Apoptotic effect of rituximab on peripheral blood B cells in rheumatoid arthritis.

Rituximab (RTX) has proven efficacious in the treatment of rheumatoid arthritis (RA). Herein, we assessed the apoptosis-inducing capability of RTX in vitro on RA peripheral blood B-cell subsets and also compared the effects of RTX on B cells from rheumatoid factor-positive (RF+) and RF- patients. The likely relevance of B cells in disease was assessed by measuring B-cell-modulating serum cytokines. Peripheral blood B cells were isolated and cultured with the presence or absence of RTX. The levels of apoptosis within the naïve, memory and IgD+CD27+ B-cell subpopulations were determined by cytofluorometric analysis and caspase 3/7 assays. Levels of serum cytokines were measured with a multiplex cytokine array system. RTX induced significant apoptosis in all B-cell subsets in both RA and controls. In naïve and memory B cells from RA patients, RTX induced significantly higher levels of apoptosis than in controls. RTX induced apoptosis of B cells in RF+ and RF- patients. Serum levels of interleukin-1beta (IL-1beta), IL-4, IL-10 and IL-13 were profoundly increased in RF+ patients compared to RF- patients and controls. Although our cohort was small (10 RA patients), the data suggest that RTX induces apoptosis in all investigated subsets of B cells from RA patients. Interestingly, memory B cells from RA patients were more sensitive to RTX than memory cells from normal controls, suggesting that the delay in treatment response to RTX observed in clinical trials may be due in part to memory cell depletion. The apoptotic effects of RTX were similar in RF+ and RF- patients, but serum levels of B-cell-activating cytokine levels were only elevated in RF+ but not RF- patients. These data suggest that RTX is less effective in RF- RA because B cells play a less significant role in RA pathogenesis in RF- patients.

Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger.

Extracellular alpha-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for approximately 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (beta-D-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex(7-26)HexHA(c2). Indicating that oligosaccharides from GlcNA(c2)Man(7), increasing in molecular size up to GlcNA(c2)Man(24) were present. Each of these were additionally substituted with up to three beta-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger.

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.

Beta-galactofuranoside glycoconjugates on conidia and conidiophores of Aspergillus niger.

Galactose in the furanoic conformation appears to be limited to bacteria and lower eukaryotes. Galactofuranoic (Galf)-containing glycoconjugates that occur in organisms pathogenic or allergenic to man are frequently antigenic and immunodominant. We have used an immunochemical approach, employing a monoclonal antibody that recognises Galf epitopes, to investigate the presence of Galf-containing glycoconjugates within conidia and conidiophores of Aspergillus niger. ELISA and immunofluorescence microscopy indicated that specific and saturable binding sites were found on both. Inhibition studies confirmed that this binding was to Galf-containing glycoconjugates. Interestingly, the conidiophore heads were particularly rich in these glycoconjugates. Western blotting identified a Galf glycoprotein of 150-200 kDa from disrupted conidia.

An extracellular beta-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis.

Aspergillus niger produces an extracellular beta-galactofuranosidase, which can specifically hydrolyse beta-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when beta-galactofuranosidase is absent, suggesting in situ beta-galactofuranosidic hydrolysis. The beta-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS-PAGE) with activity against beta-linked Galf residues, with a Km of 4 mM against p-nitrophenyl-beta-D-galactofuranoside and a pH optimum of 3-4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-beta-D- and -alpha-D-galactopyranose, and alpha-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.

Dietary choline restriction causes complex I dysfunction and increased H(2)O(2) generation in liver mitochondria.

Removal of choline from the diet results in accumulation of triglycerides in the liver, and chronic dietary deficiency produces a non-genotoxic model of hepatocellular carcinoma. An early event in choline deficiency is the appearance of oxidized lipid, DNA and protein, suggesting that increased oxidative stress may facilitate neoplasia in the choline deficient liver. In this study, we find that mitochondria isolated from rats fed a choline-deficient, L-amino acid defined diet (CDAA) demonstrate impaired respiratory function, particularly in regard to complex I-linked (NADH-dependent) respiration. This impairment in mitochondrial electron transport occurs coincidentally with alterations in phosphatidylcholine metabolism as indicated by an increased ratio of long-chain to short-chain mitochondrial phosphatidylcholine. Moreover, hydrogen peroxide (H(2)O(2)) generation is significantly increased in mitochondria isolated from CDAA rats compared with mitochondrial from normal rats, and the NADH-specific yield of H(2)O(2) is increased by at least 2.5-fold. These findings suggest an explanation for the rapid onset of oxidative stress and energy compromise in the choline deficiency model of hepatocellular carcinoma and indicate that dietary choline withdrawal may be a useful paradigm for the study of mitochondrial pathophysiology in carcinogenesis.

