Factors affecting protoplast yield of the carrageenophyte Solieria filiformis (Gigartinales,Rhodophyta)

Summary The effect of age, pH of the culture medium, pre-treatment of tissues, enzymes sources and enzymatic adaptability of phycophages fed with a monospecific diet were analyzed on the protoplast yields of the red seaweed Solieria filiformis (Kützing) Gabrielson. New apices from fast growing plants showed the highest protoplast yields. The protoplast yield decreased when the pH of the culture medium increased from 6.0 to 9.0. Crude extracts from the abalone Haliotis coccinea canariensis Nordsieck, fed with Solieria filiformis thalli for three months in combination with cellulysin, released the highest number of viable cells and protoplasts. Yields ranged from 1.0 to 8.5 x 10⁶ protoplasts per gram of fresh weight.Abbreviations AAP abalone acetone powder - Bis-Tris Bis(2hydroxyethyl)imino-tris(hydroxymethyl)methane - CMC carboxymethyl cellulose - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(ß-aminoethyl ether) NNNN-tetraacetic acid - FDA fluorescein diacetate - FW fresh weight - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane     

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

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

Protein displacement in dye-ligand chromatography using neutral and charged polymers

Displacement chromatography was demonstrated to perform separations efficiently under mass-overloaded conditions, offering advantages such as increased product recovery and purity, superior resolving power, and concentration and purification in a single processing step. The use of water-soluble polymers for protein displacement in dye-ligand chromatography was initiated in our laboratory. The polymers for displacement were selected using differences spectroscopy to monitor their interactions with a dye-ligand in solution. Non-charged polymers such as poly(N-vinyl pyrrolidone) and poly(N-vinyl caprolactam) efficiently displaced lactate dehydrogenase from porcine muscle from a Blue Sepahrose column. The latter polymer, being thermosensitive, could be easily removed from the eluate and recovered by precipitation at 45 degrees C and low-speed centrifugation. The positively charged polymer poly(ethylene imine) proved to be an even more efficient displacer. The dye-ligand column could be regenerated after application of displacer either by washing with a solution of the soluble ligand Cibacron Blue (in the case of non-charged polymers) or by washing with highly alkaline solutions containing polyanions (in the case of poly(ethylene imine)) The latter formed a soluble complex with poly(ethylene imine) and stripped the column from the polymer.     

Instant formation of molecularly imprinted poly(ethylene-co-vinyl alcohol)/quantum dot composite nanoparticles and their use in one-pot urinalysis

The high stability of quantum dots (QDots) with photoluminescence has led to their increased use as imaging approaches in biological systems to replace conventional fluorescence labels. The antibodies are generally coated on the surface of QDots to the targeting site, and molecular imprinting polymers are designed to mimic the antibodies. Hence, quantum dots can be incorporated into molecularly imprinted polymers, which provide shape and selectivity, and then respond to template rebinding by emitting quenched photoluminescence. In this study, poly(ethylene-co-ethylene alcohol) creatinine-, albumin- and lysozyme-imprinted polymers nanoparticles are synthesized via phase inversion of poly(ethylene-co-ethylene alcohol) with various ethylene mole ratios when target molecules and hydrophobic quantum dots are mixed within the polymer solution. Finally, those particles were prepared for the detection of creatinine, human serum albumin and lysozyme in real sample (urine) and compared with commercial ARCHITECT ci 8200 system.     

Hydrogels based on u.v.-crosslinked poly(ethylene oxide) – matrices for immobilization of <Emphasis Type="Italic">Candida boidinii</Emphasis> cells for xylitol production

