Grafting onto wool: 10. Quantitative analysis of the high-angle X-ray diffraction intensity from grafted and ungrafted wool keratin

The equatorial and the meridional scattering intensities at prominent reflections (0.98 nm, 0.465 nm and 0.51 nm) of X-rays by the keratin in grafted fibres stretched in water have been quantitatively determined. Introduction of poly(methyl methacrylate) into the fibres leads to a marked decrease in -content. It is suggested that the two types of -helical component present in the wool fibres are identical with respect to the ease of unfolding of the -helix during fibre extension. During extension, no significant difference was observed for the rates of production of β-materials by native and grafted fibres. On the basis of these experimental findings, the β transformation of the keratin system is discussed.     

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Comb polymers prepared by ATRP from hydroxypropyl cellulose

Hydroxypropyl cellulose (HPC) was used as a core molecule for controlled grafting of monomers by ATRP, the aim being to produce densely grafted comb polymers. HPC was either allowed to react with an ATRP initiator or the first generation initiator-functionalized 2,2-bis(methylol)propionic acid dendron to create macroinitiators having high degrees of functionality. The macroinitiators were then "grafted from" using ATRP of methyl methacrylate (MMA) or hexadecyl methacrylate. Block copolymers were obtained by chain extending PMMA-grafted HPCs via the ATRP of tert-butyl acrylate. Subsequent selective acidolysis of the tert-butyl ester moieties was performed to form a block of poly(acrylic acid) resulting in amphiphilic block copolymer grafts. The graft copolymers were characterized by 1H NMR and FT-IR spectroscopies, DSC, TGA, rheological measurements, DLS, and tapping mode AFM on samples spin coated upon mica. It was found that the comb (co)polymers were in the nanometer size range and that the dendronization had an interesting effect on the rheological properties.     

L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.     

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 2: Non-isothermal conditions

β-Galactosidase has been immobilised through spacers of different length on nylon membranes grafted with glycidyl methacrylate. Hexamethylendiamine, ethylendiamine or hydrazine have been separately used as spacers.

The behaviour of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT.

Comparison of the enzyme reaction rates under isothermal and non-isothermal conditions resulted in percentage activity increases (PAI) and reduction of the production time (τ_r) proportional to the size of the applied ΔT. Both these parameters increased with the increase of the spacer length.

Results have been discussed in the frame of reference of the process of thermodialysis which reduces the limitations to the diffusion of substrate and reaction products across the catalytic membrane, limitations introduced by the grafting and immobilisation process.

The advantages of employing non-isothermal bioreactors in biotechnological productive processes have been outlined.     

Hydroxy functional acrylate and methacrylate monomers prepared via lipase—catalyzed transacylation reactions

Candida antarctica lipase B (CAL-B, Novozyme 435) catalyzes the transacylation of methyl acrylate and methyl methacrylate with diols and triols in 2-methyl-2-butanol at 50 °C. Under the experimental conditions, up to 70 mol% of the acyl donor methyl acrylate was converted. Methyl methacrylate is the less efficient acyl donor (up to 60 mol%) due to the higher sterical hindrance in the enzymatic transacylation. Under the reaction conditions high yields of the mono-acylated products are obtained, which contain minor amounts of bis(meth)acrylates. In addition it was observed that Novozyme 435 catalyzes regioselectively the acylation of the primary hydroxyl groups. In comparison with the chemical catalyzed route no selectivity was observed for unsubstituted diols. For substituted diols more mono-acylated product was formed in the lipase-catalyzed reaction than in the chemical catalyzed reaction.     

Enhanced production of cyclomaltooctaose (γ-cyclodextrin) through selective complexation with C12 cyclic compounds

Enhanced yields of cyclomaltooctaose (γ-cyclodextrin, γ-CD) were produced by treating, incrementally, starch or maltooligosaccharide mixtures of 22 with Bacillus macerans cyclodextrin glucanotransferase (EC 2.4.1.19) in the presence of cyclododecanone, cyclododecanol, cyclododecanemethanol, cyclododecyl methyl ether, and cyclododecane. Maximum yields achieved through use of these complexants were 50, 32, 26, 34, and 17%, respectively. Cyclododecene, N-cyclododecylacetamide, and C₁₁ cyclic compounds favored production of β-CD. Complexants were removed from their CD complexes either by azeotropic distillation or by ether extraction from aqueous media at high pH.     

Wool Fibres: Sectioning and Staining, Differentiation of Ortho and Paracortex

A double embedding technique for tangential sectioning of hair and wool fibres is as follows: The cleaned fibre bundle is attached to a U-shaped, 16 gauge, tinned-copper wire frame with collodion adhesive, soaked in 6% nitrocellulose for 1 hr, and treated with chloroform for 2 hr. The hardened bundle is then cut fom the wire support and embedded in paraffin-beeswax, 95:5. Sectioning is at 6-8 μ. The use of 2% orange G or saturated aqueous picric acid for quantitative study of the fibres, and the demonstration of wool fibre cortical fractions by staining with polychrome methylene blue after oxidation of the sectioned fibres in a solution of formic acid (98/100 w/v) 25 ml; distilled water, 65 ml; and H₂O₂ (30% w/v), 10 ml, for 1 hr, is recommended.     

