Enzymatic acylation of hydroxypropyl cellulose in organic media and determination of ester formation by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%.     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

Characterization of Ca2+-activated, phospholipid-dependent protein kinase C/protein kinase M in guinea pig peritoneal exudate macrophages

Protein kinase C (C-kinase) is shown to be present in the cytosolic and particulate fractions of mineral oil induced peritoneal macrophages of guinea pigs. By omission or use of a high concentration of leupeptin, three forms of the enzyme were obtained: stimulant/Ca2+/phospholipid-dependent C-kinase, eluted from DEAE 52 cellulose at 0.08-0.16 M NaCl; stimulant/Ca2+/phospholipid-independent protein kinase M (M-kinase), and Ca2+-inhibited & stimulant/phospholipid-dependent form of protein kinase, both eluted from DEAE 52 cellulose at 0.18-0.22 M NaCl. Phorbol ester or 1,2-diacylglycerol were used as stimulants. It is suggested that Ca2+-inhibited & stimulant/phospholipid-dependent protein kinase represents the in vivo form of the M-kinase in intact cells.     

Synthesis and characterization of a trifunctional aminoamide cellulose derivative

As part of an effort to synthesize a dendronized cellulose, we have synthesized a trifunctional aminoamide derivative, which is the first generation of a dendron substituent. We anticipate that a dendronized cellulose would have applications in complexing metals and could be employed as an adjuvant for drugs. The trifunctional aminoamide substituent was introduced by coupling di-tert-butyl 4-[2-(tert-butoxycarbonyl)ethyl]-4-aminoheptanedicarboxylate, BA, directly to a (carboxymethyl)cellulose (CMC) backbone and converting the tert-butyl ester peripheral groups to aminoamide substituents by use of N,N-dimethyl-1,3-propanediamine. Confirmation of the proposed chemical structure of the intermediates as well as the water-soluble aminoamide derivative (CMCBADMPDA) was obtained by Fourier transform infrared (FT-IR) and NMR spectroscopy. The degree of substitution (DS) was determined to be 0.40 +/- 0.01 by thermogravimetric analysis. Typical weight average molecular weight (M(w)), molecular weight distribution (MWD), and molecular size of the dendronized polymers were found to be 97,000, 1.7, and 17.4 nm for derivatives of a CMC with corresponding M(w), MWD, and root-mean-square radius (RMS) of 230 000, 3.2, and 24 nm. A differential refractive index (dn/dc) for the aminoamide derivative measured in aqueous 0.40 N ammonium acetate-0.01 N NaOH was found to be 0.1473. The intrinsic viscosity of the dendronized cellulose decreased significantly when compared with that of CMC, that is, 0.40 dL/g relative to 5.60 dL/g. The hydrophobicity of the CMCBADMPDA microenvironment in aqueous solution was probed by evaluating the relative fluorescence intensities of the I(373)/I(384) pyrene bands; a slightly more hydrophobic environment was observed.     

Synthesis of cellulose adipate derivatives

Cellulose esters containing adipates and other ester groups are synthesized by the reaction of commercially available cellulose esters in solution with the benzyl monoester of adipoyl chloride. The products, cellulose adipate esters in which the distal end of the adipate moiety is a benzyl ester, were easily converted to cellulose adipate derivatives by Pd-catalyzed hydrogenation. These cellulose adipate derivatives are promising biopolymers for drug delivery and other applications in which water-dispersion or swelling are desired.     

Adsorption mechanism for xanthene dyes to cellulose granules

The xanthene dyes, erythrosine, phloxine, and rose bengal, were adsorbed to charred cellulose granules. The charred cellulose granules were preliminarily steeped in ionic (NaOH, NaCl, KOH, KCl, and sodium dodecyl sulfate (SDS)), nonionic (glucose, sucrose, and ethanol), and amphipathic sucrose fatty acid ester (SFAE) solutions, and adsorption tests on the dye to the steeped and charred cellulose granules were conducted. Almost none of the dye was adsorbed when the solutions of ionic and amphipathic molecules were used, but were adsorbed in the case of steeping in the nonionic molecule solutions. Thin-layer chromatography (TLC) and the Fourier transform infra-red (FT-IR) profiles of SFAE which was adsorbed to the charred cellulose granules and extracted by ethyl ether suggested the presence of hydrophobic sites on the surface of the charred cellulose granules. We confirmed that the xanthene dyes could bind to the charred cellulose granules by ionic and hydrophobic bonds.     

