RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

BACKGROUND: In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. RESULTS: As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. CONCLUSION: The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target.

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

'Biofilm Clippers'- enzyme formulation for bovine mastitic biofilm therapy.

Mastitis is one of the most important diseases that are threatening modern dairy farms. Biofilms of mastitic teat canal have serious clinical implications because of colonized pathogens having the ability to construct an extracellular polymeric substance (EPS) with increased tolerance to antimicrobials leads to difficulty in eradicating the infection. In this study, we investigated the synergistic biofilm disruptive effect of a combination of carbohydrate hydrolases targeting extracellular polysaccharides of biofilm matrix and we termed it as 'Biofilm Clippers (BC)'. Our findings demonstrate that the BC formulation exhibits intense biofilm-disrupting activity against Staphylococcus aureus biofilms. The results of the study showed that BC enables activity equivalent to physiologically achievable concentrations in disrupting biofilms of S. aureus in vitro. The synergistic anti-biofilm activities of BC on S. aureus biofilms demonstrated that the biofilm matrix is predominant of complex polysaccharides. Further, the confocal microscopic analysis demonstrates that the BC formulation is highly effective compared to the single treatment of either of the enzymes in disrupting the biofilm. To the best of our knowledge, this is the first report on the synergistic anti-biofilm activity of a class of enzyme formulation against mastitic biofilm mass. Even though a small study showed a promising effect on mastitic teat canal, further extensive investigation on a large number of bovines for mastitis therapeutic potential of this BC-derived product is now warranted.

The Interaction of Helicobacter pylori with TFF1 and Its Role in Mediating the Tropism of the Bacteria Within the Stomach.

Helicobacter pylori colonises the human stomach and has tropism for the gastric mucin, MUC5AC. The majority of organisms live in the adherent mucus layer within their preferred location, close to the epithelial surface where the pH is near neutral. Trefoil factor 1 (TFF1) is a small trefoil protein co-expressed with the gastric mucin MUC5AC in surface foveolar cells and co-secreted with MUC5AC into gastric mucus. Helicobacter pylori binds with greater avidity to TFF1 dimer, which is present in gastric mucus, than to TFF1 monomer. Binding of H. pylori to TFF1 is mediated by the core oligosaccharide subunit of H. pylori lipopolysaccharide at pH 5.0-6.0. Treatment of H. pylori lipopolysaccharide with mannosidase or glucosidase inhibits its interaction with TFF1. Both TFF1 and H. pylori have a propensity for binding to mucins with terminal non-reducing α- or ß-linked N-acetyl-d-glucosamine or α-(2,3) linked sialic acid or Gal-3-SO42-. These findings are strong evidence that TFF1 has carbohydrate-binding properties that may involve a conserved patch of aromatic hydrophobic residues on the surface of its trefoil domain. The pH-dependent lectin properties of TFF1 may serve to locate H. pylori deep in the gastric mucus layer close to the epithelium rather than at the epithelial surface. This restricted localisation could limit the interaction of H. pylori with epithelial cells and the subsequent host signalling events that promote inflammation.

Evaluation of Mannosidase and Trypsin Enzymes Effects on Biofilm Production of Pseudomonas aeruginosa Isolated from Burn Wound Infections.

Biofilm is an important virulence factor in Pseudomonas aeruginosa and has a substantial role in antibiotic resistance and chronic burn wound infections. New therapeutic agents against P. aeruginosa, degrading biofilms in burn wounds and improving the efficacy of current antimicrobial agents, are required. In this study, the effects of α-mannosidase, ß-mannosidase and trypsin enzymes on the degradation of P. aeruginosa biofilms and on the reduction of ceftazidime minimum biofilm eliminating concentrations (MBEC) were evaluated. All tested enzymes, destroyed the biofilms and reduced the ceftazidime MBECs. However, only trypsin had no cytotoxic effect on A-431 human epidermoid carcinoma cell lines. In conclusion, since trypsin had better features than mannosidase enzymes, it can be a promising agent in combatting P. aeruginosa burn wound infections.

Recombinant endo-mannanase (ManB-1601) production using agro-industrial residues: Development of economical medium and application in oil extraction from copra.

