Comparative efficacy of liposomes containing synthetic bacterial cell wall analogues for tumoricidal activation of monocytes and macrophages

Summary We examined the activation to the tumoricidal state of normal mouse peritoneal exudate macrophages, bone marrow macrophages, and human blood monocytes by liposomes containing either lipophilic muramyl tripeptide (CGP 19 835) or a new synthetic analogue of lipoprotein from gram-negative bacteria outer wall, CGP 31 362, or combinations of the two. The superiority of liposomes containing the synthetic lipopeptide over liposomes containing lipophilic muramyl tripeptide for in vitro activation of monocytes and macrophages was demonstrated in several experiments. First, liposome-CGP-19 835 activated monocytes only in the presence of interferon-, whereas activation with liposome-CGP 31 362 was interferon-independent. Second, activation of both mouse macrophages and human blood monocytes by liposome-CGP 31 362 occurred at a lower liposomal concentration than that by liposome-CGP 19 835. Third, monocytes incubated with liposome-CGP 31 362 released both tumor necrosis factor (TNF) and interleukin-1 activities, whereas monocytes treated with liposome-CGP 19 835 (in the absence of interferon-) released only TNF activity. These data suggest that liposomes containing the synthetic lipopeptide CGP 31 362 are superior to liposomes containing CGP 19 835 for systemic activation of macrophages.     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

     

The Induction of Colitis and Ileitis in Mice Is Associated with Marked Increases in Intestinal Concentrations of Stimulants of TLRs 2, 4, and 5

Background

Inflammatory bowel diseases (IBDs) appear to be modulated by the interaction of pathogen-associated molecular patterns (PAMPs) derived from intestinal bacteria with their respective innate immune receptors, including Toll-like receptors (TLRs). We aimed to establish if intestinal concentrations of proinflammatory bacterial ligands of TLR2, TLR4, or TLR5 may be altered in murine IBD models, and to characterize which of the major bacterial groups may contribute to each signal.

Methodology/Principal Findings

PAMPs specific for TLR2 (lipopeptide equivalents), TLR4 (lipopolysaccharide equivalents), and TLR5 (flagellin equivalents) in human and murine fecal and intestinal samples were quantified using HEK-293 cells transfected with respective TLRs and calibrated with defined standard PAMPs. The induction of colitis in mice by dextran-sodium-sulphate treatment significantly increased colonic lipopeptide (fourfold) and LPS equivalent (550-fold) concentrations, while flagellin equivalent concentrations remained similar. The induction of ileitis by oral infection with Toxoplasma gondii dramatically increased ileal concentrations of lipopeptide (370-fold), LPS (3,300-fold), and flagellin equivalents (38-fold), all P<0.01. Analysis of representative strains of the major bacterial groups of the human intestine revealed that enterobacterial species are likely to be more significant contributors of soluble TLR2 and TLR4 stimulants to the intestinal milieu than Bacteroides species or Gram-positive Firmicutes.

Conclusions/Significance

We conclude that the induction of colitis or ileitis in mice is associated with significant disease-specific alterations to the PAMP profile of the gut microbiota.     

Improvement of glidobactin A production byPolyangium brachysporum

Summary Glidobactins A, B and C are lipopeptide antitumor antibiotics produced by the gliding bacteriumPolyangium brachysporum sp. nov. No. K481-B101. The production of glidobactin A was examined in shake flasks and laboratory fermentors. Medium screening and optimization led to approximately five fold increases in glidobactin A titers in shake flasks and a ten fold increase in titers in 40-1 batch fermentations. Utilization of a stepped glucose feeding protocol resulted in glidobactin A titers of 1860 g/ml after 144 h of fermentation.     

Characterization of <Emphasis Type="Italic">lpaH2</Emphasis> gene corresponding to lipopeptide synthesis in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> HAB-2

Background

Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.

Results

A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.

Conclusion

Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.

     

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH₂ (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.     

Structural studies on equine chorionic gonadotropin

The amino acid sequence of the subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin subunit; in particular, their sequence lacked the first fourteen residues.For the subunit we have placed in sequence 104 amino acid residues by direct sequence determination and peptide overlap procedures; in addition, 37 residues have been placed provisionally by homology with the human chorionic gonadotropin (hCG) sequence and composition and/or sequence data for the peptides isolated in the present studies. Difficulties in the procurement of the hormone have stalled completion of the -subunit amino acid sequence determination. The data now available indicate that eCG -subunit is highly homologous to hCG subunit and the subunits of luteinizing hormone from the pituitary gland of the several species so far described. The proposed partial sequence of eCG is:     

Synthesis of lipopeptides of the immunodominant epitope hMBP(83–99) containing amide or C-C bond linked hydrophobic chains for the study of T cell response

We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74–85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitis in vivo strongly increased the T cell proliferative response in vitro. We extended this study to the human immunodominant epitope hMBP(83–99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83–99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited superagonist activity.     

