Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M_n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

Free radical generation during chemical depolymerization of heparin

Low-molecular weight heparins (LMWHs), as compared with unfractionated heparin (UFH), present superior bioavailability, much longer plasma half-life, and lower incidence of side effects. For these reasons, over the past two decades LMWHs have become the drugs of choice for the treatment of deep venous thrombosis, pulmonary embolism, arterial thrombosis, and unstable angina. Furthermore, their use in acute ischemic stroke is currently under study. LMWHs are obtained by UFH depolymerization, which can be performed using various methods, including nitrous acid depolymerization, cleavage by beta-elimination of benzyl ester, enzymatic depolymerization, and peroxyl radical-dependent depolymerization. This article addresses the chemical depolymerization, obtained by free radical attack (mainly hydroxyl radical), of heparin. The electron spin resonance (ESR) spectroscopy, coupled to the spin trapping technique, was employed to study this reaction. Free radical-mediated heparin depolymerization was performed under different chemical conditions. The final products of the reactions were purified and classified on the basis of their molecular weight and other characteristics. The level of heparin fragmentation was different depending on the type of depolymerization reaction used. Moreover, the level of reproducibility and the resulting radical species were different for every type of reaction performed.     

Partial depolymerization of pectin by a photochemical reaction

Complex heterogeneous polysaccharides that comprise pectin were partially depolymerized by a photochemical reaction using ultraviolet light in the presence of titanium dioxide catalyst. In a period of 6 h at pH 7, this UV/TiO₂ process decreased the average molecular weight of pectin from 400 kDa to 200 kDa. The characterization of the partially depolymerized pectin, which was fractionated by size-exclusion chromatography, was performed by ¹H NMR spectroscopy, and the spectra obtained showed that the resulting oligosaccharides and polysaccharides maintained the intact core structure of pectin. The monosaccharide content and depolymerization profile were determined by high-performance anion-exchange chromatography coupled with pulsed amperometric detection. This controlled photochemical depolymerization technique might be useful for preparation of pectin oligosaccharides as an ingredient in food and pharmaceutical products.     

On the influence of fixations on the normal hydration of rabbit cornea and the total amount of acid mucopolysaccharides in the stroma

Summary The authors studied the influence of fixations on the normal hydration of the rabbit cornea and the total amount of acid mucopolysaccharides (AMPS) in the stroma. The following fixatives were used: formol-calcium chloride at 19° C for 24 hours, formolcetylpyridinium chloride (CPC) at 19 and 28° C for 48 hours, Lillie's fixation at 19° C for 24 hours and Carnoy's fluid at 19° C for 30 minutes. The sections of the corneae were stained with Alcian blue, colloidal Fe³⁺ in the modification according to Rinehart and Abu'l Haj and with toluidine and methylene blue. The amount of AMPS was determined with the method of Rondle and Morgan and the total hydration of the stroma by weighing the corneae before and after using different fixative fluids and by calculation of obtained values on dry weight.The best results were obtained by using formol-CPC at 28° C. At the ordinary room temperature (±19° C) it was the poorest fixation, however, as the corneae in this solution became hydrated. Formol-calcium chloride was the second in the row and it was much better than Lillie's and Carnoy's fluid.The amount of AMPS in the stroma was not essentially changed by the effect of fixatives. Within 24–48 hours formol-CPC at 28° C retained the normal content and formol-calcium chloride caused the 11% decrease of AMPS maximally. The loss of AMPS after other fixatives was minimal.The intensity of staining with cationic dyes in paraffin sections was different after individual fixatives and after the kind of their application and was dependent chiefly on the state of hydration of the corneal stroma: It is impossible to interpret the results of staining reactions in terms of the quantity of AMPS as it was hitherto done.     