Adsorption Mechanism for Xanthene Dyes to Cellulose Granules

The xanthene dyes, erythrosine, phloxine, and rose bengal, were adsorbed to charred cellulose granules. The charred cellulose granules were preliminarily steeped in ionic (NaOH, NaCl, KOH, KCl, and sodium dodecyl sulfate (SDS)), nonionic (glucose, sucrose, and ethanol), and amphipathic sucrose fatty acid ester (SFAE) solutions, and adsorption tests on the dye to the steeped and charred cellulose granules were conducted. Almost none of the dye was adsorbed when the solutions of ionic and amphipathic molecules were used, but were adsorbed in the case of steeping in the nonionic molecule solutions. Thin-layer chromatography (TLC) and the Fourier transform infra-red (FT-IR) profiles of SFAE which was adsorbed to the charred cellulose granules and extracted by ethyl ether suggested the presence of hydrophobic sites on the surface of the charred cellulose granules. We confirmed that the xanthene dyes could bind to the charred cellulose granules by ionic and hydrophobic bonds.     

Photobactericidal plastic films based on cellulose esterified by chloroacetate and a cationic porphyrin

The synthesis and characterisation of pyridinium porphyrinic chloroacetyl cellulose ester chlorides, where photosensitizing agents are covalently bounded to the polymeric chain, is presented in this paper. First, cellulose was homogenously converted into chloroacetate cellulose ester in DMAc/LiCl solvent by using chloroacetyl chloride. The complete substitution of cellulose was achieved using 7equiv of chloroacetyl chloride for a 2h reaction at room temperature. The absence of base did not prove detrimental to reaction. The grafting of monopyridyltritolylporphyrin onto chloroacetate cellulose ester was then realised by alkylation of the photosensitizer in DMF. These new plastic films were found to be thermostable up to 55 degrees C; higher temperatures led to progressive deacetylation. First results of their photobactericidal activity against Staphylococcus aureus and Escherichia coli strains are very encouraging. Such materials could find applications in medical environments as an alternative to overcome the rampant bacterial multiresistance to classical antibiotics.     

Resistin and RELM-alpha gene expression in white adipose tissue of lactating mice

An ORF2 gene located upstream of the cellulose synthase (bcs) operon of Acetobacter xylinum BPR2001 was disrupted and a mutant (M2-2) was constructed. In static cultivation, the parent strain produced a tough, colorless, and insoluble cellulose pellicle, whereas M2-2 culture produced a thin, yellow, and fragile pellicle. The results of X-ray diffraction and 13C solid-state NMR indicated that the product of M2-2 is a mixture of cellulose I, cellulose II, and amorphous cellulose. The cellulose I to cellulose II ratio of the mixture was evaluated from the signal areas of C6 to be about 1:2. Electron microscopy revealed that the product of M2-2 included ribbon-like cellulose and irregularly shaped particles attached to the ribbons. On the other hand, the mutant complemented with plasmid pSA-ORF2/k containing the ORF2 gene and BPR2001 produced only cellulose I. These results indicate that the ORF2 gene is involved in the production and crystallization of cellulose I microfibrils by this microorganism.     

Measurement of biosynthetic rates and intracellular transit times for a cell-surface membrane glycoprotein, alkaline phosphatase in HeLa cells

An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 M NaCl solution, were incubated with 125I-labeled thyroid hormone (T3), and filtered on the cellulose ester membranes under suction at 2 degrees C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentrations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent Kd of 1.38 X 10(-10) M and a pH optimum of T3 binding at 8.2-8.4.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites

Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain.     

基于微滴式数字PCR方法的鱼类环境DNA样本处理与保存技术优化

以实验室内的鲫(Carassius auratus)为研究对象,利用微滴式数字PCR(Droplet Digital PCR,ddPCR)定量技术,优化了鱼类环境DNA(Environmental DNA,eDNA)样本的捕获、提取和保存方法,并对免DNA提取的PCR直扩技术进行了探索.研究结果如下:(1)在同一孔径、...     

Preparation, characterization and cytocompatibility of polyurethane/cellulose based liquid crystal composite membranes

Two types of polyurethane/liquid crystal (PU/LC) composite membranes with different LC contents, namely polyurethane/octyl hydroxypropyl cellulose ester (PU/OPC) and polyurethane/propyl hydroxypropyl cellulose ester (PU/PPC), were prepared and studied. The effects of surface properties on cell compatibility of the membranes were elucidated. PPC tended to assemble to independent phases in the composite membranes, while OPC formed uniformly distributed LC domains. As the introduction of LC, phase separation occurred, and the crystallization of PU was disrupted. The surface of PU/LC composite membranes showed fingerprint texture and two-phase morphology. Hydrophilicity of the two types of composite membranes exhibited a reversal tendency with the increase of LC contents. Cells seeded on the composite membranes presented favorable growth when the content of LC was over 30%, especially on PU/OPC complex. The surface morphology, phase separation between LC and PU as well as the type of LC showed significant effects on the cell behaviors.     

