Genetic diversity of Lactobacillus delbrueckii isolated from raw milk in Hokkaido, Japan.

Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii, one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products.

Interaction among vitamin C, vitamin E, and beta-carotene.

The effects of vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), and beta-carotene as antioxidants and their cooperative action against the oxidation of lipid in solution, membranes, and lipoproteins have been studied and reviewed. Ascorbic acid and alpha-tocopherol act as potent, and probably the most important, hydrophilic and lipophilic antioxidants, respectively. They function at their own site individually and furthermore act synergistically. beta-Carotene has lower reactivity toward radicals than does alpha-tocopherol and acts as a weak antioxidant in solution. It is more lipophilic than alpha-tocopherol and is assumed to be present at the interior of membranes or lipoproteins, which enables it to scavenge radicals within the lipophilic compartment more efficiently than does alpha-tocopherol. The cooperative interaction between vitamin C and vitamin E may be quite probable, that of vitamin C and beta-carotene is improbable, whereas that between vitamin E and beta-carotene may be possible.

The affinity of betaxolol, a beta 1-adrenoceptor-selective blocking agent, for beta-adrenoceptors in the bovine trachea and heart.

1. The specificity of betaxolol, a beta-adrenoceptor antagonist, for beta 1- and beta 2-adrenoceptors was compared with that of other beta-antagonists, atenolol, ICI-118551, butoxamine and (+/-)-propranolol, in the bovine trachea and heart by competitive interaction with [3H]-CGP12177 as a radioligand. 2. The radioligand Kd values were 0.75 +/- 0.12 and 1.60 +/- 0.11 nM in the trachea and heart, respectively, and the Bmax values were 34.00 +/- 4.41 and 21.54 +/- 2.94 fmol mg-1 protein, respectively. 3. Using ICI-118551, we determined the ratio of beta 1:beta 2-adrenoceptors in the trachea and heart to be approximately 29:71 and 56:44, respectively. 4. In the trachea, a beta 2-predominant tissue, betaxolol and atenolol were more selective for beta 1-adrenoceptor binding sites than beta 2-adrenoceptor binding sites, whereas ICI-118551 and butoxamine were more selective for beta 2-adrenoceptor binding sites. 5. The beta 1-selectivity of betaxolol was 2.2 and 2.7 fold higher than that of atenolol in the bovine trachea and heart. These findings suggest that betaxolol may be useful in the treatment of hypertension, cardiac arrhythmia and angina pectoris.

Protuberic acid-hydrolytic enzyme in Kobayasia nipponica and characterization of the hydrolytic products.

When the cold water-extract of Kobayasia nipponica containing a glycuronan, protuberic acid (PA), was allowed to stand at room temperature, PA was hydrolyzed. The optimum conditions for this PA-hydrolysis were 37 degrees C and pH 4-5 in 0.1 M acetate buffer. Characterization of the hydrolytic products was performed by chemical analysis, and by 1H- and 13C-NMR spectroscopy. They were identified as D-GlcUA and O-(alpha-L-IdUAp)-(1-4)-D-GlcUA. These results suggest that PA-hydrolytic enzyme(s) include at least endo-beta-D-glucuronidase.

Exo-beta-D-glucuronidase from the fungus Kobayasia nipponica.

The occurrence of three forms, I, II, and III, of exo-beta-D-glucuronidase of the fungus Kobayasia nipponica was demonstrated. These three forms were purified 1,905-fold, 857-fold, and 357-fold, respectively. Forms I, II, and III of exo-beta-D-glucuronidase behaved differently on heparin-Sepharose chromatography, and differed in optimum pH (3.5, 3.2, and 2.6, respectively), pH-stability, Km (0.22, 0.16, and 0.13 mM, respectively), and Vmax (V) values. Their molecular weights, as estimated by gel filtration through Sephacryl S-200, were all 70,000; polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave a value of 72,000. These three forms were very active towards 1-4 linked beta-D-glucuronans, (Formula: see text) and (Formula: see text), but weak or inactive towards protuberic acid and several glycosaminoglycans.

Isolation and characterization of an endo-beta-D-glucuronidase from the fungus Kobayasia nipponica.

An endo-beta-D-glucuronidase was isolated and characterized from Kobayasia nipponica. The enzyme was purified by ammonium sulfate fractionation, CM-Sephadex chromatography, gel filtration with Sephacryl S-200, and heparin-Sepharose chromatography. The enzyme shows the following properties: optimum pH 5.0, thermal stability below 37 degrees C, pH stability 5-6, optimum temperature 45-55 degrees C, and Km 0.12% for L-idurono-D-glucuronan (protuberic acid (PA), L-IdUA:D-GlcUA = 1:2) from Kobayasia nipponica, 0.19% for PF (L-IdUA:D-GlcUA = 1:3) from Pseudocolus fusiformis, and 0.23% for (1-4)-beta-D-glucuronan(mucoric acid) from Mucor mucedo as determined from Hofstee plots. The molecular weight values estimated by gel filtration through Sephacryl S-200 and Sephadex G-50 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were 10,500 and 10,200, respectively. The endo-beta-D-glucuronidase was inactive towards several glycosaminoglycans.

