Hairy Regions Research Articles

A New Demodecidae Species (Acari) from the Yellow-Necked MouseApodemus flavicollis(Rodentia: Muridae)—Description with Data on Parasitism

This paper describes morphological characteristics and the occurrence of Demodex corniculatus n. sp., which was found on Apodemus flavicollis in Poland. The yellow-necked murid mouse is a common European rodent; until now, it was parasitized by a single demodectic mite species, Demodex rosus, which occurs in the oral cavity and esophagus. The new species was found in hair follicles, particularly within hairy regions of the head and genital-anal area. Demodex corniculatus occurred in 33% of the yellow-necked mice examined. The new species is small (adult stages 140 μm in length); characteristic features of these mites are massive supracoxal spines (ca. 5-6 μm long) on the dorsal side of the gnathosoma and palps with thick-set, bifurcated terminal spines. This paper also contains a checklist of demodecids in European and cosmopolitan murids.

Effect of extraction time and temperature on the characteristics of loosely bound pectins from Japanese plum

The cell wall composition of Japanese plums (Prunus salicina) at six developmental stages was previously evaluated (Ponce et al., J. Agric. Food Chem. 2010, 58, 2562–2570). This fruit is an interesting source of pectins, polysaccharides of valuable functionality for pharmaceutical and food formulations. In the present work it was investigated how the different conditions for the aqueous extraction of pectins from Japanese plums affect the yield as well as their chemical and rheological characteristics. It has been determined that extraction with water at room temperature for periods longer than 2h did not produce additional increment of yield (12%) but decreased the average molecular weights of the extracted pectins. Pectins with a degree of methylation ≈40% with high viscosity in water and with adequate molecular weights (≈72,000) were obtained. Conversely, utilization of boiling water for extraction increased considerably the yields (33–38%) but the extracted pectins showed significant lower viscosity in water in spite of their higher molecular weights. The poorer thickening ability was associated to the lower proportion of arabinose residues present in the hairy regions of the pectin macromolecules extracted by hot water, which led the polymers to interact more transiently in a 2% w/v water solution.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.

Structural characteristics of a hyperbranched acidic polysaccharide from the stems of Ephedra sinica and its effect on T-cell subsets and their cytokines in DTH mice

An acidic hetero-polysaccharide (ESP-B4) was acquired from the stems of Ephedra sinica. Its structure was characterized by periodate oxidation, Smith degradation, partial acid hydrolysis, methylation analysis combined with HPCE, GC–MS and FT-IR spectra. The results indicated that ESP-B4 contained a repeating unit of [→4)-GalpA-(1→2)-Rhap-(1→] as hairy region, substituted by arabinose, xylose, galactose, glucose, mannose and glucuronic acid. To better understand the mechanism of bioactivity for this compound, the quantities of ESP-B4 was further enriched by ion exchange and gel filtration chromatography. Immunological tests in vivo showed that ESP-B4 exhibits compelling immunosuppressive effects by hematoxylin and eosin staining, enzyme-linked immunosorbent assay and flow cytometric analysis. The mechanism of action might be attributed to its inhibition of IL-2 and IL-4 levels in serum, and also effectively suppressed the ratio of CD4+/CD8+ in serum of T cell mediated delayed-type hypersensitivity mice. The results suggest that ESP-B4 could be a valuable lead material for immunosuppressive drug development.

Effect of Macerating Enzymes on the Oligosaccharide Profiles of Merlot Red Wines

Commercial pectinase preparations are applied in winemaking to improve wine processing and final quality. These preparations contain pectolytic enzyme activities such as polygalacturonases, pectin esterases, pectin lyases, and rhamnogalacturonases. These enzymes modify the polysaccharide and oligosaccharide composition of wines. The influence of various commercial enzyme preparations on wine oligosaccharide composition was studied, on Merlot wines from the Bordeaux area. Wine oligosaccharides were isolated by high-resolution size-exclusion chromatography on a Superdex-30 HR column. The glycosyl residue and glycosyl linkage compositions of the oligosaccharide fractions obtained were determined. The MS spectra of the Merlot oligosaccharide fractions from control and enzyme-treated wines were recorded on an AccuTOF mass spectrometer equipped with an electrospray ionization (ESI) source and a time-of-flight (TOF) mass analyzer. Oligosaccharides in the control wines were partly methylated homogalacturonans, corresponding to smooth regions of pectins, whereas those of the enzyme-treated wines were mostly rhamnogalacturonan-like structures linked with neutral lateral chains, arising from the hairy regions. The enzyme preparations used thus cleaved the rhamnogalacturonan backbone of the hairy zones and demethylated and hydrolyzed the smooth regions. Besides, different structures were detected, depending on the enzyme preparation used, indicating that they contained rhamnogalacturonase activities with different specificities. The oligosaccharide profiles can serve as a marker of enzymatic treatments.

