytic system, in the skeletal muscle of rats. The rate of proteolysis was evaluated from 3-methylhisitidine (MeHis) released from isolated muscles, in plasma, and excreted in urine. Supplementing lysine with the 10% casein diet decreased the rate of proteolysis induced by intake of a low-protein diet. The upregulated autophagy activity [light chain 3 (LC3)-II/total LC3] caused by a low-protein diet was reduced, and the Akt/mTOR signaling pathway was activated by Lys. Importantly, continuous feeding of a Lys-rich 10% casein diet for 15 days increased the masses of the soleus and gastrocnemius muscles. Taken together, supplementation of Lys to a low-protein diet suppresses autophagic proteolysis through the Akt/mTOR signaling pathway, and continuous feeding of a Lys-rich diet may increase skeletal muscle mass.

  4. Aminocella lysinolytica gen. nov., sp. nov., a L-lysine-degrading, strictly anaerobic bacterium in the class Clostridia isolated from a methanogenic reactor of cattle farms.

    PubMed

    Ueki, Atsuko; Shibuya, Toru; Kaku, Nobuo; Ueki, Katsuji

    2015-01-01

    A strictly anaerobic bacterial strain (WN037(T)) was isolated from a methanogenic reactor. Cells were Gram-positive rods. Strain WN037(T) was asaccharolytic. The strain fermented L-lysine in the presence of B-vitamin mixture or vitamin B12 and produced acetate and butyrate. L-arginine and casamino acids poorly supported the growth. Strain WN037(T) used neither other amino acids nor organic acids examined. The strain had C18:1 ω7c, C16:0 and C18:1 ω7c DMA as the predominant cellular fatty acids. The genomic DNA G + C content was 44.2 mol %. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN037(T) in the family Eubacteriaceae in the class Clostridia. The closest relative was Eubacterium pyruvativorans (sequence similarity, 92.8 %). Based on the comprehensive analyses, the novel genus and species, Aminocella lysinolytica gen. nov., sp. nov. was proposed to accommodate the strain. The type strain is WN037(T) (= JCM 19863(T) = DSM 28287(T)).

  5. Effects of high-intensity ultrasound on Maillard reaction in a model system of d-xylose and l-lysine.

    PubMed

    Yu, Hang; Seow, Yi-Xin; Ong, Peter K C; Zhou, Weibiao

    2017-01-01

    This study compared the effects of high-intensity ultrasound on Maillard reaction (MR) with those of thermally produced MR using a model system of d-xylose and l-lysine. The ultrasonic MR process had higher depletion rates of reactants and higher generation rates of intermediate MR products (MRPs) and melanoidins under relatively low processing temperatures (55 and 60°C). However, the rates were lower for ultrasonic MR than thermal MR when the processing temperature increased to 65, 70 and 75°C. Overall, ultrasonic MR had relatively low activation energy (Ea) compared to thermal MR (e.g. 55.59 vs. 80.42kJmol(-1) for d-xylose depletion). Moreover, ultrasonic MR could produce at least one N-containing pyrazine (3-ethyl-2,5-dimethylpyrazine), one N-containing amine (butyl amine) and one O-containing volatile compound (maltol) that were absent from thermal MR. The difference in flavour generation might be a result of the extremely high, albeit momentary, temperature and pressure condition produced by high-intensity ultrasound.

  6. Star-Shaped Amphiphilic Hyperbranched Polyglycerol Conjugated with Dendritic Poly(l-lysine) for the Codelivery of Docetaxel and MMP-9 siRNA in Cancer Therapy.

    PubMed

    Zhou, Xiaoyan; Zheng, Qianqian; Wang, Changyong; Xu, Jiake; Wu, Jian-Ping; Kirk, Thomas Brett; Ma, Dong; Xue, Wei

    2016-05-25

    The drug/gene codelivery is a promising strategy for cancer treatment. Herein, to realize the codelivery of docetaxel and MMP-9 siRNA plasmid efficiently into tumor cells, a star-shaped amphiphilic copolymer consisting of hyperbranched polyglycerol derivative (HPG-C18) and dendritic poly(l-lysine) (PLLD) was synthesized by the click reaction between azido-modified HPG-C18 and propargyl focal point PLLD. The obtained HPG-C18-PLLD could form the nanocomplexes with docetaxel and MMP-9, and the complexes showed good gene delivery ability in vitro by inducing an obvious decrease in MMP-9 protein expression in MCF-7 cells. The apoptosis assay showed that the complex could induce a more significant apoptosis to breast cancer cells than that of docetaxel or MMP-9 used alone. In vivo assay indicated that the codelivery strategy displayed a better effect on tumor inhibition. Moreover, HPG-C18-PLLD displayed lower toxicity as well as better blood compatibility compared to polyethylenimine PEI-25k, which may be the result of that HPG-C18-PLLD showed the comparative MMP-9 delivery ability in vivo compared with PEI-25k even if it showed the slight lower transfection efficiency in vitro. Therefore, HPG-C18-PLLD is a safe and effective carrier for the codelivery of drug/gene, which should be encouraged in tumor therapy.

  7. Immobilization of heparin/poly-(L)-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior.

    PubMed

    Liu, Tao; Liu, Yang; Chen, Yuan; Liu, Shihui; Maitz, Manfred F; Wang, Xue; Zhang, Kun; Wang, Jian; Wang, Yuan; Chen, Junying; Huang, Nan

    2014-05-01

    Restenosis, thrombosis formation and delayed endothelium regeneration continue to be problematic for coronary artery stent therapy. To improve the hemocompatibility of the cardiovascular implants and selectively direct vascular cell behavior, a novel kind of heparin/poly-l-lysine (Hep/PLL) nanoparticle was developed and immobilized on a dopamine-coated surface. The stability and structural characteristics of the nanoparticles changed with the Hep:PLL concentration ratio. A Hep density gradient was created on a surface by immobilizing nanoparticles with various Hep:PLL ratios on a dopamine-coated surface. Antithrombin III binding quantity was significantly enhanced, and in plasma the APTT and TT times as coagulation tests were prolonged, depending on the Hep density. A low Hep density is sufficient to prevent platelet adhesion and activation. The sensitivity of vascular cells to the Hep density is very different: high Hep density inhibits the growth of all vascular cells, while low Hep density could selectively inhibit smooth muscle cell hyperplasia but promote endothelial progenitor cells and endothelial cell proliferation. These observations provide important guidance for modification of surface heparinization. We suggest that this method will provide a potential means to construct a suitable platform on a stent surface for selective direction of vascular cell behavior with low side effects.

  8. In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium.

    PubMed

    Galli, Daniela; Benedetti, Laura; Bongio, Matilde; Maliardi, Valentina; Silvani, Giulia; Ceccarelli, Gabriele; Ronzoni, Flavio; Conte, Silvio; Benazzo, Francesco; Graziano, Antonio; Papaccio, Gianpaolo; Sampaolesi, Maurilio; De Angelis, Maria Gabriella Cusella

    2011-05-01

    Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through β1-INTEGRIN, because the presence of β1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.

  9. Staphylococcus aureus Resistance to Human Defensins and Evasion of Neutrophil Killing via the Novel Virulence Factor Mprf Is Based on Modification of Membrane Lipids with l-Lysine

    PubMed Central

    Peschel, Andreas; Jack, Ralph W.; Otto, Michael; Collins, L. Vincent; Staubitz, Petra; Nicholson, Graeme; Kalbacher, Hubert; Nieuwenhuizen, Willem F.; Jung, Günther; Tarkowski, Andrej; van Kessel, Kok P.M.; van Strijp, Jos A.G.

    2001-01-01

    Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria. PMID:11342591

  10. RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion.

    PubMed

    VandeVondele, Stephanie; Vörös, Janos; Hubbell, Jeffrey A

    2003-06-30

    A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.

  11. Effects of poly(L-lysine), poly(acrylic acid) and poly(ethylene glycol) on the adhesion, proliferation and chondrogenic differentiation of human mesenchymal stem cells.

    PubMed

    Lu, Hongxu; Guo, Likun; Kawazoe, Naoki; Tateishi, Tetsuya; Chen, Guoping

    2009-01-01

    Microenvironments, composed of many kinds of cytokines and growth factors plus extracellular matrices with diverse electrostatic properties, play key roles in controlling cell functions in vivo. In this study, three kinds of water-soluble polymers, positively charged poly(L-lysine) (PLL), negatively charged poly(acrylic acid) (PAAc) and neutral poly(ethylene glycol) (PEG), were compared based on their effects on the adhesion, spread, proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). The MSCs were seeded and cultured in the presence of polymers of different concentrations applied by methods using coating, mixing or covering. The effects of the water-soluble polymers depended on their electrostatic properties and method of application. The methods were in the order of coating, mixing and covering in terms of high to low influence. A low concentration of PLL promoted MSC adhesion, spread, proliferation and chondrogenic differentiation, while a high concentration of PLL was toxic. The PEG-coated surface facilitated cell aggregation and spheroid formation by inhibiting cell adhesion. A high concentration of mixed PEG (10 microg/ml) promoted cell proliferation in serum-free medium. PAAc showed no obvious effects on MSC adhesion, spread, proliferation, or chondrogenic differentiation.

  12. Quaternary complexes modified from pDNA and poly-l-lysine complexes to enhance pH-buffering effect and suppress cytotoxicity.

    PubMed

    Kodama, Yukinobu; Yatsugi, Yuiko; Kitahara, Takashi; Kurosaki, Tomoaki; Egashira, Kanoko; Nakashima, Mikiro; Muro, Takahiro; Nakagawa, Hiroo; Higuchi, Norihide; Nakamura, Tadahiro; Sasaki, Hitoshi

    2015-04-01

    We developed a modified complex of pDNA and poly-l-lysine (PLL) by the addition of poly-l-histidine (PLH) and γ-polyglutamic acid (γ-PGA) to enhance its pH-buffering effect and suppress cytotoxicity. The binary and ternary complexes of pDNA with PLL or/and PLH showed particle sizes of approximately 52-76 nm with cationic surface charge. The ternary complexes showed much higher gene expression than the binary complexes with PLL. The mixed solution of PLL and PLH showed higher buffering capacity than PLL solution. The high gene expression of ternary complexes was reduced by bafilomycin A1 . These results indicated the addition of PLH to PLL complexes promoted endosomal escape by enhancing the pH-buffering effect. The binary and ternary complexes showed cytotoxicity and blood agglutination because of their cationic surface charge. We therefore developed quaternary complexes by the addition of anionic γ-PGA, which was reported to decrease the toxicity of cationic complexes. In fact, quaternary complexes showed no cytotoxicity and blood agglutination. Also, quaternary complexes showed higher gene expression than ternary complexes regardless of their anionic surface charge. Quaternary complexes showed selectively high gene expression in the spleen after their intravenous administration. Thus, we successfully developed the quaternary complexes with high gene expression and no toxicity.

  13. Injectable supramolecular hydrogel formed from α-cyclodextrin and PEGylated arginine-functionalized poly(l-lysine) dendron for sustained MMP-9 shRNA plasmid delivery.

    PubMed

    Lin, Qianming; Yang, Yumeng; Hu, Qian; Guo, Zhong; Liu, Tao; Xu, Jiake; Wu, Jianping; Kirk, Thomas Brett; Ma, Dong; Xue, Wei

    2017-02-01

    Hydrogels have attracted much attention in cancer therapy and tissue engineering due to their sustained gene delivery ability. To obtain an injectable and high-efficiency gene delivery hydrogel, methoxypolyethylene glycol (MPEG) was used to conjugate with the arginine-functionalized poly(l-lysine) dendron (PLLD-Arg) by click reaction, and then the synthesized MPEG-PLLD-Arg interacted with α-cyclodextrin (α-CD) to form the supramolecular hydrogel by the host-guest interaction. The gelation dynamics, hydrogel strength and shear viscosity could be modulated by α-CD content in the hydrogel. MPEG-PLLD-Arg was confirmed to bind and deliver gene effectively, and its gene transfection efficiency was significantly higher than PEI-25k under its optimized condition. After gelation, MMP-9 shRNA plasmid (pMMP-9) could be encapsulated into the hydrogel matrix in situ and be released from the hydrogels sustainedly, as the release rate was dependent on α-CD content. The released MPEG-PLLD-Arg/pMMP-9 complex still showed better transfection efficiency than PEI-25k and induced sustained tumor cell apoptosis. Also, in vivo assays indicated that this pMMP-9-loaded supramolecular hydrogel could result in the sustained tumor growth inhibition meanwhile showed good biocompatibility. As an injectable, sustained and high-efficiency gene delivery system, this supramolecular hydrogel is a promising candidate for long-term gene therapy.

  14. A pH-responsive drug nanovehicle constructed by reversible attachment of cholesterol to PEGylated poly(l-lysine) via catechol-boronic acid ester formation.

