Production of poly-D(-)-3-hydroxybutyrate and poly-D(-)-3-hydroxyvalerate by strains ofAlcaligenes latus

Alcaligenes latus strains can accumulate poly-D(-)-3-hydroxybutyrate (PHB) up to about 85% of cell dry weight. The abilities to store poly-D(-)-3-hydroxyvalerate (PHV) of three strains ofA. latus were investigated. With Na-propionate as PHV precursor, strainA. latusDSM 1122 had better PHV accumulation ability than strainsA. latusDSM 1123 and 1124. StrainA. latus DSM 1123 could store PHV when Na-valerate but not Na-propionate served as the PHV precursor. PHB and PHV accumulation byA. latus DSM 1124 rapidly increased when propionic acid and acetic acid were together added to the fermentor. This increase was not obtained in the culture shaker flask and fermentor growing the same strain when Na-propionate alone served as a PHV precursor.     

Nanocomposites based on poly-D,L-lactide and multiwall carbon nanotubes

A possibility of poly-D,L-lactide modification by multiwall carbon nanotubes (MWCNT) has been shown. MWCNT were prepared from methane-air mixture upon atmospheric pressure without catalyst on high voltage atmospheric pressure discharge plasma set-up. According to scanning and transmission electronic microscope data carbon nanotubes diameters were within 12-60 nm. Quantities of MWCNT incorporated did not exceed 0.5%. Nanocomposites were obtained by sonification of mixture of a poly-D,L-lactide solution in chloroform and MWCNT followed by film casting on glass substrates. Tensile strength and thermomechanical properties of the dried composite films were investigated. Introduction of MWCNT into poly-D,L-lactide has been shown to cause the enhanced polymer stability to thermal oxidative destruction. Taking into account the results obtained one could anticipate that implants from nanocomposites of poly-D,L-lactide with MWCNT would be dispersed in a living organism more slowly as compared to implants from pure poly-D,L-lactide without additives.     

Hydrogen-tritium exchange of the random chain polypeptide

The Hydrogen–tritium exchange character of poly-D ,L -alanine was studied in detail as a model for the hydrogen exchange behavior of the unhindered, polymeric peptide group. The random chain nature of poly-D ,L -alanine was evident in the uniformity of exchange rate of all its hydrogens and in the similarity between this rate and that of random chain poly-D ,L -lysine and other known, unhindered secondary amide groups. An equilibrium isotope effect favoring the binding of tritium over protium to the extent of 21% was measured. Specific acid and base catalysis of the exchange and the absence of detectable general catalysis were demonstrated. Apparent energy of activation is 17 kcal/mole for deprotonation, largely due to dependence of K_w on temperature, and 15 kcal/mole for protonation, which correlates with the extreme apparent pK. The hydrogen –tritium exchange half-time rate; of poly-D ,L -alamine at any pH and temperature (T: °C) is given by the equation:      

Structural investigation of poly-L,D-proline, a synthetic ion channel across bilayer membranes: crystal structure and molecular conformation of two tetra-D,L-proline derivatives

The crystal structures and molecular conformations of two tetraproline derivatives with alternating configurations Boc(D -Pro,L -Pro)₂OH and Boc(D -Pro,L -Pro)₂OCH₃ are investigated in connection with the ability of the homologous polymer to selectively increase (as an ion channel) the ion permeability across bilayer membranes. Both tetramers are characterized by the cis-trans alternating conformation of the peptide bonds, which formally transforms in a turn of the poly-D ,L -proline channel after a cis-trans change of the central peptide residue.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study

The theoretical yield of poly-D(-)-3-hydroxybutyrate (PHB) has been estimated from the biochemical pathway leading to PHB when a carbohydrate (glucose), a C(1) compound (methanol), a C(2) compound (acetic acid), or a C(4) compound (butyric acid) is used as a carbon source. In estimating the yield, recycling (or regeneration) of NADP(+)/ (NADPH + H(+)) and NAD(+) /(NADH + H(+)) have been taken into account. A special emphasis is made on te regeneration of NADPH, which is the coenzyme of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADPH-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. An equation which predicts the overall yield of PHB when non-PHB residual biomass is actually formed has been derived as a function of both the theoretical yield and PHB content of the dry cell mass. The ratio of the overall yield to the theoretical yield is roughly proportional to the PHB content. (c) 1993 John Wiley & Sons, Inc.     