Al-induced inhibition of root elongation in corn,Zea mays L. is overcome by Si addition

The effect of silicic acid on Al-induced inhibition of root elongation was investigated in corn roots (Zea mays L. cv. golden cross bantam) in 100 t M CaCl2 solution at pH 4.3. Twenty t M Al inhibited root elongation (20 h) about 70%, however, inhibition was alleviated by addition of silicic acid. The alleviative effect increased with higher silicic acid concentrations. The concentration of Al3+, the toxic species, in solution was decreased to about 15, 10, and 5 t M, respectively, from the initial concentration of 20 t M by addition of silicic acid at 500, 1000, and 2000 t M Si. Under the same concentration of Al3+, Al-induced inhibition of root elongation showed the same extent regardless of the addition of silicic acid or not by comparing 5 t M Al treatment with 20 t M Al + 2000 t M Si treatment, and 10 t M Al treatment with 20 t M Al +1000 t M Si treatment. Viability of cells on the root tip surface was decreased by Al addition. Cell viability was not improved by addition of silicic acid under the same concentration of Al3+. All these facts suggest that the alleviative effect of silicic acid on Al toxicity resulted from decreasing toxic Al3+ concentration by forming Al-Si complexes rather than from other physiological effects of silicic acid in corn roots.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

P2Y6 nucleotide receptor activates PKC to protect 1321N1 astrocytoma cells against tumor necrosis factor-induced apoptosis

1. We recently reported that the activation by UDP of rat P2Y₆ nucleotide receptors expressed in 1321N1 astrocytoma cells protected them from TNF-induced apoptosis by suppressing activation of caspase 3 and 8. This study aims to characterize the involvement of intracellular signaling pathways, including kinases, involved in the antiapoptotic effect of UDP.2. Cell death was induced in 1321N1 astrocytoma cells permanently expressing the rat P2Y₆ receptor by exposure to TNF in the presence of cycloheximide. The apoptotic fraction was analyzed using flow cytometry.3. The activation of P2Y₆ receptors by UDP both protected the astrocytes from TNF- induced apoptosis and activated protein kinase C (PKC) isotypes. The phorbol ester PMA also activated PKC and protected the cells from TNF-induced cell death. The - and -isotypes of PKC were both activated in a persistent fashion upon 5-min exposure to either UDP (10 M) or the phorbol ester PMA (100 nM). The PKC isotype was markedly activated upon UDP treatment.4. The addition of PKC inhibitors, GF109203X or Gö6976, partially antagonized the protective effect of UDP and reduced the UDP-induced phosphorylation of extracellular signal-regulated protein kinases (Erk). The inhibitors of Erk, PD98,059 or U0126, antagonized UDP-induced protection.5. The antiapoptotic protein, Akt, was not affected by P2Y₆ receptor activation. Incubation of the astrocytes with calcium modifiers, BAPTA-AM or dantrolene, did not affect the UDP-induced protection from apoptosis.6. The addition of phospholipase C (PLC) inhibitors, D609 or U73122, partially antagonized both UDP-induced protection and PKC activation.7. Therefore, it is suggested that P2Y₆ receptors in 1321N1 cells, through coupling to PC-PLC and PI-PLC, activate PKC to protect against TNF -induced apoptosis, in which the activation of Erk is involved in part.     

Antimutagenic Role of Autonomous 3′ → 5′-Exonucleases

Original and published data on the antimutagenic role of autonomous 3 5-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but are often involved in replicative complexes. AE overproduction in bacterial cells is accompanied by a sharp suppression of mutagenesis, whereas AE inactivation in bacteria and higher fungi results in the increase in mutation rates by two to three orders of magnitude. The combined action of AE and DNA polymerases substantially improves the fidelity of their functioning in vitro. The fidelity of nuclease-free DNA polymerases and increases by two to three orders of magnitude in the presence of AE. The fidelity of moderately processive DNA polymerase I increases by two orders of magnitude, and that of highly processive DNA polymerase increases by a factor of 5–10, although both these polymerases possess their own 3 5-exonucleolytic activity. In biochemical experiments, AE was shown to participate directly in the correction of errors made by DNA polymerase I. The presence of AE in multienzyme DNA polymerase complexes increases their fidelity by a factor of 5–10. A model of extrinsic proofreading by AE in DNA biosynthesis is proposed. An investigation of thirty objects from all three kingdoms of life (from archaea and bacteria to mammals, including humans) has shown that AE account for 30–90% of the total cellular 3 5-exonucleolytic activity. Therefore, AE increase significantly the intracellular ratio of 3 5-exonuclease to DNA polymerase activities in a wide phylogenetic variety of species, which always leads to the increasing fidelity of DNA biosynthesis.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