    PubMed

    Yang, Bin; Lv, Yin; Zhu, Jing-Yi; Han, Yun-Tao; Jia, Hui-Zhen; Chen, Wei-Hai; Feng, Jun; Zhang, Xian-Zheng; Zhuo, Ren-Xi

    2014-08-01

    The present work reports the construction of a drug delivery nanovehicle via a pH-sensitive assembly strategy for improved cellular internalization and intracellular drug liberation. Through spontaneous formation of boronate linkage in physiological conditions, phenylboronic acid-modified cholesterol was able to attach onto catechol-pending methoxypoly(ethylene glycol)-block-poly(l-lysine). This comb-type polymer can self-organize into a micellar nanoconstruction that is able to effectively encapsulate poorly water-soluble agents. The blank micelles exhibited negligible in vitro cytotoxicity, yet doxorubicin (DOX)-loaded micelles could effectively induce cell death at a level comparable to free DOX. Owing to the acid-labile feature of the boronate linkage, a reduction in environmental pH from pH 7.4 to 5.0 could trigger the dissociation of the nanoconstruction, which in turn could accelerate the liberation of entrapped drugs. Importantly, the blockage of endosomal acidification in HeLa cells by NH4Cl treatment significantly decreased the nuclear uptake efficiency and cell-killing effect mediated by the DOX-loaded nanoassembly, suggesting that acid-triggered destruction of the nanoconstruction is of significant importance in enhanced drug efficacy. Moreover, confocal fluorescence microscopy and flow cytometry assay revealed the effective internalization of the nanoassemblies, and their cellular uptake exhibited a cholesterol dose-dependent profile, indicating the contribution of introduced cholesterol functionality to the transmembrane process of the nanoassembly.

  15. Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes. Identity of receptor for IL 1-. cap alpha. and IL 1-. beta

    SciTech Connect

    Matsushima, K.; Akahoshi, T.; Yamada, M.; Furutani, Y.; Oppenheim, J.J.

    1986-01-01

    The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-..beta.. from a myelomonocytic cell line (THP-1) was labeled with /sup 125/I. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of /sup 125/I-IL 1-..beta... The binding of /sup 125/I-IL 1-..beta.. to VDS-O cells was inhibited by F(ab)'/sub 2/ fragments of anti-human IL 1 and recombinant human IL 1-..cap alpha.., as well as by unlabeled human IL 1-..beta.. but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-..cap alpha.. and IL 1-..beta.. bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.

  16. Characterization of the temporary anion states of metal carbonyl complexes: an MS-X. cap alpha. and ETS study of Cr(CO)/sub 6/ and Fe(CO)/sub 5/

    SciTech Connect

    Guerra, M.; Jones, D.; Distefano, G.; Foffani, A.; Modelli, A.

    1988-01-20

    The electron affinities in the 0-5 eV energy range have been determined for chromium hexacarbonyl and iron pentacarbonyl by means of multiple scattering X..cap alpha.. calculations. The total electron scattering cross sections have been estimated with the continuum MS-X..cap alpha.. method. The computational results are compared with the electron transmission spectra and used for the assignment of the experimental features. The ET spectrum of Fe(CO)/sub 5/ is presented and assigned for the first time and that of Cr(CO)/sub 6/ is reassigned. For both complexes, all the anion states associated with electron capture into the orbitals deriving from the ligand 2..pi..* MOs and into the metal empty d orbitals are assigned to resonances occurring in the 0.4-2.6 eV energy region of the ET spectra. A charge density analysis reveals that a net electronic charge transfer occurs from the ligands to the metal, owing to the acceptor capability of the metal 4s and 4p orbitals. This is consistent with the experimentally observed average stabilization of the anion states localized at the CO ligands with respect to the II anion state of free CO.

  17. Residual Cap

    NASA Image and Video Library

    2006-05-10

    This MOC image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions

  18. Cradle Cap

    MedlinePlus

    ... and hair follicles. Another factor may be a yeast (fungus) called malassezia (mal-uh-SEE-zhuh) that ... ketoconazole, are often effective, supporting the idea that yeast is a contributing factor. Cradle cap isn't ...

  19. Cervical Cap

    MedlinePlus

    ... Staff The cervical cap is a birth control (contraceptive) device that prevents sperm from entering the uterus. ... more times a week, you've had previous contraceptive failure with vaginal barrier methods or you're ...

  20. Cusp Cap

    NASA Astrophysics Data System (ADS)

    Murdin, P.

    2000-11-01

    A brightening at one or other of the tips—cusps—of the crescent phase of Venus, as seen from Earth. Cusp caps were first reported by the German amateur astronomer Baron Franz Paula von Gruithuisen in 1813, and have been recorded by telescopic observers ever since. They were named by analogy with the Earth's polar caps; early observers fancied they were seeing glimpses of a possibly Earth-like sur...

  1. An Investigation of the Influence of Chain Length on the Interfacial Ordering of L-Lysine and L-Proline and Their Homopeptides at Hydrophobic and Hydrophilic Interfaces Studied by Sum Frequency Generation and Quartz Crystal Microbalance

    SciTech Connect

    York, R.L.; Holinga, G.J.; Somorjai, G.A.

    2009-02-23

    Sum frequency generation vibrational spectroscopy (SFG) and quartz crystal microbalance with dissipation monitoring (QCM-D) are employed to study the interfacial structure and adsorbed amount of the amino acids l-lysine and l-proline and their corresponding homopeptides, poly-l-lysine and poly-l-proline, at two liquid-solid interfaces. SFG and QCM-D experiments of these molecules are carried out at the interface between phosphate buffered saline at pH 7.4 (PBS) and the hydrophobic deuterated polystyrene (d{sub 8}-PS) surface as well as the interface between PBS and hydrophilic fused silica (SiO{sub 2}). The SFG spectra of the amino acids studied here are qualitatively similar to their corresponding homopeptides; however, the SFG signal from amino acids at the solid/PBS interface is smaller in magnitude relative to their more massive homopeptides at the concentrations studied here. Substantial differences are observed in SFG spectra for each species between the hydrophobic d{sub 8}-PS and the hydrophilic SiO{sub 2} liquid-solid interfaces, suggesting surface-dependent interfacial ordering of the biomolecules. Over the range of concentrations used in this study, QCM-D measurements also indicate that on both surfaces poly-l-lysine adsorbs to a greater extent than its constituent amino acid l-lysine. The opposite trend is demonstrated by poly-l-proline which sticks to both surfaces less extensively than its corresponding amino acid, l-proline. Lastly, we find that the adsorption of the molecules studied here can have a strong influence on interfacial water structure as detected in the SFG spectra.

  2. Copolymer of poly(ethylene glycol) and poly(l-lysine) grafting polyethylenimine through a reducible disulfide linkage for siRNA delivery

    NASA Astrophysics Data System (ADS)

    Li, Jingguo; Cheng, Du; Yin, Tinghui; Chen, Weicai; Lin, Yujie; Chen, Jifeng; Li, Ruitang; Shuai, Xintao

    2014-01-01

    siRNA therapy research has primarily focused on the synthesis and development of effective siRNA delivery vectors with easy biodegradability and low toxicity. In the present study, we synthesized a ternary copolymer mPEG-b-PLL-g-(ss-lPEI), denoted as PLI, by introducing disulfide bond linkages to graft low molecular weight linear polyethylenimine (lPEI) to the block copolymer of poly(l-lysine) (PLL) and poly(ethylene glycol) (PEG) for siRNA delivery. The PLL block and disulfide linkage rendered the carrier biodegradability, while lPEI grafting brought about the proton buffering capacity for lysosomal siRNA release and low cationic toxicity. Conjugation of a single chain monoclonal antibody (Herceptin) to the carrier as a targeting ligand for the Her2/neu receptor significantly increased the transfection activity of the copolymer/siRNA nanocomplex (i.e. the polyplex) in Skov-3, a human ovarian cancer cell line. Determination of gene expression at both the mRNA and protein levels demonstrated that Her2-targeted delivery of siRNA (XIAP siRNA) effectively downregulated the targeted XIAP (X-linked inhibitor of apoptosis protein) gene, resulting in enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. The distinct features of low cytotoxicity, easy degradability, and high siRNA transfection efficiency make the copolymer a promising candidate for siRNA therapy in tumors.siRNA therapy research has primarily focused on the synthesis and development of effective siRNA delivery vectors with easy biodegradability and low toxicity. In the present study, we synthesized a ternary copolymer mPEG-b-PLL-g-(ss-lPEI), denoted as PLI, by introducing disulfide bond linkages to graft low molecular weight linear polyethylenimine (lPEI) to the block copolymer of poly(l-lysine) (PLL) and poly(ethylene glycol) (PEG) for siRNA delivery. The PLL block and disulfide linkage rendered the carrier biodegradability, while lPEI grafting brought about the proton

  3. Gold Nanoparticles Stabilized with MPEG-Grafted Poly(l-lysine): in Vitro and in Vivo Evaluation of a Potential Theranostic Agent

    PubMed Central

    2015-01-01

    As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with 99mTc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers “stealth” properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy. PMID:25496453

  4. Role of Polymeric Endosomolytic Agents in Gene Transfection: A Comparative Study of Poly(l-lysine) Grafted with Monomeric l-Histidine Analogue and Poly(l-histidine)

    PubMed Central

    2015-01-01

    Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(l-lysine) (PLL), a stacking form of poly(l-histidine) (PLL-g-PHis) and a mono- l-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups from PHis also provided higher gene expression than mHis due to its ability to escape the endosome. mHis or PHis grafting reduced the cytotoxicity of PLL and changed the rate of cellular uptake by changing the quantity of free ε-amines available for gene condensation. The subcellular localization of PLL-g-PHis/pDNA measured by YOYO1-pDNA intensity was highest inside the nucleus, while the lysotracker, which stains the acidic compartments was lowest among these polymers. Thus, the polymeric histidine arrangement demonstrate the ability to escape the endosome and trigger rapid release of polyplexes into the cytosol, resulting in a greater amount of pDNA available for translocation to the nucleus and enhanced gene expression. PMID:25144273

  5. In vivo distribution of avidin-conjugated MX35 and (211)At-labeled, biotinylated poly-L-lysine for pretargeted intraperitoneal α-radioimmunotherapy.

    PubMed

    Frost, Sofia Helena Linnea; Bäck, Tom; Chouin, Nicolas; Jensen, Holger; Hultborn, Ragnar; Jacobsson, Lars; Lindegren, Sture

    2011-12-01

    Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity. (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc). We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose. To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.

  6. Gold nanoparticles stabilized with MPEG-grafted poly(l-lysine): in vitro and in vivo evaluation of a potential theranostic agent.

    PubMed

    Bogdanov, Alexei A; Gupta, Suresh; Koshkina, Nadezhda; Corr, Stuart J; Zhang, Surong; Curley, Steven A; Han, Gang

    2015-01-21

    As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with (99m)Tc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers "stealth" properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy.

  7. Role of polymeric endosomolytic agents in gene transfection: a comparative study of poly(L-lysine) grafted with monomeric L-histidine analogue and poly(L-histidine).

    PubMed

    Hwang, Hee Sook; Hu, Jun; Na, Kun; Bae, You Han

    2014-10-13

    Endosomal entrapment is one of the main barriers that must be overcome for efficient gene expression along with cell internalization, DNA release, and nuclear import. Introducing pH-sensitive ionizable groups into the polycationic polymers to increase gene transfer efficiency has proven to be a useful method; however, a comparative study of introducing equal numbers of ionizable groups in both polymer and monomer forms, has not been reported. In this study, we prepared two types of histidine-grafted poly(L-lysine) (PLL), a stacking form of poly(L-histidine) (PLL-g-PHis) and a mono-L-histidine (PLL-g-mHis) with the same number of imidazole groups. These two types of histidine-grafted PLL, PLL-g-PHis and PLL-g-mHis, showed profound differences in hemolytic activity, cellular uptake, internalization, and transfection efficiency. Cy3-labeled PLL-g-PHis showed strong fluorescence in the nucleus after internalization, and high hemolytic activity upon pH changes was also observed from PLL-g-PHis. The arrangement of imidazole groups from PHis also provided higher gene expression than mHis due to its ability to escape the endosome. mHis or PHis grafting reduced the cytotoxicity of PLL and changed the rate of cellular uptake by changing the quantity of free ε-amines available for gene condensation. The subcellular localization of PLL-g-PHis/pDNA measured by YOYO1-pDNA intensity was highest inside the nucleus, while the lysotracker, which stains the acidic compartments was lowest among these polymers. Thus, the polymeric histidine arrangement demonstrate the ability to escape the endosome and trigger rapid release of polyplexes into the cytosol, resulting in a greater amount of pDNA available for translocation to the nucleus and enhanced gene expression.

  8. Surface modification with dopamine and heparin/poly-L-lysine nanoparticles provides a favorable release behavior for the healing of vascular stent lesions.