Structural basis of the inhibition of class C acid phosphatases by adenosine 5'-phosphorothioate

The inhibition of phosphatases by adenosine 5'-phosphorothioate (AMPS) was first reported in the late 1960s; however, the structural basis for the inhibition has remained unknown. Here, it is shown that AMPS is a submicromolar inhibitor of class C acid phosphatases, a group of bacterial outer membrane enzymes belonging to the haloacid dehalogenase structural superfamily. Furthermore, the 1.35-? resolution crystal structure of the inhibited recombinant Haemophilus influenzae class C acid phosphatase was determined; this is the first structure of a phosphatase complexed with AMPS. The conformation of AMPS is identical to that of the substrate 5'-AMP, except that steric factors force a rotation of the thiophosphoryl out of the normal phosphoryl-binding pocket. This conformation is catalytically nonproductive, because the P atom is not positioned optimally for nucleophilic attack by Asp64, and the O atom of the scissile O-P bond is too far from the Asp (Asp66) that protonates the leaving group. The structure of 5'-AMP complexed with the Asp64→Asn mutant enzyme was also determined at 1.35-? resolution. This mutation induces the substrate to adopt the same nonproductive binding mode that is observed in the AMPS complex. In this case, electrostatic considerations, rather than steric factors, underlie the movement of the phosphoryl. The structures not only provide an explanation for the inhibition by AMPS, but also highlight the precise steric and electrostatic requirements of phosphoryl recognition by class C acid phosphatases. Moreover, the structure of the Asp64→Asn mutant illustrates how a seemingly innocuous mutation can cause an unexpected structural change.     

Evaluation of rice straw hemicellulose hydrolysate in the production of xylitol byCandida guilliermondii

Summary Rice straw was used as a lignocellulosic source to provide rich pentose media. By using a well characterized yeast strain,Candida guilliermondii FTI 20037, the hydrolysate obtained was converted to xylitol with an efficiency of 75% and production of 27 g of xylitol per liter in 48 hours. The satisfactory results reported here can be attributed to the low concentrations of toxic components generated throughout the chemical depolymerization of this raw material.     

Characterization of enzymatic degradation products of carboxymethyl cellulose by gel chromatography

In order to investigate the molecular weight distribution of depolymerization products obtained by enzymatic degradation of carboxymethyl cellulose (CMC), a high resolution size exclusion chromatographic (SEC) system was developed.The SEC system using Fractogel® TSK-HW and an eluent containing sodium sulfate and sodium acetate enables an effective separation of the anionic cleavage products to be carried out. The experimental set-up was equipped with a sensitive detection system based on the post-column reaction of carbohydrates with orcinol.The elution patterns of enzymatic depolymerization products obtained from CMC with different degrees of substitution make it feasible to infer parts of the sequence and the distribution of carboxymethyl groups.     

A monte carlo simulation of the depolymerization of linear homopolymers by endo-enzymes exhibiting random-attack probability and single-attack mechanism: application to the (1-->3), (1-->4)-beta-D-glucan/endo-(1-->3),(1-->4)-beta-D-glucanase system

A Monte Carlo simulation of the depolymerization of linear homopolymers by specific endo-enzymes exhibiting random-attack probability and a single-attack mechanism has been developed. The program simulates the "real" depolymerization versus time of a polydisperse sample of substrate by a specific endo-enzyme. Given the initial mass distribution and concentration of the substrate, the initial concentration of the enzyme, and its Michaelis-Menten constant, the program simulates the evolution of the mass distribution of the substrate with the depolymerization time. When tested against experimental data from the depolymerization of barley (1-->3),(1-->4)-beta-D-glucan by malt endo-(1-->3), (1-->4)-beta-D-glucanase, monitored using the Calcofluor-FIA method with fluorescent detection, excellent results were obtained. Copyright 1998 John Wiley & Sons, Inc.     

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.     

The Golgi complex in the esophageal mucous glands of the newly hatched chick

The general morphology of the mucous gland cell and the nature of the secretory granule in esophageal glands of the newly hatched chick have been described. Lightly basophilic supporting cells, attached to secretory cells by desmosomes and containing tonofilaments, are located on the basal lamina. Electron microscopic studies showed a morphological polarity of the Golgi complex which suggests that mucous precursors are transported from other sites within the cell to the Golgi complex for further packaging into secretory granules. Finally, acid mucopolysaccharides (AMPS) were specifically stained using the Thorotrast technique and not detected in the rough endoplasmic reticulum, the transitional elements, or in the lamellae at the forming face of the Golgi complex. Conversely, AMPS are found in the vicinity of the mature face of the Golgi complex, and in the secretory granules. The acquisition of cytochemical reactivity for AMPS within the Golgi complex is discussed.     

