Escherichia coli is normally the most common facultative anaerobe in the large bowel and usually nonpathogenic for man. However, some E. coli strains which cause distinct syndromes of diarrhea diseases have been proved to be pathogenic. These organisms are one of the most common causes of food poisoning in Japan. In developing countries, these pathogens are known to be main causative agents of diarrhea which is the major cause of infantile morbidity and mortality. These E. coli strains are divided into four groups : enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enterohemorrhagic E. coli (EHEC). The four groups are distinguished on the basis of pathogenic, clinical, and epidemiologic features. Moreover, the fifth group of diarrheagenic E. coli, termed enteroaggregative E. coli (EAggEC), has been recently proposed. The strains of EAggEC exhibit characteristic patterns of adherence when bound to cultured epithelial cells. But the epidemiologic significance of EAggEC as diarrheal pathogens has not been yet determined. Although the features of the four groups are different, they have certain underlying commonalities with respect to pathogenesis as follows. (1) Critically virulent properties are encoded in plasmids. (2) Characteristic interactions with intestinal mucosa occurred. (3) Enterotoxins or cytotoxins are produced. In this review, microbiological features and etiological significances of these strains, are described.

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Bacterial endotoxin, which is thought to be a direct cause of shock or tissue damage of multi-organs in Gram-negative septis, has been studied on their biological activities for a long time. In the last several years, the advance in endotoxin research has been very rapid. Macrophage activation is the first and essential step in the course of endotoxin action and is followed by the release of many kinds of mediators including cytokines such as tumor necrosis factor α (TNFα)/cachectin, interleukin-1 (IL-1), interleukin-6 (IL-6), interferons, and colony-stimulating factors, and lipid derivatives such as prostaglandines, leukotrienes, and platelet-activating factor. Of these mediators, TNF α/cachectin and IL-1 play a central role in the multiple activities of endotoxin. TNF α and IL-1 have many similar activities and exert alone or cooperatively the endotoxin-like activities such as lethal toxicity, pyrogenesis, promotion of coagulation and leukocyte adherence to endothelial cells, and induction of acute phase proteins. Endotoxin tolerance and Shwartzman reaction are also mediated through these inflammatory cytokines. On the basis of these findings, some remedies using anti-TNF α monoclonal antibody or antagonist of IL-1 are under research for septic shock as well as clinical use of TNF α for cancer therapy.

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Urinary excretion of hydroxylysine diglycoside (GGH) or monoglycoside (GH) is known to be a reliable indicator of metabolic status of bone collagen. Although some preparation methods of the standard GGH or GH from different species have been reported, this study demonstrated that the preparation of the GGH or GH from the human urine was more suitable for the analyses of these glycosides. GGH and GH were prepared in two ways : one from the normal human urine and the other from the alkaline hydrolyzate of marine sponge. The adequacy for their use as the standard substances was evaluated by proton NMR and HPLC. The glycosides obtained from the human urine were confirmed to be normal forms. As for the glycosides obtained from marine sponge, it was demonstrated that the glycosides were diastereoisomers. The urinary levels of GGH and GH of the inhabitants in Cd-polluted area were compared with the values obtained from non-polluted subjects. The measurements of GGH and GH were performed by using the glycosides prepared from the normal human urine as the standard substanses. As the results, the urinary GGH or GH levels of the subjects in the Cd-polluted area had wider distribution ranges than those of controls, although there was no significant difference between Cd-polluted subjects and controls.

