Thiamin is decomposed due to Anaphe spp. entomophagy in seasonal ataxia patients in Nigeria.

A fairly high activity of a relatively heat-resistant thiaminase was detected and characterized from the pupae of an African silkworm Anaphe spp. which had been the putative cause of a seasonal ataxia and impaired consciousness in Nigerians. The thiaminase in the buffer extract of Anaphe pupae was type I (thiamin: base 2-methyl-4-aminopyrimidine methyl transferase EC 2.5.1.2), and the optimal temperature and pH were 70 degrees C and 8.0-8.5, respectively. Based on gel filtration chromatography, the molecules were estimated to be 200 kDa. Second substrates which could be utilized by the thiaminase were pyridoxine, amino acids, glutathione, taurine and 4-aminopyridine. Thiamin phosphate esters were inactive as substrates. This is the first report describing an insect thiaminase. Our results indicate the necessity of thorough heat treatment for the detoxification of the African silkworm, making the worm a safe source of high-quality protein.

Intake ratio of water-insoluble dietary fiber to the water-soluble one in Japanese: an estimation on dietary records and the secular consideration on the national nutrition survey.

Thirty-four foods were analyzed in order to determine the content of water-soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). Using the results with the standard table for 227 foods, the intake ratio of IDF/SDF of an average Japanese was calculated for the period 1946-1990. The ratio was 3.22 in 1990 as calculated on the food intakes shown in the national nutrition survey, and the secular change was not detected since 1946 when the ratio was 3.30. The ratio was also shown to be well preserved between types of households including the age of the head. Using dietary records of 60 healthy city workers (average 42.8 years) for 4 weeks, however, the weekly average ratio for an individual was found to vary in the range of 2.25-5.13 although the total average for 60 individuals was 3.33. Thus, the well preserved IDF/SDF intake ratio for an average Japanese showed, on the contrary, a wide variation of food selection between each person.

Enzyme immunoassay for phycocyanin as the main component of spirulina color in foods.

An enzyme immunoassay (EIA) for phycocyanin in foods was developed. Anti-phycocyanin monoclonal antibodies were obtained from A/J mice immunized with phycocyanin. The phycocyanin in a food was extracted by dissolving the sample in a borate buffer solution, pH 8.0 (BBS) and adjusting the pH value of this solution to 8.0 with NaOH. The extract was then diluted more than 10 fold with 1% gelatin in BBS. Phycocyanin was determined by avidin-biotin sandwich EIA, using the P26-8 monoclonal antibody as the solid-phase antibody and the P277-4 monoclonal antibody as the enzyme-labeled antibody. The working range for a quantitative analysis was 100-1000 ng/ml, and the detection limit was 10 micrograms/g of the original sample. Recoveries of phycocyanin from foods by this assay were > 71% for candy, and > 66% for ice cream and sherbet. Phycocyanin was assayed in 22 blue-, green-, purple-, and brown-colored commercial foods, and detected in one green colored-jelly at 49 micrograms/g.

Enzyme immunoassay for carminic acid in foods.

A competitive enzyme immunoassay (EIA) for carminic acid was investigated. Monoclonal anticarminic acid antibody was obtained from A/J mice immunized with carminic acid-human immunoglobulin G (IgG) conjugate. Carminic acid was extracted with distilled water from beverage, jelly, candy, pasta sauce, yogurt, or ice cream samples. Ham or fish paste samples were digested with pronase, then carminic acid was extracted from samples with sodium hydroxide solution. The extract was diluted more than 10-fold with 1% gelatin in borate buffer solution. Microtiter plates were coated with carminic acid-bovine serum albumin (BSA) conjugate or just BSA. Goat anti-mouse IgG(H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 0.3-10 ng/mL, and the detection limit was 0.2 micrograms/g original sample. Recoveries of carminic acid by this assay were > 95% for milk beverage and jelly, and > 85% for yogurt and fish paste. Carminic acid was detected in 7 of 26 red-colored commercial food products and ranged from 3.5 to 356 micrograms/g. This EIA system also responded to the structural analogue of carminic acid, laccaic acid.

Aspergillus ochraceus Production of 5,6-Dihydropenicillic Acid in Culture and Foods.