Pectic Polysaccharides from Panax ginseng as the Antirotavirus Principals in Ginseng

To evaluate the antidiarrheal effect of ginseng, the active principals of ginseng were studied in vitro model of rotavirus infection, the leading cause of severe diarrhea. Two pectic polysaccharides, named as GP50-dHR (56.0 kDa) and GP50-eHR (77.0 kDa), were purified from hot water extract of ginseng by bioassay-linked fractionation. Both polysaccharides rescued cell viability from rotavirus infection dose-dependently (IC50 are 15 and 10 microg/mL, respectively). Both polysaccharides had common structural features of homogalacturonan backbone with hairy regions of rhamnogalacturonan type I. Arabinose-rich side chains with abundant branch points were unique in GP50-eHR and may contribute to a greater antirotavirus effect of GP50-eHR than GP50-dHR. Because homogalacturonan itself did not show an antirotavirus effect, hairy regions might be functional sites. Of note, the antirotavirus effect of both polysaccharides resulted from inhibiting rotavirus attachment to cells. Together with a wide range of noncytotoxicity, these findings suggest that ginseng polysaccharides are viable therapeutic options for rotavirus diarrhea.

Composition and properties of biologically active pectic polysaccharides from leek (Allium porrum)

Leek (Allium porrum) is very commonly used vegetable in Bulgaria and is distinctive with high content of bioactive components. Previously we obtained five crude pectic polysaccharides from leek through consecutive extraction. Some of them appeared to be good stimulators of the immune system. Schols and Voragen investigated the composition of modified hairy regions of pectic polysaccharides isolated from leek cell walls. Samuelson et al. identified the polysaccharide structures encountered in hairy regions as bioactive. The aim of this work was to study the isolation, composition and biological activities of pectic polysaccharides from leek. Two pectic polysaccharides from leek were isolated through consecutive water and acid extraction. The water extractable pectin had higher polyuronic content, higher protein content and lower neutral sugar content. It was found that next to galacturonic acid they also contain glucuronic acid in ratio 9:1 for the water- and 3:1 for the acid-extractable polysaccharide. The main neutral sugar was galactose. The water-extractable pectic polysaccharide had higher molecular weight (10(6) Da) and homogeneity. It was shown that the pectic polysaccharides from leek have considerable immunostimulating activities. Leek polysaccharides have relatively high galacturonic and glucuronic acid content and are distinguished with high biological activity.

Structural Characterization and Cytotoxic Properties of an Apiose-Rich Pectic Polysaccharide Obtained from the Cell Wall of the Marine Phanerogam Zostera marina

Zosterin, an apiose-rich pectic polysaccharide, was extracted and purified from the sea grass Zostera marina. Structural studies conducted by gas chromatography and NMR spectroscopy on a purified zosterin fraction (AGU) revealed a typical apiogalacturonan structure comprising an alpha-1,4-d-galactopyranosyluronan backbone substituted by 1,2-linked apiofuranose oligosaccharides and single apiose residues. The average molecular mass of AGU was estimated to be about 4100 Da with a low polydispersity. AGU inhibited proliferation of A431 human epidermoid carcinoma cells with an approximate IC(50) value of 3 microg/mL (0.7 microM). In addition, AGU inhibited A431 cell migration and invasion. Preliminary experiments showed that inhibition of metalloproteases expression could play a role in these antimigration and anti-invasive properties. Autohydrolysis of AGU, which eliminated apiose and oligo-apiose substituents, led to a virtual disappearance of cytotoxic properties, thus suggesting a direct structure-function relationship with the apiose-rich hairy region of AGU.

Monoclonal antibodies to rhamnogalacturonan I backbone

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides--BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [-->2)-alpha-L-rhamnosep-(1-->4)-alpha-D-galacturonic acid p-(1-->](7) structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose-galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.

Structural features of pectic polysaccharide from Angelica sinensis (Oliv.) Diels

The structure of the pectic polysaccharide (ASP3) isolated from roots of Angelica sinensis (Oliv.) Diels was investigated using partial acid hydrolysis, enzymic digestion combined with methylation analysis, and further supported by 1 H and 13 C NMR spectroscopy techniques. The results indicated that ASP3 contained a backbone of linear homogalacturonan fragments as “smooth regions” and rhamnogalacturonan fragments as “hairy regions” with repeating unit of [→ 4)-α- d -Gal p A-(1 → 2)-α- l -Rha p -(1 →]. A total of 58.8% rhamnopyranose residues in the backbone were substituted at O -4 position by the side chains. The side chains contained mainly β-1,6- and β-1,4-galactopyranan bearing 3,6- and 4,6-substituted β- d -galactopyranose residues as branched points and short α-1,5-arabinofuranan possessing 3,5-substituted α- l -arabinofuranose residues as branching points. In addition, β-1,6-galactopyranan side chains were highly branched with α-1,5-arabinofuranan carrying 3- O -substituents (1,3,6-Gal) and terminated by the α-arabinofuranose residues which form arabinogalactan.