Synthesis and characterization of polyamides containing unnatural amino acids

We describe the synthesis of several polyamides that retain the secondary structure of proteins and contain derivatizable side chains. The derivatizable side chain allows for further reaction of the polymer chain (e.g., chain cross-linking or addition of pendant groups). Polymers of α-amino acids containing a terminal unsaturated bond on the side chain have been synthesized. Poly-L -pentenyl glycine, poly-L -propargyl glycine, and poly-L -allyl glycine were synthesized chemically via Leuchs' anhydrides and enzymatically using subtilisin Carlsberg. Poly-L -propargyl glycine and poly-D ,L -allyl glycine folded into the β-sheet configuration whereas poly-L -pentenyl glycine assumed a helical conformation. The secondary structure of poly-L -allyl glycine and poly-D ,L -pentenylglycine could not be determined conclusively. Comparison of properties between the polymers obtained chemically and enzymatically is provided. © 1995 John Wiley & Sons, Inc.     

Gelatin-based microcarriers as embryonic stem cell delivery system in bone tissue engineering: an in-vitro study

Mouse embryonic stem cells were cultured on commercially available biodegradable macroporous microcarriers. A culture period of 1-2 weeks was needed to colonize the microcarriers. Embryonic stem cells retained their pluripotency for up to 14 days when cultured in medium supplemented with leukemia inhibitory factor. Replacing this medium by differentiation medium for 2 weeks initiated osteogenic differentiation. Encapsulation of the cell-loaded microcarriers in photopolymerizable polymers (methacrylate-endcapped poly-D,L-lactide-co-caprolactone), triacetin/hydroxyethylmethacrylate (HEMA) as solvent and with/without gelatin as porogen, resulted in a homogeneous distribution of the microcarriers in the polymer. As observed by transmission electron microscopy, viability of the cells was optimal when gelatin was omitted and when using triacetin instead of HEMA.     

Vibrational frequencies and modes of alpha-helix

Dichroic properties of the far-infrared absorption bands of the right-handed α-helix of poly-L -alanine were measured. The normal vibration frequencies of this structure were calculated. The assignments of bands were made and the vibrational modes dis-cussed. The frequencies of the α-helix vibrations with various phase differences were calculated. The frequencies of accordionlike vibrations and Young's modulus of the α-helix were estimated. The vibrational frequency for the right-handed α-helices of poly-D -alanine and poly(L -α-amino-n-butyric acid) were calculated, and the results were used for the interpretation of the spectra of copoly-D ,L -alanines and poly(L -α-amino-n-butyric acid). For the latter compound the existence of the rotational isomers in the side chain was strongly suggested. The vibrational modes of the bands characteristic of the α-helix were discussed with regard to the results of the normal coordinate treatment.     

Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material

Copper particles were incorporated into nanofibers during the electrospinning of poly-D,L-lactide (PDLLA) and poly(ethylene oxide) (PEO). The ability of the nanofibers to prevent Pseudomonas aeruginosa PA01 and Staphylococcus aureus (strain Xen 30) to form biofilms was tested. Nanofibers containing copper particles (Cu-F) were thinner (326 ± 149 nm in diameter), compared to nanofibers without copper (CF; 445 ± 93 nm in diameter). The crystalline structure of the copper particles in Cu-F was confirmed by X-ray diffraction (XRD). Copper crystals were encapsulated, but also attached to the surface of Cu-F, as shown scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM), respectively. The copper particles had no effect on the thermal degradation and thermal behaviour of Cu-F, as shown by thermogravimetric analysis (TGA) and differential scanning calorimeter (DSC). After 48 h in the presence of Cu-F, biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 was reduced by 41% and 50%, respectively. Reduction in biofilm formation was ascribed to copper released from the nanofibers. Copper-containing nanofibers may be incorporated into wound dressings.     