An unambiguous microassay of galactofuranose residues in glycoconjugates using mild methanolysis and high pH anion-exchange chromatography.

An original, unambiguous microassay of galactofuranose (Galf) residues in glycoconjugates is described. The method involves mild acid methanolysis (5 mM HCl) for 3 h at 84 degrees C followed by high pH anion-exchange chromatography using a routine monosaccharide system. The methanolysis products Mealpha-Galf and Mebeta-Galf were characterized chromatographically by comparison with the authentic compounds and by their response to treatment with mild acid and with beta-galactofuranosidase. Testing against p-nitrophenyl-beta-Galf and UDPalpha-Galf showed the method to be applicable to both alpha- and beta- galactofuranosides over the range 10-200 pmol. The results of partial mild methanolysis over shorter periods were consistent with initial inversion of anomeric configuration at methylation followed by anomerization to an equilibrium mixture of alpha- and beta-forms. When applied to a sample of invertase from Aspergillus nidulans, the method indicated that all of the mild acid-labile galactose (78% of the total galactose present) was in the form of a galactofuranoside and that much of this was in the beta-configuration. As expected, when applied to asialofetuin (known to contain galactose only in the pyranoside form, Galp), NPalpha-Galp, NPbeta-Galp, or UDPalpha-Galp, mild acid methanolysis failed to produce any galactofuranoside.

Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation.

We have studied the effects of overexpression and secretion of a homologous model glycoprotein, glucoamylase (GAM-1), on glycosylation in a single gene copy wild-type parent and multiple gene copy transformants of Aspergillus niger. In batch culture the B36 strain, which possess 80 additional copies of the GAM glaA gene, secreted about 5-8-fold more protein and GAM-1 than the parent strain (N402). A comparison of the glycosylation of GAM-1 secreted by the parent strain with that secreted by the multiple copy and hyper-secreting B36 strain showed that both the N-linked and O-linked glycan composition was very similar. Short oligomannose N-linked glycans were found (Man(7-8)GlcNAc(2)). O-Linked glycans were comprised of short (1-3 residues) oligosaccharide chains of mannose and galactose. Evidence is presented that this galactose is present in the novel galactofuranose conformation. This glycan composition of GAM-1 differed from that of a commercially available (A. niger) GAM source. Microsomes prepared from the mycelium showed a 2-3-fold co-ordinated increase in the activity of the dolichol phosphate:glycosyltransferases. Similar results were obtained from strains B1 (20 copies of glaA) and N402 when grown at a low dilution rate in a chemostat, although both the levels of GAM secretion and the activities of the dolichol phosphate:glycosyltransferases were lower than found in batch culture. These data suggest that A. niger is capable of secreting large amounts of a single glycoprotein combined with higher activity levels of the dolichol phosphate:glycosyltransferases without an increase in the heterogeneity of the glycan structures. Thus, from a biotechnological viewpoint, protein glycosylation may not be a bottleneck to enhanced glycoprotein production using A. niger.

Expression of cytokines and activation of transcription factors in lipopolysaccharide-administered rats and their inhibition by phenyl N-tert-butylnitrone (PBN).

The spin-trapping compound phenyl N-tert-butylnitrone (PBN) affords protection from the lethality of septic shock in rodents. Previous studies have shown that PBN elicits its protection by inhibiting inducible nitric oxide synthase (iNOS) induction. In the present study, using the lipopolysaccharide (LPS) rat septic shock model, we determined the expression of various cytokine genes (tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma, interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-10) and the activation of transcription factors nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) in the liver tissue, 30 min and 3 h after LPS administration. The effects of PBN preadministration on the production levels were also investigated. The results show that LPS (4 mg/kg, ip) induced the production of the cytokine genes and increased the nuclear protein level of NF-kappaB within 30 min after LPS administration. Preadministration of PBN (150 mg/kg, ip) significantly down-regulated the production of cytokine genes (TNF-alpha by 94%, IL-1 by 63%, and IL-1 by 70%) and reduced the nuclear protein level of NF-kappaB by 75% and AP-1 by 72% at 3 h after LPS injection. These results demonstrate that PBN, in addition to its iNOS induction inhibition, also has multiple anti-inflammatory effects in septic shock, via modulation of the production of the key inflammatory mediators.

Investigation of the glycosyltransferase enzymes involved in the initial stages of the N-linked protein glycosylation pathway in Aspergillus niger.