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A sensitive method for the determination of four anthranilic acid derivatives (diclofenac sodium, aluminium flufenamate, mefenamic acid and tolfenamic acid) of analgesic and antiinflammatory drugs was developed by using high performance liquid chromatography (HPLC). It was found that the four drugs were converted into methylphthalimide (MPI) derivatives in a constant yield by reaction with N-chloromethylphthalimide at 60°C for 30 min. The productions of the MPI derivatives were confirmed by mass spectrometry. The MPI derivatives of the four drugs tested showed satisfactory results of separation and sensitivity by HPLC using C₁₈ bonded phase with acetonitrile : H₂O (80 : 20, v/v) as mobile phase at 282nm. The calibration curves of the MPI derivatives of the four drugs were linear from 1.0 to 5.0μg/ml. The detection limits of the four drugs were 0.5-5ng. The extraction procedure of the four anthranilic acid derivatives added in the plasma and urine was performed by using Extrelut^[○!R] 1 column. Yields of column extraction of 100μl of plasma and urine samples (containing 0.5μg of anthranilic acid derivatives) with 6ml of ethyl acetate were 84-106%. The present procedure may be an easier and more convenient method for the analysis of anthranilic acid derivatives of analgesic and anti-inflammatory drugs and could be applicable for the determinations of these drugs in forensic samples.

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In examining substances causing hemoglobin (Hb) denaturation in vitro obtained from lipophilic fractions of fishes, the transformation of oxyhemoglobin (O₂Hb) to methemoglobin (MetHb) was observed in the ether extracts of Engraulis japonica (anchovy) and the viscera of Cololabis saira in six fish species. Removal of acidic substances from ether extracts of anchovy viscera by pretreatment with Na₂CO₃ resulted in suppression of Hb denaturation activity, suggesting that the substance causing Hb denaturation was acidic. The transformation of hemoglobin was observed in three fractions separated by thin layer chromatography : Fr. 1, found at the origin contained primarily phospholipids ; Fr. 2 consisted of two bands between fractions of phospholipids and free fatty acids ; and Fr. 3 was composed of free fatty acids. Addition of α-tocopherol to the sample completely suppressed the transformation of O₂Hb to MetHb, and the usual brownish precipitation caused by protein denaturation did not appear. The concentrations of free eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contained in ether extracts were directly proportional to magnitude of the MetHb formation index. These results indicated that the main acidic substances causing blood Hb denaturation in vitro were polyunsaturated fatty acids such as EPA and DHA which were present in abundance in anchovy viscera.

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Enantiomeric compositions of methamphetamine (MA) and its major metabolite, amphetamine (AP), in the urine of mice administered with l-deprenyl (DPN), d-methamphetamine and dl-methamphetamine were analysed by the selected ion monitoring of gas chromatographmass spectrometry (GC-MS) using a chiral reagent. Each drug was intraperitoneally administered to mouse at 0.1, 0.5, 1mg/kg and urine samples of each two mice in a cage were collected at 0-12, 12-24, 24-48 and 48-72h, respectively. Urine added with deuterium-labelled MA and AP as the internal standards were applied to Bond Elut C18 and eluted with acidic methanol. The extract was evaporated to dryness and the residue dissolved in 0.1ml of chloroform containing 10% triethylamine and added with 0.05ml of 0.1M N-trifluoroacetyl-l-proryl chloride in chloroform. The derivatives were analysed by GC-MS on an OV-1 bonded capillary column (10m×0.25mm). The apparent d/l of MA excreted in the urine of mice by the administration of l-DPN, d-MA and dl-MA were 0-0.06, 3.20-6.76 and 1.04-1.71, respectively. The ratio of AP/MA in the urine by the administration of l-DPN, d-MA and dl-MA were approximately 1 : 2, 1 : 20 and 1 : 20, respectively. By use of these two factors it is possible to discriminate l-DPN use from MA use.

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Determination of 9 kinds of herbicides containing nitrogen in agricultural products was studied by GC and HPLC. FTD-GC and UV-HPLC methods were compared for adaptability, sensitivity of the pesticides, and for interfering peaks from food components. Swep (N-phenylcarbamate), diuron, linuron (urea type) and propyzamide (acid amide type) were decomposed during determination by GC. Alachlor and butachlor were not able to be determined by HPLC. Sensitivities of the pesticides by GC were 5 times (50 times by the same injection volume) that by HPLC, and GC was more sustainable for in terfering peaks than HPLC. Preparation method for test solution was systematically established according to the kind of agricultural products. Pesticides in common vegetables and fruits were directly determined for the extracts, and recoveries of the pesticides added at 1 ppm were 77.1-96.5%. Extracts from onion, garlic, etc. were purified on a florisil column before determination, and recoveries of the pesticides added at 1 ppm were 77.2-89.7%. Extracts from grains, beans, etc. were defatted by coagulation, purified on a florisil column before determination, and recoveries of the pesticides added at 1 ppm were 77.5-89.6%.

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Low dose (10mg/kg as rare earth elements) or high dose (50 mg/kg as rare earth elements) of 4 kinds of rare earth element solutions, i. e. dysprosium chloride (DyCl₃), europium chloride (EuCl₃), ytterbium chloride (YbCl₃) and yttrium chloride (YCl₃), were administered intravenously from the caudal vein of rat, and the distribution and the histological change in the tissues were investigated at the 8th days after the administration. The rusults were as follows : 1) The administered rare earth elements were mainly accumulated in the liver, spleen and bone. The concentrations of rare earth elements in the kidney, pancreas and heart were low. No rare earth elements was detected from the muscle and whole blood. 2) More than 3 times of amounts of the rare earth elements were accumulated in the spleen of rats when administered low dose of YCl₃, and higher amounts of the rare earth elements were accumulated in the bone of the rats administered YbCl₃ compared to the rats administered other rare earth elements of low dose. On the contrary, the difference in the pattern of accumulation in the various organs was not observed when administered high dose. The distribution (% of dose) of the rare earth elements in the spleen tended to elevate but those in the bone tended to be lower in the bone of the rats administered high dose. 3) No obvious change was observed in the liver of the rat administered low dose. The deposition of the brown substance or xenobiotics was observed in the spleens of the rats administered low dose of DyCl₃ and YCl₃. The reaction of macrophage was observed in the spleen of the rats administered low dose of YCl₃. 4) The formation of xenobiotic glanuloma was observed in the spleen when the rats administered high dose of rare earth elements. Therefore, when the chlorides of Dy, Eu, Yb and Y were administered to the rats intravenously, the administered rare earth elements accumulated mainly in the liver, spleen and bone and the formation of glanuloma was observed in the spleen when high dose administered.

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Four kinds of rare earth element solutions [(10mg/kg as rare earth elements), i. e. dysprosium chloride (DyCl₃), europium chloride (EuCl₃), ytterbium chloride (YbCl₃) and yttrium chloride (YCl₃)], were administered intravenously from the caudal vein of rat. The excretion into the feces and urine, and the concentration and the distribution (% of dose) of rare earth elements in the whole blood, liver, kidney, lung, spleen and bone were investigated as a function of time. The results were as follows : 1) The administered rare earth elements were excreted gradually into the feces for 7d. The excretion rate accounts for 5.4-18.4%. The excretion of Y was lower than the other 3 kinds of rare earth elements. No rare earth elements were detected from the urine. 2) Rare earth elements in the whole blood disappeared within 1d. Y in the whole blood disappeared within 4h after the administration. 3) The major organs for accumulation of the administered rare earth elements were liver, spleen and bone. The distribution (% of dose) of rare earth elements in the liver was the highest level at 8h to 2d after the administration, then decreased gradually. The levels of distribution of rare earth elements in the kidney and lung were low during the experimental period. The distributions of Dy, Eu and Yb in these organs increased till 1d after the administration, and then almost unchanged. On the contrary, the distribution of Y in the spleen was larger than those of Dy, Eu and Yb, and increased as a function of time during the experimental period. The distribution of rare earth elements in bone increased gradually as a function of time. The highest level was observed in the case of Yb administration. Therefore, it was suggested that the rare earth elements in the blood disappeared within 1d, and that the rare earth elements were accumulated mainly in the liver, spleen and bone and retained for a long time.

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Comparison of pharmacological activity of two cannabis extracts from cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) strains in mice was evaluated using catalepsy, hypothermia, prolongation of pentobarbital (PB)-induced sleep and locomotor activity as indices. ED₅₀s of catalepsy of CBDA strain extract (CBDA-E) and THCA strain extract (THCA-E) were 41.0 and 20.1 mg/kg, i. v., respectively. The most effective cannabis extract inducing hypothermia was CBDA-E at a dose of 50mg/kg, i. v.. By treatments (25mg/kg, i. v.) with CBDA- and THCA-Es, PB-induced sleeping time was prolonged 4.1 and 1.8 fold, as compared with that of 1% Tween 80-saline control. In its effect on locomotive activity CBDA-E showed a tendency to decrease and THCA-E at a low dose (5 and 25mg/kg) a tendency to increase, and at a high dose (50mg/kg) to decrease horizontal activity. CBDA- and THCA-Es tested, except for a dose of 5mg/kg, significantly decreased vertical motor activity of mice. From these results, CBDA-E containing CBDA as a major component and THCA-E containing THCA as a major component were found to have different pharmacological activity.

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m-Phenylenediamine (PD) is one of the components of hair dye used in our country. In order to evaluate the modulating effect of other hair dye components on hydrogen peroxide (H₂O₂) oxidation of m-PD, 8 kinds of phenol derivatives and p-PD were treated with H₂O₂ in the presence or absence of m-PD. Before and after treatment with H₂O₂, their mutagenicity was tested by using Salmonella typhimurium TA98 with or without S9mix. The mutagenic potency of m-aminophenol (AP) was most remarkably enhanced with H₂O₂ among 8 phenol derivatives tested. p-AP and phloroglucinol (PH) were slightly mutagenic in the presence of S9mix after treatment with H₂O₂, but the other phenol derivatives were not mutagenic with or without S9mix both before and after treatment with H₂O₂. As major mutagenic products after the H₂O₂ oxidation of the mixture of m- and p-PD, 2, 7-and 2, 8-diaminophenazine (diNH₂-Pz) were identified by TLC and HPLC comparing with authentic samples. The yield of 2, 7-diNH₂-Pz obviously decreased with an increase in p-PD, but the formation of 2, 7-diNH₂-Pz was not completely inhibited by the addition of a 9-fold molar ratio of p-PD to m-PD. The mutagenic potency of the oxidation mixture obtained from m-PD and phenol derivatives decreased and the production of 2, 7-diNH₂-Pz from m-PD was inhibited by the addition of phenol derivatives to the m-PD oxidation system. From the HPLC analysis of the oxidation mixture, the declined mutagenic potency of the oxidized mixture from m-PD and phenol derivatives was found to be mainly due to a decrease in 2, 7-diNH₂-Pz. The modulating effect of m-AP on the oxidation of m-PD was the strongest among 8 phenol derivatives tested.

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A gas chromatographic (GC) method for the determination of trace amounts of oxinecopper (Ox-Cu) in water is described. A 500ml volume of sample water (pH 6.5-6.8) was passed through Sep Pak C₁₈ cartridge at a flowrate of 5ml/min. Then Ox-Cu absorbed on the cartridge was eluted with 4ml of methanol. After removing the solvent from the eluate, 2ml of 0.01M disodium ethylenediaminetetraacetate solution was added to the residue and the formation of 8-hydroxyquinoline (8-HQ) was facilitated by ultrasonic treatment and vigorous shaking. 8-HQ was extracted twice with 1 ml of benzene and determined with a flame-thermoionic detector (FTD)-GC using a megabore capillary column (DB-1). The recoveries of Ox-Cu from 6 kinds of sample water were 81-97% and the relative standard deviation were 2.5-5.7%. The limit of detection was 0.5μg/l.

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The biodegradation of fungicide pentachloronitrobenzene (PCNB) in water environment was evaluated. The biodegradability of PCNB was different in each region. However, seasonal and yearly variations in biodegradation of PCNB were not found. The biodegradation of PCNB was rapid in river water polluted by PCNB. Pentachloroaniline (PCA) was formed with the biodegradation of PCNB under anaerobic conditions, but PCA was slightly biodegraded under anaerobic conditions. Furthermore, it was assumed that PCNB degrading bacteria isolated from the river water belonged to Pseudomonas sp., which were capable of biodegrading PCNB to pentachlorothioanisole (PCTA). According to the survey of water qualities at Tsumagoi in Gunma prefecture and at Sugadaira in Nagano prefecture, there were differences in concentration ratios of PCNB and PCA. This observed results were consistent with the results of the PCNB biodegradation test, by which the differences between PCNB biodegradation ratios and PCA formation ratios were observed at both areas.

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A high-performance liquid chromatographic rapid analytical method with β-naphthoquinone (BNQ) prelabelling for the simultaneous determination of methamphetamine (MA) and its metabolite, amphetamine (AP), in saliva has been studied. Without pretreatment, the direct derivatization of amphetamines in saliva has been developed. As a result of study on the optimization of the derivatization, a reaction mixture of 100μl of saliva containing MA and AP, 200μl of 0.5% Na₂CO₃, 200μl of aq. solution of 0.5% BNQ-sulfonate, 200μl of methoxyphenamine (1μg/ml) as an internal standard and 100μl of methanol, as a shift agent of interference peak was warmed at 40°C for 20 min. The reaction solution was extracted with n-hexane-ether (1 : 1) and the solvent was evaporated under N₂. The residue dissolved in acetonitrile was analysed by high-performance liquid chromatography on a column of μBondasphere C₁₈ with acetonitrilemethanol-0.01M H₂SO₄ (20 : 20 : 67) as a mobile phase (0.8ml/min) and electrochemical detection at -0V. Calibration curves were rectilinear for 50 to 10000ng/ml of MA and AP in saliva. By this method, 6 saliva samples of 4 male and 1 female human suspects who had taken i. v. dose of MA hydrochloride at 7.3, 7.8, 10.2, 20.4, 31 and 66.5h earlier were analysed to confirm the use of MA. Until at least 31h after use, MA and AP were detected at 0.1 to 24.8μg/ml. Comparing with urine and blood samples, the usefulness of the saliva sample was disccused.

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A systematic method was developed for the analyses of 15 kinds of artificial dyes and 19 kinds of natural dyes in foods. Dyes were extracted from the sample with water and dichloromethane. Water soluble dyes in the aqueous extract were purified and fractionated into fractions I and II by use of polyamide. Oil soluble dyes in the dichloromethane extract were purified and fractionated into fractions III-V by liquid-liquid partition, silica gel column and alumina column chromatographies. The dyes in each fraction were identified by thin-layer chromatography. This method was applied for the detection of artificial and natural dyes in various commercial foods.

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The variation of fenitrothion (MEP) concentration in the atmosphere was investigated after the aerial application of MEP for the control of pine sawyer beetles in Niigata City. In the applied area, MEP in air (C_A, μg/m³) was 5.2-100μg/m³ at maximal concentrations ; MEP was detected on 12d after the application. MEP was drifted to the points located 50-1000m from the applied area ; atmospheric MEP (C, μg/m³) were 0.19-1.1μg/m³ at maximal concentrations. C was mainly dependent on passing time after the application, wind direction, air temperature and the distance from the applied area. C_A (n=37) was represented by multiple regression analysis as log C_A=-1.007×log T+0.01290×cos θ-0.07283×υ^-1+6.033×log t-6.416 (γ²=0.736) where T (h), v (m), θ and t (°C) are passing time after the application, wind speed, wind direction (0⪈θ⪈180 ; θ=0 at a NW wind) and air temperature, respectively. C (n=122) was represented as log C=-0.3165×log T+0.2154×cos θ+0.6329×υ^-1+3.094×log t-0.2642×log D+0.2833×C_A-4.570 (γ²=0.763) where D (m) was 100 + the distance from the applied area.

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