Active site directed inhibitors and mechanism of action of glycosidases

Summary A review is given on the inactivation of glycosidases by active site directed inhibitors, their use to identify functional groups at the active site, and deductions with respect to the mechanism of catalysis. The following enzymes are discussed:-glucosidases from various sources,-glucosidase from S. cerevisiae,-galactosidases from E. coli and Helix pomatia, cellulase and lysozyme. Epoxide type inhibitors appear especially suitable because their reactivity is enhanced selectively by acidic groups at the active site.In those cases where definite conclusions regarding the mechanism can be drawn, catalysis seems to occur by a carboxylate ion as base and an acid that has been identified as a carboxyl group with-glucosidase from Asp. wentii. The hydrolysis probably proceeds via a glycosyl-enzyme intermediate, which may be a stabilized carbonium ion or a covalent intermediate.an invited article     

Screening of white-rot fungi for biological pretreatment of wheat straw for biogas production

Summary Twenty two basidiomycetes, mostly white rot fungi, were grown on wheat straw. Lignin-, cellulose-, and hemicellulose-degradation was recorded in order to find a species growing on lignin preferably. The oyster-mushroomPleurotus sp. florida showed fastest delignification of all tested fungi.Straw pretreated by this fungus was fermented anaerobically to biogas. The gas yield produced from myco-straw is twice the amount from untreated straw.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

Nucleotide sequence of the maize chloroplast rpo B/C1/C2 operon: Comparison between the derived protein primary structures from various organisms with respect to functional domains

Summary The genes (rpo B/C₁/C₂) coding for the , , subnits of maize (Zea mays) chloroplast RNA polymerase have been located on the plastome and their nucleotide sequences established. The operon is part of a large inversion with respect to the tobacco and spinach chloroplast genomes and is flanked by the genes trnC and rps2. Notable features of the nucleotide sequence are the loss of an intron in rpoC₁, and an insertion of approximately 450 by in rpOC₂ compared to the dicotyledons tobacco, spinach and liver-wort. The derived amino acid sequence of this additional monocotyledon specific sequence is characterized by acidic heptameric repeat units containing stretches of glutamic acid, tyrosines and leucines with regular spacing. Other structural motifs, such as a nucleotide binding domain in the subunit and a zinc finger in the subunit, are compared at the amino acid level throughout the RNA polymerase subunits with the enzymes from other organisms in order to identify functionally important conserved regions.The sequence data presented in this paper will appear in the EMBL/Gen Bank/DDBJ Nucleotide Databases under the accession number X17318     

A membrane-bound enzyme complex synthesising glucan and glucomannan in pine tissues

Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [¹⁴C]glucans using either guanosine 5-diphosphate (GDP)-D-[U-¹⁴C]glucose or uridine 5-diphosphate (UDP)-D-[U-¹⁴C]glucose as glycosyl donors. Although these glucans had -(13) and -(14) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed -(13) and -(14) glucan from GDP-D-[U-¹⁴C]glucose was changed to that of -(14) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-¹⁴C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K _m and V _max of the glucan synthase for GDP-D-glucose were 6.38 M and 5.08 M·min^-1, respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.Abbreviations GDP guanosine 5-diphosphate - GLC gasliquid chromatography - UDP trridine 5-diphosphate     

Kallikrein 6 signals through PAR1 and PAR2 to promote neuron injury and exacerbate glutamate neurotoxicity

CNS trauma generates a proteolytic imbalance contributing to secondary injury, including axonopathy and neuron degeneration. Kallikrein 6 (Klk6) is a serine protease implicated in neurodegeneration, and here we investigate the role of protease‐activated receptors 1 (PAR1) and PAR2 in mediating these effects. First, we demonstrate Klk6 and the prototypical activator of PAR1, thrombin, as well as PAR1 and PAR2, are each elevated in murine experimental traumatic spinal cord injury (SCI) at acute or subacute time points. Recombinant Klk6 triggered extracellular signal‐regulated kinase (ERK1/2) signaling in cerebellar granule neurons and in the NSC34 spinal cord motoneuron cell line, in a phosphoinositide 3‐kinase and MEK‐dependent fashion. Importantly, lipopeptide inhibitors of PAR1 or PAR2, and PAR1 genetic deletion, each reduced Klk6‐ERK1/2 activation. In addition, Klk6 and thrombin promoted degeneration of cerebellar neurons and exacerbated glutamate neurotoxicity. Moreover, genetic deletion of PAR1 blocked thrombin‐mediated cerebellar neurotoxicity and reduced the neurotoxic effects of Klk6. Klk6 also increased glutamate‐mediated Bim signaling, poly‐ADP‐ribose polymerase cleavage and lactate dehydrogenase release in NSC34 motoneurons and these effects were blocked by PAR1 and PAR2 lipopeptide inhibitors. Taken together, these data point to a novel Klk6‐signaling axis in CNS neurons that is mediated by PAR1 and PAR2 and is positioned to contribute to neurodegeneration.