David S. Adams

PGG-Glucan, a soluble β-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-κB-like factor in human PMN: Evidence for a glycosphingolipid β-(1,3)-glucan receptor

PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.

PGG‐Glucan activates NF‐κB‐like and NF‐IL‐6‐like transcription factor complexes in a murine monocytic cell line

PGG‐Glucan (Betafectin®) is a novel soluble β‐glucan immunomodulator that enhances leukocyte microbicidal activities without inducing inflammatory cytokines. Although several different receptors for soluble and particulate β‐glucans have been described, the signal transduction pathway(s) used by soluble β‐glucans have not been elucidated. We report that in a murine monocytic cell line (BMC2.3) PGG‐Glucan activates nuclear factor‐κB (NF‐κB)‐like and NF‐interleukin‐6 (IL‐6)‐like transcription factors. Electrophoretic mobility shift assays showed that PGG‐Glucan activation of the factors is time‐ and concentration‐dependent. The NF‐κB‐like complex includes subunit p65 (rel‐A) as one of its components, but apparently not p50 (κB1), p52 (κB2), p68 (rel‐B), or p75 (C‐rel) family members. The NF‐IL‐6‐like complex contains subunit C/EBP‐β (NF‐IL‐6α) as one of its components, but apparently not C/EBP‐α or C/EBP‐δ (NF‐IL‐6β). As expected, lipopolysaccharide (LPS) activated p65/p50 NF‐κB and C/EBP‐β NF‐IL‐6 complexes, increased the nuclear titer of p65 and p50 antigens, and increased cytokine (IL‐1β, tumor necrosis factor α) mRNA production. In contrast, PGG‐Glucan increased the nuclear titer of p65, but apparently not p50, and did not induce cytokine mRNA production, These data demonstrate that PGG‐Glucan utilizes signal transduction pathways different from those used by LPS. The data suggest that activation of the PGG‐Glucan‐stimulated factors is not sufficient to stimulate cytokine mRNA transcription. J. Leukoc. Biol. 62: 865–873; 1997.

Activation of a rel-A/CEBP-beta-related transcription factor heteromer by PGG-glucan in a murine monocytic cell line.

PGG‐Glucan is a soluble β‐glucan immunomodulator that enhances a variety of leukocyte microbicidal activities without activating inflammatory cytokines. Although several different cell surface receptors for soluble (and particulate) β‐glucans have been described, the signal transduction pathway(s) used by these soluble ligands have not been elucidated. Previously we reported that PGG‐Glucan treatment of mouse BMC2.3 macrophage cells activates a nuclear factor κ‐B‐like (NF‐κB) transcription factor complex containing subunit p65 (rel‐A) attached to an unidentified cohort. In this study, we identify the cohort to be a non‐rel family member: a CCAAT enhancer‐binding protein‐β (C/EBP‐β)‐related molecule with an apparent size of 48 kDa, which is a different protein than the previously identified C/EBP‐β p34 also present in these cells. C/EBP‐β is a member of the bZIP family whose members have previously been shown to interact with rel family members. This rel/bZIP heteromer complex activated by PGG‐Glucan is different from the p65/p50 rel/rel complex induced in these cells by lipopolysaccharide (LPS). Thus, our data demonstrate that PGG‐Glucan uses signal transduction pathways different from those used by LPS, which activates leukocyte microbicidal activities and inflammatory cytokines. We further show that heteromer activation appears to use protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways, but not mitogen‐activated protein kinase p38. Inhibitor κ‐B‐α (IκB‐α) is associated with the heteromer; this association decreases after PGG‐Glucan treatment. These data are consistent with a model whereby treatment of BMC2.3 cells with PGG‐Glucan activates IκB‐α via PKC and/or PTK pathways, permitting translocation of the rel‐A/CEBP‐β heteromer complex to the nucleus and increases its DNA‐binding affinity. J. Cell. Biochem. 77:221–233, 2000.

CMX-8933, a peptide fragment of the glycoprotein ependymin, promotes activation of AP-1 transcription factor in mouse neuroblastoma and rat cortical cell cultures

An 8-amino acid peptide fragment (CMX-8933) of Ependymin, a glycoprotein component of the extracellular fluid and cerebrospinal fluid of goldfish brain, was synthesized and tested for its capacity to activate AP-1 transcription factor in cell cultures. Dose-response and time-course studies of AP-1s binding to DNA were carried out in neuroblastoma (NB2a/dl) and primary rat brain cortical cultures using an electrophoretic mobility shift assay (EMSA). A 13-14-fold increase in AP-1s DNA binding was obtained when NB2a cells were incubated for 4 h with 6-10 microg/ml CMX-8933. Primary rat brain cortical cultures were much more sensitive to the effects of CMX-8933 than transformed (NB2a) cultures; here a 26.7+/-5.2-fold increase in binding was observed following a 3-h treatment with as little as 10 ng/ml peptide. These findings are consistent with an activation of this transcription factor, a characteristic that has been previously correlated with functional aspects of full-sized neurotrophic factors (nerve growth factor and brain-derived nerve growth factor) in neuronal differentiation and regeneration. Such data suggest a role for Ependymin in transcriptional control.