ATRP grafting from cellulose fibers to create block-copolymer grafts

Cellulose fibers, in the form of a conventional filter paper, have been modified by reacting the hydroxyl groups on the fiber surface with 2-bromoisobutyryl bromide, followed by grafting using ATRP conditions. The papers were first grafted with methyl acrylate (MA), rendering the paper very hydrophobic as reported in an earlier work. The papers were analyzed by gravimetry, FT-IR, ESCA, and AFM. To verify that the polymerization from the surface was "living", a second layer of another, hydrophilic, polymer, 2-hydroxyethyl methacrylate (HEMA), was grafted upon the PMA layer, creating a block-copolymer graft from the fibers. After the layer of PHEMA had been attached, contact angle measurements were no longer possible, because of the absorbing nature of PHEMA-grafted layer. This indicates that a copolymer had indeed been formed on the surface. FT-IR showed a large increase in carbonyl content after the PHEMA-grafting, which further proves that a layer of PHEMA was attached to the PMA layer. This goes to show that the hydrophilic/hydrophobic behavior of a cellulose surface can be tailored by the use of "living"/controlled radical polymerization methods such as ATRP.     

Incorporation of the antibacterial agent, miconazole nitrate into a cellulosic fabric grafted with β-cyclodextrin

The aim of the study was to investigate the incorporation of the antibacterial agent, miconazole nitrate into cyclodextrin cavities covalently bonded onto cloth fibers. The cellulosic fabric was grafted with β-cyclodextrin molecules through reaction with monochlorotriaziny β-cyclodextrin (MCT-β-CD). The suitable bonded reaction conditions were found to be MCT-β-CD 60–100 g/L, catalyst Na₂CO₃ 50–60 g/L, the reaction temperature 150–160 °C and the reaction time 5–8 min.

The modified and unmodified fabrics were characterized by UV spectrophotometry. The level of miconazole nitrate entrapped in the fabrics were determined by HPLC and was founded to be much higher (0.458% w/w) for the textile functionalized with MCT-β-CD compared to the unmodified fabric (0.056% w/w). The antibacterial abilities measured by shaker flask method showed that the antibacterial property was markedly enhanced by impregnation with miconazole nitrate of the MCT-β-CD grafted textile. The finished fabric kept the antibacterial abilities more than 70% even after washing 10 times, while the antibacterial activity of the unmodified textile was almost lost.     

Enzyme-mediated coupling of a bi-functional phenolic compound onto wool to enhance its physical,mechanical and functional properties

Wool fibres have been modified with nordihydroguaiaretic acid (NDGA) to improve their performance at use. This water insoluble bi-functional phenolic compound has been grafted on wool through a laccase enzyme catalyzed reaction in an aqueous–ethanol mixture. The capacity of laccase to oxidise NDGA in this aqueous–organic medium has been studied electrochemically. The increase of CH₂, CH₃ and aromatic groups signal in the DRIFT spectra, together with SEM images of the enzymatically modified fabrics confirmed the covalent grafting of NDGA on wool. This one step enzymatic process for grafting of NDGA improved the physical and mechanical properties of wool fabrics such as shrink resistance, crease recovery and tensile strength. Furthermore, the NDGA imparted to the textile material strong antioxidant activity and UV protection.     

Effects of alkyl chain lengths of gallates upon enzymatic wool functionalisation

The covalent grafting of alkyl gallates on wool through a laccase catalysed reaction in 80/20 (v/v, %) aqueous–ethanol mixture provided in a one-step process a multifunctional textile material with antioxidant, antibacterial and water repellent properties. Gallic acid and its alkyl esters ethyl, propyl, octyl and dodecyl gallate have been enzymatically grafted on wool fibres in order to study the effect of alkyl chain length on wool functional modification. The capacity of laccase to oxidise these phenolic compounds in an aqueous–organic medium has been verified by electrochemical techniques. The increase of CH₂, CH₃ groups in the FTIR spectra, together with the XPS analysis of the enzymatically modified fabrics confirmed the covalent grafting of ester gallates on wool. The result obtained in this work for antibacterial, water repellent as well as antioxidant properties show that the length of the alkyl chain of gallates molecule play an important role on wool functionalisation.     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of - and β-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native -subunits of both enzymes were primarily ₂-dimers and tended to aggregate. Most E. coli enzyme β-subunits were monomeric and only a small fraction was represented by β₂-dimers. All thermophilic β-subunits were β-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA^Phe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β-subunits. -Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.     