In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates produced by Pseudomonas putida Bet001

In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid was studied. Both processes were studied under optimum conditions for mcl-PHA depolymerization viz. 0.2?M Tris-HCl buffer, pH 9, ionic strength (I)?=?0.2?M at 30°C. For in vitro depolymerization studies, cell-free system was obtained from lysing bacterial cells suspension by ultrasonication at optimum conditions (frequency 37?kHz, 30% of power output, <25°C for 120?min). The comparison between in vivo and in vitro depolymerizations of intracellular mcl-PHA was made. In vitro depolymerization showed lower depolymerization rate but higher yield compared to in vivo depolymerization. The monomer liberation rate reflected the mol% distribution of the initial polymer subunit composition, and the resulting direct individual products of depolymerization were identical for both in vivo and in vitro processes. It points to exo-type reaction for both processes, and potential biological route to chiral molecules.     

On the occurrence of acid mucopolysaccharides in the spermatophores of two marine prawns,Penaeus indiens (Milne-Edwards) and Metapenaeus monoceros (Fabricius) (Crustacea: Macrura)

In marine prawns, spermatophores aid in sperm transfer. The prawn spermatophores, in general, consist of two parts, sperm sac and wing. The former encloses spermatozoa and the latter serves as an adhesive pad during transfer to the female's thelycum. This paper reports on the occurrence of acid mucopolysaccharides (AMPS) in the spermatophores of two penaeid prawns, Penaeus indicus (Milne-Edwards) and Metapenaeus monoceros (Fabricius). They, being the principal component of the spermatophores, have been characterized and quantified in the wing, sperm mass, sperm sac, and crystalline structures. In both prawns, the spermatophore is a simple structure. In P. indicus, it consists of sperm mass, enclosed within sperm sac, to which is attached the foliacious wing. In M. monoceros, the freshly extruded spermatophore consists of sperm mass and milky-white crystalline structures. Isolation and purification of AMPS resulted in the resolution of two toluidine blue-positive AMPS fractions in agarose-gel electrophoresis. Densitometric study of the standard and sample AMPS fractions reveals further that the two fractions correspond to chondroitin sulfate and hyaluronic acid. Quantitative assay on total AMPS shows that the sperm sac of P. indicus contains the maximum amount of AMPS (195.50 μg/mg), whereas its wing contains the least quantity of AMPS (43.68 μg/mg). The qualitative and quantitative variations found in the AMPS content of spermatophoric components of two prawns have been discussed in relation to their adhesion to thelycum as well as sperm storage and maintenance pending fertilization.     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.     

The effect of hydration of the rabbit cornea on the histochemical demonstration of acid mucopolysaccharides

Summary The influence of hydration on the histochemical staining of acid mucopolysaccharides (AMPS) in the rabbit cornea was studied. The rabbit cornea was immersed in distilled water for 4, 8, 12 and 16 hour intervals, and the water content of the hydrated stroma was investigated. Simultaneously the hexosamine content was examined. For the histochemical identification of AMPS the corneae were fixed with formol-calcium chloride and embedded in paraffin.Various degrees of the corneal hydration were without influence on the content of AMPS determined by biochemical methods. On the other hand the results of all histochemical staining reactions for AMPS were extremely dependent on the state of hydration. The quantitative evaluation of the content of AMPS in the corneal stroma on the basis of staining reactions is thus impossible.     

Acute Modulation of Brain Connectivity in Parkinson Disease after Automatic Mechanical Peripheral Stimulation: A Pilot Study

Objective

The present study shows the results of a double-blind sham-controlled pilot trial to test whether measurable stimulus-specific functional connectivity changes exist after Automatic Mechanical Peripheral Stimulation (AMPS) in patients with idiopathic Parkinson Disease.