Pectin‐coated titanium implants are well‐tolerated in vivo

Multiform coated titanium implants are widely used in orthopedic and dental surgery. In this study, we have investigated the reactivity of pectin-coated titanium samples implanted under the latissimus dorsi-muscle fascia of rats. Samples were coated with two enzyme treated apple pectins; modified hairy regions (MHR-A and MHR-B) that differed in chemical structure. Aminated (AMI) and uncoated titanium (Ti) served as controls. The thicknesses of the peri-implant fibrous tissue capsules formed 1 or 3 weeks after implantation were measured as indicative of possible inflammatory reactions toward the biomaterials. After 1 week, the MHR-B implant was surrounded by a thicker fibrous capsule (42.9 microm) than any of the other sample types: MHR-A (33.2 microm), AMI (32.5 microm), and Ti (32.3 microm), the last one being the only statistically significant difference. After 3 weeks, however, this difference disappeared; the capsule thicknesses around MHR-B and Ti implants had decreased to the values found for AMI and MHR-A. Additionally, the capsule formation represents merely a stromal rather than an inflammatory reaction, as indicated by the absence of activated macrophages or foreign body giant cells in the capsules. These results indicate for the first time the in vivo tolerability of covalently linked pectins, and suggest the feasibility of pectin-coated bone and dental implants for clinical use.

Development of a High Methoxyl Pectin Edible Film for Retention of l-(+)-Ascorbic Acid

An edible film to carry l-(+)-ascorbic acid (AA) was formulated for natural antioxidant food protection. Considering previous works where films based on the "rigid" structure of gellan (deacylated) or on a mixture of acylated-deacylated (more "disordered") gellan were used for network development, pectin was herein chosen by considering that the alternating presence of "disordered" (hairy) regions together with ordered (homogalacturonan) ones could sufficiently immobilize water for better AA retention and lower browning. High methoxyl pectin (HMP) was first investigated. AA stability and browning were studied during film storage at 33.3, 57.7, or 75.2% relative humidity (RH) and 25 degrees C; their dependence on water mobility determined through (1)H NMR analysis as well as the correlation between browning and AA degradation were again found. Network characteristics and glycerol (plasticizer) interactions were analyzed through X-ray diffraction and Fourier transform infrared spectroscopy as well as through uniaxial tensile assay. From all results obtained, it was hypothesized that browning development in solidlike systems may be directly related to the water molecules more closely adsorbed on the hydroxyl-polymeric (active) surfaces. The HMP film microstructure produced the best immobilization of water molecules except at 75.2% RH, where it showed lower AA stability than acylated-deacylated gellan film. It is suggested that disordered regions of this pectin network may not be adequately counterbalanced by more transient junction zones of alternating hydrophilic (water) and hydrophobic (methyl ester) interactions, also disturbed by glycerol molecules, for accomplishing enough water immobilization in the whole network at 75.2% RH.

Humans Can Integrate Force Feedback to Toes in Their Sensorimotor Control of a Robotic Hand

Tactile sensory feedback is essential for dexterous object manipulation. Users of hand myoelectric prostheses without tactile feedback must depend essentially on vision to control their device. Indeed, improved tactile feedback is one of their main priorities. Previous research has provided evidence that conveying tactile feedback can improve prostheses control, although additional effort is required to solve problems related to pattern recognition learning, unpleasant sensations, sensory adaptation, and low spatiotemporal resolution. Still, these studies have mainly focused on providing stimulation to hairy skin regions close to the amputation site, i.e., usually to the upper arm. Here, we explored the possibility to provide tactile feedback to the glabrous skin of toes, which have mechanical and neurophysiological properties similar to the fingertips. We explored this paradigm in a grasp-and-lift task, in which healthy participants controlled two opposing digits of a robotic hand by changing the spacing of their index finger and thumb. The normal forces applied by the robotic fingertips to a test object were fed back to the right big and second toe. We show that within a few lifting trials, all the participants incorporated the force feedback received by the foot in their sensorimotor control of the robotic hand.