Hydrogels based on high molecular weight poly(ethylene oxide) were synthesized by u.v.-irradiation of aqueous solutions in presence of the photoinitiator, (4-benzoylbenzyl)trimethylammonium chloride and different crosslinkers, poly(ethylene glycol), diacrylates and N,N′-methylenebisacrylamide. Candida boidinii cells were immobilized in these hydrogels and the gels were characterized in regards to gel fraction yield, degree of equilibrium swelling, shear storage and loss moduli. In addition, the number average molecular weight between crosslinks and the mesh size were estimated. The incorporated yeast cells considerably affected the viscoelastic properties of the gels. Immobilized C. boidinii cells were used for conversion of xylose to xylitol. Of the immobilized systems tested, only the system with poly(ethylene oxide) crosslinked with N,N′-methylenebisacrylamide exhibited xylitol production. The operational stability of this system was evaluated by seven repeated-batch runs performed in Erlenmeyer flasks in duration of 55 days. The progressive improvement of xylose consumption, up to 73.5%, stopped in the fifth cycle, after which it dropped to 42.7%. Although xylitol concentration never reached more than 4.2 g l⁻¹, xylitol was produced in each of the seven cycles. The cell leakage of 1.8 g l⁻¹ during the first 45 days, indicated very good stability of the system.     

Formulation and Performance Characterization of Radio-Sterilized “Progestin-Only” Microparticles Intended for Contraception

The aim of this study was to formulate and characterize a microparticulate system of progestin-only contraceptive. Another objective was to evaluate the effect of gamma radio-sterilization on in vitro and in vivo drug release characteristics. Levonorgestrel (LNG) microspheres were fabricated using poly(lactide-co-glycolide) (PLGA) by a novel solvent evaporation technique. The formulation was optimized for drug/polymer ratio, emulsifier concentration, and process variables like speed of agitation and evaporation method. The drug to polymer ratio of 1:5 gave the optimum encapsulation efficiency. Speed of agitation influenced the spherical shape of the microparticles, lower speeds yielding less spherical particles. The speed did not have a significant influence on the drug payloads. A combination of stabilizers viz. methyl cellulose and poly vinyl alcohol with in-water solvent evaporation technique yielded microparticles without any free drug crystals on the surface. This aspect significantly eliminated the in vitro dissolution “burst effect”. The residual solvent content was well within the regulatory limits. The microparticles passed the test for sterility and absence of pyrogens. In vitro dissolution conducted on the product before and after gamma radiation sterilization at 2.5 Mrad indicated no significant difference in the drug release patterns. The drug release followed zero-order kinetics in both static and agitation conditions of dissolution testing. The in vivo studies conducted in rabbits exhibited LNG release up to 1 month duration with drug levels maintained within the effective therapeutic window.     

Thermocontrol of solute permeation across polymer membrane composed of poly(N,N-dimethylaminoethyl methacrylate) and its copolymers

Polymer membranes composed ofN,N-dimethylaminoethyl methacrylate (DMAEMA) and acrylamide (AAm) (or ethyl acrylamide (EAAm)) were prepared to demonstrate the thermocontrol of solute permeation. Poly DMEMA has a lower critical solution temperature (LCST) at around 50°C in water. With the copolymerization of DMAEMA with AAm (or EAAm), a shift in the LCST to a lower temperature was observed, probably due to the formation of hydrogen bonds between the amide andN,N-dimethylamino groups. However, the temperature-induced phase transition of poly (DMAEMA-co-EAAm) did not show a similar trend to that of poly (DMAEMA-co-AAm) in the gel state. The hydrogen bonds in poly (DMAEMA-co-EAAm) were significantly disrupted with the formation of a gel network, which led to a difference in the swelling behavior of polymer gels in response to temperature. To apply these polymers to temperature-sensitive solute permeation, polymer membranes were prepared. The permeation pattern of hydrocortisone, used as the model solute, was explained based on the temperature-sensitive swelling behavior of the polymer membranes.     

Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of <Emphasis Type="Italic">mdx</Emphasis> mice

Background  

Exon skipping oligonucleotides (ESOs) of 2'O-Methyl (2'OMe) and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD). However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) are regarded as effective agents for enhanced delivery of nucleic acids in various applications.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Effect of octanoic acid on ethylene-mediated processes in Arabidopsis

We have examined whether octanoic acid (OA) one of the short chain saturated fatty acids (SCSFA), increases ethylene response in the following three ethylene-mediated processes: a) hypocotyl growth in darkness; b) formation of new flowers; c) flower abscission. These processes were examined in the presence or absence of exogenous ethylene in Arabidopsis wild type (WT) and in the ethylene-insensitive mutants, etr1-3 and ein2-1 and in the ethylene over-producer mutant eto1-1. Our results show that OA decreased hypocotyl length of WT in the absence or presence of exogenous ethylene, apparently showing that OA acts via augmentation of ethylene action. However, the hypocotyl growth inhibition could not be ascribed to increased ethylene sensitivity since application of inhibitors of ethylene synthesis (aminoethoxyvinylglycine; AVG) or action (1-methylcyclopropene;1-MCP) to WT seedlings did not prevent specifically the OA-induced growth inhibition. Also, OA inhibited hypocotyl growth in the mutants etr1-3 and ein2-1 in a similar pattern to that obtained in WT. On the other hand, OA had no effect on flower formation neither in WT, etr1-3 and eto1-1, in which ethylene reduced flower formation, nor in the ein2-1 mutant, in which ethylene had no effect. OA also did not increase flower abscission in WT or in the mutants etr1-3 and ein2-1 neither in the absence nor in the presence of ethylene. However, OA has augmented flower abscission in the mutant eto1-1 only in the absence of exogenous ethylene. This result might indicate that the effect of OA on eto1-1 is specific to this mutant and is not due to general deleterious effects inflicted by OA. Taken together, our results show that in general OA does not augment ethylene response in Arabidopsis, but it might affect ethylene action in flower abscission of the ethylene-overproducer mutant.     

Polymer displacement/shielding in protein chromatography

An overview of different applications of polymer interactions with ion-exchange and dye-affinity chromatographic matrices is presented here. The strength of interaction between the ligand and the polymer plays a crucial role in deciding the mode of chromatographic application. Charged, non-ionic and thermosensitive polymers such as poly(ethylene imine), poly(N-vinyl pyrrolidone) and poly(vinyl caprolactam) respectively, show different degrees of interaction with the dye molecules in dye ligand chromatography. Polymers, with their ability of multipoint and hence strong attachment to the chromatographic matrices, were used as efficient displacers in displacement chromatography. The polymer displacement resulted in better recoveries and sharper elution profiles than traditional salt elutions. The globule–coil transition of the thermosensitive reversible soluble–insoluble polymer, poly(vinyl caprolactam), can be exploited in dye-affinity columns for the temperature induced displacement of the bound protein. In another situation, prior to the column chromatography of crude protein extract, polymers formed complexes with the dye matrix and “shielded” the column. The polymer shielding decreased the nonspecific interactions without affecting the specific interactions of the target protein to the dye matrix.     

Effect of 8-hydroxyquinoline on the uptake of uridine and incorporation into RNA

8-hydroxyquinoline has been previously used as an inhibitor in studies on porphyrin metabolism, where it is thought to act by chelating iron. It is shown that this compound also rapidly inhibits uridine uptake of seedlings or cotyledons of the crucifer Matthiola incana R.Br. RNA synthesis is also affected but the inhibition is not as severe as reported for fission yeast.Abbreviations oligo (dT)-cellulose cellulose with oligo-deoxythymidylic acid attached - poly (A) polyadenylic acid     