Expression of pRSETA manb-1601 construct in Hi-Control Escherichia coli BL21 (DE3) cells improved recombinant endo-mannanase (ManB-1601) production by 2.73-fold (1821±100U/ml). A low-cost, agro-industrial residue supplemented industrial medium for enhanced and economical production of ManB-1601 was developed in two mutual phases. Phase-I revealed the potential of various pre- (induction time: 5h, induction mode: lactose 0.5mM) and post-induction [peptone supplementation: 0.94%(w/v), glycerol 0.123%(v/v)] parameters for enhanced production of ManB-1601 and resulted in 4.61-fold (8406±400U/ml) and 2.53-fold (3.30g/l) higher ManB-1601 and biomass production, respectively. Under phase-II, economization of phase-I medium was carried out by reducing/replacing costly ingredients with solubilized-defatted flax seed meal (S-DFSM), which resulted in 3.25-fold (5926U/ml) higher ManB-1601 production. Industrial potential of ManB-1601 was shown in oil extraction from copra as enzyme treatment led to cracks, peeling, fracturing and smoothening of copra, which facilitated higher (18.75%) oil yield.

Effects of mannanase and distillers dried grain with solubles on growth performance, nutrient digestibility, and carcass characteristics of grower-finisher pigs.

Four experiments were conducted to determine the effects of dietary supplementation of corn distillers dried grain with solubles (DDGS) diets with mannanase on performance, apparent total tract digestibility (ATTD) of energy and nutrients, blood metabolites, and carcass characteristics of grower-finisher pigs. In Exp. 1, 96 grower pigs (initial BW, 57.6 kg), 6 pigs per pen and 4 pens per treatment, were fed corn-soybean meal-based diets containing 10% DDGS and 0, 200, 400, or 600 units (U) of mannanase/kg. The ADG and blood glucose increased (linear, P < 0.05) with increasing concentrations of dietary mannanase. Pigs fed diets containing increasing levels of mannanase had improved ATTD of DM and CP (quadratic, P < 0.05). In Exp. 2, 64 finisher pigs (initial BW, 92.7 kg) were allotted to 4 treatment groups with 4 pigs per pen and 4 pens per treatment. Pigs were fed corn-soybean meal-based diets containing 15% DDGS and 0, 200, 400, or 600 U of mannanase/kg. Linear increases (P < 0.05) in ADG, blood glucose, and ATTD of DM, GE, and CP were observed with increasing levels of dietary mannanase supplementation. In Exp. 3, 208 grower pigs (initial BW, 60.5 kg) were allotted to 4 treatment groups with 13 pigs per pen and 4 pens per treatment. Pigs were fed diets containing 0 or 10% DDGS and 0 or 400 U of mannanase/kg in a 2 x 2 factorial arrangement. An increase (P < 0.05) in ADG and blood glucose for pigs fed diets containing mannanase was observed. The ATTD of DM and CP (P < 0.05) was decreased with the inclusion of DDGS, whereas pigs fed the mannanase-supplemented diets had an increased (P < 0.05) ATTD of CP. In Exp. 4, 208 finisher pigs (initial BW, 86.5 kg), with 13 pigs per pen and 4 pens per treatment, were fed diets containing 0 or 15% DDGS and 0 or 400 U of mannanase/kg in a 2 x 2 factorial arrangement. The ADG and blood glucose increased (P < 0.05) when mannanase was included in the diets. The ATTD of DM (P < 0.05), GE (P < 0.10), and CP (P < 0.05) increased by the supplementation with mannanase in the diets of finisher pigs. The carcass characteristics and meat quality were not affected by the DDGS or mannanase inclusion. These results indicated that including 10 and 15% DDGS in conventional swine grower and finisher diets had no detrimental effects on growth performance or carcass characteristics. In addition, supplementation with 400 U of mannanase/kg to diets containing 10 and 15% DDGS fed to grower and finisher pigs may improve growth performance and the ATTD of CP.

Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness.

The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity.

An evaluation of endo-beta-D-mannanase (Hemicell) effects on broiler performance and energy use in diets varying in beta-mannan content.

Two experiments were conducted to evaluate the effects of a commercial endo-beta-D-mannanase (Hemicell) on overall performance, MEn, net energy for gain, and some serum parameters of broilers fed diets varying in beta-mannan level (experiment 1) and to evaluate effects of enzyme level on the same variables in broilers fed diet high in beta-mannan (experiment 2). As a semipurified beta-mannan source, guar gum was used to alter the dietary beta-mannan level. In experiment 1, guar gum was added at 0, 0.5, 1, and 2% in a corn-soy-based starter diet with (0.05%) and without endo-beta-D-mannanase supplementation in a 4 x 2 factorial design. Enzyme supplementation improved (P < 0.01) feed efficiency at control and each guar gum inclusion level, whereas 2% guar gum supplementation reduced (P < 0.01) BW and increased (P < 0.01) 14-d feed:gain ratio. Enzyme supplementation also increased dietary MEn and net energy gain. In experiment 2, endo-beta-D-mannanase was added at 0, 0.5, 1, and 1.5% in a corn-soy-based starter diet containing 1% guar gum. Increasing endo-beta-D-mannanase supplementation did not affect (P > 0.10) final BW but improved 14-d feed:gain ratio at all inclusion levels. As in the first experiment, ME improved (P < 0.05) with increasing enzyme inclusion. Dietary endo-beta-D-mannanase inclusion significantly reduced water:feed ratio and total dry fecal output (P < 0.01). Taken together, the results of these 2 experiments indicate that endo-beta-D-mannanase supplementation may improve the utilization of nutrients in diets containing beta-mannan.