On-line solid-phase extraction liquid chromatography-continuous flow frit fast atom bombardment mass spectrometric and tandem mass spectrometric determination of hydrolysis products of nerve agents alkyl methylphosphonic acids by p-bromophenacyl derivatization.

For proof of the presence of chemical warfare agents sarin, soman and VX, a rapid, accurate and sensitive method which allows us to determine their hydrolysis products ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methyl phosphonic acid was explored by using continuous flow frit fast atom bombardment (FAB) LC-MS and LC-MS-MS. After derivatization of analytes with p-bromophenacyl bromide, LC-MS-MS analyses for screening were performed by a flow injection method. The three alkyl methylphosphonic acids (AMPAs) were eluted within 5 min, and the detection limits for the three AMPAs ranged from 1 to 5 ng/ml. For confirmation of the screening results, LC-MS-MS analysis with chromatographic separation was conducted by using a narrow bore column. The three AMPAs were all eluted with excellent separation within 25 min, and the detection limits ranged from 1 to 20 ng/ml. Quantitative measurement was performed by LC-MS in selected ion monitoring (SIM) mode with chromatographic separation. Linear calibration curves were obtained for the three AMPAs and the detection limits ranged from 0.5 to 3 ng/ml. The relative standard deviation for peak area ranged from 3.4 to 6.0% at 50 ng/ml for the three AMPAs.

Binding of [3H]haloperidol to dopamine D2 receptors in the rat striatum.

The present study was designed to examine the properties of [3H]haloperidol binding to dopamine D2-receptors in rat striatum membranes, displacement potencies of various chemicals and differences between the affinities of various chemicals and two new 5-hydroxytryptamine (5-HT2) receptor antagonists, MCI-9042, (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenetyl)phenoxy]methyl]et hyl hydrogen succinate hydrochloride and one of its metabolites. The plots of specific binding for the striatum membranes obtained from the Scatchard analysis using [3H]haloperidol were monophasic when non-specific binding was determined with 10 microM chlorpromazine, and the Kd and Bmax values were 7.42 +/- 1.03 nM and 1.58 +/- 0.20 pmol (mg protein)-1 (n = 10), respectively. The displacement potencies of D2 receptor, 5-HT2 receptor, histamine H1-receptor, and adrenoceptor antagonists were characterized by [3H]haloperidol binding to D2 receptors. The pKi values of a new antiplatelet agent, MCI-9042, and its metabolite were 5.02 and 5.53, respectively, and these values were lower than those of the D2-receptor antagonists, fluphenazine, spiperone, haloperidol, prochlorperazine, chlorpromazine, thioridazine, and sulpiride. They were also lower than the pKi values of the 5-HT2-receptor antagonists, pirenperone, ketanserin, methysergide, and mianserin. We conclude that the binding site of [3H]haloperidol in the rat striatum is the D2 receptor, that MCI-9042 and its metabolite have lower affinities for D2 receptors than for 5-HT2 receptors, and that this radioreceptor assay is useful for assessing the affinities of various agents.

Alpha 1-adrenoceptor subtypes in bovine prostate.

The object of this study was to examine the existence and characteristics of alpha 1-adrenoceptor subtypes in the bovine prostate using the radioligand binding assay method. [3H]Prazosin was used as the radioligand and its binding sites in bovine prostate were classified into two subtypes. One subtype showed a high affinity (alpha 1High, Kd: 101.1 pM and Bmax: 11.8 fmol (mg protein)-1) and the other had a low affinity (alpha 1Low, Kd: 3371.4 pM and Bmax: 50.5 fmol (mg protein)-1). Although the same pKi values of clorethylclonidine, p-aminoclonidine, benoxathian and dibenamine to both alpha 1High and alpha 1Low binding sites in bovine prostate tissue were observed, other alpha 1 antagonists used in this study had different pKi values for the two alpha 1-adrenoceptor subtypes. The existence and binding characteristics of alpha 1-adrenoceptor subtypes in bovine prostate were clarified. It is possible that agents selective for one site may contribute to the development of better drugs for the treatment of bladder outlet obstructions of men with benign prostatic hyperplasia.

Alpha 1-adrenoceptor subtypes in the rat ventricular muscle.