Ethylene-binding characteristics in Phaseolus,Citrus, and Ligustrum plants

A number of plants were tested for their ability to bind ethylene and the number of binding sites present in each was calculated. Primary leaves of laboratory-grown beans (Phaseolus vulgaris) bound 140 dpm/g fwt (1794 dpm/g dry wt) when exposed to 1.0 Ci/1 of [¹⁴C]ethylene (110 ci/mol). Phytotron-grown leaves were less succulent but only bound 90 dpm/g fwt (1046 dpm/g dry wt). Bean roots bound 30 dpm/g fwt. Citrus and Ligustrum bound 207 and 240 dpm/g fwt, respectively. The time required to achieve equilibrium of leaves with the gas phase was 15 min for bean, 30 min for Citrus, and 30–60 min for Ligustrum. The time for 1/2 of the bound ethylene to diffuse out of the leaves was 20 min for bean, 10 min for Citrus, and 30 min for Ligustrum. The amount of ethylene needed to occupy 1/2 of the binding sites was obtained from Scatchard plots. This value (K_d) was 0.2 l/1 for bean, 0.15 for Citrus, and 0.31 for Ligustrum. The quantity of binding sites in the tissues was 2.0×10^-9 mol of binding sites/kg tissue for bean leaves, 5.7×10^-9 for Citrus leaves, and 6.8×10^-9 for Ligustrum. Pretreatment with indoleacetic acid (IAA), ehtylene, and cycloheximide (1 mg/1) had little effect on the level of ethylene-binding sites in Citrus.Contribution from the Department of Biochemistry, School of Agriculture and Life Sciences and School of Physical and Mathematical Sciences, North Carolina State University. Paper No. 8445 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601.North Carolina-Israel exchange Scholar for 1981 at the Department of Biochemistry, North Carolina State University Raleigh, North Carolina, USA     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Aminooxyacetic acid inhibits antheridiogenesis and development of Anemia phyllitidis gametophytes

Cytomorphological studies of the development of young fern gametophytes (Anemia phyllitidis) have been used to investigate combined effects of gibberellic acid and ethylene on male sex expression. ACC (the key by-product in ethylene biosynthesis pathway) was found to exert a synergetic effect on the gibberellic acid-induced antheridia formation, and this phenomenon could be related with the specific stimulation of cell growth and activity of their differentiation. To complete and verify those observations male sex expression in the fern gametophytes treated with ACC-biosynthesis inhibitor was reinvestigated. Aminooxyacetic acid (AOA) restrained antheridia formation via inhibition of cell divisions. AOA influenced the arrangement and flexibility of cellulose microfibrils in the antheridial zone cells, thus affecting cell expansion. On the other hand, the level of DNA synthesis was not reduced. Transient increase in the number of S-phase cells, followed by the accumulation of G2-phase cells led to the enhancement of cell polyploidization. All these findings correspond with the previous observations and support participation of ethylene in gibberellic acid-induced male sex expression in ferns.Abbreviations AOA Aminooxyacetic acid - CPA Cell profile area - GA Gibberellin - GA₃ Gibberellic acid     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Protein immobilization to polystyrene via long poly(ethylene lycol) chains

Human albumin has been attached to 24-hole polystyrene plates via branched poly(ethylene lycol) (PEG) spacer arms. A tetraepoxude of PEG of molecular weight (1.4-1.5) x 10(4) g/mol was reacted with the protein in solution allowing approximately one-third of the oxirane rings to react. The protein conjugate was then coupled to the long, cationic polymer poly(ethylene imine) (PEI), and the protein-PEG-PEI adduct was subsequently adsorption to unmodified polystyrene. Since the protein is linked to the surface via long, hydrophilic and nonchargedchains, interactions between the biomolecule and the surface is minimized.     

New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)–dextran derivative conjugate

Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble–insoluble poly(N-isopropylacrylamide)–dextran derivative conjugate, named poly(NIPAAm)–DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)–DD conjugation and characterization. On the other hand, the poly(NIPAAm)–DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)–DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.     

Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics

Microorganisms isolated from soil samples were screened for their ability to degrade various biodegradable polyester-based plastics. The most active strain, designated as strain TB-13, was selected as the best strain for degrading these plastics. From its phenotypic and genetic characteristics, strain TB-13 was closely related to Paenibacillus amylolyticus. It could degrade poly(lactic acid), poly(butylene succinate), poly(butylene succinate-co-adipate), poly(caprolactone) and poly(ethylene succinate) but not poly(hydroxybutylate-co-valerate). However, it could not utilize these plastics as sole carbon sources. Both protease and esterase activities, which may be involved in the degradation of plastic, were constitutively detected in the culture broth.