The significance of N-linked glycosylation in pig endogenous retrovirus infectivity.

The significance of the envelope glycoprotein in the transmission of pig endogenous retrovirus (PERV) to human cells was investigated. Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection and then infected with PERV subtype B. Culture supernatants of the infected PEC previously incubated with several types of drugs were inoculated into HEK293 cells. The inoculated cells were then stained and the number of LacZ-positive foci was counted. PERV from tunicamycin treated PEC was not transmitted to human cells, indicating the importance of N-linked sugars in this process. Moreover, while inhibition of the terminal alpha-glucose residues from the precursor N-glycan by castanospermine and 1-deoxynojirimycin attenuated PERV infectivity, the mannosidase inhibitors, 1-deoxymannojirimycin and swainsonine, upregulated the infectivity. In addition, treatment with alpha-mannosidase and incubation with concanavalin A completely abrogated the transmission of PERV to HEK293. These data imply that the high-mannose type of N-glycan plays a key role in PERV infectivity.

Effect of mannan-endo-1,4-beta-mannosidase on the growth performance of turkeys fed diets containing 44 and 48% crude protein soybean meal.

Soybean meal (SBM) contains heat-resistant mannans. Domesticated turkeys are sensitive to mannans because of the high inclusion rate of SBM in their diets, causing increased chyme viscosity, wet droppings, and reduced feed conversion. Three experiments of similar design were conducted to determine the effect of mannan-endo-1,4-f'-mannosidase supplementation of corn-SBM diets on market turkeys. Experiment 1 was conducted at North Carolina State University using Nicholas hens raised from 1 to 98 d of age. Experiments 2 and 3 were conducted at PARC Institute Inc. using Large White turkey toms raised from 1 to 126 d of age. In each experiment, birds were randomly assigned to litter floor pens. Each pen was assigned to one of four experimental treatments in 2 x 2 factorial arrangement of two basal diets containing 44% CP and 48% CP SBM (SBM-44 and SBM-48, respectively) with or without 100 million units (MU) Hemicell/tonne (1 MU = 106 enzyme activity U). Birds fed SBM-44 had lower final BW (14.9 vs. 14.56 kg 18 wk BW / tom; 7.66 vs. 7.46 kg 14 wk BW/hen, P < 0.05) and higher final cumulative feed/gain than those fed the SBM-48. Hemicell supplementation generally improved performance of all birds, with a greater response in birds fed SBM-44. Hemicell improved BW and feed/gain by 1% (P = 0.779) and 3% (P = 0.377) in hens and 2.5% (P = 0.0016) and 4% (P = 0.0001) in toms, respectively. The results of these experiments indicate that some of the adverse effects of antinutritional factors of SBM of on turkey growth performance can be alleviated by dietary mannan-endo-1,4-beta-mannosidase supplementation.

Transfer specificity of detergent-solubilized fenugreek galactomannan galactosyltransferase.

The current experimental model for galactomannan biosynthesis in membrane-bound enzyme systems from developing legume-seed endosperms involves functional interaction between a GDP-mannose (Man) mannan synthase and a UDP-galactose (Gal) galactosyltransferase. The transfer specificity of the galactosyltransferase to the elongating mannan chain is critical in regulating the distribution and the degree of Gal substitution of the mannan backbone of the primary biosynthetic product. Detergent solubilization of the galactosyltransferase of fenugreek (Trigonella foenum-graecum) with retention of activity permitted the partial purification of the enzyme and the cloning and sequencing of the corresponding cDNA with proof of functional identity. We now document the positional specificity of transfer of ((14)C)Gal from UDP-((14)C)Gal to manno-oligosaccharide acceptors, chain lengths 5 to 8, catalyzed by the detergent-solubilized galactosyltransferase. Enzymatic fragmentation analyses of the labeled products showed that a single Gal residue was transferred per acceptor molecule, that the linkage was (1-->6)-alpha, and that there was transfer to alternative Man residues within the acceptor molecules. Analysis of the relative frequencies of transfer to alternative Man residues within acceptor oligosaccharides of different chain length allowed the deduction of the substrate subsite recognition requirement of the galactosyltransferase. The enzyme has a principal recognition sequence of six Man residues, with transfer of Gal to the third Man residue from the nonreducing end of the sequence. These observations are incorporated into a refined model for enzyme interaction in galactomannan biosynthesis.