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

Distinct role of phosphatidylinositol 3-kinase and Rho family GTPases in Vav3-induced cell transformation, cell motility, and morphological changes

Vav3 is a member of the Vav family of guanine nucleotide exchange factors (GEFs) for the Rho family GTPases. Deleting the N-terminal calponin homology (CH) domain to generate Vav3-(5-10) or deleting both the CH and the acidic domain to generate Vav3-(6-10) results in activating the transforming potential of Vav3. Expression of either the full-length Vav3 or its truncation mutants led to activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), and Stat3. We investigated the requirement of these signaling molecules for Vav3-induced focus formation and found that PI3K and its downstream signaling molecules, Akt and p70 S6 kinase, are required, albeit to varying degrees. Inhibition of PI3K had a more dramatic effect than inhibition of MAPK on Vav3-(6-10)-induced focus formation. Activated PI3K enhanced the focus-forming activity of Vav3-(6-10). Wild type FAK but not Y397F mutant FAK enhanced Vav3-(6-10)-induced focus formation. Dominant negative (dn) mutant of Stat3 resulted in a 60% inhibition of the focus-forming activity of Vav3-(6-10). Moreover, Rac1, RhoA, and to a lesser extent, Cdc42, are important for Vav3-(6-10)-induced focus formation. Constitutively activated (ca) Rac synergizes with Vav3-(6-10) in focus formation. This synergy requires signaling via Rho-associated kinase (ROK) and p21-activated kinase (PAK), downstream effectors of Rac. Consistently, a ca PAK mutant enhanced, whereas a dn PAK mutant inhibited the focus-forming ability of Vav3-(6-10). Despite having potent focus-forming ability, Vav3-(6-10) has very weak colony-forming ability. This colony-forming ability of Vav3-(6-10) can be enhanced dramatically by co-expressing an activated PI3K and to some extent by co-expressing an activated PAK mutant or c-Myc. Interestingly, inhibition of PI3K and MAPK had no effect on the ability of either wild type or Vav3-(6-10) to induce cytoskeletal changes including formation of lamellipodia and filopodia in NIH 3T3 cells. Over expression of Vav3 or Vav3-(6-10) resulted in an enhancement of cell motility. This enhancement was dependent on PI3K, Rac1, and Cdc42 but not on Rho. Overall, our results show that signaling pathways of PI3K, MAPK, and Rho family GTPases are differentially required for Vav3-induced focus formation, colony formation, morphological changes, and cell motility.     

Polyionic regulation of cartilage development: promotion of chondrogenesis in vitro by polylysine is associated with altered glycosaminoglycan biosynthesis and distribution.

The development of cartilage nodules in cultures of chick limb bud mesenchyme (Hamburger-Hamilton stages 23/24) is significantly promoted when the culture medium is supplemented with (poly-L-lysine (PL) (M(r) greater than or equal to 14K) (San Antonio and Tuan, 1986. Dev. Biol. 115: 313). Here we present findings consistent with the hypothesis that PL may promote chondrogenesis by interacting electrostatically with sulfated glycosaminoglycans (GAGs): (1) poly-L-ornithine, poly-L-histidine, poly-D,L-lysine, and lysine-containing heteropolypeptides stimulate chondrogenesis in proportion to their contents of cationic residues; (2) the effects of PL are diminished when limb mesenchyme cultures are supplemented with exogenous GAGs, including heparin, dermatan sulfate, and chondroitin sulfate; (3) in high density cultures of limb bud mesenchyme, the release of sulfated macromolecules, but not of proteins in general, into the culture medium was significantly inhibited by PL (398K M(r)) treatment, and a net increase in total GAG content of the PL-treated cultures was observed; and (4) in monolayer cultures of cells derived from other chick embryonic tissues, including liver, skeletal muscle, and calvaria, PL treatment promoted the cell layer-associated retention of sulfated GAG. These effects were not observed using the nonstimulatory, low M(r) PL (4K). Based on the above findings and those from previous studies, it is proposed that PL may promote chondrogenesis by interacting electrostatically with cartilage GAGs, thus trapping the extracellular matrix around the newly emerging cartilage nodules and thereby stabilizing their growth and differentiation.     