    PubMed

    Liu, Tao; Zeng, Zheng; Liu, Yang; Wang, Jian; Maitz, Manfred F; Wang, Yuan; Liu, Shihui; Chen, Junying; Huang, Nan

    2014-06-11

    Surface biofunctional modification of coronary artery stents to prevent thrombosis and restenosis formation, as well as accelerate endothelialization, has become a new hot spot. However, bioactive coatings on implants are not yet sufficiently developed for long-term activity, as they quickly lose efficiency in vivo and finally fail. On the basis of a novel time-ordered concept of biofunctionality for vascular stents, heparin/poly l-lysine nanoparticle (NP) was developed and immobilized on a polydopamine-coated titanium surface, with the aim of regulating and maintaining the intravascular biological response within the normal range after biomaterial implantation. An in vitro dynamic release model was established to mimic the blood flow condition in vivo with three phases: (1) An early phase (1-7 days) with release of predominantly anticoagulant and anti-inflammatory substances and to a minor degree antiproliferative effects against smooth muscle cells (SMCs); (2) this is followed by a phase (7-14 days) of supported endothelial cell (ECs) proliferation and suppressed SMC proliferation with persisting high antithrombogenicity and anti-inflammatory properties of the surface. (3) Finally, a stable stage (14-28 days) with adequate biomolecules on the surface that maintain hemocompatibility and anti inflammation as well as inhibit SMCs proliferation and promote ECs growth. In vivo animal tests further confirmed that the NP-modified surface provides a favorable release behavior to apply a stage-adjusted remedy. We suggested that these observations provide important guidance and potential means for reasonable and suitable platform construction on a stent surface.

  9. Autologous aldrithiol-2-inactivated HIV-1 combined with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose as a vaccine platform for therapeutic dendritic cell immunotherapy.

    PubMed

    Miller, Elizabeth; Spadaccia, Meredith; Sabado, Rachel; Chertova, Elena; Bess, Julian; Trubey, Charles Mac; Holman, Rose Marie; Salazar, Andres; Lifson, Jeffrey; Bhardwaj, Nina

    2015-01-03

    Therapeutic interventions for HIV-1 that successfully augment adaptive immunity to promote killing of infected cells may be a requisite component of strategies to reduce latent cellular reservoirs. Adoptive immunotherapies utilizing autologous monocyte-derived dendritic cells (DCs) that have been activated and antigen loaded ex vivo may serve to circumvent defects in DC function that are present during HIV infection in order to enhance adaptive immune responses. Here we detail the clinical preparation of DCs loaded with autologous aldrithiol-2 (AT-2)-inactivated HIV that have been potently activated with the viral mimic, Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC). HIV is first propagated from CD4+ T cells from HIV-infected donors and then rendered non-replicative by chemical inactivation with aldrithiol-2 (AT-2), purified, and quantified. Viral inactivation is confirmed through measurement of Tat-regulated β-galactosidase reporter gene expression following infection of TZM-bl cells. In-process testing for sterility, mycoplasma, LPS, adventitious agents, and removal of AT-2 is performed on viral preparations. Autologous DCs are generated and pulsed with autologous AT-2-inactivated virus and simultaneously stimulated with Poly-ICLC to constitute the final DC vaccine product. Phenotypic identity, maturation, and induction of HIV-specific adaptive immune responses are confirmed via flow cytometric analysis of DCs and cocultured autologous CD4+ and CD8+ T cells. Lot release criteria for the DC vaccine have been defined in accordance with Good Manufacturing Practice (GMP) guidelines. The demonstrated feasibility of this approach has resulted in approval by the FDA for investigational use in antiretroviral (ART) suppressed individuals. We discuss how this optimized DC formulation may enhance the quality of anti-HIV adaptive responses beyond what has been previously observed during DC immunotherapy trials for HIV infection. Copyright

  10. Ligand-specific targeting of microspheres to phagocytes by surface modification with poly(L-lysine)-grafted poly(ethylene glycol) conjugate.

    PubMed

    Faraasen, Sofia; Vörös, János; Csúcs, Gábor; Textor, Marcus; Merkle, Hans P; Walter, Elke

    2003-02-01

    The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.

  11. The Natural Antimicrobial Subtilosin A Synergizes with Lauramide Arginine Ethyl Ester (LAE), ε-Poly-L-lysine (Polylysine), Clindamycin Phosphate and Metronidazole, Against the Vaginal Pathogen Gardnerella vaginalis.

    PubMed

    Cavera, Veronica L; Volski, Anna; Chikindas, Michael L

    2015-06-01

    Bacterial vaginosis (BV) is a common, recurrent vaginal infection linked to increased chances of preterm delivery, incidence of sexually transmitted infections and fertility problems. BV is caused by a shift of the vaginal ecosystem from predominately Lactobacillus to a multispecies Actinomyces biofilm with the most common representatives identified as Gardnerella vaginalis and Prevotella spp. Current treatments have been associated with increased resistance as well as negative effects on healthy microbiota. The objective of this study was to evaluate the synergistic potential of ten two-antimicrobial combinations against G. vaginalis and four representative lactobacilli. The four tested antimicrobials were lauramide arginine ethyl ester, ε-poly-L-lysine, clindamycin phosphate, metronidazole and the bacteriocin subtilosin A. The use of bacteriocins as either synergist or alternative treatment positions bacteriocins as an excellent alternative to current antibiotics. The microdilution method was used to determine the minimum inhibitory concentration (MIC) of each of the antimicrobials individually, and the checkerboard assay was used to evaluate these MICs in combination. Clindamycin and subtilosin (CS), and metronidazole and subtilosin were synergistic against G. vaginalis in terms of fractional inhibitory concentration index (FICI). All tested combinations were found to have Bliss synergy. The combination of clindamycin and polylysine (CP) was identified as antagonistic against L. acidophilus in terms of both FICI and Bliss synergy. The combination of clindamycin and metronidazole (CM) was antagonistic against L. vaginalis for both FICI and Bliss synergy. The combinations of CP, clindamycin and LAE, CS, and LAE and polylysine were identified as Bliss antagonistic against L. vaginalis but did not indicate FICI antagonism.

  12. Cradle Cap (For Parents)

    MedlinePlus

    ... Kids to Be Smart About Social Media Cradle Cap (Infantile Seborrheic Dermatitis) KidsHealth > For Parents > Cradle Cap ( ... many babies develop called cradle cap. About Cradle Cap Cradle cap is the common term for seborrheic ...

  13. Interfacial electron-transfer equilibria and flat-band potentials of. cap alpha. -Fe/sub 2/O/sub 3/ and TiO/sub 2/ colloids studied by pulse radiolysis

    SciTech Connect

    Dimitrijevic, N.M.; Savic, D.; Micic, O.I.; Nozik, A.J.

    1984-09-13

    The kinetics and equilibria of electron transfer between methylviologen cation radicals and ..cap alpha..-Fe/sub 2/O/sub 3/ or TiO/sub 2/ colloidal particles were studied with the pulse-radiolysis technique. The rates of electron transfer to both colloids are lower than those predicted for a diffusion-controlled reaction. For higher pHs (TiO/sub 2/, pH > 2; ..cap alpha..-Fe/sub 2/O/sub 3/, pH > 9) the established equilibrium MV/sup +/ in equilibrium MV/sup 2 +/ + (e/sup -/)/sub coll/ is strongly influenced by the MV/sup 2 +/ concentration and pH. The MV/sup +/ equilibrium concentration can be exploited to derive the flat-band potential of the semiconductor colloids. The method for determining the flat-band potential of the particles is independent of whether the injected electrons are free or trapped, and whether the electrons raise the bulk Fermi level toward the conduction band or just produce a space charge. The flat-band potentials for both colloids appear to be somewhat more negative (-0.1 to -0.2 V) than the corresponding single-crystal electrodes. Also, the flat-band potentials become slightly more negative with increasing radiation dose (initial MV/sup +/ concentration). The effect of absorbed radiation dose is explained by the corresponding changes in the ratio of oxidized to reduced forms of the redox couple, which in turn changes the adsorbed ionic charge on the semiconductor surface. For colloidal particles of TiO/sub 2/ stabilized by poly(vinyl alcohol) (PVA), the flat-band potentials were almost the same as those for PVA-free TiO/sub 2/ sols. The decrease of particle diameter from 800 to 70 A does not affect the value of the flat-band potentials for TiO/sub 2/ and ..cap alpha..-Fe/sub 2/O/sub 3/ colloids. 28 references, 9 figures.

  14. Synthesis of tritium labeled Ac-(Nle/sup 4/, D-Phe/sup 7/)-. cap alpha. -MSH/sub 4-11/-NH/sub 2/: a superpotent melanotropin with prolonged biological activity

    SciTech Connect

    Wilkes, B.D.; Hruby, V.J.; Yamamura, H.I.; Akiyama, K.; Castrucci, A.M. de; Hadley, M.E.; Andrews, J.R.; Wan, Y.P.

    1984-03-05

    Ac-(Nle/sup 4/, D-Phe/sup 7/)-..cap alpha..-MSH/sub 4-11/-NH/sub 2/ an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH/sub 2/), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of (/sup 3/H)-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors.

  15. Cytochrome P450c17 (steroid 17. cap alpha. -hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues

    SciTech Connect

    Chung, B.; Picado-Leonard, J.; Haniu, M.; Bienkowski, M.; Hall, P.F.; Shively, J.E.; Miller, W.L.

    1987-01-01

    P450c17 is the single enzyme mediating both 17..cap alpha..-hydroxylase (steroid 17..cap alpha..-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. The authors sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac 17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17.

  16. Cradle cap

    MedlinePlus

    ... known. Doctors think the condition is due to oil glands in the baby's scalp producing too much oil. Cradle cap is not spread from person to ... each day to remove any scales and scalp oil. If scales do not easily loosen and wash ...

  17. EPR, ENDOR, and DFT study of free radicals in L-lysine·HCl·2H2O single crystals X-irradiated at 298 K.

    PubMed

    Zhou, Yiying; Nelson, William H

    2011-10-27

    With K-band EPR (Electron Paramagnetic Resonance), ENDOR (Electron-Nuclear DOuble Resonance), and EIE (ENDOR-induced EPR) techniques, three free radicals (RI-RIII) in L-lysine hydrochloride dihydrate single crystals X-irradiated at 298 K were detected at 298 K, and six radicals (R1, R1', R2-R5) were detected if the temperature was lowered to 66 K from 298 K. R1 and RI dominated the central portion of the EPR at 66 and 298 K, respectively, and were identified as main chain deamination radicals, (-)OOCĊH(CH(2))(4)(NH(3))(+). R1' was identified as a main chain deamination radical with the different configuration from R1 at 66 K, and it probably formed during cooling the temperature from 298 to 66 K. The configurations of R1, R1', and RI were analyzed with their coupling tensors. R2 and R3 each contain one α- and four β-proton couplings and have very similar EIEs at three crystallographic axes. The two-layer ONIOM calculations (at B3LYP/6-31G(d,p):PM3) support that R2 and R3 are from different radicals: dehydrogenation at C4, (-)OOCCH(NH(3))(+)CH(2)ĊH(CH(2))(2)(NH(3))(+), and dehydrogenation at C5, (-)OOCCH(NH(3))(+)(CH(2))(2)ĊHCH(2)(NH(3))(+), respectively. The comparisons of the coupling tensors indicated that R2 (66 K) is the same radical as RII (298 K), and R3 is the same as RIII. Thus, RII and RIII also are the radicals of C4 and C5 dehydrogenation. R4 and R5 are minority radicals and were observed only when temperature was lowered to 66 K. R4 and R5 were only tentatively assigned as the side chain deamination radical, (-)OOCCH (NH(3))(+)(CH(2))(3)ĊH(2), and the radical dehydrogenation at C3, (-)OOCCH(NH(3))(+)ĊH(CH(2))(3)(NH(3))(+), respectively, although the evidence was indirect. From simulation of the EPR (B//a, 66 K), the concentrations of R1, R1', and R2-R5 were estimated as: R1, 50%; R1', 11%; R2, 14%; R3, 16%; R4, 6%; R5, 3%.

  18. Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

    PubMed Central

    Buchholz, Jens; Schwentner, Andreas; Brunnenkan, Britta; Gabris, Christina; Grimm, Simon; Gerstmeir, Robert; Takors, Ralf; Eikmanns, Bernhard J.

    2013-01-01

    Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a YP/S of 0.24 mol per mol of glucose and a QP of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products. PMID:23835179

  19. Apical cap

    SciTech Connect

    McLoud, T.C.; Isler, R.J.; Novelline, R.A.; Putman, C.E.; Simeone, J.; Stark, P.