Intestinal dipeptidyl peptidase IV is efficiently sorted to the apical membrane through the concerted action of N- and O-glycans as well as association with lipid microdomains.

The apical sorting of human intestinal dipeptidyl peptidase IV (DPPIV) occurs through complex N-linked and O-linked carbohydrates. Inhibition of O-linked glycosylation by benzyl-N-acetyl-alpha-d-galactosaminide affects significantly the sorting behavior of DPPIV in intestinal Caco-2 and HT-29 cells. However, random delivery to the apical and basolateral membranes and hence a more drastic effect on the sorting of DPPIV in both cell types is only observed when, in addition to O-glycans, the processing of N-glycans is affected by swainsonine, an inhibitor of mannosidase II. Together the data indicate that both types of glycosylation are critical components of the apical sorting signal of DPPIV. The sorting mechanism of DPPIV implicates its association with detergent-insoluble membrane microdomains containing cholesterol and sphingolipids, whereas an efficient association largely depends on the presence of a fully complex N- and O-linked glycosylated DPPIV. Interestingly, cholesterol is a more critical component in this context than sphingolipids, because cholesterol depletion by beta-cyclodextrin affects the detergent solubility and the sorting behavior of DPPIV more strongly than fumonisin, an inhibitor of sphingolipid synthesis.

The glycosylation status and the role of carbohydrate moieties in the heterogeneity of cucumber anionic virus-inducible peroxidase.

Three forms of anionic peroxidase (PRX) from hypersensitively reacting cucumber cotyledons were purified to homogeneity and different methods were used to analyze the nature of their carbohydrate chains. Immunoblot analysis with betaF1 antiserum showed that all three forms are highly glycosylated and contain asparagine N-linked glycans commonly found in other plant glycoproteins. Mobility shift analysis showed that chemical deglycosylation converted PRXs 1, 2 and 3 to the same-sized (35 K) products. Enzymatic deglycosylation with alpha-mannosidase converted PRX1 and PRX2 to immunoreactive products migrating in mobility shift polyacrylamide gels at the positions of PRX2 and PRX3, respectively. PRX3 treated with alpha-mannosidase yielded a product with Mr similar to that obtained with the chemical deglycosylation. Cleavage of the PRXs 1, 2 and 3 by formic acid at the Asp-Pro site resulted in peptide maps and the putative glycopeptide(s) were recognized using betaF1 antiserum. Only one glycopeptide was observed for each of the forms. Lectin-affinity blot analysis using biotin-conjugated lectins suggested that virus-inducible PRX contains complex-type N-glycosyl carbohydrate chain(s). These results indicate that heterogeneity of cucumber virus-inducible PRX is not caused mainly by differences in the terminal alpha-linked mannose residues.

Mannosidase action, independent of glucose trimming, is essential for proteasome-mediated degradation of unassembled glycosylated Ig light chains.

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain kappaNS1, which is unfolded in unassembled molecules. We investigated the fate of kappaNS1 glycosylated at position 70 (K70) and of a double mutant (kappa18/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappaNS1 chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.

Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography-electrospray mass spectrometry and Edman degradation.

An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with alpha-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry.

Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.

Hcs77 is a putative cell surface sensor for cell integrity signaling in Saccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39 degrees C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated the MID2 gene as a dosage suppressor of the cell lysis defect of an hcs77 null mutant. MID2 encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog of MID2, designated MTL1, which provides a partially redundant function with MID2 for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.

Family-10 and family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps.

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.

Affinity sites for beta-glucuronidase on the surface of human spermatozoa.

Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

Mycophenolic acid suppresses protein N-linked glycosylation in human monocytes and their adhesion to endothelial cells and to some substrates.

Mycophenolic acid (MPA) is the active part of the corresponding morpholinoethyl ester pro-drug Mycophenolate Mofetil. MPA, an inhibitor of IMP dehydrogenase, depletes GTP and thereby suppresses transfer of mannose and fucose to proteins. Treatment of human monocytes with a clinically attainable concentration of MPA (10 microM) decreases their attachment to endothelial cells and to laminin, but not to type I collagen or fibronectin. Our results not only elucidate a major role of mannose/fucose residues in homing of monocytes on activated endothelium but also explain in part the beneficial effects of MPA in rheumatoid arthritis and organ graft rejection.