Epitope specificity of hemagglutinating monoclonal anti-B antibodies]

Fine epitope specificity of ten monoclonal antibodies (MA) agglutinating red blood cells B was studied. Three methods were used: 1) inhibition of MA binding to natural antigen by synthetic oligosaccharides (OS) and their polyacrylamide conjugates, 2) direct MA binding to a series of synthetic OS-polyacrylamide conjugates differing in carbohydrate epitope density, 3) direct MA binding to the affinity sorbents. It is shown that all antibodies studied prefer trisaccharide B determinant Gal alpha 1-3(Fuc alpha 1-2) Gal independently of their ability to discriminate serological subgroups of B erythrocytes (B, Bweak, B3). The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells B subgroups is discussed. Of an interest is that MAs which are able to agglutinate any B subgroups also bing the synthetic tetrasaccharide Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc, a B type 3 determinant.     

Effect of Zn2+ on Bacterial Conjugation: Increase in Ability of F− Cells to Form Mating Pairs

Incubation of F(-) cells at 37 C with 10(-3) M Zn(2+) before mating was found to increase the ability of F(-) cells to form mating pairs when mated. This increased pair-forming ability is persistent, at least for the duration of mating. The F(-) cells with increased pair-forming ability obtained by the 10(-3) M Zn(2+) treatment can form mating pairs efficiently with males from which F pili were removed or inactivated with 10(-3) M Zn(2+).     

The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system

Sucrose-6-phosphate synthase (SPS) is a target for 14-3-3 protein binding in plants. Because several isoforms of the 14-3-3 protein are expressed in plants, I investigated which isoforms have the ability to bind SPS. Two 14-3-3 isoforms (T14-3d and a novel isoform designated T14-3 g) were found to interact with SPS from tobacco (Nicotiana tabacum L.) in a two-hybrid screen. To further address the question of isoform specificity of 14-3-3s, four additional isoforms were tested for their ability to interact with SPS in the yeast two-hybrid system. The results clearly revealed large differences in affinity between individual 14-3-3 isoforms toward SPS. Deletion analysis suggested that these differences were mediated by the variable C-terminus of 14-3-3s. Site-directed mutagenesis of candidate 14-3-3 binding sites on SPS demonstrated that interaction could be independent of a phosphorylated serine residue within conserved binding motifs in the yeast system. These findings suggest that the large number of 14-3-3 isoforms present in plants reflects functional specificity.     

Structural and functional variations in human apolipoprotein E3 and E4

There are three major apolipoprotein E (apoE) isoforms. Although APOE-epsilon3 is considered a longevity gene, APOE-epsilon4 is a dual risk factor to atherosclerosis and Alzheimer disease. We have expressed full-length and N- and C-terminal truncated apoE3 and apoE4 tailored to eliminate helix and domain interactions to unveil structural and functional disturbances. The N-terminal truncated apoE4-(72-299) and C-terminal truncated apoE4-(1-231) showed more complicated or aggregated species than those of the corresponding apoE3 counterparts. This isoformic structural variation did not exist in the presence of dihexanoylphosphatidylcholine. The C-terminal truncated apoE-(1-191) and apoE-(1-231) proteins greatly lost lipid binding ability as illustrated by the dimyristoylphosphatidylcholine turbidity clearance. The low density lipoprotein (LDL) receptor binding ability, determined by a competition binding assay of 3H-LDL to the LDL receptor of HepG2 cells, showed that apoE4 proteins with N-terminal (apoE4-(72-299)), C-terminal (apoE4-(1-231)), or complete C-terminal truncation (apoE4-(1-191)) maintained greater receptor binding abilities than their apoE3 counterparts. The cholesterol-lowering abilities of apoE3-(72-299) and apoE3-(1-231) in apoE-deficient mice were decreased significantly. The structural preference of apoE4 to remain functional in solution may explain the enhanced opportunity of apoE4 isoform to display its pathophysiologic functions in atherosclerosis and Alzheimer disease.     