    1981-08-01

    Apical caps, either unilateral or bilateral, are a common feature of advancing age and are usually the result of subpleural scarring unassociated with other diseases. Pancoast (superior sulcus) tumors are a well recognized cause of unilateral asymmetric apical density. Other lesions arising in the lung, pleura, or extrapleural space may produce unilateral or bilateral apical caps. These include: (1) inflammatory: tuberculosis and extrapleural abscesses extending from the neck; (2) post radiation fibrosis after mantle therapy for Hodgkin disease or supraclavicular radiation in the treatment of breast carcinoma; (3) neoplasm: lymphoma extending from the neck or mediastinum, superior sulcus bronchogenic carcinoma, and metastases; (4) traumatic: extrapleural dissection of blood from a ruptured aorta, fractures of the ribs or spine, or hemorrhage due to subclavian line placement; (5) vascular: coarctation of the aorta with dilated collaterals over the apex, fistula between the subclavian artery and vein; and (6) miscellaneous: mediastinal lipomatosis with subcostal fat extending over the apices.

  20. Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans

    PubMed Central

    Geng, Weitao; Yang, Chao; Gu, Yanyan; Liu, Ruihua; Guo, Wenbin; Wang, Xiaomeng; Song, Cunjiang; Wang, Shufang

    2014-01-01

    ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24–30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis. PMID:24423427

  1. Influence of the foundation layer on the layer-by-layer assembly of poly-L-lysine and poly(styrenesulfonate) and its usage in the fabrication of 3D microscale features.

    PubMed

    Zhou, Dejian; Bruckbauer, Andreas; Batchelor, Matthew; Kang, Dae-Joon; Abell, Chris; Klenerman, David

    2004-10-12

    The layer-by-layer (LBL) assembly of a polypeptide, poly-L-lysine (PLL), with poly(styrenesulfonate) sodium salt (PSS) on flat template-stripped gold (TSG) surfaces precoated with a self-assembled monolayer of alkanethiols terminated with positive (pyridinium), negative (carboxylic acid), and neutral [hexa(ethylene glycol)] groups is investigated. Both the topography and the rate of film thickness growth are found to be strongly dependent on the initial surface foundation layer. LBL assembly of PLL and PSS on patterned TSG surfaces produced by micro contact printing leads to structurally distinct microscale features, including pillars, ridges, and wells, whose height can be controlled with nanometer precision.

  2. Effect of l-lysine-assisted surface grafting for nano-hydroxyapatite on mechanical properties and in vitro bioactivity of poly(lactic acid-co-glycolic acid).

    PubMed

    Liuyun, Jiang; Lixin, Jiang; Chengdong, Xiong; Lijuan, Xu; Ye, Li

    2016-01-01

    It is promising and challenging to study surface modification for nano-hydroxyapatite to improve the dispersion and enhance the mechanical properties and bioactivity of poly(lactic acid-co-glycolic acid). In this paper, we designed an effective new surface grafting with the assist of l-lysine for nano-hydroxyapatite, and the nano-hydroxyapatite surface grafted with the assist of l-lysine (g-nano-hydroxyapatite) was incorporated into poly(lactic acid-co-glycolic acid) to develop a series of g-nano-hydroxyapatite/poly(lactic acid-co-glycolic acid) nano-composites. The surface modification reaction for nano-hydroxyapatite, the mechanical properties, and in vitro human osteoblast-like cell (MG-63) response were characterized and investigated by Fourier transformation infrared, thermal gravimetric analysis, dispersion test, electromechanical universal tester, differential scanning calorimeter measurements, and in vitro cells culture experiment. The results showed that the grafting amount on the surface of nano-hydroxyapatite was enhanced with the increase of l-lysine, and the dispersion of nano-hydroxyapatite was improved more, so that it brought about better promotion crystallization and more excellent mechanical enhancement effect for poly(lactic acid-co-glycolic acid), comparing with the unmodified nano-hydroxyapatite. Moreover, the cells' attachment and proliferation results confirmed that the incorporation of the g-nano-hydroxyapatite into poly(lactic acid-co-glycolic acid) exhibited better biocompatibility than poly(lactic acid-co-glycolic acid). The above results indicated that the new surface grafting with the assist of l-lysine for nano-hydroxyapatite was an ideal novel surface modification method, which brought about better mechanical enhancement effect and in vitro bioactivity for poly(lactic acid-co-glycolic acid) with adding higher g-nano-hydroxyapatite content, suggesting it had a great potential to be used as bone fracture internal fixation materials

  3. Sulfur Ylides. Communication 1. Cyclopropanation of. cap alpha. ,. beta. -unsaturated Ketones with Ethyl (dimethylsulfuranylidene) acetate generated in the presence of phase-transfer catalysts

    SciTech Connect

    Tolstikov, G.A.; Galin, F.Z.; Iskandarova V.N.; Khalilov, L.M.; Panasenko, A.A.

    1986-04-01

    This paper presents a modified method for the cyclopropanation of alpha, beta-unsaturated ketones with ethyl (dimethylsulfuranylidene) acetate, generated in situ from a sulfonium salt with 85% KOH in the presence of a phase-transfer catalyst, and studies the sterochemistry of the polysubstituted cyclopropanes. The chemical shifts of the carbon atoms of the cyclopropane rings of the isomer pairs are close together in the C 13 NMR spectra, which makes the assignment of the signals of C/sup 2/ and C/sup 3/ and the sterochemical assignment of each isomer to the cis and the trans series difficult. It is shown that the signals of the carboxyl carbon atoms differ not more than 0.65 ppm in the isomer pairs.

  4. Distinct Paths for Basic Amino Acid Export in Escherichia coli: YbjE (LysO) Mediates Export of L-Lysine.

    PubMed

    Pathania, Amit; Sardesai, Abhijit A

    2015-06-15

    In Escherichia coli, argO encodes an exporter for L-arginine (Arg) and its toxic analogue canavanine (CAN), and its transcriptional activation and repression, by Arg and L-lysine (Lys), respectively, are mediated by the regulator ArgP. Accordingly argO and argP mutants are CAN supersensitive (CAN(ss)). We report the identification of ybjE as a gene encoding a predicted inner membrane protein that mediates export of Lys, and our results confirm the previous identification with a different approach of YbjE as a Lys exporter, reported by Ueda and coworkers (T. Ueda, Y. Nakai, Y. Gunji, R. Takikawa, and Y. Joe, U.S. patents 7,629,142 B2 [December 2009] and 8,383,363 B1 [February 2013] and European patent 1,664,318 B1 [September 2009]). ybjE was isolated as a multicopy suppressor of the CAN(ss) phenotype of a strain lacking ArgO. The absence of YbjE did not confer a CAN(ss) phenotype but instead conferred hypersensitivity to the lysine antimetabolite thialysine and led to growth inhibition by the dipeptide lysylalanine, which is associated with elevated cellular Lys content. YbjE overproduction resulted in Lys excretion and syntrophic cross-feeding of a Lys auxotroph. Constitutive overexpression of argO promoted Lys cross-feeding that is indicative of a latent Lys export potential of ArgO. Arg modestly repressed ybjE transcription in an ArgR-dependent manner, and ArgR displayed Arg-sensitive binding to the ybjE promoter region in vitro. Our studies suggest that the reciprocal repression of argO and ybjE, respectively, by Lys and Arg confers the specificity for basic amino acid export by distinct paths and that such cross-repression contributes to maintenance of cytoplasmic Arg/Lys balance. We propose that YbjE be redesignated LysO. This work ascribes a lysine export function to the product of the ybjE gene of Escherichia coli, leading to a physiological scenario wherein two proteins, ArgO and YbjE, perform the task of separately exporting arginine and lysine

  5. Mammalian CAP interacts with CAP, CAP2, and actin.

    PubMed

    Hubberstey, A; Yu, G; Loewith, R; Lakusta, C; Young, D

    1996-06-01

    We previously identified human CAP, a homolog of the yeast adenylyl cyclase-associated protein. Previous studies suggest that the N-terminal and C-terminal domains of CAP have distinct functions. We have explored the interactions of human CAP with various proteins. First, by performing yeast two-hybrid screens, we have identified peptides from several proteins that interact with the C-terminal and/or the N-terminal domains of human CAP. These peptides include regions derived from CAP and BAT3, a protein with unknown function. We have further shown that MBP fusions with these peptides can associate in vitro with the N-terminal or C-terminal domains of CAP fused to GST. Our observations indicate that CAP contains regions in both the N-terminal and C-terminal domains that are capable of interacting with each other or with themselves. Furthermore, we found that myc-epitope-tagged CAP coimmunoprecipitates with HA-epitope-tagged CAP from either yeast or mammalian cell extracts. Similar results demonstrate that human CAP can also interact with human CAP2. We also show that human CAP interacts with actin, both by the yeast two-hybrid test and by coimmunoprecipitation of epitope-tagged CAP from yeast or mammalian cell extracts. This interaction requires the C-terminal domain of CAP, but not the N-terminal domain. Thus CAP appears to be capable of interacting in vivo with other CAP molecules, CAP2, and actin. We also show that actin co-immunoprecipitates with HA-CAP2 from mammalian cell extracts.

  6. Waning Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    14 June 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows the outer edge of the south polar residual cap of Mars. During summer, the scarps that delineate the sides of the mesas, retreat (on average) by about 3 meters (10 feet) owing to the sublimation of solid carbon dioxide.

    Location near: 85.6oS, 349.8oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  7. Waning Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    14 June 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows the outer edge of the south polar residual cap of Mars. During summer, the scarps that delineate the sides of the mesas, retreat (on average) by about 3 meters (10 feet) owing to the sublimation of solid carbon dioxide.

    Location near: 85.6oS, 349.8oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  8. The effect of insulin-loaded linear poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer on renal ischemia/reperfusion-induced lung injury through downregulating hypoxia-inducible factor.

    PubMed

    Tong, Fei; Tang, Xiangyuan; Li, Xin; Xia, Wenquan; Liu, Daojun

    2016-01-01

    The aim of this study was to observe the therapeutic effect of insulin-loaded linear poly(ethylene glycol)-brush-like poly(l-lysine) block copolymer poly(ethylene glycol)-b-(poly(ethylenediamine l-glutamate)-g-poly(l-lysine)) (PEG-b-(PELG-g-PLL) on renal ischemia/reperfusion-induced lung injury through downregulating hypoxia-inducible factor (HIF) as compared to free insulin. Sprague Dawley rats were pretreated with 30 U/kg insulin or insulin/PEG-b-(PELG-g-PLL) complex, and then subjected to 45 minutes of ischemia and 24 hours of reperfusion. The blood and lungs were collected, the level of serum creatinine and blood urea nitrogen were measured, and the dry/wet lung ratios, the activity of superoxide dismutase and myeloperoxidase, the content of methane dicarboxylic aldehyde and tumor necrosis factor-α, and the expression of HIF-1α and vascular endothelial growth factor (VEGF) were measured in pulmonary tissues. Both insulin and insulin/PEG-b-(PELG-g-PLL) preconditioning improved the recovery of renal function, reduced pulmonary oxidative stress injury, restrained inflammatory damage, and downregulated the expression of HIF-1α and VEGF as compared to ischemia/reperfusion group, while insulin/PEG-b-(PELG-g-PLL) significantly improved this effect.

  9. Polarization of the angular momentum of electrons in calculations of the electronic structure of the NiO/sub 6//sup 10 -/ cluster by the X. cap alpha. -scattered-wave method

    SciTech Connect

    Gagarin, S.G.; Teterin, Yu.A.; Plekhanov, Yu.V.

    1986-05-01

    The x-ray photoelectron spectra of the core 2p/sub 1/2/, 2p/sub 3/2/, 3s/sub 1/2/, 3p/sub 1/2/, and 3/p/sub 3/2/ electrons of nickel in the oxide NiO and in an aluminum - nickel catalyst have been discussed on the basis of the results of a self-consistent calculation of the electronic states of the NiO/sub 6//sup 10 -/ cluster in the spin-polarized variation of the X..cap alpha..-scattered-wave method with complete consideration of the relativistic properties of the core electrons in the angular-momentum-polarized variant. Partial consideration of the relativistic properties of the valence-band electrons (consideration of the Darwin terms and the dependence of the mass on the velocity during the averaging of the wave functions and the density with respect to the angular momentum) does not alter the basic conclusions of the work.

  10. Residual Cap

    NASA Technical Reports Server (NTRS)

    2006-01-01

    10 May 2006 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a summertime view of the south polar residual cap of Mars. In this image, mesas composed largely of solid carbon dioxide are separated from one another by irregularly-shaped depressions. The variation in brightness across this scene is a function of several factors including, but not limited to, varying proportions of dust and solid carbon dioxide, undulating topography, and differences in the roughness of the slopes versus the flat surfaces.

    Location near: 86.7oS, 343.3oW Image width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  11. Enrichment of Bifidobacterium longum subsp. infantis ATCC 15697 within the human gut microbiota using alginate-poly-L-lysine-alginate microencapsulation oral delivery system: an in vitro analysis using a computer-controlled dynamic human gastrointestinal model.