The production of 5,6-dihydropenicillic acid (DHPA) and penicillic acid (PA) by Aspergillus ochraceus was studied. DHPA has been revealed to be genotoxic in the Drosophila DNA-repair test in our previous report. The two compounds were produced by various type strains of A. ochraceus and related strains of Aspergillus in nutrient cultures. When A. ochraceus cells were inoculated into nutrient medium, PA was rapidly produced followed by a steady increase of DHPA and a decrease in the PA level. On the contrary, A. ochraceus produced almost exclusively PA in potato dextrose agar medium, which is low in nutrients. A. ochraceus cells produced DHP A when inoculated into grain, especially in rice flour at 20 to 24°C, and the DHPA level increased as the incubation was prolonged. These results suggested that PA was converted to DHPA under highly nutritive conditions. In vitro mutagenicity tests, the Ames test and the umu test, showed that DHPA was not mutagenic.

Mutagenicity of 5,6-Dihydropenicillic Acid (DHPA) in Drosophila melanogaster and Bacterial Conversion of Penicillic Acid to DHPA.

We studied the mutagenicity of 5,6-dihydropenicillic acid (DHPA) by means of the Drosophila wing-spot test. DHPA (10 mg/g of medium) significantly increased the number of small single and twin spots. Since twin spots were exclusively caused by mitotic crossing-over, the results demonstrated that DHPA can induce chromosome recombination in Drosophila somatic cells. Penicillic acid (PA) was converted to DHPA by an incubation with Agrobacterium radiobactor . The conversion occurred with other spoilage bacteria, such as Pseudomonas aeruginosa and P. cepacia . PA was completely converted to DHPA within a 24-h incubation with P. aeruginosa or P. cepacia in medium containing yeast extract. The results suggested that mutagenic DHPA is produced by environmental bacteria when foods are contaminated by fungi which produce PA.

Direct detection using the Drosophila DNA-repair test and isolation of a DNA-damaging mycotoxin, 5,6-dihydropenicillic acid, in fungal culture.

The Drosophila DNA-repair test was used in an attempt to detect fungal production of DNA-damaging mycotoxins without an extraction process. 29 species of fungi, 13 Aspergillus, 12 Penicillium and four Fusarium were inoculated directly to a Drosophila medium, and the larvae were then bred in the mouldy medium. Production of DNA-damaging mycotoxin was detected directly by counting the decrease in the survival of DNA-repair-deficient flies. With the direct detection method, Aspergillus ochraceus, A. parasiticus and A. versicolor produced DNA-damaging mycotoxins. The same results were obtained with the mouldy medium extract using the standard DNA-repair test. The direct detection method was convenient for surveying the fungal production of DNA-damaging mycotoxins. The extracts of A. parasiticus and A. versicolor contained aflatoxin B1 and sterigmatocystin, respectively. The DNA-damaging compound in the extract of A. ochraceus was isolated and purified to clear, colourless 'needles'. With nuclear magnetic resonance-mass spectroscopy spectra, the compound was confirmed to be 5,6-dihydropenicillic acid, the DNA-damaging potency of which has not been previously reported.

Dietary fiber intake of Japanese younger generations and the recommended daily allowance.

A method was introduced for the estimation of total dietary fiber (TDF) intake of a population using a menu-oriented questionnaire and a menu-based calculation table. TDF intake correlated well with age in a population investigated, and in younger generations TDF consumption was very low (less than 11.5 g/day in teenagers). The similar results were obtained from the calculation using data of National Nutrition Survey (10.7 g/day). The foodstuffs they consumed were more processed and refined. This fact suggested that in younger generations a future resumption of their present eating habits might produce a serious lack of TDF intake in later years. To clarify the optimal level of TDF intake for the upper limit of recommended daily allowance (RDA) of an average Japanese, the following were measured and calculated. (I) Estimation of recent TDF intake (1990) and of 30 and 50 years ago (1955, 1935), based on TDF data of foodstuffs by the enzymatic-gravimetric method. (II) Measurement of the TDF of model duplicate meals and model composite diets for the average Japanese in 1985 using the same assay method. (III) Conversion of a recommendation of 20-35 g/day for American into RDA for Japanese considering energy consumption and lower fat intake. (IV) Re-estimation of the literature data on the adverse effects of DF on the human mineral balance considering the insufficient calcium intake of Japanese. The results indicated an RDA of 10-12 g TDF/1,000 kcal fit better for an average Japanese.

Glycemic response and fiber content of some foods.