Structure analysis of an acidic polysaccharide isolated from green tea

An acidic polysaccharide (ATPS-2) was isolated and purified from coarse green tea. ATPS-2 with peak molecular weight of 4430 Da was composed of rhamnose, arabinose, galactose and galacturonic acid. Structure features of the purified ATPS-2 were investigated by a combination of chemical and instrumental analysis, such as periodate oxidation, Smith degradation, 1H NMR, 13C NMR, COSY, HSQC, HMBC and ROESY spectrum. It was found that ATPS-2 was a pectic polysaccharide. The backbone proved to consist of the smooth region of α-1,4-D-galactopyranosyluronan blocks interconnected by 1,2-linked rhamnose residues involved in the linear sugar chain. The ramified hairy regions were shown to contain the following of the backbone: →4)-α-D-GalpA-(1 → 2)-α-L-Rhap-(1 → 4)α-D-GalpA-(1→ and the side chains attached to the residues of rhamnopyranose of these fragments. The backbone galacturonic acid residues of ATPS-2 were partially O-methyl esterified and acetylated. The side chains contain linear and branched α-L-Araf and β-D-Galp residues.

Inhibition of LPS-induced proinflammatory responses of J774.2 macrophages by immobilized enzymatically tailored pectins

The surface of an implant device can be modified by immobilizing biological molecules on it to improve its integration into the host tissue. We have previously demonstrated that enzymatically tailored plant pectins are promising nanocoatings for biomaterials. This study investigates whether a coating of modified hairy region (rhamnogalacturonan-I) from apple pectin (MHR-α) which has anti-adhesive properties can inhibit the generation of inflammatory mediators by lipopolysaccharide (LPS)-activated macrophages. For that purpose, J774.2 murine macrophages were cultured for 24 h on MHR-α-coated Petri dishes and tissue culture polystyrene controls, with and without LPS. Cell morphology, cell growth, nitrite and TNF-α secretion were studied. The results indicate that MHR-α coating inhibits the LPS-induced activation of macrophages.

Arabinogalactan endo-1,4-β-galactosidase: a putative plant cell wall-degrading enzyme of plant-parasitic nematodes

Abstract Plant-parasitic nematodes secrete a plethora of enzymes to degrade polysaccharides of the recalcitrant plant cell wall. Here we report on the presence of a putative endo-1,4-β-galactosidase (EC 3.2.1.89) in cyst nematodes of the genus Heterodera. This enzyme hydrolyses β-1,4-galactan in the hairy regions of pectin and to our knowledge it is the first report of this class of enzymes in animals. The gene was cloned from H. schachtii and subjected to a detailed molecular characterisation. The deduced protein contains a putative signal peptide for secretion, being in agreement with the presumed extracellular function of the mature protein. It has a molecular mass of 33.78 kDa and folds into an (α/β)8 barrel structure typical for glycosyl hydrolases. The two glutamic acids that function as electron donor and acceptor in the active site are conserved. Whole mount in situ hybridisation revealed that the gene is expressed in the subventral pharyngeal glands and the expression was correlated with the onset of parasitism.

Investigating the binding of β‐1,4‐galactan to Bacillus licheniformis β‐1,4‐galactanase by crystallography and computational modeling

Microbial beta-1,4-galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH-A of glycoside hydrolases, which cover many different poly- and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis beta-1,4-galactanase and its inactive nucleophile mutant have been obtained with methyl-beta(1-->4)-galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the beta-1,4-galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a beta(1-->4)-galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite -4 to +5. In particular, this analysis newly identified a conserved beta-turn, which contributes to subsites -2 to +3. This beta-turn is unique to family 53 beta-1,4-galactanases among all clan GH-A families that have been structurally characterized and thus might be a structural signature for endo-beta-1,4-galactanase specificity.

Fingerprinting complex pectins by chromatographic separation combined with ELISA detection

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-α- l-arabinan and (1,4)-β- d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts.

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.

Differentiation of Osteoblasts on Pectin-Coated Titanium

The gold standard for implant metals is titanium, and coatings such as collagen-I, RGD-peptide, chondroitin sulfate, and calcium phosphate have been used to modify its biocompatibility. We investigated how titanium coated with pectins, adaptable bioactive plant polysaccharides with anti-inflammatory effects, supports osteoblast differentiation. MC3T3-E1 cells, primary murine osteoblasts, and human mesenchymal cells (hMC) were cultured on titanium coated with rhamnogalacturonan-rich modified hairy regions (MHR-A and MHR-B) of apple pectin. Alkaline phosphatase (ALP) expression and activity, calcium deposition, and cell spreading were investigated. MHR-B, but not MHR-A, supports osteoblast differentiation. The MHR-A surface was not mineralized, but on MHR-B, the average mineralized area was 14.0% with MC3T3-E1 cells and 26.6% with primary osteoblasts. The ALP activity of hMCs on MHR-A was 58.3% at day 7 and 9.3% from that of MHR-B at day 10. These data indicate that modified pectin nanocoatings may enhance the biocompatibility of bone and dental implants.