A peptide fragment of ependymin neurotrophic factor uses protein kinase C and the mitogen-activated protein kinase pathway to activate c-Jun N-terminal kinase and a functional AP-1 containing c-Jun and c-Fos proteins in mouse NB2a cells.

Ependymin (EPN) is a goldfish brain neurotrophic factor previously shown to function in a variety of cellular events related to long‐term memory formation and neuronal regeneration. CMX‐8933, an 8‐amino‐acid synthetic peptide fragment of EPN, was designed for aiding an investigation of the biological properties of this glycoprotein. We reported from previous studies that treatment of mouse neuroblastoma (NB2a) cultures with CMX‐8933 promotes activation of transcription factor AP‐1, a characteristic previously associated with the following full‐length neurotrophic factors: nerve growth factor, neurotropin‐3, and brain‐derived neurotrophic factor. The CMX‐8933‐activated AP‐1 specifically bound an AP‐1 consensus probe and appeared to contain c‐Jun and c‐Fos protein components in antibody supershift experiments. Because AP‐1 influences a variety of positive and negative cellular processes, determined in part by its exact protein composition and mechanism of activation, we extended these initial AP‐1 observations in the current study to confirm the identity of the CMX‐8933‐activated c‐Jun and c‐Fos components. CMX‐8933 increases the enzymatic activity of c‐Jun N‐terminal kinase (JNK), increases the phosphorylation of JNK and c‐Jun proteins, and increases the cellular titers of c‐Jun and c‐Fos mRNAs. Furthermore, the AP‐1 activated by CMX‐8933 is functional, insofar as it transactivates both synthetic and natural AP‐1‐dependent reporter plasmids. Inhibition studies indicate that activation of the 8933‐induced AP‐1 occurs via the mitogen‐activated protein kinase pathway. These data are in agreement with the recently proposed model for the conversion of short‐ to long‐term synaptic plasticity and memory, in which a JNK‐activated transcription factor AP‐1, containing c‐Jun and c‐Fos components, functions at the top of a hierarchy of transcription factors known to regulate long‐term neural plasticity.

Cloning and sequencing the genes encoding goldfish and carp ependymin

Ependymins (EPNs) are brain glycoproteins thought to function in optic nerve regeneration and long-term memory consolidation. To date, epn genes have been characterized in two orders of teleost fish. In this study, polymerase chain reactions (PCR) were used to amplify the complete 1.6-kb epn genes, gf-I and cc-I, from genomic DNA of Cypriniformes, goldfish and carp, respectively. Amplified bands were cloned and sequenced. Each gene consists of six exons and five introns. The exon portion of gf-I encodes a predicted 215-amino-acid (aa) protein previously characterized as GF-I, while cc-I encodes a predicted 215-aa protein 95% homologous to GF-I.

Comparison of U-small nuclear RNA levels in tissues of the silkmoth, Bombyx mori

Abstract 1. 1. Tissue-specific abundance of the capped small RNAs in the silkmoth Bombyx mori was compared using preparative immunoprecipitation with anti-trimethylguanosine antibody. 2. 2. The yields of total capped small RNAs from larval posterior silk gland, 1. early, 2. late in the fifth-instar, and 3. immortal ovarian-derived cells in culture, were determined to be 187, 50 and 218 ng, respectively, per mg of total cellular RNA. 3. 3. Separation of immunoprecipitated RNAs by polycrylamide gel electrophoresis, followed by densitometric analysis of the bands, allowed the quantitation of individual capped molecules. 4. 4. This analysis revealed tissue-specific patterns. 5. 5.|The data indicate that the total abundance of capped small RNAs in Bombyx is highest in rapidly-dividing cells.

Genes encoding giant danio and golden shiner ependymin

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, trueepn genes have been characterized in one order of teleost fish,Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the completeepn genes,gd (1480 bp), andsh (2071 bp), fromCypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid, genome. Each gene was found to contain six exons and five introns. Genegd encodes a predicted 218-amino acid (aa) protein GD 93% conserved to goldfish EPN, whilesh encodes a predicted 214-aa protein SH 91% homologous to goldfish. Evidence is presented classifying proteins previously termed “EPNs” into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark features of true EPNs.