    PubMed

    Rodes, Laetitia; Tomaro-Duchesneau, Catherine; Saha, Shyamali; Paul, Arghya; Malhotra, Meenakshi; Marinescu, Daniel; Shao, Wei; Kahouli, Imen; Prakash, Satya

    2014-01-01

    This study evaluates alginate-poly-L-lysine-alginate Bifidobacterium longum subsp. infantis ATCC 15697-loaded microcapsules to enrich the human gut microbiota. The cell survival of alginate-poly-L-lysine-alginate microencapsulated B. infantis ATCC 15697 in gastric acid, bile, and through human gastrointestinal transit was investigated, as well as the formulation's effect on the gut microbiota. Results show that microencapsulation increases B. infantis ATCC 15697 cell survival at pH1.0 (33.54 ± 2.80% versus <1.00 ± 0.00%), pH1.5 (41.15 ± 2.06% versus <1.00 ± 0.00%), pH2.0 (60.88 ± 1.73% versus 36.01 ± 2.63%), pH3.0 (75.43 ± 1.23% versus 46.30 ± 1.43%), pH4.0 (71.40 ± 2.02% versus 47.75 ± 3.12%) and pH5.0 (73.88 ± 3.79% versus 58.93 ± 2.26%) (p < 0.05). In addition, microencapsulation increases cell survival at 0.5% (76.85 ± 0.80% versus 70.77 ± 0.64%), 1.0% (59.99 ± 0.97% versus 53.47 ± 0.58%) and 2.0% (53.10 ± 1.87% versus 44.59 ± 1.52%) (p < 0.05) (w/v) bile. Finally, daily administration of alginate-poly-L-lysine-alginate microencapsulated B. infantis ATCC 15697 in a human gastrointestinal model induces a significant enrichment of B. infantis within the ascending (184.51 ± 17.30% versus 53.83 ± 17.82%; p < 0.05), transverse (174.79 ± 25.32% versus 73.17 ± 15.30%; p < 0.05) and descending (94.90 ± 25.22% versus 46.37 ± 18.93%; p > 0.05) colonic microbiota.

  12. Ca/sup 2 +/-stimulated catecholamine release from. cap alpha. -toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G protein

    SciTech Connect

    Ahnert-Hilger, G.; Braeutigam, M.; Gratzl, M.

    1987-12-01

    Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in ..cap alpha..-toxin-permeabilized cells with micromolar levels of free Ca/sup 2 +/. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca/sup 2 +/. Both types of experiments provide direct evidence for the operation of Ca/sup 2 +/-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca/sup 2 +/ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below ..mu..M free Ca/sup 2 +/ and plateaus at 1-5 ..mu..M free Ca/sup 2 +/, while a second occurs in the presence of larger amounts of free Ca/sup 2 +/ (10-100 ..mu..M). Ca/sup 2 +/-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg/sup 2 +//ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca/sup 2 +/-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.

  13. Development of non-pyrogenic magnetosome minerals coated with poly-l-lysine leading to full disappearance of intracranial U87-Luc glioblastoma in 100% of treated mice using magnetic hyperthermia.

    PubMed

    Alphandéry, Edouard; Idbaih, Ahmed; Adam, Clovis; Delattre, Jean-Yves; Schmitt, Charlotte; Guyot, François; Chebbi, Imène

    2017-10-01

    Magnetic hyperthermia was reported to increase the survival of patients with recurrent glioblastoma by 7 months. This promising result may potentially be further improved by using iron oxide nanoparticles, called magnetosomes, which are synthesized by magnetotactic bacteria, extracted from these bacteria, purified to remove most endotoxins and organic material, and then coated with poly-l-lysine to yield a stable and non-pyrogenic nanoparticle suspension. Due to their ferrimagnetic behavior, high crystallinity and chain arrangement, these magnetosomes coated with poly-l-lysine (M-PLL) are characterized by a higher heating power than their chemically synthesized counterparts currently used in clinical trials. M-PLL-enhanced antitumor efficacy was demonstrated by administering 500-700 μg in iron of M-PLL to intracranial U87-Luc tumors of 1.5 mm(3) and by exposing mice to 27 magnetic sessions each lasting 30 min, during which an alternating magnetic field of 202 kHz and 27 mT was applied. Treatment conditions were adjusted to reach a typical hyperthermia temperature of 42 °C during the first magnetic session. In 100% of treated mice, bioluminescence due to living glioblastoma cells fully disappeared 68 days following tumor cell implantation (D68). These mice were all still alive at D350. Histological analysis of their brain tissues revealed an absence of tumor cells, suggesting that they were fully cured. In comparison, antitumor efficacy was less pronounced in mice treated by the administration of IONP followed by 23 magnetic sessions, leading to full tumor bioluminescence disappearance in only 20% of the treated mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Targeting inflammasome by the inhibition of caspase-1 activity using capped mesoporous silica nanoparticles.

    PubMed

    García-Fernández, Alba; García-Laínez, Guillermo; Ferrándiz, María Luisa; Aznar, Elena; Sancenón, Félix; Alcaraz, María José; Murguía, José Ramón; Marcos, María D; Martínez-Máñez, Ramón; Costero, Ana M; Orzáez, Mar

    2017-02-28

    Acute inflammation is a protective response of the body to harmful stimuli, such as pathogens or damaged cells. However, dysregulated inflammation can cause secondary damage and could thus contribute to the pathophysiology of many diseases. Inflammasomes, the macromolecular complexes responsible for caspase-1 activation, have emerged as key regulators of immune and inflammatory responses. Therefore, modulation of inflammasome activity has become an important therapeutic approach. Here we describe the design of a smart nanodevice that takes advantage of the passive targeting of nanoparticles to macrophages and enhances the therapeutic effect of caspase-1 inhibitor VX-765 in vivo. The functional hybrid systems consisted of MCM-41-based nanoparticles loaded with anti-inflammatory drug VX-765 (S2-P) and capped with poly-L-lysine, which acts as a molecular gate. S2-P activity has been evaluated in cellular and in vivo models of inflammation. The results indicated the potential advantage of using nanodevices to treat inflammatory diseases.

  15. The cervical cap (image)

    MedlinePlus

    The cervical cap is a flexible rubber cup-like device that is filled with spermicide and self-inserted over the cervix ... left in place several hours after intercourse. The cap is a prescribed device fitted by a health ...

  16. Cradle Cap: Treatment

    MedlinePlus

    Cradle cap Treatment Cradle cap usually doesn't require medical treatment. It clears up on its own within a few months. In the meantime, wash ... tips can help you control and manage cradle cap. Gently rub your baby's scalp with your fingers ...

  17. Peroxidation of the dried thin film of lipid by high-energy alpha particles from a cyclotron

    SciTech Connect

    Agarwal, S.; Chatterjee, S.N.

    1984-11-01

    High-energy ..cap alpha.. particles produced a dose-dependent linear increase in different lipid peroxidation products (e.g., malondialdehyde (MDA), conjugated dienes, and hydroperoxides) in the dried thin film state. An inverse dose-rate effect was observed when the dose rate was varied by changing either the ..cap alpha..-particle fluence rate or the ..cap alpha..-particle energy. The antioxidants ..cap alpha..-tocopherol and butylated hydroxytoluene (BHT) suppressed the ..cap alpha..-particle-induced lipid peroxidation in the dried thin film state, and in this respect ..cap alpha..-tocopherol was found superior to BHT. It was found that ..cap alpha..-tocopherol was equally efficient in inhibiting lipid peroxidations by ..cap alpha.. particles and ultraviolet light.

  18. Hapten-specific delayed hypersensitivity to epsilon-2,4-dinitrophenyl-L- lysine-Ficoll in guinea pigs immunized with 2,4-dinitrophenyl-keyhole limpet hemocyanin

    PubMed Central

    1977-01-01

    After active immunization with 2,4-dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH), 2,4-dinitropheynl-L-lysine (DNPL)-Ficoll may elicit indurated, erythematous skin reactions lasting 24-72 h. Histological sections of these reactions, examined by microscope techniques, showed they contained polymorphonuclear leukocytes and perivascularly situated lymphocytes and macrophages, but had very few basophils. Consequently, the reaction was interpreted as having an immediate component and a component typical of delayed hypersensitivity; this indicated that the delayed reaction could be specific for the DNP hapten. Although this delayed type of skin reaction was not transferred to recipients with anti-DNP-KLH serum, one pool of that serum did sensitize guinea pigs so that they could respond with a different skin reaction after challenge with DNPL-Ficoll. This reaction was soft, pale pink, and lasted for 24 h. Histologically, it contained only a few polymorphonuclear leukocytes. It differed from the delayed reaction in actively immunized animals in that it lacked induration, and was devoid of lymphocytes and macrophages. PMID:858997

  19. A comparison of the effectiveness of cationic polymers poly-L-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC).

    PubMed

    Farrell, Laura-Lee; Pepin, Joel; Kucharski, Cezary; Lin, Xiaoyue; Xu, Zhenghe; Uludag, Hasan

    2007-03-01

    Bone marrow stromal cells (BMSC) represent an important cell phenotype for pursuit of successful gene therapy. Non-viral methods to enable expression of exogenous genes in BMSC will accelerate clinical application of gene therapy, without the concerns associated with the viral means of gene transfer. Towards this end, this study investigated the potential of cationic polymers poly-L-lysine (PLL) and branched polyethylenimine (PEI) as gene carriers for modification of BMSC. Both polymers rapidly (approximately 30 min) condensed a 4.2 kb Enhanced Green Fluorescent Protein (pEGFP-N2) plasmid into 100-200 nm particles. PLL and PEI were both readily internalized with BMSC with >80% of BMSC exhibiting polymer uptake by flow cytometric analysis. The relative uptake of PEI, however, was significantly higher as compared to the PLL. The majority of the BMSC (>60%) exhibited nuclear presence of the polymers as analyzed by fluorescent microscopy. Although both polymers were able to deliver the pEGFP-N2 into the cells under microscopic evaluation, only a small fraction of the cells (<10%) displayed nuclear localization of the plasmid. Consistent with better uptake, PEI gave a higher delivery of pEGFP-N2 into the BMSC, which resulted in a more sustained expression of the model gene EGFP in short-term (7-day) culture. We conclude that both PLL and PEI readily displayed cellular uptake, but PEI was more effective in delivering plasmid DNA intracellularly, which was likely the underlying basis for a more sustained gene expression.

  20. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    PubMed

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-04

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  1. The PPII-to-α-helix transition of poly-l-lysine in methanol/water solvent mixtures accompanied by fibrillar self-aggregation: An influence of fluphenazine molecules.

    PubMed

    Cieślik-Boczula, Katarzyna

    2017-08-01

    Fourier-transform infrared, vibrational circular dichroism spectroscopy and transmission electron microscopy are used to follow the structural changes of pure and fluphenazine (FPh)-mixed poly-l-lysine (PLL) triggered by variations of the methanol to water ratio in solvent mixtures. FPh molecules are used as an effective psychotic drug but with a strong Parkinson's-related side effect. To answer the question whether FPh molecules can modify the fibril development, the PLL polypeptide was used as a model of α-helix- and PPII-rich fibrils. It was stated that the presence of FPh molecules did not inhibit the creation of both types of PLL fibrils with clustering features. The methanol-poor aqueous solutions promote the formation of extended polyproline II (PPII) helices; however, the methanol-rich aqueous solutions induce the development of α-helices of both pure and FPh-mixed PLL. Unpredicted and interesting features of PLL fibrillogenesis are evidenced by the formation of uncommon fibrillar aggregates, which are developed in methanol/water solvents from PLL molecules rich in either α-helix or PPII structures. Possibility of PLL molecules to form β-sheet-, α-helix- and PPII-rich fibrils demonstrating that fibrillogenesis is a common phenomenon, and fibrillar aggregates can be based on all of the basic protein secondary structures. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Mixed polyelectrolyte coatings on glassy carbon electrodes: Ion-exchange, permselectivity properties and analytical application of poly-l-lysine-poly(sodium 4-styrenesulfonate)-coated mercury film electrodes for the detection of trace metals.

    PubMed

    Monterroso, Sandra C C; Carapuça, Helena M; Duarte, Armando C

    2006-02-28

    The present work describes the preparation, optimization and characterization of mixed polyelectrolyte coatings of poly-l-lysine (PLL) and poly(sodium 4-styrenesulfonate) (PSS) for the modification of thin mercury film electrodes (MFEs). The novel-modified electrodes were applied in the direct analysis of trace metals in estuarine waters by square-wave anodic stripping voltammetry (SWASV). The effects of the coating morphology and thickness and also of the monomeric molar ratio PLL/PSS on the cation-exchange ability of the PLL-PSS polyelectrolyte coatings onto glassy carbon (GC) were evaluated using target cationic species such as dopamine (DA) or lead cation. Further, the semi-permeability of the PLL-PSS-coated electrodes based both on electrostatic interactions and on molecular size leads to an improved anti-fouling ability against several tensioactive species. The analytical usefulness of the PLL-PSS-mixed polyelectrolyte coatings on thin mercury film electrodes is demonstrated via SWASV measurements of trace metals (lead, copper and cadmium at the low nanomolar level; accumulation time of 180s) in estuarine waters containing moderate levels of dissolved organic matter, resulting in a fast and direct methodology requiring no sample pretreatment.

