Chemistry and biology of angiitis inducer,Candida albicans water-soluble mannoprotein-beta-glucan complex (CAWS)

Deep mycoses have been clearly demonstrated to release beta-glucans into the blood. Structure of the beta-glucan was, at least in part, suggested to be a mannoprotein beta-glucan complex (CAWS) as assessed by biochemical and immunochemical analyses of the extracellular macromolecular fraction of Candida albicans. Half clearance time of i.v. administered CAWS was about 30 min in mice. In addition to the reactivity with limulus G-test, CAWS was found to exhibit various biological activities, such as cytokine synthesis by leukocyte, platelet aggregation, lethal toxicity, enhancement of side effect of indomethacin, induction of coronary arteritis in mice, and so on. In this review, the chemical properties and biological activities of CAWS are discussed.     

Chemical and immunochemical characterization of limulus factor G-activating substance of Candida spp.

The limulus test is a well-established method for the diagnosis of both gram (-) sepsis and invasive fungal infection. To diagnose deep-seated fungal infections, a (1-->3)-beta-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. It is suggested that the limulus reactive substance was released from the fungi to the blood, however, its chemical properties were not precisely examined in detail because of the limited quantity available. In this study, we used chemically defined liquid medium to culture Candida spp. and collected the water soluble fraction, CAWS. The yield of CAWS was circa 100 mg/l, independent of the strain of Candida. CAWS reacted with limulus factor G (Fungitec G test MK) at concentrations as low as 100 ng/ml. Limulus factor G reactivity of CAWS was sensitive to (1-->3)-beta-glucanase, zymolyase and was, at least in part, bound to ConA-agarose. The ConA-bound fraction also reacted with anti-beta-glucan antibody. CAWS is mainly composed of mannan and (1-->6)-beta-glucan, in addition to protein, assessed by 1H-NMR spectroscopy. CAWS also reacted with typing sera of Candida spp., specific for cell wall mannan. Chemical, immunochemical and biochemical analyses of CAWS strongly suggested that the limulus factor G-activating substance was a mannan-beta-glucan complex, present within the architecture of the yeast cell wall.     

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice

The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.     

Induction of IFN-γ by a highly branched 1,3-β-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways

We have previously elucidated the precise structure of a unique type of 1,3-β-D-glucan, AP-FBG (Aureobasidium pullulans-fermented β-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-β-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.     

The kappa-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases

We report here cloning from the marine gliding bacterium Cytophaga drobachiensis of kappa-carrageenase, a glycoside hydrolase involved in the degradation of kappa-carrageenan. Structural features in the nucleotide sequence are pointed out, including the presence of an octameric omega sequence similar to the ribosome-binding sites of various eukaryotes and prokaryotes. The cgkA gene codes for a protein of 545 aa, with a signal peptide of 35 aa and a 229-aa-long posttranslationaly processed C-terminal domain. The enzyme displays the overall folding and catalytic domain characteristics of family 16 of glycoside hydrolases, which comprises other beta-1,4-alpha-1,3-D/L- galactan hydrolases, beta-1,3-D-glucan hydrolases (laminarinases), beta- 1,4-1,3-D-glucan hydrolases (lichenases), and beta-1,4-D-xyloglucan endotransglycosylases. In order to address the origin and evolution of CgkA, a comprehensive phylogenetic tree of family 16 was built using parsimony analysis. Family-16 glycoside hydrolases cluster according to their substrate specificity, regardless of their phylogenetic distribution over eubacteria and eukaryotes. Such a topology suggests that the general homology between laminarinases, agarases, kappa- carrageenases, lichenases, and xyloglucan endotransglycosylases has arisen through gene duplication, likely from an ancestral protein with laminarinase activity.      

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.     

Comprehensive conformational study of key interactions involved in zearalenone complexation with beta-D-glucans

The beta-D-glucans from the cell wall of Saccharomyces cerevisiae have shown in vitro affinity for zearalenone. For this reason, their utilization as dietary adsorbent, to reduce the bioavailability of zearalenone, is of practical interest. Our study used powerful devices to elucidate the spatial conformation and molecular sites of interaction between ZEN and beta-D-glucans. In this respect, 1H NMR spectroscopy implicated the hydroxyl groups of the phenol moiety of zearalenone in the complexation by laminarin, a pure beta-(1,3)-D-glucan. X-ray diffraction determined that laminarin displays the conformation of a single-helix with six beta-D-glucopyranose residues per turn. At this stage, molecular modeling was useful to locate the interaction sites and to propose highly probable complexes of zearalenone with laminarin fragment. Interestingly, the beta-(1,3)-D-glucan chain favors a very stable intra-helical association with zearalenone, nicely stabilized by beta-(1,6)-D-glucans side chains. Both hydrogen bonds and van der Waals interactions were precisely identified in the complex and could thus be proposed as driving interactions to monitor the association between the two molecules.     