The proposed mechanisms for the action of dietary fiber (DF) on the glycemic response suggest a nonlinear relationship between glycemic index (GI) and DF content. This relationship was analyzed by using the newly reported total DF (TDF) values, assuming a nonlinear regression curve. The empirical equation obtained was Y = 19.9X-0.322. Similar regression curves were also obtained for soluble DF (SDF) and insoluble DF (IDF). The two regression curves indicated that the correlation coefficient between observed GI and GI calculated from the IDF regression curve (-0.781) was virtually identical to that for SDF (-0.780), but a given content (eg, 5% vs available carbohydrate) of SDF gave a lower GI (39) than did IDF (48). This stronger dependency of GI on SDF suggests a major function of SDF in the TDF hypoglycemic effect. From the regression curve of GI vs TDF, we propose a supplemental GI to predict the glycemic response to foods with no published GI.

Effects of metal elements on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3).

Immediate allergy is caused by a chemical mediator released from basophile and mast cells via cell degranulation due to reaction between an immunoglobulin E (IgE) antibody, bound with the IgE receptor on the cell membrane, and an antigen. The present authors have established a new method for assaying the enzyme activity of beta-hexosaminidase as an index of chemical mediator release. Using cultured cells instead of conventional methods based on histamine release from mast cells, the present method permits highly accurate mass screening since it uses a well-established cell line of rat basophilic leukemia cells (RBL-2H3). The effects of metal elements on immediate allergic reaction were evaluated using a newly developed assay system. A total of 38 metal elements were investigated for effects on immediate allergic reactions in vitro. These elements were classified by five types on the basis of action on beta-hexosanimidase release: 1) those which showed very strong inhibitory action, such as ZnCl2 and ZrCl4, 2) those which showed relatively strong inhibitory action, such as CdCl2 and CuCl2, 3) those which showed relatively weak inhibitory action, such as CoCl2 and Pb(NO3)2, 4) those which showed neither inhibitory nor promoting action, such as MnCl2 and SrCl2, and 5) AgNO3, which alone showed promoting action.

Enzyme immunoassay for hen egg white lysozyme used as a food additive.

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.

Determination of total dietary fiber in Japanese foods.

Total dietary fiber was determined in Japanese foods by the Prosky-AOAC method. To accomplish the analyses of unsuitable samples, we introduced a few minor modifications to the versions for (i) seaweed and fruits, (ii) cereals, and (iii) fish and meats. These modified methods were used together with the standard method to obtain results with reasonably good relative standard deviation for 231 foods and 21 groups of mixed foods. In this study, dietary fiber was defined so as not to exclude the nondigestible polysaccharide portions of animal foods. A method was proposed which could estimate more accurately the fiber components of animal foods by measuring the "nondigestible protein" of the fiber sample of the fiber sample by the Biuret colorimetric method, instead of the Kjeldahl method, to avoid deducting the values for aminopolysaccharides. In Japanese diets, the amount of fiber obtained from animals foods was less than 5% of the total intake of dietary fiber.

Conditions for thiamin assay by cyanogen bromide oxidation.

Optimum conditions for the assay of thiamin were studied using a cyanogen bromide (BrCN) oxidation method. The adopted procedure included neither pre-purification of samples through an ion exchanger nor extraction of the thiochrome into an organic solvent. The 0.25 M BrCN (the concentration before the addition of alkali) and the final NaOH concentration of approx. 1% gave the highest yield of thiochrome by a laboratory-prepared BrCN. To obtain the highest intensity of fluorescence, a concentrated BrCN (1.8 M) was introduced in place of the conventional BrCN (0.11 M), obtaining 300% or more intensity of fluorescence. For the oxidation of thiamin diphosphate, 0.15-0.2 M of laboratory-prepared BrCN gave the highest intensity of fluorescence instead of the 0.25 M for free thiamin. For simultaneous oxidation of free thiamin and thiamin diphosphate, therefore, 0.23-0.24 M of laboratory-prepared BrCN was deduced to give the best yield of fluorescence. With a solution of commercially obtained solid CNBr, optimum concentrations for the oxidation of thiamin were about 0.04 M for CNBr and about 0.16% for NaOH. When the sample contained in inhibitor of oxidation, such as ascorbic acid, the percentage of inhibition decreased inversely proportional to the concentration of the sample in a rough approximation. The degree of inhibition was not reduced by the increased amount of BrCN reagent. Thus the possibility was indicated that thiamin in an ascorbic acid-contaminated sample could be determined accurately by extrapolating values for serially diluted samples.

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase.

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45 degrees C, and the K(m) values for the substrates are 2.7 x 10(-6) molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 x 10(-6) molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.

Molecular studies on thiaminase I.

The thiaminase I gene of Bacillus thiaminolyticus was cloned on a 1.6 kb DNA fragment (enzyme molecular weight 42,000), and was expressed in both Escherichia coli and Bacillus subtilis. When a selection drug was absent, the plasmid was maintained stably for approx. 100 generations in wild-type E. coli. Instability of the thiaminase gene was demonstrated in the thiamin pyrophosphate-requiring mutant of E. coli from which the plasmid was deleted rapidly. Wild-type E. coli accumulated the enzyme in its periplasm. A method for the detection of thiaminase I enzyme in SDS-polyacrylamide gel was developed. Thiaminase I of B. thiaminolyticus was found to exist in two sizes, 44 and 42 kDa, among different strains. Moreover, thiaminase of 42 kDa became approximately 41 kDa after a long-term culture, most likely because of the action of proteinases. Thiaminase expressed in E. coli from a thiaminase-positive recombinant plasmid was 42 kDa, and showed the same mobility on SDS-polyacrylamide gele electrophoresis as the enzyme isolated from the young culture of the parent strain of B. thiaminolyticus used for cloning. This value was, therefore, considered to represent intact thiaminase that had escaped from the attack of bacilli proteinases.

Hydrolysis and synthesis of thiamin triphosphate in bacteria.

Thiamin triphosphate (ThTP) in early stationary phase cells of Escherichia coli grown in nutrient broth with 0.1% yeast extract was found to constitute approximately 5-7% of cellular thiamin diphosphate (ThDP) or around 5 nmol/g cell. Nearly the same level of ThTP was obtained in a Bacillus strain. When E. coli was loaded with an excess of ThTP or ThDP, cellular ThTP was found to be controlled in the course of the long term to maintain its ratio to the amount of cellular ThDP. The ThTP vs. ThDP ratio in E. coli cells after short-term ThDP uptake was found to be a function of the cellular growth phase. The ratio in early exponential phase E. coli cells was found to be approximately 4% and it became lower (less than 3%) when cell growth proceeded to the late exponential stage. Two phosphatases specific for ThTP (ThTPase) among thiamin phosphates were detected in E. coli. One required Mg2+ and was found mainly in the soluble fraction, while the other was Mg2+-independent and originated from the membrane. The two ThTPases were similar to their rat tissue counterparts.

Nucleoside-triphosphatase and hydrolysis of thiamin triphosphate in Escherichia coli.

A membrane-bound nonspecific triphosphatase of E. coli was solubilized and purified to a homogeneous SDS-acrylamide gel electrophoresis band. It was found to be a single polypeptide of 16 kDa requiring no Mg2+, with an optimal pH at 6.5. The substrate specificity was broad and a nonspecific Mg2+-independent ribonucleoside-triphosphatase (NTPase) activity was expressed together with thiamin-triphosphatase activity. The molecular size and characteristics were clearly different from the known NTPase (EC 3.6.1.15). Using the purified thiamin-triphosphatase II, ATP:thiamin-diphosphate phosphoryl transferase (EC 2.7.4.15) activity was demonstrated with an optimal pH of approx. 5.3. Considering its kinetic parameters and other characteristics, however, the thiamin triphosphate synthesizing activity was not thought to take part in cellular thiamin triphosphate synthesis. The possibility that thiamin-triphosphatase II plays a part in the hydrolysis of thiamin triphosphate to control its cellular level is suggested.

Bacteriocinlike killing action of a temperate bacteriophage phiBA1 of Bacillus aneurinolyticus.

A new temperate phage, phiBA1, was isolated from Bacillus aneurinolyticus, phiBA1 had an icosahedral head with a diameter of about 70 nm and a tail about 20 nm long and contained a circularly permuted, linear duplex DNA of about 38 x 106 daltons. This phage showed two activities: bacteriocin-like killing activity against five strains of B. aneurinolyticus and normal temperate phage activity against three other strains. phiBA1 killed sensitive cells by a single-hit process. After adsorption of phiBA1 to cells sensitive to killing, the content of intracellular ATP increased for the first 5 min and then gradually decreased. Phage DNA injected into the cell immediately after infection was degraded rapidly. Killing was also caused by heavily UV-irradiated phiBA1. Killing-resistant mutants showed normal adsorption of phiBA1 and normal injection of the DNA with its instantaneous restriction. Our results indicate that the killing action of phiBA1 is different from the phenomenon of abortive infection and suggest that the killing might be caused by a proteinaceous component of phiBA1.

Counterflow efflux of thiamin in Escherichia coli.

A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux). This C-efflux could be separated frm an energy-dependent exit by a selective inhibition of exit by 2 x 10(-2) M NaN3. The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low. This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent. Basic characteristics of free thiamin uptake and exit in E. coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit. The low rate of exit in E. coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.