  3. Synthesis of water-dispersible poly-l-lysine-functionalized magnetic Fe3O4-(GO-MWCNTs) nanocomposite hybrid with a large surface area for high-efficiency removal of tartrazine and Pb(II).

    PubMed

    Hu, Dan; Wan, Xiaodong; Li, Xiaohui; Liu, Jianguo; Zhou, Chunhua

    2017-03-07

    In this study, a novel, effective and environment-friendly methods was used to prepare poly-l-lysine (PLL)-functionalized magnetic Fe3O4-(GO-MWCNTs) hybrid composite with large surface area and abundant hydroxyl and amino groups. The as-prepared PLL-Fe3O4-(GO-MWCNTs) nanocomposite was systematically characterized by FT-IR, XRD, TGA, SEM, TEM, VSM and EDX. The PLL-Fe3O4-(GO-MWCNTs) hybrid composite exhibited excellent adsorption performance for the removal of a dye (tartrazine) and a heave metal (Pb(II)). The result showed that adsorption of Pb(II) reached equilibrium in 30min and adsorption of tartrazine reached equilibrium in approximately 60min. Most importantly, PLL-Fe3O4-(GO-MWCNTs) hybrid possesses high adsorption capacity, rapid separation, and less time-consuming. The equilibrium adsorption capacity was 1038.42mgg(-1) for Pb(II) and 775.19mgg(-1) for tartrazine under the optimal conditions. These two pollutants removal were found to obey Langmuir adsorption model, while the kinetics of adsorption followed pseudo-second-order kinetic model. A possible adsorption mechanism has been proposed where the chelation between PLL and Pb(II) or electrostatic interaction between GO and tartrazine. These results demonstrated the potential applications of PLL-Fe3O4-(GO-MWCNTs) hybrid composite in deep-purification of polluted water. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

    PubMed

    Gu, Yanyan; Wang, Xiaomeng; Yang, Chao; Geng, Weitao; Feng, Jun; Wang, Yuanyuan; Wang, Shufang; Song, Cunjiang

    2016-04-01

    ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.

  5. Cell-targeted, dual reduction- and pH-responsive saccharide/lipoic acid-modified poly(L-lysine) and poly(acrylic acid) polyionic complex nanogels for drug delivery.

    PubMed

    How, Su-Chun; Chen, Yu-Fon; Hsieh, Pin-Lun; Wang, Steven S-S; Jan, Jeng-Shiung

    2017-05-01

    A cell-targeted, reduction-/pH-responsive polyionic complex (PIC) nanogel system was developed by simply mixing cationic lactobionolatone/lipoic acid-modified poly(L-lysine) (PLL-g-(Lipo-Lac)) and anionic poly(acrylic acid) (PAA), followed by disulfide cross-linking. The nanogels with sizes smaller than 150nm can be prepared at certain mixing ratio via forming interchain disulfide cross-link and helical PAA/PLL complexes. In vitro drug release study showed that Doxorubicin (Dox) release from the nanogels was significantly enhanced by increasing acidity and/or introducing disulfide cleaving agent. Carbohydrate-lectin binding and cellular uptake studies confirmed that Lac-conjugated nanogels can effectively bind to the cells bearing asialoglycoprotein receptors and subsequently afford efficient cell internalization. Cytotoxicity assays showed that Dox-loaded, Lac-conjugated nanogels exhibited efficient cell proliferation inhibition toward HepG2 cells, whereas the nanogels exhibited excellent biocompatibility. Furthermore, TUNEL assay was employed to detect apoptosis pertaining to the mechanism of cell death. This study highlights that polyionic complexation with subsequent cross-linking can be a simple approach to prepare multifunctional nanogels as drug delivery vehicles. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The cervical cap.

    PubMed

    1988-10-07

    The US Food and Drug Administration has approved marketing of the Prentif cavity-rim cervical cap. This contraceptive device is being distributed in the US and Canada by Cervical Cap Ltd, Los Gatos, California. The Prentif cap is available in 4 sizes: 22, 25, 28, and 31 mm inside diameter, with a length of 1 1/4-1 1/2 inches. In a multicenter trial involving 522 diaphragm users and 581 cap users followed for 2 years, the cap was 82.6% effective and the diaphragm was 83.3% effective in preventing pregnancy. When pregnancies attributable to user failure were excluded, these rates were increased to 93.6% for the cap and 95.4% for the diaphragm. 4% of cap users compared with only 1.7% of diaphragm users in this study developed abnormal Pap smears after 3 months of use; in addition, a higher proportion of cap users became infected with Gardnerella vaginalis and Monilia. Theoretical hazards include toxic shock syndrome and endometriosis due to backflow of menstrual fluids. Cap users are advised to undergo a Pap test after 3 months of use and discontinue cap use if the results are abnormal. The cap should not be used during menstruation. Although the cap can be left in place for up to 48 hours, its position should be checked before and after each episode of intercourse. The cervical cap requires less spermicide than the diaphragm and is not as messy. In addition, it can be left in the vagina twice as long as the diaphragm, without additional spermicide. Since the cap is smaller than the diaphragm and does not cover the vaginal wall, some women find intercourse more pleasurable with this device.

  7. Vinyl capped addition polyimides

    NASA Technical Reports Server (NTRS)

    Vannucci, Raymond D. (Inventor); Malarik, Diane C. (Inventor); Delvigs, Peter (Inventor)

    1991-01-01

    Polyimide resins (PMR) are generally useful where high strength and temperature capabilities are required (at temperatures up to about 700 F). Polyimide resins are particularly useful in applications such as jet engine compressor components, for example, blades, vanes, air seals, air splitters, and engine casing parts. Aromatic vinyl capped addition polyimides are obtained by reacting a diamine, an ester of tetracarboxylic acid, and an aromatic vinyl compound. Low void materials with improved oxidative stability when exposed to 700 F air may be fabricated as fiber reinforced high molecular weight capped polyimide composites. The aromatic vinyl capped polyimides are provided with a more aromatic nature and are more thermally stable than highly aliphatic, norbornenyl-type end-capped polyimides employed in PMR resins. The substitution of aromatic vinyl end-caps for norbornenyl end-caps in addition polyimides results in polymers with improved oxidative stability.

  8. Claymax landfill cap

    SciTech Connect

    Selby, C.L.

    1989-12-15

    A commercial product called Claymax'' consisting of one-quarter inch of bentonite clay between two geotextile sheets is a candidate landfill cap to replace kaolin caps. A permeability apparatus incorporating a 20 foot water head was operated for 56 days to estimate a Claymax permeability of 2 {times} 10{sup {minus}9} cm/sec compared with 10{sup {minus}8}, the EPA max for a burial site cap. 1 fig.

  9. Health-care cap.

    PubMed

    1996-05-03

    Dallas Avionics agreed to discontinue its cap on HIV-related medical expenses. The Texas company offered employees $1 million worth of lifetime medical benefits, with the exception of HIV-related expenses. Lambda Legal Defense and Education Fund intervened, demanding that the cap be removed and the company pay an employee's $82,000 outstanding HIV-related medical bills. According to Lambda, the cap violates the Americans with Disabilities Act (ADA).

  10. Reaction of. cap alpha. ,. cap alpha. , omega-trihydroperfluoroalkanols with phosphorus tribromide

    SciTech Connect

    Krolevets, A.A.; Ragulin, L.I.; Shevchenko, V.I.; Lyubimova, E.I.

    1986-08-20

    The authors investigate the bromination and phosphorylation of the title alkanol by phosphorus tribromide and determine the structure and composition of the resulting phosphorus and bromine compounds using IR and NMR spectroscopy with phosphorus 31, fluorine 19, and hydrogen 1. An extensive analysis of chemical shifts and spin-spin coupling constants is performed. The reaction of fluorine-containing trialkyl phosphites with sulfuric acid anhydride was found to lead to the formation of the corresponding fluorinated trialkyl phosphates in a quantitative yield.

  11. Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat.

    PubMed

    Hester, Kirstin; Liu, Jing; Flynn, Nicholas; Sultatos, Lester G; Geng, Liyi; Brimijoin, Stephen; Ramsey, Joshua D; Hartson, Steven; Ranjan, Ashish; Pope, Carey

    2017-09-25

    We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced kcat (30-33% reduction), but no significant changes in Km. Inhibitory potency (as reflected by the bimolecular rate constant, ki) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Biodegradable tri-block copolymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a non-viral vector to enhance gene transfection.

    PubMed

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-02-23

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH(2)-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.

  13. Adsorption properties of poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) at a hydrophobic interface: influence of tribological stress, pH, salt concentration, and polymer molecular weight.

    PubMed

    Lee, Seunghwan; Spencer, Nicholas D

    2008-09-02

    The adsorption properties of a graft copolymer of poly(ethylene glycol) (PEG) with a polycationic backbone, namely, poly( l-lysine)- graft-poly(ethylene glycol) (PLL- g-PEG), onto nonpolar, hydrophobic PDMS surfaces from aqueous solution and the lubrication properties of the self-mated sliding contacts of PDMS surfaces modified with PLL- g-PEG have been investigated. Whereas PLL- g-PEG is spontaneously attracted to negatively charged surfaces as a result of the polycationic PLL backbone, the collective interaction of (CH 2) 4 hydrocarbon moieties on the lysine units in the PLL backbone with nonpolar, hydrophobic surfaces also enables the adsorption of PLL- g-PEG onto hydrophobic surfaces such as PDMS. The adsorption and lubrication properties of PLL- g-PEG have been investigated by varying the aqueous solution parameters, such as pH (2, 7, and 12) and KCl concentration (0, 0.01, 0.1, and 1 M) as well as the length of the PLL backbone of the copolymer (20 vs 375 kDa). In the absence of tribological stress, the adsorption of PLL- g-PEG onto PDMS surfaces was mainly governed by the KCl concentration, whereas the role of pH or the molecular weight of the copolymer was of relatively minor importance; for all pH values, the adsorbed mass decreased with increasing KCl concentration. Under tribological stress, however, a clear dependence of the lubrication properties of PLL- g-PEG on all of the studied parameters, including pH, KCl concentration, and backbone molecular weight, was observed. The adsorption strength of PLL- g-PEG on PDMS surfaces, rather than the adsorbed mass itself, appeared to be the most critical parameter in determining the lubrication properties.

  14. Acidic pH shock induced overproduction of ε-poly-L-lysine in fed-batch fermentation by Streptomyces sp. M-Z18 from agro-industrial by-products.

    PubMed

    Ren, Xi-Dong; Chen, Xu-Sheng; Zeng, Xin; Wang, Liang; Tang, Lei; Mao, Zhong-Gui

    2015-06-01

    ε-Poly-L-lysine (ε-PL) is produced by Streptomyces as a secondary metabolite with wide industrial applications, but its production still needs to be further enhanced. Environmental stress is an important approach for the promotion of secondary metabolites production by Streptomyces. In this study, the effect of acidic pH shock on enhancing ε-PL production by Streptomyces sp. M-Z18 was investigated in a 5-L fermenter. Based on the evaluation of acidic pH shock on mycelia metabolic activity and shock parameters optimization, an integrated pH-shock strategy was developed as follows: pre-acid-shock adaption at pH 5.0 to alleviate the damage caused by the followed pH shock, and then acidic pH shock at 3.0 for 12 h (including pH decline from 4.0 to 3.0) to positively regulate mycelia metabolic activity, finally restoring pH to 4.0 to provide optimal condition for ε-PL production. After 192 h of fed-batch fermentation, the maximum ε-PL production and productivity reached 54.70 g/L and 6.84 g/L/day, respectively, which were 52.50 % higher than those of control without pH shock. These results demonstrated that acidic pH shock is an efficient approach for improving ε-PL production. The information obtained should be useful for ε-PL production by other Streptomyces.

  15. Polyion complex micelle MRI contrast agents from poly(ethylene glycol)-b-poly(l-lysine) block copolymers having Gd-DOTA; preparations and their control of T(1)-relaxivities and blood circulation characteristics.