Antioxidant activity of an aminothiazole compound: possible mechanisms

Oceans are among the richest natural sources of many bioactive compounds. Several of these compounds have shown pharmacological activities for many diseases. Dendrodoine (5-[(3-N-dimethylamino)-1,2,4-thiadiazolyl]-3-indanyl methanone) is an alkaloid extracted from the marine tunicate Dendrodoa grossularia. Aminothiazoles have a wide range of biological activities including anti-tumor and antioxidant properties. The aim of our study was to examine the antioxidant ability of an aminothiazole derivative, dendrodoine analogue (DA) [(4-amino-5-benzoyl-2-(4-methoxy phenylamino) thiazole] which has been chemically synthesized and is similar to dendrodoine. In all the biochemical assays used in our study, corresponding to different levels of protection, DA showed concentration dependent antioxidant ability. DA (3.07 microM) showed an ability to inhibit 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical formation to the extent of 0.17 microM of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). The ferric complex reducing ability of 3.07 microM DA was equivalent to 110 microM Trolox. 3.07 microM DA gave 84% protection against deoxyribose degradation, a measure of hydroxyl radical scavenging. DA also has an ability to scavenge NO radical, 3.07 microM DA effecting 20% scavenging. Concentration dependent inhibition of lipid peroxidation and protein oxidation induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and ascorbate-Fe2+ was observed with low concentrations of DA (1.5-3.07 microM). Mechanistic studies using pulse radiolysis revealed that DA scavenges peroxyl radicals with a bimolecular rate constant of 3 x 10(8)M(-1)s(-1). Moreover, the initially formed nitrogen-centered radical gets transformed into sulfur-centered radical before furnishing any final product. Our results indicated that DA can be a free radical scavenger and potential antioxidant for future application.     

Radical trapping properties of imidazolyl nitrones

The ability of ten imidazolyl nitrones to directly scavenge free radicals (R(*)) generated in polar ((*)OH, O(*)(2)(-), SO(*)(3)(-) cysteinyl, (*)CH(3)) or in apolar (CH(3)-(*)CH-CH(3)) media has been studied. When oxygen or sulfur-centered radicals are generated in polar media, EPR spectra are not or weakly observed with simple spectral features. Strong line intensities and more complicated spectra are observed with the isopropyl radical generated in an apolar medium. Intermediate results are obtained with (*)CH(3) generated in a polar medium. EPR demonstrates the ability of these nitrones to trap radicals to the nitrone C(alpha) atom (alpha radical adduct) and to the imidazol C(5) atom (5-radical adduct). Beside the nucleophilic addition of the radical to the C(alpha) atom, the EPR studies suggest a two-step mechanism for the overall reaction of R(*) attacking the imidazol core. The two steps seem to occur very fast with the (*)OH radical obtained in a polar medium and slower with the isopropyl radical prepared in benzene. In conclusion, imidazolyl nitrones present a high capacity to trap and stabilize carbon-centered radicals.     

Identification of the bile acid-binding region in the soy glycinin A1aB1b subunit

Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114-161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the delta[138-291] and delta[130-291] mutants. The 130-138 region is rich in hydrophobic residues. In this regard, when we constructed the delta[129-134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129-134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.