The fungal cell wall and the mechanism of action of anidulafungin

The fungal cell wall is a structure with a high plasticity that protects the cell from different types of environmental stresses including changes in osmotic pressure. In addition to that, the cell wall allows the fungal cell to interact with its environment, since some of its proteins are adhesins and receptors. Some of its components are highly immunogenic. The structure of the fungal cell wall is unique to the fungi, and it is composed of glucan, chitin and glycoproteins. Since humans lack the components present in the cell walls of fungi, this structure is an excellent target for the development of antifungal drugs. Anidulafungin, like the rest of echinocandins acts on beta-1,3-D-glucan synthase inhibiting the formation of beta-1,3-D-glucan and causing, depending on the type of fungus, a fungicidal or either a fungistatic effect.     

Anidulafungin: a new therapeutic approach in antifungal therapy. Pharmacology of anidulafungin

Anidulafungin is a new echinocandin antifungal agent which inhibits beta-1,3-D-glucan synthase and disrupts fungal cell-wall synthesis. It has marked antifungal activity against Candida spp. and Aspergillus spp., including amphotericin B and triazole resistant strains. Due to the limited oral availability, anidulafungin in clinical use is available for parenteral administration only. Elimination of anidulafungin takes place via slow non-enzymatic degradation to inactive metabolites. Less than 10% and 1% of the initially administered drug is excreted unchanged into feces and urine, respectively. It does not require dosage adjustment in subjects with hepatic or renal impairment established. Anidulafungin is generally well tolerated. Adverse events appear not to be dose or infusion related. The most common treatment related adverse events are phlebitis, headache, nausea, vomiting and pyrexia. The lack of interactions with tacrolimus, cyclosporine and corticosteroids and its limited toxicity profile places anidulafungin as an attractive new option for the treatment of invasive fungal infections especially in transplant patients.     

β-Glucan Derived from Aureobasidium pullulans Is Effective for the Prevention of Influenza in Mice

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches produced by mushrooms, yeast and fungi are known to be an immune activation agent, and are used in anti-cancer drugs or health-promoting foods. In this report, we demonstrate that oral administration of Aureobasidium pullulans-cultured fluid (AP-CF) enriched with the β-(1→3),(1→6)-D-glucan exhibits efficacy to protect mice infected with a lethal titer of the A/Puerto Rico/8/34 (PR8; H1N1) strain of influenza virus. The survival rate of the mice significantly increased by AP-CF administration after sublethal infection of PR8 virus. The virus titer in the mouse lung homogenates was significantly decreased by AP-CF administration. No significant difference in the mRNA expression of inflammatory cytokines, and in the population of lymphocytes was observed in the lungs of mice administered with AP-CF. Interestingly, expression level for the mRNA of virus sensors, RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated protein 5) strongly increased at 5 hours after the stimulation of A. pullulans-produced purified β-(1→3),(1→6)-D-glucan (AP-BG) in murine macrophage-derived RAW264.7 cells. Furthermore, the replication of PR8 virus was significantly repressed by pre-treatment of AP-BG. These findings suggest the increased expression of virus sensors is effective for the prevention of influenza by the inhibition of viral replication with the administration of AP-CF.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Prevention of 5-fluorouracil-induced infection with indigenous Escherichia coli in tumor-bearing mice by nonspecific immunostimulation.

We have previously reported that the lethal toxicity of 5-fluorouracil (5-FU) in specific-pathogen-free mice is due to an indigenous infection with Escherichia coli (K. Nomoto, T. Yokokura, Y. Yoshikai, et al. Can. J. Microbiol. 37:244-247, 1991). In the present study, we demonstrate that nonspecific immunostimulation augments host resistance against the lethal toxicity of 5-FU in tumor-bearing mice. Intravenous administration of a preparation of heat-killed Lactobacillus casei (LC 9018), a nonspecific immunostimulant, at a dose of 20 mg/kg to BALB/c mice augmented their resistance against the lethal toxicity of 5-FU if the preparation was injected into the mice 10-40 days before administration of 5-FU. Injection of LC 9018 into BALB/c mice bearing Meth A fibrosarcoma also enhanced their resistance against the lethality of 5-FU. Systemic infection with E. coli was induced in all of the 5-FU-treated tumor-bearing mice 10 days or more after administration of the drug at a lethal dose of 500 mg/kg, and it was accompanied by an overgrowth of the bacteria in the intestine. Treatment of tumor-bearing mice with LC 9018 resulted in decreased rates of occurrence of systemic infection with E. coli and inhibition of overgrowth of the bacteria in the intestine after administration of 5-FU. A single administration of either LC 9018 or 5-FU significantly inhibited the growth of Meth A cells in vivo, and a combined antitumor effect was shown in the mice treated with both 5-FU and LC 9018.     

Studies of factors influencing the mutagenicity of EMS in mice

Three different routes of administration of ethyl methanesulphonate (EMS) (i.p., oral, i.t.) were compared for their relative efficiencies in the induction of dominant lethal effects. Included in the comparisons between oral and i.p. injections, was a preliminary study into the existence of strain differences in sensitivity to EMS between C3D2 F1 hybrid mice and strain DBA/2J mice. No route of administration dependent effects were found between oral and i.p. injections regardless of the test animal used. I.t. injections of EMS did not induce dominant lethal effects. One treatment related strain difference was observed.     

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.     

Anidulafungin: a new echinocandin for the treatment of mycosis

The latest antifungal drugs introduced for clinical use are echinocandins; they possess a distinctive mechanism of action based on the inhibition of the beta-1,3-D-glucan sintesis, through the damage of the fungal cell wall without impairment of human cells because these do not contain beta-1,3-D-glucan. Among echinocandins, anidulafungin is the last that has received the FDA approval in the USA for the treatment of candidemia in non-neutropenic patients, intra-abdominal abscesses, peritonitis and esofagitis caused by Candida. In Europe, the EMEA has also approved its use for invasive candidiasis in non-neutropenic patients and for candidal esofagitis. The characteristics of anidulafungin are close to those of the ideal antifungal since it has a wide spectrum, is active at low minimal inhibitory concentrations and it is fungicidal for Candida. In addition, it is well tolerated, has few pharmacological interactions, is active intravenously, has a long half life and is auto-biodegradable. Finally, anidulafungin has shown a higher therapeutic efficiency when compared with the conventional treatment of candidemia, since although it is more expensive, the treatment with anidulafungin is highly cost effective.     

Protective effect of sulfhydryl compounds on acute toxicity of morphinone

The ability of sulfhydryl compounds to provide protection against the acute toxicity of morphinone was investigated in mice. Subcutaneous administration of morphinone produced a reduction of hepatic non-protein sulfhydryl concentration. Pretreatments of mice with glutathione or cysteine significantly increased the survival rate of mice given a lethal dose of morphinone, whereas morphinone lethality was markedly potentiated by diethyl maleate. On the other hand, the administration of morphine produced a dose dependent reduction of hepatic non-protein sulfhydryl contents. However, neither glutathione nor cysteine protected mice from the acute toxicity of morphine. A possible explanation for these observations was proposed as follows: morphine is oxidized by morphine 6-dehydrogenase to morphinone, and the morphinone thus produced decreases the sulfhydryl contents in the liver. This mechanism is supported by the fact that morphinone reacts easily with glutathione and cysteine $\text{in vitro}$.     

Antibodies to catecholamines and serotonin during alcoholic intoxication in animals with different predispositions to the development of experimental alcoholism

Experiments on C57Bl/6, CBA and DBA/2 mice characterized by different preferences for ethanol have shown that during chronic administration of alcohol to animals with natural ethanol motivation (strain C57Bl/6) the level of antibodies to catecholamines and serotonin was increased on the 3rd month of ethanol intoxication, with the voluntary alcohol consumption in mice decreased by this time. On the contrary in mice rejecting alcohol (strains DBA/2, CBA) no antibodies to catecholamines and serotonin have been found.     

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.     

Genetic factors influencing neurosensitivity to early phenobarbital administration in mice

This study was designed to assess the possible genetic determinants of neurosensitivity to early (neonatal) phenobarbital (PhB) administration and to conduct a strain comparison for the cerebellar histology of both inbred and outbred mice. HS/Ibg, C57BL/10 and DBA/1 pups were injected with 50 mg PhB/kg daily on neonatal days 2-21. On day 50, treated animals (B) of all strains had smaller brains than controls (C). Moreover, the cerebellar area was decreased in HS and C57 B mice but not in DBA mice, suggesting genotype-environment interaction. B mice from all strains had similar Purkinje cell losses. Strain comparison showed that control C57 mice had smaller brains than control HS, and DBA had smaller brains than both HS and C57. Similarly, C57 had smaller cerebellar layers than HS and DBA had smaller cerebellar layers than both HS and C57. DBA and C57 mice had fewer Purkinje cells than HS but did not differ from each other.     

Resistance/susceptibility to lethal Sendai virus infection genetically linked to a mucociliary transport polymorphism.

Linkage was tested between a mucociliary transport polymorphism and resistance/susceptibility to lethal Sendai virus infection in segregant hybrid mice of C57BL/6J and DBA/2J parents. The distribution of paired phenotypes for tracheal mucociliary transport rates and susceptibility to lethal Sendai virus infection in 171 F1 X DBA/2J mice showed strong interaction of the parental phenotypes.