    PubMed

    Shiraishi, Kouichi; Kawano, Kumi; Maitani, Yoshie; Yokoyama, Masayuki

    2010-12-01

    The current study synthesized macromolecular magnetic resonance imaging (MRI) contrast agents constituted of the poly(ethylene glycol)-b-poly(L-lysine) block copolymer (PEG-P(Lys)). A chelate group, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), was attached to the primary amino group of the block copolymer in desired contents. Gd-DOTA-based macromolecular contrast agents were prepared from PEG-P(Lys) having DOTA (PEG-P(Lys-DOTA) and Gd(III) ions. All of the PEG-P(Lys) block copolymers having gadolinium ions (PEG-P(Lys-DOTA-Gd)) showed higher T(1) relaxivity (per gadolinium), r(1)=5.6-7.3mM(-1)s(-1), than that of a low-molecular-weight gadolinium-chelate, diethylenetriaminepentaacetic acid-gadolinium(III) (Gd-DTPA) at 9.4T. The study prepared the polyion complex (PIC) micelles from the amino groups of the lysine units and an oppositely charged polyanion, poly(methacrylic acid) or dextran sulfate, in an aqueous medium. In contrast, the fully DOTA-attached PEG-P(Lys-DOTA-Gd) formed a PIC with a polycation. Compared with partially DOTA-attached cationic PEG-P(Lys-DOTA-Gd), this PIC micelle yielded a forty percent decrease of r(1). This r(1) decrease was considered to result from a change in the accessibility of water molecules to gadolinium ions in the micelles' inner core. The r(1) was decreased upon formation of the PIC micelle, and this change proved that our concept worked in vitro. Blood-circulation characteristics of PIC micelles were controlled by means of changing the molecular weight of the counter anion. The PIC micelles accumulated in tumor tissues, and MRI study showed T1W image of axial slice of tumor area was significantly enhanced at 24h after the injection.

  16. Simultaneous determination of trace Cd(II), Pb(II) and Cu(II) by differential pulse anodic stripping voltammetry using a reduced graphene oxide-chitosan/poly-l-lysine nanocomposite modified glassy carbon electrode.

    PubMed

    Guo, Zhuo; Li, Dong-di; Luo, Xian-Ke; Li, Ya-Hui; Zhao, Qi-Nai; Li, Meng-Meng; Zhao, Yang-Ting; Sun, Tian-Shuai; Ma, Chi

    2017-03-15

    The reduced graphene oxide (RGO) and Chitosan (CS) hybrid matrix RGO-CS were coated onto the glassy carbon electrode (GCE) surface, then, poly-l-lysine films (PLL) were prepared by electropolymerization with cyclic voltammetry (CV) method to prepare RGO-CS/PLL modified glassy carbon electrode (RGO-CS/PLL/GCE) for the simultaneous electrochemical determination of heavy metal ions Cd(II), Pb(II) and Cu(II). Combining the advantageous features of RGO and CS, RGO and CS are used together because the positively charged CS can interact with the negatively changed RGO to prevent their aggregation. Furthermore, CS has many amino groups along its macromolecular chains and possessed strongly reactive with metal ions. Moreover, PLL modified electrodes have good stability, excellent permselectivity, more active sites and strong adherence to electrode surface, which enhanced electrocatalytic activity. The RGO-CS/PLL/GCE was characterized voltammetrically using redox couples (Fe(CN)6(3-/4-)), complemented with electrochemical impedance spectroscopy (EIS). Differential pulse anodic stripping voltammetry (DPASV) has been used for the detection of Cd(II), Pb(II) and Cu(II). The detection limit of RGO-CS/PLL/GCE toward Cd(II), Pb(II) and Cu(II) is 0.01μgL(-1), 0.02μgL(-1) and 0.02μgL(-1), respectively. The electrochemical parameters that exert influence on deposition and stripping of metal ions, such as supporting electrolytes, pH value, deposition potential, and deposition time, were carefully studied.

  17. A simple strategy for the immobilization of catalase on multi-walled carbon nanotube/poly (L-lysine) biocomposite for the detection of H2O2 and iodate.

    PubMed

    Ezhil Vilian, A T; Chen, Shen-Ming; Lou, Bih-Show

    2014-11-15

    Herein, we report a novel third-generation H2O2 and IO3- biosensor, which was fabricated by loading catalase (CAT) onto l-lysine/multiwalled carbon nanotube (PLL/f-MWCNT) film modified glassy carbon electrode (GCE). The UV-visible (UV-vis) and Fourier-transform infrared (FTIR) spectra show that the catalase encapsulated in the PLL/f-MWCNT film can effectively retain its bioactivity. The immobilized CAT retained its bioactivity with a high protein loading of 4.072 × 10(-10) mol cm(-2), thus exhibiting a surface-controlled reversible redox reaction, with a fast heterogeneous electron transfer rate of 5.48 s(-1). The immobilized CAT shows a couple of reversible and well-defined cyclic voltammetry peaks with a formal potential (E(0)) of -0.471 V (vs. Ag/AgCl) in a pH 6.5 phosphate buffer solution (PBS). Moreover, the modified film exhibited high electrocatalytic activity for the reduction of hydrogen peroxide (H2O2). It exhibited a wide linear response to H2O2 in the concentration range of 1 × 10(-6) - 3.6 × 10(-3), with higher sensitivity (392 mA cm(-2) M(-1)) and a lower Michaelis-Menten constant (0.224 mM). It provided high-catalytic activity towards H2O2 in a shorter time (5s), with a detection limit of 8 nM. These results indicate great improvement in the electrochemical and electrocatalytic properties of the CAT/PLL/f-MWCNT biosensor, offering a new idea for the design of third-generation electrochemical biosensors.

  18. Synthesis of Leishmania cap-4 intermediates, cap-2 and cap-3.

    PubMed

    Lewdorowicz, Magdalena; Stepinski, Janusz; Kierzek, Ryszard; Jemielity, Jacek; Zuberek, Joanna; Yoffe, Yael; Shapira, Michal; Stolarski, Ryszard; Darzynkiewicz, Edward

    2007-01-01

    Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).

  19. Mars ice caps.

    PubMed

    Leovy, C

    1966-12-02

    Minimum atmospheric temperatures required to prevent CO(2) condensatio in the Mars polar caps are higher than those obtained in a computer experiment to simulate the general circulation of the Mars atmosphere. This observation supports the view that the polar caps are predominantly solid CO(2). However, thin clouds of H(2)0 ice could substantially reduce the surface condensation rate.

  20. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  1. Biodegradable Tri-Block Copolymer Poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)(PLA-PEG-PLL) as a Non-Viral Vector to Enhance Gene Transfection

    PubMed Central

    Fu, Chunhua; Sun, Xiaoli; Liu, Donghua; Chen, Zhijing; Lu, Zaijun; Zhang, Na

    2011-01-01

    Low cytotoxicity and high gene transfection efficiency are critical issues in designing current non-viral gene delivery vectors. The purpose of the present work was to synthesize the novel biodegradable poly (lactic acid)-poly(ethylene glycol)-poly(l-lysine) (PLA-PEG-PLL) copolymer, and explore its applicability and feasibility as a non-viral vector for gene transport. PLA-PEG-PLL was obtained by the ring-opening polymerization of Lys(Z)-NCA onto amine-terminated NH2-PEG-PLA, then acidolysis to remove benzyloxycarbonyl. The tri-block copolymer PLA-PEG-PLL combined the characters of cationic polymer PLL, PLA and PEG: the self-assembled nanoparticles (NPs) possessed a PEG loop structure to increase the stability, hydrophobic PLA segments as the core, and the primary ɛ-amine groups of lysine in PLL to electrostatically interact with negatively charged phosphate groups of DNA to deposit with the PLA core. The physicochemical properties (morphology, particle size and surface charge) and the biological properties (protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in HeLa and HepG2 cells) of the gene-loaded PLA-PEG-PLL nanoparticles (PLA-PEG-PLL NPs) were evaluated, respectively. Agarose gel electrophoresis assay confirmed that the PLA-PEG-PLL NPs could condense DNA thoroughly and protect DNA from nuclease degradation. Initial experiments showed that PLA-PEG-PLL NPs/DNA complexes exhibited almost no toxicity and higher gene expression (up to 21.64% in HepG2 cells and 31.63% in HeLa cells) than PEI/DNA complexes (14.01% and 24.22%). These results revealed that the biodegradable tri-block copolymer PLA-PEG-PLL might be a very attractive candidate as a non-viral vector and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo. PMID:21541064

  2. Polyion complex micelles from plasmid DNA and poly(ethylene glycol)-poly(L-lysine) block copolymer as serum-tolerable polyplex system: physicochemical properties of micelles relevant to gene transfection efficiency.

    PubMed

    Itaka, Keiji; Yamauchi, Kyosuke; Harada, Atsushi; Nakamura, Kozo; Kawaguchi, Hiroshi; Kataoka, Kazunori

    2003-11-01

    Polyion complex (PIC) micelles composed of the poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) block copolymer and plasmid DNA (pDNA) were investigated in this study from a physicochemical viewpoint to get insight into the structural feature of the PIC micellar vector system to show practical gene transfection efficacy particularly under serum-containing medium. The residual ratio (r) of the lysine units in PEG-PLL to the phosphate units of pDNA in the system significantly affects the size of the PIC micelles evaluated from dynamic light scattering, being decreased from approximately 120 to 80 nm with an increase in the r value for the region with r > or = 1.0. The zeta potential of the complexes slightly increased with r in the same region, yet maintained a very small absolute value and leveled off to a few mV at r approximately 2.0. These results suggest that the micelles are most likely to take the core-shell structure with dense PEG palisades surrounding the PIC core to compartmentalize the condensed pDNA. Furthermore, an increasing r value in the region of r > or = 1 induces a rearrangement of the stoichiometric complex formed at r=1.0 to the non-stoichiometric complex composed of the excess block copolymer. The association number of pDNA and the block copolymer in the micelle was estimated from the apparent micellar molecular weight determined by static light scattering measurements, indicating that a single pDNA molecule was incorporated in each of the micelles prepared from the PEG (Mw=12,000 g/mol)-PLL (polymerization degree of PLL segment: 48) (12-48) block copolymer at r=2.0. These 12-48/pDNA micelles showed a gene expression comparable to the lipofection toward cultured 293 cells, though 100 microM chloroquine was required in the transfection medium. Notably, even in the presence of serum, the PIC micelles achieved appreciable cellular association to attain a high gene expression, which is in sharp contrast with the drastic decrease in the gene

  3. Influence of chemical modification of N alpha-cocoyl-L-arginine ethyl ester on its hepatitis B surface antigen-inactivating effect.

    PubMed Central

    Sugimoto, Y; Toyoshima, S

    1980-01-01

    We have reported previously that N alpha-cocoyl-L-arginine ethyl ester (CAE) strongly inactivates hepatitis B surface antigen (HBsAg; Sugimoto and Toyoshima, Antimicrob. Agents Chemother. 16:329--332, 1979). Replacement of the L-arginine moiety of CAE by L-lysine did not decrease the HBsAg-inactivating effect of CAE, whereas replacement by some neutral amino acids and L-ornithine decreased it. Esterification of the carboxyl group of N alpha-acyl-L-arginine enhanced its inactivating effect. When the ethyl ester of CAE was converted to an amide group, the effect was appreciably decreased. Modification of the carboxyl group was essential for the inactivation. The effectiveness of N alpha-acyl-L-arginine ethyl ester depends upon the length of the acyl group, with the optimum length for the inactivation of HBsAg being C12 to C14. In addition to CAE, N alpha-lauroyl-L-lysine ethyl ester and N alpha-cocoyl-L-arginine amide were found to be strong inactivators of HBsAg. Significant inactivating effects on HBsAg were not observed in many anionic detergents containing an amino acid. These results suggest that for strongly inactivating HBsAg, a compound should contain a special amino acid, such as L-arginine, and a long acyl group and exhibit a cationic property. PMID:7447415

  4. CCiCap: Boeing

    NASA Image and Video Library

    NASA announced today its plans to partner with The Boeing Company for the next phase of the agency's Commercial Crew Program (CCP). Called Commercial Crew integrated Capability (CCiCap), the initia...

  5. CENTRIFUGE END CAP

    DOEpatents

    Beams, J.W.; Snoddy, L.B.

    1960-08-01

    An end cap for ultra-gas centrifuges is designed to impart or remove angular momentum to or from the gas and to bring the entering gas to the temperature of the gas inside the centrifuge. The end cap is provided with slots or fins for adjusting the temperature and the angular momentum of the entering gas to the temperature and momentum of the gas in the centrifuge and is constructed to introduce both the inner and the peripheral stream into the centrifuge.

  6. ROTOR END CAP

    DOEpatents

    Rushing, F.C.

    1959-02-01

    An improved end cap is described for the cylindrical rotor or bowl of a high-speed centrifugal separator adapted to permit free and efficient continuous counter current flow of gas therethrough for isotope separation. The end cap design provides for securely mounting the same to the hollow central shaft and external wall of the centrifuge. Passageways are incorporated and so arranged as to provide for continuous counter current flow of the light and heavy portions of the gas fed to the centrifuge.

  7. CAPS Simulation Environment Development

    NASA Technical Reports Server (NTRS)

    Murphy, Douglas G.; Hoffman, James A.