A protocol has been developed to produce functional microsomes from mycelium of Aspergillus niger. Using these preparations, radioactive biochemical assays have been designed and optimised to measure the in vitro activities of three of the enzymes of the N-linked dolichol phosphate (Dol-P) glycosylation pathway: UDP-N-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), Dol-P mannose synthase (DPMS) and Dol-P glucose synthase (DPGS). Exogenous Dol-P and Triton X-100 are essential for in vitro activity. All three Dol-P:glycosyl transferases had alkaline pH optima and activity rapidly decreased below neutrality. Characterisation of the glycolipid products by anion-exchange chromatography and TLC showed that they were Dol-P-P-GlcNAc, Dol-P-Glc and Dol-P-Man for GPT, DPGS and DPMS, respectively. The antibiotic tunicamycin completely inhibited GPT activity at a concentration of 100-200 ng ml(-1) and an IC50 of 40 ng ml(-1), but had little effect on the other two enzymes. The ratio of the activity of the three enzymes to each other suggested that DPMS may be involved in other cellular activities and is probably under different control mechanisms than the other two.

Inhibition of NF-kappaB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl-N-tert-butylnitrone (PBN).

Previously, the spin trapping agent phenyl-N-tert-butylnitrone (PBN) has been shown to decrease the level of nitric oxide synthase mRNA in vivo. This inhibition is suggested to be an underlying mechanism for PBN's wide variety of pharmacological actions in animal models. However, the determination of PBN's cellular pharmacological activities has not been carried out, but is necessary for the understanding of the effects in vivo. Since the known pharmacological effects of PBN are primarily anti-inflammatory in nature, in this study we determined the inhibitory activities of PBN against two inflammatory factors: inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX2). We show here that PBN decreases steady state COX2 mRNA level and COX2 catalytic activity in macrophage cell culture at supra-pharmacological concentrations. While PBN decreases iNOS mRNA, it does not inhibit iNOS catalytic activity, which is consistent with previous in vivo studies. We also studied nuclear factor kappaB (NF-kappaB), a transcription factor that can rapidly activate the expression of genes involved in inflammatory, immune and acute phase responses. The binding of NF-kappaB to iNOS gene has been shown to be critical for iNOS gene expression, and the promoter region of COX2 gene contains NF-kappaB consensus sequence. We show that PBN inhibits lipopolysaccharide-mediated increase of NF-kappaB DNA binding activity with a lower concentration than that for the non-steroidal anti-inflammatory drug (NSAID), salicylate. Furthermore, we show that PBN inhibits COX2 catalytic activity, suggesting that PBN has an NSAID-like function.

Secretion of two beta-fructofuranosidases by Aspergillus niger growing in sucrose.

Aspergillus niger is induced to secrete two invertases, named SUC 1 and SUC 2, when grown on a minimal medium containing sucrose. Although, both have been classified as beta-D-fructofuranoside fructohydrolases, SUC 2 also possesses inulin hydrolytic activity (sucrose/inulin activity ratio of 4). These activities have been separated from each other and almost completely purified by anion-exchange, lectin affinity chromatography, and chromatofocusing. SUC 1 appeared as a single glycoprotein band on PAGE and SDS-PAGE corresponding in size to 250 and 125 kDa, respectively, compared with a much broader band (suggesting greater glycan heterogeneity) of 210-240 and 90-120 kDa for SUC 2. Therefore, both may be dimers, in their natural conformation. The glycan part of both contained the same monosaccharides: mannose, glucose, galactose, and N-acetylglucosamine; however, SUC 1 had approximately 10-fold more mannose and this was utilized to separate it from SUC 2 by Galanthus nivalis lectin affinity. Both the apparent Km values and the pH activity curves were different; SUC 1 did not show normal Michaelis-Menten kinetics to sucrose and apparent Michaelis constants of 30 and 160 mM were obtained. Activity was observed over a large range of pH 4.5-9 with a maximum at pH 6. In contrast, SUC 2 exhibited a Km of 40 and 1.7 mM to sucrose and inulin, respectively, with a pH optimum of 5.0 for both. Treatment with endo-beta-N-acetylglucosaminidase suggests that in both SUC 1 and SUC 2 some of the glycan present was N-linked glycan but that the differences in enzyme activities were not due to the N-linked moiety.

Catechol adrenergic agents enhance hydroxyl radical generation in xanthine oxidase systems containing ferritin: implications for ischemia/reperfusion.

Iron chelators have been reported to protect tissues against reperfusion injury. This implies that iron is being released into the plasma or is made accessible in tissues for oxidation-reduction reactions. It has been postulated that ferritin is a likely source for this iron. This report demonstrates that adrenergic agents with the catechol structure, which includes the endogenous catecholamines norepinephrine and epinephrine, are capable of releasing iron from ferritin. It is shown that the net release of iron from ferritin by epinephrine is significantly enhanced under anaerobic conditions. The findings suggest that catecholamines can mediate iron release from ferritin under conditions that can occur during ischemia/reperfusion. Catecholamines are also shown to interact with the released iron and xanthine oxidase to produce highly reactive hydroxyl radicals. The implications of this interaction for ischemia/reperfusion are discussed.