A Simple Preparative Method for the Isolation of Amylose and Amylopectin from Potato Starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8, 0.1 M) ± urea (2 M) ± 1-butanol (8.5 %, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and β -amylolysis limit.     

A method for the identification of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Two-dimensional electrophoresis on cellulose acetate membrane permits the clean separation of sulfated glycopeptide in a mixture of acidic glycans (glycosaminoglycans and acidic glycopeptides). Two systems were used. In system 1, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M barium acetate (pH 8.0) in the second dimension. In system 2, 0.1 M pyridine-0.47 M formic acid buffer (pH 3.0) was used in the first and 0.1 M HCl in the second dimension. All of the acidic glycans on electrophoretogram were stained with alcian blue in 70% ethanol. On the other hand, sulfated glycans alone were made visible with alcian blue in 0.1 M HCl. Alcian blue in 70% ethanol or 0.1 M HCl, when combined with periodic acid-Schiff's reagent identified sulfated glycopeptides on cellulose acetate membrane.     

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.     

The use of natural abundance stable isotopic ratios to indicate the presence of oxygen-containing chemical linkages between cellulose and lignin in plant cell walls

Qualitative and quantitative understanding of the chemical linkages between the three major biochemical components (cellulose, hemicellulose and lignin) of plant cell walls is crucial to the understanding of cell wall structure. Although there is convincing evidence for chemical bonds between hemicellulose and lignin and the absence of chemical bonds between hemicellulose and cellulose, there is no conclusive evidence for the presence of covalent bonds between cellulose and lignin. This is caused by the lack of selectivity of current GC/MS-, NMR- and IR-based methods for lignin characterisation as none of these techniques directly targets the possible ester and ether linkages between lignin and cellulose. We modified the widely-accepted “standard” three-step extraction method for isolating cellulose from plants by changing the order of the steps for hemicellulose and lignin removal (solubilisation with concentrated NaOH and oxidation with acetic acid-containing NaClO₂, respectively) so that cellulose and lignin could be isolated with the possible chemical bonds between them intact. These linkages were then cleaved with NaClO₂ reagent in aqueous media of contrasting ¹⁸O/¹⁶O ratios. We produced cellulose with higher purity (a lower level of residual hemicellulose and no detectable lignin) than that produced by the “standard” method. Oxidative artefacts may potentially be introduced at the lignin removal stage; but testing showed this to be minimal.Cellulose samples isolated from processing plant-derived cellulose–lignin mixtures in media of contrasting ¹⁸O/¹⁶O ratios were compared to provide the first quantitative evidence for the presence of oxygen-containing ester and ether bonds between cellulose and lignin in Zea mays leaves. However, no conclusive evidence for the presence or lack of similar bonds in Araucaria cunninghamii wood was obtained.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of salbutamol sulphate and ketoprofen enantiomers from matrices containing HPMC and cellulose derivatives

We studied the release of salbutamol and ketoprofen enantiomers from HPMC K100M matrices containing two types of cellulose derivatives: cellulose tris (3,5-dimethylphenylcarbamate) and cellulose tris (2,3-dichlorophenylcarbamate), chiral excipients used as stationary phases for liquid chromatography. These matrices provided an extended release of both drugs. Ketoprofen release from formulations elaborated with cellulose tris (2,3-dichlorophenylcarbamate) was by anomalous transport, because the value of n (release exponent of the diffusion equation) ranged between 0.60-0.68, whereas for all other formulations the value of exponent n ranged from 0.50-0.54. The drug thus diffuses through the matrix and is released following a quasi-Fickian diffusion mechanism (stereoselective process). The matrices preferentially retained R-salbutamol and S-ketoprofen and cellulose tris (3,5-dimethylphenylcarbamate) showed more capacity of chiral discrimination for both drugs than cellulose tris (2,3-dichlorophenylcarbamate). Moreover, we observed that stereoselectivity is dependent on the amount of chiral excipient in the formulation. Diffusion tests confirmed the chiral interaction between drugs and cellulose derivatives observed in the dissolution assays except for matrices elaborated with ketoprofen and cellulose tris (2,3-dichlorophenylcarbamate), where the low stereoselectivity observed with the matrices is due to the presence of HPMC K100M. We conclude that the inclusion of these cellulose derivatives in HPMC matrices does not result in a relevant stereoselectivity with respect to the two drugs studied.