Association of immunological disorders in lethal side effect of NSAIDs on beta-glucan-administered mice

(1-->3)-beta-D-Glucan (beta-glucan) is a biological response modifier that regulates host immune response. We have found that the combination of a beta-glucan and a non-steroidal anti-inflammatory drug (NSAID), indomethacin (IND), induced lethal toxicity in mice [Yoshioka et al. (1998) FEMS Immunol. Med. Microbiol., 21, 171-179]. This study was undertaken to analyze the mechanism of the lethal side effect. Combination of a beta-glucan and IND increased the number of leukocytes, especially macrophages and neutrophils, in various organs and these cells were activated. The activated state of these cells was supported by the enhanced production of interferon-gamma in the presence of IND in vitro culture of the peritoneal exudate cells. Intestinal bacterial flora was translocated into the peritoneal cavity in these mice to cause peritonitis. Comparing the toxicity of various NSAIDs, nabumetone, a partially cyclooxygenase-2-selective NSAID with weaker toxicity to the gastrointestinal tract, did not exhibit a lethal side effect. These facts strongly suggested that gastrointestinal damage by NSAIDs was more severe in beta-glucan-administered mice, resulting in peritonitis by enteric bacteria and leading to death.     

Congo red inhibits in vitro β-glucan synthases of Saprolegnia

Abstract 1,3-β- and 1,4-β-glucan synthases from Saprolegnia were inhibited in vitro by Congo red. This dye was found to be a non competitive inhibitor which prevented initiation and elongation of polymer chains.     

Effect of β-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3- β -glucanase, a purified β -glucosidase, and of partially purified endo-1,3- β -glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied.
Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F₁, an α -glucan; F₃, a β -glucan; F₄, a chitin-glucan; and F_4b, a β -glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation.
The enzymes were found to degrade fraction F_4b ( β -glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

β,β''-Iminodipropionitrile Impairs Retrograde Axonal Transport

beta,beta'-Iminodipropionitrile (IDPN), a neurotoxin, causes redistribution of neurofilaments in axons followed by the development of proximal axonal swellings and, in chronic intoxication, a distal decrease in axonal caliber. The latter changes are caused by a selective impairment in the slow anterograde axonal transport of neurofilament proteins. To assess the role of retrograde axonal transport in IDPN toxicity, we used [3H]N-succinimidyl propionate ([3H]NSP) to label covalently endogenous axonal proteins in sciatic nerve of the rat and measured the accumulation of radioactively labeled proteins in the cell bodies of motor and sensory neurons over time. IDPN was injected intraneurally 6 h or intraperitoneally 1 day before subepineurial injection of [3H]NSP into the sciatic nerve, and the animals were killed 1, 2, and 7 days after [3H]NSP injection. Neurotoxicity was assessed by electron microscopic observation of the nerves of similarly treated animals. Both intraneural and intraperitoneal injection of IDPN caused an acute reduction in the amount of labeled proteins transported back to the cell bodies. The early appearance of these changes suggests that alterations in retrograde transport may play a role in the production of the neuropathic changes.     

Antigen-specific response of murine immune system toward a yeast β-glucan preparation, zymosan

Zymosan, a particulate beta-glucan preparation from Saccharomyces cerevisiae, shows various biological activities, including anti-tumor activity. We have previously shown that soluble beta-glucan initiated anti-tumor activity was long-lived and was effective even by prophylactic treatment at 1 month prior to tumor challenge. However, the activity by zymosan was relatively short-lived. Antigen-specific responses of mice to zymosan might be a causative mechanism. In this paper, mice were immunized with zymosan and antibody production and antigen-specific responses of lymphocytes to zymosan were analyzed. Sera of zymosan immune mice contained zymosan-specific IgG assessed by enzyme-linked immunosorbent assay and FACS. Spleen and bone marrow cells of zymosan-immune mice showed higher cytokine production in response to zymosan. Specificity of zymosan-specific responses were also analyzed using various derivatives prepared from zymosan. These facts strongly suggested that mice recognize zymosan as antigen in addition to non-specific immune stimulant.     

Effect of GM-CSF on cytokine induction by soluble β-glucan SCG in vitro in β-glucan-treated mice

SCG is a 6-branched 1,3-β- d -glucan, which are major cell wall structural components in fungi. Leukocytes from DBA/1 and DBA/2 mice are highly sensitive to SCG, producing cytokines such as GM-CSF, IFN-γ, TNF-α and IL-12p70, but not IL-6. GM-CSF plays a key biological role in this activity. In the present study, we examined the effect of giving i.p. SCG to DBA/2 mice on cytokine production in vitro . SCG was given i.p. to DBA/2 mice on day 0. Splenocytes were prepared on day 7 and cultured in the presence of SCG in vitro . The levels of cytokine production induced by SCG in vitro were lower in the cells from SCG-treated mice than in control mice. Expression of the β-glucan receptor, dectin-1, in SCG-treated mice was comparable with that shown in control mice. However, the consumption of exogenously added rmGM-CSF in vitro was observed in SCG-treated mice. The addition of a large amount of rmGM-CSF to the culture medium resulted in larger amounts of TNF-α and IL-6 in SCG-treated mice than in normal mice. These results suggested that GM-CSF was closely related with the reactivity of β-glucan. Giving SCG increased the number of macrophages and granulocytes in the spleen. These results suggested that in SCG-treated mice, a change of cell population would be related to modulation of the profile of cytokine production induced by SCG in vitro .     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Phosphorylation-Dependent Immunoreactivity of Neurofilaments Increases During Axonal Maturation and β,β''-Iminodipropionitrile Intoxication

The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with beta,beta'-iminodipropionitrile (IDPN). A maturational increase in the relative binding of phosphorylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phosphorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal transport of NF proteins.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

The Dietary Excitotoxins β-N-Methylamino-l-Alanine and β-N-Oxalylamino-l-Alanine Induce Necrotic- and Apoptotic-Like Death of Rat Cerebellar Granule Cells

Abstract: The neurotoxic properties of the dietary excitotoxins β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine have been studied in rat cerebellar granule cells and compared with those of glutamate. Glutamate caused dose-dependent death of cerebellar granule cells after a 30-min exposure when viability was assessed 24 h later. β- N -Methylamino- l -alanine and β- N -oxalylamino- l -alanine, however, were toxic only after 24 or 48 h of exposure. The neurotoxic effects of β- N -methylamino- l -alanine were blocked by d (−)-2-amino-5-phosphonopentanoic acid, and those of β- N -oxalylamino- l -alanine were blocked by kynurenic acid, which demonstrated that these excitotoxins caused cerebellar granule cell death through the activation of glutamate receptors. The features of this death were examined morphologically (fluorescent dyes, electron microscopy) and biochemically (conventional agarose gel electrophoresis, effect of aurintricarboxylic acid). Characteristics of apoptosis were identified by transferring cerebellar granule cells from a high K⁺ (30 m M )- to a low K⁺ (10 m M )-containing medium. In cerebellar granule cells exposed to β- N -methylamino- l -alanine or β- N -oxalylamino- l -alanine (3 m M ), hallmarks of necrotic- and apoptotic-like death were observed at various time points over a 72-h period. Therefore, in cerebellar granule cells, β- N -methylamino- l -alanine and β- N -oxalylamino- l -alanine induce death over 12–72 h of exposure via a mechanism that involves both necrotic- and apoptotic-like cell death.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.