Methods

Eleven patients (6 women and 5 men) with idiopathic Parkinson Disease underwent brain fMRI immediately before and after sham or effective AMPS. Resting state Functional Connectivity (RSFC) was assessed using the seed-ROI based analysis. Seed ROIs were positioned on basal ganglia, on primary sensory-motor cortices, on the supplementary motor areas and on the cerebellum. Individual differences for pre- and post-effective AMPS and pre- and post-sham condition were obtained and first entered in respective one-sample t-test analyses, to evaluate the mean effect of condition.

Results

Effective AMPS, but not sham stimulation, induced increase of RSFC of the sensory motor cortex, nucleus striatum and cerebellum. Secondly, individual differences for both conditions were entered into paired group t-test analysis to rule out sub-threshold effects of sham stimulation, which showed stronger connectivity of the striatum nucleus with the right lateral occipital cortex and the cuneal cortex (max Z score 3.12) and with the right anterior temporal lobe (max Z score 3.42) and of the cerebellum with the right lateral occipital cortex and the right cerebellar cortex (max Z score 3.79).

Conclusions

Our results suggest that effective AMPS acutely increases RSFC of brain regions involved in visuo-spatial and sensory-motor integration.

Classification of Evidence

This study provides Class II evidence that automatic mechanical peripheral stimulation is effective in modulating brain functional connectivity of patients with Parkinson Disease at rest.

Trial Registration

Clinical Trials.gov NCT01815281     

Dynamics of microtubule depolymerization in monocytes

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.     

Low molecular weight chitosans: preparation with the aid of papain and characterization

Low molecular weight chitosans (LMWC) of different molecular weight (4.1-5.6 kDa) were obtained by the depolymerization of chitosan using papain (from Carica papaya latex, EC. 3.4.22.2) at optimum conditions of pH 3.5 and 37 degrees C for 1-5 h. Scanning electron microscopy (SEM) showed approximately 15-fold decrease in the particle size after depolymerization. Decrease in the molecular weight was associated with decrease in the degree of acetylation (DA) as evidenced by circular dichroism (CD), FT-IR and solid-state CP-MAS 13C-NMR data. X-ray diffraction pattern revealed slight decrease in the crystallinity index (CrI) whereas the 13C-NMR data showed molecular inhomogeneity. LMWC showed lytic effect towards Bacillus cereus and Escherichia coli more efficiently than native chitosan. The growth inhibitory effect was maximal towards B. cereus, with minimum inhibitory concentration (MIC) of 0.01% (w/v).     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Water content, body weight and acid mucopolysaccharides, hyaluronidase and beta-glucuronidase in response to aestivation in Australian desert frogs

This study investigates the effects of aestivation on body water content, body mass, acid mucopolysaccharide (AMPS) and some of its degrading enzymes in different tissues for some Australian desert frogs. The AMPS component of the liver, kidney, skin and cocoon alter during aestivation to help retain water, which is unchanged in most tissues of all frog species, and to protect the frogs from desiccation during extended periods of aestivation. Hepatic AMPS was unaltered in Cyclorana maini, C. platycephala and Neobatrachus sutor but increased significantly after 2 months of aestivation in C. australis. The level of AMPS in the kidney was elevated in all four frog species after 5 months of aestivation. Skin AMPS content in the skin of awake frogs decreases with aestivation period and increases in the cocoon. AMPS in the cocoon probably works as a cement between the cocoons' layers and its physical presence presumably contributes to preventing water flux. Changes in AMPS content in different tissues were accompanied by significant changes in both hyaluronidase and beta-glucuronidase activities, which play an important role in AMPS metabolism. Alcian blue staining of control and digested skin of C. australis and C. platycephala with testicular hyaluronidase indicated the presence of AMPS, concentrated in a thin layer (called ground substance, GS) located between stratum compactum and stratum spongiosum, and acid mucin concentrated in the mucous glands and in a 'tubular' structure which could be observed in the epidermal layer. Hyaluronidase digestion of the cocoon slightly changed the Alcian Blue colour, suggesting the presence of a large amount of acid mucin similar to that found in the skin mucous gland. The results of this study present data for the redistribution of AMPS, which may help in reducing water loss across the cocoon and reabsorption of water in the kidney during aestivation.