Scatchard analyses of [3H]prazosin binding in rat ventricular muscle membranes showed biphasic curves, which identified alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 1 microM phenoxybenzamine. The displacement potencies of alpha 1-adrenergic antagonists were characterized by [3H]prazosin binding to alpha 1High- and alpha 1Low-affinity sites in the absence and presence of 1 microM phenoxybenzamine. The affinities of most chemicals for alpha 1Low-affinity sites were significantly lower than those for alpha 1High-affinity sites, but WB-4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane), arotinolol, cinanserin, nifedipine, and p-aminoclonidine had the same affinities for both alpha 1Low- and alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat heart, and that there are physical variations in alpha 1-adrenoceptor binding sites, based on their selectivity to antagonists.

Analysis of methylbenactyzium bromide in human urine by thin-layer chromatography and pyrolysis gas chromatography.

A rapid and simple method of utilizing thin-layer chromatography (TLC) and pyrolysis gas chromatography (PyGC) for the identification and determination of methylbenactyzium bromide in human urine was studied in this report. Methylbenactyzium bromide was extracted from urine with ODS-cartridge (Sep-Pak C18), then spotted onto a silica gel 60 F254 TLC plate. After development, the separated spot of methylbenactyzium bromide was scraped and wrapped with a ferromagnetic foil without extraction by any organic solvents. The sample was applied into PyGC analysis. The optimum temperature for pyrolysis was 590 degrees C. The main degradation product of methylbenactyzium bromide was identified as diphenylmethane in this procedure by gas chromatography/mass spectrometry (GC/MS). A calibration graph prepared by absolute calibration method showed a good linearity over the concentration range of 1-75 micrograms/spot for methylbenactyzium bromide. The coefficient of variation obtained for eleven replicate analyses of the 3 micrograms/spot of standard methylbenactyzium bromide was 3.8%. The detection limit of this compound by this procedure was 0.1 micrograms/spot.

The analysis of quaternary ammonium compounds in human urine by direct inlet electron impact ionization mass spectrometry.

An accurate and rapid screening test for nine quaternary ammonium compounds (suxamethonium chloride, pancuronium bromide, ambenonium chloride, benzethonium chloride, distigmine bromide, methylbenactyzium bromide, neostigmine bromide, propantheline bromide and pyridostigmine bromide) by direct inlet electron impact ionization mass spectrometry (DI/EI-MS) was investigated. Each compound was extracted from urine as an ion pair with KI3 into dichloromethane. The reliability of the identification of these compounds was verified by the mass chromatographic analysis of their characteristic fragment ions. The analysis of these drugs by DI/EI-MS could be performed within 5 min. The detection limits were between 20-150 ng/ml for the nine compounds. This method appears to be efficient, rapid and suitable as a screening procedure for the quaternary ammonium compounds found in urine.

The analysis of cocaine and its metabolites by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS).

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

Detection of designer drugs in human hair by ion mobility spectrometry (IMS).

Since its inception in the early 1970s under the name plasma chromatography, ion mobility spectrometry (IMS) has undergone great changes. It is now utilized more and more in forensic science laboratories where it is used to detect explosives and environmental pollutants [1-4] as well as its use in detecting drugs of abuse [5-8]. Although IMS is known for nearly 30 years now [9], relatively few cases of the application of ion mobility spectrometry to the analysis of human hair have been reported [10-12]. The authors report a new and quick method to rapidly screen and determine MDMA ('ecstasy', 'Adam') and MDEA ('Eve') in human hair. The proposed method using trihexylamine as internal standard resulted in a rapid procedure useful in screening human hair specimens for designer drugs.

Screening test of stimulants in human urine utilizing headspace gas chromatography for field test.

An accurate and simple screening method of stimulants in human urine using headspace gas chromatography utilizing heat of dissolution of potassium carbonate was developed. A 4.9-g portion of potassium carbonate was put into the vial prior to sending to the field, and a 5-ml aliquot of urine, suspected of containing stimulants and internal standard components was pipetted. After the vial was sealed and shaken by hand, 1 ml of its headspace gas was taken by disposable syringe and injected into the gas chromatograph. A compact gas chromatograph device with flame ionization detector and fused silica capillary column was developed for this experiment. Detection limits of methamphetamine and amphetamine were 1.0 micrograms/ml and 1.5 micrograms/ml, respectively.

GC and GC-MS determination of fluoroacetic acid and phenoxy acid herbicides via triphasal extractive pentafluorobenzylation using a polymer-bound phase-transfer catalyst.

A simple and sensitive gas chromatography and gas chromatography-mass spectrometry (GC-MS) procedure has been developed for fluoroacetic acid (FA) and phenoxy acid herbicides (PAHs) via triphasal extractive pentafluorobenzylation. The triphasal system consisted of an aqueous sample, the extraction solvent toluene containing pentafluorobenzyl bromide as the derivatization reagent, and polymer-bound tri-n-butyl-methylphosphonium bromide as a phase-transfer catalyst, FA spiked in beverages, such as orange juice and milk, was extracted as its pentafluorobenzyl (PFB) derivative under moderate conditions (i.e., at a pH value of 6.5 at 60 degrees C). The detection limits were 0.10-0.20 microgram/mL by GC with electron-capture detection (GC-ECD), and 0.42-0.50 microgram/mL by full-scan GC-MS. PAHs were also detectable in the same manner within the detection limits of 0.05-0.10 microgram/mL by GC-ECD and 0.13-0.25 microgram/mL by full-scan GC-MS. Urine and serum which both contained 2,4-dichlorophenoxyacetic acid could also be analyzed by GC-MS after the triphasal pentafluorobenzylation. The detection limit was 0.20 microgram/mL in the full-scan mode and 10 ng/mL in the selected ion monitoring mode both for the urine and serum.

Simultaneous determination of illicit drugs in human urine by liquid chromatography-mass spectrometry.

The method for simultaneous determination and confirmation of illicit drugs (e.g., methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-acetylmorphine, cocaine, and benzoylecgonine) in human urine by thermospray liquid chromatography-mass spectrometry (LC-MS) was studied. The LC-MS separation was performed on a reversed phase column (L-column ODS; 150 mm x 4.6-mm i.d.) using a gradient mobile phase system of 100mM ammonium acetate for 1 min then linear ramps to 100mM ammonium acetate including 40% acetonitrile at 20 min. Extraction was conducted by solid-phase extraction using a Sep-pak C18 cartridge. The drugs were eluted with 2 mliters of 40% acetonitrile in 100 mM ammonium acetate, pH3 (adjusted with acetic acid), from the cartridge. A 50-microL volume of the eluate was injected into the LC-MS. The recoveries by this extraction were 88 to 99%. The mass spectra of methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, 6-acetylmorphine, cocaine, and benzoylecgonine showed the quasi-molecular [M + H]+ ion as the base peak, whereas morphine-3-glucuronide and morphine-6-glucuronide showed [MH-glucuronide]+ ion as the base peak. The calibration graphs were linear and reproducible. Detection limits of these drugs ranged from 2 to 40 ng/mliters by selected ion monitoring (SIM) mode and from 50 to 400 ng/mliters by scan mode. The coefficients of variation for the analysis of these drugs ranged from 4.5 to 9.5% at a concentration of 0.4 micrograms/mliters (n = 10).

Determination of alkylmethylphosphonic acids, the main metabolites of organophosphorus nerve agents, in biofluids by gas chromatography-mass spectrometry and liquid-liquid-solid-phase-transfer-catalyzed pentafluorobenzylation.

A simple gas chromatography-mass spectrometry (GC-MS) procedure has been developed for the main metabolites of organophosphorus nerve agents, alkylmethylphosphonic acids (AMPAs; alkyl = Et, i-Pr, and pinacolyl) in biofluids via extractive pentafluorobenzylation. The derivatization was carried out under liquid-liquid-solid-phase-transfer conditions using a polymer-bound tri-n-butylmethylphosphonium bromide as a catalyst. AMPAs in aqueous samples were semiquantitatively extracted into a small-volume organic layer as their pentafluorobenzyl derivatives at pH 4.5 (85 degrees C). Sample pretreatments for urine, serum, and saliva were each examined to minimize matrix interference. The detection limits of APMAs by electron-impact ionization GC-MS were around 50 ng/mL and 2.5-10 ng/mL in the full-scan and selected-ion monitoring modes, respectively. In order to detect trace-level AMPAs, negative-ion chemical ionization (NICI) was also employed to enhance sensitivity. The detection limits of AMPAs in biofluids were typically 60 pg/mL by GC-NICI-MS.

Identification of metabolites of nerve agent VX in serum collected from a victim.

A human serum sample collected from a victim of the Osaka VX incident was analyzed according to our developed technique for metabolites of VX. Gas chromatography-mass spectrometry (GC-MS) in full-scan electron impact and chemical ionization modes were used, and, for more reliable confirmation, GC-MS-MS was also employed. In the serum sample, both ethyl methylphosphonic acid and 2-(diisopropylamino-ethyl)methyl sulfide were detected. These results indicated that the techniques using GC-MS and GC-MS-MS were applicable to biological samples such as serum. These results also provide the first documented, unequivocal identification of the specific metabolites of VX in victim's serum and, furthermore, clarify a part of the metabolic pathway of VX in the human body.