The biotransformation of methyl ent-15-oxokaur-16-en-19-oate by Rhizopus stolonifer and Mucor plumbeus

Incubation of methyl ent-15-oxokaur-16-en-19-oate with Rhizopus stolonifer and Mucor plumbeus gave methyl ent-7β,11-dihydroxy-15-oxokauran-19-oate and methyl ent-7β,16β-dihydroxy-15-oxokauran-19-oate, respectively.     

Methacrylate as an Embedding Medium for Woody Tissues

Methacrylate can be readily infiltrated into woody tissues. After infiltration, the tissue is transferred to a polymerizing mixture of 95:5 butyl methacrylate to methyl methacrylate by volume. For each 100 ml of polymerizing mixture, 2 grams of Luperco CDB catalyst are added. The hardness of the matrix may be increased by increasing the proportion of methyl methacrylate. Polymerization is accomplished by 2 days in a 50° C oven or 2 days in a small ultraviolet radiation chamber (42° C), the latter being the technique of choice. The blocks can be sectioned readily at 6 μ to more than 30 μ. Sectioning is facilitated by keeping the block wet with a 1:1 glycerol-alcohol solution. Best preparations are obtained when the matrix is removed after sectioning; however, staining in safranin O-fast green FCF may be-accomplished through the matrix. The technique is very rapid, convenient to use, and has produced excellent preparations from several species of woody plants.     

Effects of intravenous infusion of mimosine on wool growth of Merino sheep.

Merino sheep were given continuous intravenous infusions of L-mimosine for periods of 1 1/2, 2 or 21 days; efficacy as a defleecing procedure and effects on subsequent wool growth were measured. In addition, the amino acids tyrosine, phenylalanine and cystine were investigates as antagonists to the effects of mimosine. Infusions for 1 1/2 or 2 days at the daily rate of 80-120 mg/kg caused a cessation of wool growth by 1 1/2-2 days from the start of infusion, and all sheep were subsequently defleeced. It was estimated that, on average, fibre growth stopped for 10 1/2-13 days in four sheep after a 2-day infusion, and for 5 1/2 and 9 1/2 days in two sheep after an infusion for 1 1/2 days. There was considerable variation in the time taken for new fibres to recommence growth. During the period 3-5 weeks after infusion of mimosine, length growth rate was consistently greater than the pretreatment rate. Likewise, fibre diameter was greater in three out of the four sheep. As a result, the volume growth rate of fibres was greater post-treatment than it was pretreatment. Infusion for 3 weeks at the daily rate of 21-24 mg/kg did not stop wool growth. However, both length growth rate and fibre diameter were considerably depressed, and after 12 days' infusion, fibre diameter and volume growth rate were reduced to less than half the pretreatment values, and wool fibres were very weak. After the mimosine infusion stopped, fibre diameter increased to above pretreatment values and remained ther for the period of 2-3 weeks studied. The concurrent infusion of tyrosine, phenylalanine or cystine with mimosine failed to prevent any of the effects of mimosine on wool growth.     

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 mug/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.     

Single nucleotide polymorphism for animal fibre identification

Animal fibres are highly valuable industrial products often adulterated during marketing. Currently, there is no precise method available to identify and differentiate the fibres. In this study, a PCR-RFLP technique was exploited to differentiate cashmere and wool fibres derived from goat and sheep, respectively. The presence of DNA in animal hair shafts has enabled the isolation of DNA from scoured cashmere and wool fibres. The mitochondrial cytochrome b sequences of both species were amplified by PCR using primers designed from conserved regions. The polymorphism observed between the two species was detected by restricting the amplified product by endonucleases viz., BamH1 and Ssp1. The RFLP profile clearly distinguishes the cashmere and wool fibres and this technique can also be exploited to test adulteration in animal fibres qualitatively.     

Cell fouling resistance of polymer brushes grafted from ti substrates by surface-initiated polymerization: effect of ethylene glycol side chain length

This paper presents a comparative study on the antifouling properties of poly(ethylene glycol) (PEG)-based polymer coatings prepared by surface-initiated polymerization (SIP). Three types of poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMEMA) polymer thin films of approximate 100 nm thickness were grafted from a catechol initiator that was immobilized on a Ti substrate. OEGMEMA monomers containing side chains of 4, 9, and 23 EG units were used in surface-initiated atom transfer radical polymerization (SI-ATRP) to form POEGMEMA-4, -9, and -23 polymer brushes. The chemical composition, thickness, and wettability of the polymer brushes were characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, and static water contact angle measurements, respectively. The dependence of antifouling performance on EG side chain length was systemically tested and compared by 3T3 fibroblast cell adhesion assays. Results from 4-h cell culture experiments revealed the complete absence of cell attachment on all the grafted Ti substrates. Excellent cell fouling resistance continued with little dependence on EG side chain length up to three weeks, after which long-term antifouling performance depended on the EG chain length as the grafted samples reached confluent cell coverage in 7, 10, and 11 weeks for POEGMEMA-4, -9, and -23, respectively.