    2005-01-01

    The final design for an effective Comet/Asteroid Protection System (CAPS) will likely come after a number of competing designs have been simulated and evaluated. Because of the large number of design parameters involved in a system capable of detecting an object, accurately determining its orbit, and diverting the impact threat, a comprehensive simulation environment will be an extremely valuable tool for the CAPS designers. A successful simulation/design tool will aid the user in identifying the critical parameters in the system and eventually allow for automatic optimization of the design once the relationships of the key parameters are understood. A CAPS configuration will consist of space-based detectors whose purpose is to scan the celestial sphere in search of objects likely to make a close approach to Earth and to determine with the greatest possible accuracy the orbits of those objects. Other components of a CAPS configuration may include systems for modifying the orbits of approaching objects, either for the purpose of preventing a collision or for positioning the object into an orbit where it can be studied or used as a mineral resource. The Synergistic Engineering Environment (SEE) is a space-systems design, evaluation, and visualization software tool being leveraged to simulate these aspects of the CAPS study. The long-term goal of the SEE is to provide capabilities to allow the user to build and compare various CAPS designs by running end-to-end simulations that encompass the scanning phase, the orbit determination phase, and the orbit modification phase of a given scenario. Herein, a brief description of the expected simulation phases is provided, the current status and available features of the SEE software system is reported, and examples are shown of how the system is used to build and evaluate a CAPS detection design. Conclusions and the roadmap for future development of the SEE are also presented.

  8. Binary combination of epsilon-poly-L-lysine and isoeugenol affect progression of spoilage microbiota in fresh turkey meat, and delay onset of spoilage in Pseudomonas putida challenged meat.

    PubMed

    Hyldgaard, Morten; Meyer, Rikke L; Peng, Min; Hibberd, Ashley A; Fischer, Jana; Sigmundsson, Arnar; Mygind, Tina

    2015-12-23

    Proliferation of microbial population on fresh poultry meat over time elicits spoilage when reaching unacceptable levels, during which process slime production, microorganism colony formation, negative organoleptic impact and meat structure change are observed. Spoilage organisms in raw meat, especially Gram-negative bacteria can be difficult to combat due to their cell wall composition. In this study, the natural antimicrobial agents ε-poly-L-lysine (ε-PL) and isoeugenol were tested individually and in combinations for their activities against a selection of Gram-negative strains in vitro. All combinations resulted in additive interactions between ε-PL and isoeugenol towards the bacteria tested. The killing efficiency of different ratios of the two antimicrobial agents was further evaluated in vitro against Pseudomonas putida. Subsequently, the most efficient ratio was applied to a raw turkey meat model system which was incubated for 96 h at spoilage temperature. Half of the samples were challenged with P. putida, and the bacterial load and microbial community composition was followed over time. CFU counts revealed that the antimicrobial blend was able to lower the amount of viable Pseudomonas spp. by one log compared to untreated samples of challenged turkey meat, while the single compounds had no effect on the population. However, the compounds had no effect on Pseudomonas spp. CFU in unchallenged meat. Next-generation sequencing offered culture-independent insight into population diversity and changes in microbial composition of the meat during spoilage and in response to antimicrobial treatment. Spoilage of unchallenged turkey meat resulted in decreasing species diversity over time, regardless of whether the samples received antimicrobial treatment. The microbiota composition of untreated unchallenged meat progressed from a Pseudomonas spp. to a Pseudomonas spp., Photobacterium spp., and Brochothrix thermosphacta dominated food matrix on the expense of low

  9. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    7 September 2004 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a 1.4 m/pixel (5 ft/pixel) view of a typical martian north polar ice cap texture. The surface is pitted and rough at the scale of several meters. The north polar residual cap of Mars consists mainly of water ice, while the south polar residual cap is mostly carbon dioxide. This picture is located near 85.2oN, 283.2oW. The image covers an area approximately 1 km wide by 1.4 km high (0.62 by 0.87 miles). Sunlight illuminates this scene from the lower left.

  10. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    7 September 2004 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a 1.4 m/pixel (5 ft/pixel) view of a typical martian north polar ice cap texture. The surface is pitted and rough at the scale of several meters. The north polar residual cap of Mars consists mainly of water ice, while the south polar residual cap is mostly carbon dioxide. This picture is located near 85.2oN, 283.2oW. The image covers an area approximately 1 km wide by 1.4 km high (0.62 by 0.87 miles). Sunlight illuminates this scene from the lower left.

  11. Magnetospheric polar cap

    NASA Astrophysics Data System (ADS)

    Akasofu, S. I.; Kan, J. R.

    Mount Denali (McKinley), the Alaska Range, and countless glaciers welcomed all 86 participants of the Chapman Conference on the Magnetospheric Polar Cap, which was held on the University of Alaska, Fairbanks campus (UAF), on August 6-9, 1984. The magnetospheric polar cap is the highest latitude region of the earth which is surrounded by the ring of auroras (the auroral oval). This particular region of the earth has become a focus of magnetospheric physicists during the last several years. This is because a number of upper atmospheric phenomena in the polar cap are found to be crucial in understanding the solar wind—magnetosphere interaction. The conference was opened by J. G. Roederer, who was followed by the UAF Chancellor, P. J. O'Rourke, who officially welcomed the participants.

  12. [Capping strategies in RNA viruses].

    PubMed

    Bouvet, Mickaël; Ferron, François; Imbert, Isabelle; Gluais, Laure; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; Decroly, Etienne

    2012-04-01

    Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential. © 2012 médecine/sciences – Inserm / SRMS.

  13. Ice caps on venus?

    PubMed

    Libby, W F

    1968-03-08

    The data on Venus obtained by Mariner V and Venera 4 are interpreted as evidence of giant polar ice caps holding the water that must have come out of the volcanoes with the observed carbon dioxide, on the assumption that Earth and Venus are of similar composition and volcanic history. The measurements by Venera 4 of the equatorial surface temperature indicate that the microwave readings were high, so that the polar ice caps may be allowed to exist in the face of the 10-centimeter readings of polar temperature. Life seems to be distinctly possible at the edges of the ice sheets.

  14. South Polar Cap

    NASA Technical Reports Server (NTRS)

    2005-01-01

    8 December 2005 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows landforms created by sublimation processes on the south polar residual cap of Mars. The bulk of the ice in the south polar residual cap is frozen carbon dioxide.

    Location near: 86.6oS, 342.2oW Image width: width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  15. Commercialization Assistance Program (CAP)

    SciTech Connect

    Jenny C. Servo, Ph.D.

    2004-07-12

    In order to fulfill the objective of Small Business Innovation Research Program (SBIR), the Department of Energy funds an initiative referred to as the Commercialization Assistance Program (CAP). The over-arching purpose of the CAP is to facilitate transition of the SBIR-funded technology to Phase III defined as private sector investment or receipt of non-sbir dollars to further the commercialization of the technology. Phase III also includes increased sales. This report summarizes the stages involved in the implementation of the Commercialization Assistance Program, a program which has been most successful in fulfilling its objectives.

  16. South Polar Cap

    NASA Technical Reports Server (NTRS)

    2005-01-01

    8 December 2005 This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows landforms created by sublimation processes on the south polar residual cap of Mars. The bulk of the ice in the south polar residual cap is frozen carbon dioxide.

    Location near: 86.6oS, 342.2oW Image width: width: 3 km (1.9 mi) Illumination from: upper left Season: Southern Summer

  17. Capping risk adjustment?

    PubMed

    Eugster, Patrick; Sennhauser, Michèle; Zweifel, Peter

    2010-07-01

    When premiums are community-rated, risk adjustment (RA) serves to mitigate competitive insurers' incentive to select favorable risks. However, unless fully prospective, it also undermines their incentives for efficiency. By capping its volume, one may try to counteract this tendency, exposing insurers to some financial risk. This in term runs counter the quest to refine the RA formula, which would increase RA volume. Specifically, the adjuster, "Hospitalization or living in a nursing home during the previous year" will be added in Switzerland starting 2012. This paper investigates how to minimize the opportunity cost of capping RA in terms of increased incentives for risk selection.

  18. Immunochemical studies of early alpha chain crosslinking in human fibrin

    SciTech Connect

    Sobel, J.H.

    1987-01-01

    Immunochemical studies were conducted to characterize the earliest covalent ..cap alpha.. chain associations catalyzed by Factor XIII/sub a/ during in vitro fibrin formation. Preparations of purified fibrinogen and Factor XIII were incubated under crosslinking conditions for increasing periods of time and the resulting fibrins subjected to immunoblotting using the characterized monoclonal antibodies, F-103 (anti-A..cap alpha.. number259-276) and F-102 /anti-A..cap alpha.. number540-554). Data obtained for the incorporation of monomeric ..cap alpha.. chains into higher molecular weight immunoreactive species indicate that ..cap alpha.. chain crosslinking begins within the first ten minutes of clotting. Early crosslinked ..cap alpha.. chain species that formed within thirty minutes of in vitro crosslinking were isolated from preparations of reduced, /sup 3/H-S-carboxymethylated fibrin by Sepharose CL-4B gel filtration. Cyanogen bromide treatment of this material resulted in the release of crosslinked and non-crosslinked fragments which could be separated from one another by Sephadex G-150 gel filtration. Derivatives of interest were identified in the column effluent by immunoblotting with F-103 and F-102 and by radioimmunoassays in which polyclonal antisera that detect the known (A) ..cap alpha.. chain crosslinking regions, number241-476 (CNRr VIII) and number518-584 (CNBr X), were employed.

  19. 4. CAP; SHOWS TRANSITIONAL FRAMING OF CAP ROOF WITH THREE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. CAP; SHOWS TRANSITIONAL FRAMING OF CAP ROOF WITH THREE PAIR OF RAFTERS MORTISED INTO A BOSS; BRAKE WHEEL AND WINDSHAFT - Hook Windmill, North Main Street at Pantigo Road, East Hampton, Suffolk County, NY

  20. 3. CAP; CONICAL CAP HAS BOWED RAFTERS MORTISED INTO A ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. CAP; CONICAL CAP HAS BOWED RAFTERS MORTISED INTO A BOSS; ALSO SEEN ARE THE BRAKE WHEEL, WINDSHAFT AND TOP BEARING OF THE UPRIGHT SHAFT - Hayground Windmill, Windmill Lane, East Hampton, Suffolk County, NY

  1. Cradle Cap (For Parents)

    MedlinePlus

    ... cap will have skin that is a little red or itchy, and some might even have hair loss, though the hair usually grows back after ... parts of the body the rash is causing hair loss or becomes itchy the affected skin becomes firm and red, starts to drain fluid, or feels warm, which ...

  2. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site]

    This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.

    In the images of the Martian north polar cap we can see these same type of winds. Notice the streamers of dust moving downslope over the darker trough sides, these streamers show the laminar flow regime coming off the cap. Within the trough we see turbulent clouds of dust, kicked up at the trough base as the winds slow down and enter a chaotic flow regime.

    The horizontal lines in these images are due to framelet overlap and lighting conditions over the bright polar cap.

    Image information:VIS instrument. Latitude 86.5, longitude 57.4 East (302.6 West). 40 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is

  3. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site]

    This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.

    In the images of the Martian north polar cap we can see these same type of winds. Notice the streamers of dust moving downslope over the darker trough sides, these streamers show the laminar flow regime coming off the cap. Within the trough we see turbulent clouds of dust, kicked up at the trough base as the winds slow down and enter a chaotic flow regime.

    The horizontal lines in these images are due to framelet overlap and lighting conditions over the bright polar cap.

    Image information: VIS instrument. Latitude 86.5, Longitude 64.5 East (295.5 West). 40 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation

  4. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site]

    This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.

    In the images of the Martian north polar cap we can see these same type of winds. Notice the streamers of dust moving downslope over the darker trough sides, these streamers show the laminar flow regime coming off the cap. Within the trough we see turbulent clouds of dust, kicked up at the trough base as the winds slow down and enter a chaotic flow regime.

    The horizontal lines in these images are due to framelet overlap and lighting conditions over the bright polar cap.

    Image information: VIS instrument. Latitude 84.2, Longitude 57.4 East (302.6 West). 40 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation

  5. North Polar Cap

    NASA Technical Reports Server (NTRS)

    2004-01-01

    [figure removed for brevity, see original site]

    This week we will be looking at five examples of laminar wind flow on the north polar cap. On Earth, gravity-driven south polar cap winds are termed 'catabatic' winds. Catabatic winds begin over the smooth expanse of the cap interior due to temperature differences between the atmosphere and the surface. Once begun, the winds sweep outward along the surface of the polar cap toward the sea. As the polar surface slopes down toward sealevel, the wind speeds increase. Catabatic wind speeds in the Antartic can reach several hundreds of miles per hour.

    In the images of the Martian north polar cap we can see these same type of winds. Notice the streamers of dust moving downslope over the darker trough sides, these streamers show the laminar flow regime coming off the cap. Within the trough we see turbulent clouds of dust, kicked up at the trough base as the winds slow down and enter a chaotic flow regime.

    The horizontal lines in these images are due to framelet overlap and lighting conditions over the bright polar cap.

    Image information: VIS instrument. Latitude 84.3, Longitude 314.4 East (45.6 West). 40 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically