Growth of moulds inoculated into commercial mineral water.

The growth of mould spores of Penicillium sp. and Cladosporium sp. inoculated in a commercial mineral water product was studied. The strains had been isolated as fungal foreign bodies in commercial mineral waters. In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage. In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies. The viable colony counts and the beta-glucans concentration in the samples increased during storage. These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies.

Development of a stopped flow reactor for the rapid, high sensitivity measurement of endotoxin concentration in dialysate.

Continuous measurement of endotoxin concentration in dialysate, using a separated endotoxin-specific limulus reagent, promises rapid measurement without the complex operating procedures of the limulus reagent. To achieve high sensitivity measurements in a short period of time, an improved system featuring stopped-flow operation was developed. To prevent dispersion of the limulus reagent and residence of reacting solution containing the limulus reagent in the system reactor, the circuit in the reactor was changed from a coil configuration to a straight line, and its length was reduced. An endotoxin test solution was supplied at 760 microl/min, into which 40 microl of limulus reagent was pulse-injected. Flow was stopped at the point where the test solution entered the reactor. After the completion of the reaction, the solution was passed through a spectrophotometer and the relationship between reaction time and absorbance was determined. Peak tailing was less than that obtained by the conventional technique, good correlation was obtained from the peak height, and a decrease in sensitivity caused by broadening of the peak was suppressed. The lower detection limit of dialysate was 100 endotoxin units (EU)/L at a reaction time of 20 minutes, and 60 EU/L at 30 minutes. Change from the monitoring system to stop-flow operation made high sensitivity monitoring of endotoxin concentration with a short reaction time possible.

Purification and characterization of a (1-->3)-beta-D-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes.

A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.

Blood clearance of (1-->3)-beta-D-glucan in MRL lpr/lpr mice.

(1-->3)-beta-D-Glucans have a variety of biological and immunopharmacological properties, and they are used clinically as biological response modifiers (BRMs). Clinically, these glucans have often been used for long periods by multiple dosing. During studies on the clearance and metabolism of the glucans in mice, we have found that, in the case of a single dose, the glucan was cleared from blood eventually, and remained constant in the organs for at least one month. Here, we investigated the clearance of glucans from the blood following multiple dosing using MRL lpr/lpr mice with an autoimmune disease. Two kinds of glucans, GRN from Grifola frondosa and SSG from Sclerotinia sclerotiorum, were administered to the mice once a week for more than 35 weeks (250 micrograms/week/mouse by the intraperitoneal route). Examination of the blood clearance of the glucans in these mice revealed that the glucan concentrations were always high (about 20 micrograms/ml for GRN and 200 micrograms/ml for SSG). It is also shown that the glucans were significantly deposited in the liver and spleen of these mice. These findings suggest that administration of a large quantity of the glucan saturated the reticuloendothelial system, resulting in circulation of the glucan in the blood.

Development of a new endotoxin sensor with intermittent injection of limulus reagent for continuous monitoring of dialysate fluid.

This report describes a method of continuously, stably, and inexpensively measuring endotoxin (ET) concentrations in dialysate fluid using an ET sensor with intermittent injection of limulus reagent. An ET solution simulating dialysate fluid was sampled in a single tube at a flow rate of 260 microliters/min and mixed with 30 microliters of limulus reagent intermittently injected into the tube. The absorbance of the solution was measured after the limulus reaction at 313 or 318 degrees K at 26 min. A good linear relationship (r = 0.98) between peak area of absorbance and ET concentration at ET concentrations ranging from 0 to 0.12 endotoxin unit (EU)/ml was obtained, using a spectrophotometer with a cell volume of 8 microliter. The baseline rose after the measurements were taken because the cell volume was so small that the cell was stuffed with gel. A good linear relationship (r = 1.00) at ET concentration of 0.1-0.25 EU/ml was also obtained, and the baseline was unchanged after measurements, using a metal free spectrophotometer with a cell volume of 420 microliters. In conclusion, to measure ET concentrations below 0.1 EU/ml, the cell volume of a metal free spectrophotometer should be minimal.

Comparison of the blood clearance of triple- and single-helical schizophyllan in mice.

(1-->3)-beta-D-Glucans exhibit a variety of biological and immunopharmacological activities, and the degree of these activities depends on the nature of the individual glucans e.q. molecular weight, degree of branching and conformation. Based on the generally accepted evidence that the conformation of Sonifilan (SPG) used clinically is a triple helix, we prepared alkali-denatured SPG (SPG-OH) as a single helix conformer. In this report, we measured the concentration of beta-glucan administered to mice by using a beta-glucan-specific reagent prepared from limulus amebocyte lysate (Gluspecy [G test], Seikagaku Corporation, Tokyo) and discuss the blood clearance of SPG and SPG-OH following intraperitoneal (i.p.) or intravenous (i.v.) administration. Comparing the clearance of SPG-OH from the blood with that of SPG, SPG-OH was removed faster than SPG following both i.p. and i.v. administration. This strongly suggests that the clearance of beta-glucans is dependent on their conformation.

A new procedure for stable quantification of endotoxin in dialysate fluid using Limulus reagent.

For reliable determination of endotoxin, the activation of enzymes in lysate before measurement should be prevented, and the authors have designed a new procedure to effect this by dissolving the enzymes in lysate in a buffer solution of low pH. A given amount of the enzymes in lysate was dissolved in a lower pH buffer solution (pH 6.1-6.3) and the substrate was dissolved in a higher pH buffer solution (pH 8.0). After standing for 0-24 hr, both solutions were mixed with the sample solution. Data on blank absorbance and calibration line slope obtained by the new procedure were compared with those obtained by the conventional procedure. In the conventional procedure, blank absorbance increased with standing time, reaching approximately seven times the initial value in 24 hr, whereas in the improved procedure, it increased by 1.5 times at a standing time of 3 hr, after which it was independent of standing time. The change in slope of the calibration line with standing time was more gradual in the improved procedure than in the conventional procedure. The authors conclude that the activation of enzymes in lysate can be prevented by dissolving the enzymes in a buffer solution of low pH, and that this procedure is effective for long-term monitoring of endotoxin concentration.

Application of limulus test (G pathway) for the detection of different conformers of (1-->3)-beta-D-glucans.

The reactivity of factor G mediated coagulation pathway in limulus amebocyte lysate which is triggered by (1-->3)-beta-D-glucans is thought to depend on the structure of the glucans, especially on the ultrastructure: triple helix, single helix and random coil. We used Sonifilan (SPG) and grifolan (GRN) as parent compounds to compare the reactivities of these three conformers. Under a neutral condition, alkaline treated SPG (SPG-OH, single helix) and polycarboxylated SPG (PC-SPG, random coil) showed significantly stronger reactivity than untreated SPG (triple helix). After the alkaline treatment, all three conformers showed comparable reactivities. It is suggested that the pretreatment of the glucan preparations by sodium hydroxide is quite important to compare quantitatively the reactivity of the glucans by limulus test, and comparing the data of untreated and alkaline treated glucans would provide information about their conformations. Using this approach, it was found that after heat treatment at around 150 degrees C, the conformation of GRN was changed to rich in the triple helix, and that following sodium hydroxide treatment and dialysis of GRN, the conformation of GRN was changed to single helix rich conformer. About half of the single helix conformer was gradually changed to triple helix conformer over one week at 4 degrees C.

Activation of limulus coagulation factor G by several (1-->3)-beta-D-glucans: comparison of the potency of glucans with identical degree of polymerization but different conformations.

It has been demonstrated that both linear and branched (6-O-beta-D-glucosyl) (1-->3)-beta-D-glucans taking a single helical conformation are more effective than those taking a triple helical conformation for the activation of factor G from horseshoe crab amebocytes, as revealed by high-resolution solid-state 13C-NMR spectroscopy [Saitô, H. et al. (1991) Carbohydr. Res. 217, 181-190]. Annealing the linear glucan at 180 degrees C was essential to convert the conformation from the single helix to the triple helix. We found that heating of the glucan at such a high temperature resulted in depolymerization of the sample to molecular weight smaller than 10,000, which may influence the conformation and the above-mentioned biological activity. To eliminate ambiguity arising from the depolymerization of the glucan during annealing, we aimed to relate the biological activity to the conformation of samples whose chain lengths are identical, because the potency is known to depend on the molecular weight of the glucans. This molecular weight dependency of the potency, however, was found to be not the dominant factor, provided that the molecular weight is large enough to allow formation of the single helix conformation. Therefore, the single helical conformation of (1-->3)-beta-D-glucans is clearly demonstrated to be the dominant contributor to the activation of limulus coagulation factor G.

Inhibition of high-molecular-weight-(1-->3)-beta-D-glucan-dependent activation of a limulus coagulation factor G by laminaran oligosaccharides and curdlan degradation products.

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Solubilization of limulus test reactive material(s) from Candida cells by murine phagocytes.

Solubilization of limulus test reactive materials from Candida was examined in the presence or absence of phagocytic cells. Solubilized limulus test reactive materials (LTRM) were detected in culture supernatant, and hot water and sodium hydroxide extracts of the acetone dried cells of Candida parapsilosis. Suspensions of Candida cells also reacted with limulus test, and LTRM were released from the acetone dried cells by serum treatment. After treatment of the acetone dried cells with polymorphonuclear leucocytes (PMN) or macrophages (M phi), a significant amount of LTRM was solubilized. Significant amounts of LTRM were also released by PMN during treatment of live and growing C. parapsilosis. The reactivity of LTRM was completely inhibited by the addition of excess amount of purified (1----3)-beta-D-glucan, suggesting LTRM from Candida cells as described above would contain (1----3)-beta-D-glucan. These results suggested that LTRM during fungal infection would come from the extracellular water soluble polysaccharide fraction as well as the insoluble cell wall fraction solubilized by the action of phagocytes.

Determination of plasma (1-->3)-beta-D-glucan: a new diagnostic aid to deep mycosis.

We present additional evidence that plasma from patients with deep-seated mycoses contains (1-->3)-beta-D-glucan. Digestion of such samples with endo-(1-->3)-beta-D-glucanase completely abolished the ability of the plasma to activate factor-G, a horseshoe crab coagulation enzyme that is extremely sensitive to this polysaccharide. Measurement of plasma (1-->3)-beta-D-glucan is a promising method for the diagnosis of deep-seated mycoses and for monitoring the response of these infections to antifungal therapy.

Relationship between conformation and biological response for (1----3)-beta-D-glucans in the activation of coagulation factor G from limulus amebocyte lysate and host-mediated antitumor activity. Demonstration of single-helix conformation as a stimulant.

The relationship between the conformation of (1----3)-beta-D-glucans in gel or hydrated form and the stimulation of two types of biological responses, namely, activation of coagulation Factor G from limulus amebocyte lysate (LAL) and host-mediated antitumor activity was examined. Both types were activated by the single-helical conformation, as revealed by high-resolution, solid-state 13C-n.m.r. spectroscopy. The potency of activation of Factor G was increased over 100-fold by treatment with a NaOH solution which leads to a complete or partial conversion from the triple to the single helix. Such a single-helix specific response was also demonstrated for the antitumor activity of curdlan, although the distinction was less pronounced for branched (1----3)-beta-D-glucans. The presence of the single-helix conformation was observed in schizophyllan gel, even though the triple helix is the most stable form of branched glucans in aqueous media.

Primary structure of limulus anticoagulant anti-lipopolysaccharide factor.

A potent anticoagulant, anti-lipopolysaccharide (LPS) factor, found in limulus hemocytes inhibits the LPS-mediated activation of limulus coagulation cascade and shows an antibacterial action against R-types of Gram-negative bacteria (Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M. (1985) J. Biochem. (Tokyo) 97, 1611-1620). The complete amino acid sequence of this substance was determined by sequencing the peptides obtained by selective proteolytic cleavage. The NH2-terminal end of anti-LPS factor was pyroglutamic acid. Anti-LPS factor had two variant residues at position 36 and the COOH-terminal end, respectively. The following sequence was assigned to anti-LPS factor, and it was also confirmed by fast atom bombardment mass spectrometry. less than EGGIWTQLALALVKNLATLWQSGDFQFLGHE (formula; see text) Limulus anti-LPS factor consisted of a single chain of 102 residues with 2 half-cystines in disulfide linkage. Its NH2-terminal region up to 20 residues was highly hydrophobic, and positively charged residues were clustered mainly within the disulfide loop. By searching the homologous sequence in known protein sequences with that of anti-LPS factor, we found a structural homology between anti-LPS factor and alpha-lactalbumin/lysozyme family.

Purification and properties of thiosulfate reductase from Desulfovibrio vulgaris, Miyazaki F.

Thiosulfate reductase was purified to an almost homogeneous state from Desulfovibrio vulgaris, strain Miyazaki F, by ammonium sulfate precipitation, chromatography on DEAE-Toyopearl, Ultrogel AcA 34, and hydroxylapatite, and disc electrophoresis. The specific activity was increased 580-fold over the crude extract. The molecular weight was determined by gel filtration to be 85,000-89,000, differing from those reported for thiosulfate reductases from other Desulfovibrio strains. The enzyme had no subunit structure. When coupled with hydrogenase and methyl viologen, it stoichiometrically reduced thiosulfate to sulfite and sulfide with consumption of hydrogen. It did not reduce sulfite or trithionate. Cytochrome c3 was active as an electron donor. More than 0.75 mM thiosulfate inhibited the enzyme activity. o-Phenanthroline and 2,2'-bipyridine inhibited the enzyme and ferrous ion stimulated the reaction.

Characterization of periplasmic hydrogenase from Desulfovibrio vulgaris Miyazaki K.

Periplasmic hydrogenase [hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki K (MK) was purified to homogeneity. Its chemical and immunological properties were examined and compared with those of other Desulfovibrio hydrogenases. The pure enzyme showed a specific activity of 1,000 mumol H2 evolution min-1 (mg protein)-1. The enzyme had a molecular weight of 50,000 as estimated by gel filtration and consisted of a single polypeptide chain. The absorption spectrum of the enzyme was characteristic of an iron-sulfur protein and the extinction coefficients at 400 and 280 nm were 34 and 104 mM-1. cm-1, respectively. It contained 9.4 mol iron and 6.9 mol of acid-labile sulfide per mol. The amino acid composition of the preparation was very similar to the value reported for D. desulfuricans NRC 49001 hydrogenase. Rabbit antisera were prepared against the enzyme of D. vulgaris MK. Ouchterlony double diffusion and immunotitration tests of crude extracts from several strains of Desulfovibrio revealed that the enzyme from MK cells was immunologically identical with those from D. vulgaris Hildenborough and D. desulfuricans NRC 49001, but different from those from D. vulgaris Miyazaki F (MF) and Miyazaki Y, and D. desulfuricans Essex 6 strains. It is concluded that among Desulfovibrio hydrogenases, those from D. vulgaris MK, D. vulgaris Hildenborough and D. desulfuricans NRC 49001 form one group in terms of both subunit structure and antigenicity.