Analysis of the vibrioferrin biosynthetic pathway of Vibrio parahaemolyticus.

Siderophores are small-molecule iron chelators produced by many microorganisms that capture and uptake iron from the natural environment and host. Their biosynthesis in microorganisms is generally performed using non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) enzymes. Vibrio parahaemolyticus secretes its cognate siderophore vibrioferrin under iron-starvation conditions. Vibrioferrin is a dehydrated condensate composed of α-ketoglutarate, L-alanine, aminoethanol, and citrate, and pvsA (the gene encoding the ATP-grasp enzyme), pvsB (the gene encoding the NIS enzyme), pvsD (the gene encoding the NIS enzyme), and pvsE (the gene encoding decarboxylase) are engaged in its biosynthesis. Here, we elucidated the biosynthetic pathway of vibrioferrin through in vitro enzymatic reactions using recombinant PvsA, PvsB, PvsD, and PvsE proteins. We also found that PvsD condenses L-serine and citrate to generate O-citrylserine, and that PvsE decarboxylates O-citrylserine to form O-citrylaminoethanol. In addition, we showed that O-citrylaminoethanol is converted to alanyl-O-citrylaminoethanol by amidification with L-Ala by PvsA and that alanyl-O-citrylaminoethanol is then converted to vibrioferrin by amidification with α-ketoglutarate by PvsB.

Synthesis of Biosynthetic Intermediates of Vibrioferrin and Enzyme Reactions Using Them as Substrates.

Biosynthetic intermediates of siderophore vibrioferrin (VF), O-citryl-L-serine, 2-aminoethyl citrate, and alanine-2-amidoethyl citrate were respectively synthesized as a mixture of stereoisomers. These compounds were used as substrates for enzyme reactions using recombinant PvsA, PvsB, and PvsE proteins as corresponding enzyme equivalents. The results of our study show that each enzyme reacts with a respective substrate and produces VF along the proposed biosynthetic pathway. Furthermore, the results of this study will contribute to the understanding of VF biosynthetic enzymes and may help in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

Preparation of [1'-13 C]citric acid as a probe in a breath test to evaluate tricarboxylic acid cycle flux.

[1'-13 C]Citric acid (1) was efficiently prepared from dimethyl 1,3-acetonedicarboxylate in two steps as a probe for a breath test. The synthetic method was selected because of the yield and reproducibility. Compound 1 was orally administrated to rats, and the time course of the increase of 13 CO2 /12 CO2 ratios (Δ13 CO2 ) in their breath was successfully followed, indicating the metabolism of 1. Thus, the 13 C-breath test using 1 is a promising method to evaluate tricarboxylic acid (TCA) cycle flux.

Degradation and inactivation efficacy of ozone water for antineoplastic drugs in hospital settings.

PURPOSE: Occupational exposure to antineoplastic drugs in hospital settings is recognized to be hazardous, and as such environmental decontamination including degradation and inactivation of such drugs is recommended. To data, although various agents such as oxidants have been reported to be useful for decontamination, simpler, safer, and more convenient methods are required. In this study, the degradation and inactivation efficacy of ozone water, which has newly been introduced for decontamination of antineoplastic drugs in spills, was investigated for formulations of gemcitabine, irinotecan, and paclitaxel. METHODS: Antineoplastic formulations (medicinal ingredient: ∼1.5 µmol) were mixed with 50 mL of ozone water (>4 mg/L). The reactions were monitored by high-performance liquid chromatography, and the degradation mixtures were analyzed by 1H nuclear magnetic resonance spectroscopy in order to obtain the structural information of the degradation products. The formulations of gemcitabine and irinotecan and those degradation mixtures were evaluated for their mutagenicity using the Ames test and cytotoxicity against human cancer cells. RESULTS: gemcitabine and irinotecan were found to be readily degraded by the ozone treatment, and their active sites were suggested to be degraded. In contrast, paclitaxel was hard to be decomposed, possibly owing to the consumption of ozone by the polyoxyethylene castor oil added as a pharmaceutical additive of the formulation. No significant mutagenic changes of Salmonella typhimurium strains used for the Ames test were observed for the samples within the concentration ranges examined. The ozone treatment showed obvious increases in cell viability for gemcitabine formulation, and mild increases for irinotecan formulation. CONCLUSIONS: Ozone water was shown to be effective as a decomposition agent for the antineoplastic drug formulations examined, although the efficacy depends on the chemical structures of the drugs and the pharmaceutical additives. It was also suggested that ozone treatment has a tendency to decrease the toxicity of the antineoplastic drug formulations. As such, further studies are required in order to clarify the effects and application limitations of ozone water.

Application of Quantitative NMR Spectroscopy to the Quality Evaluation of Diclofenac Gargles as Hospital Preparations.

Hospital preparations are frequently prepared in Japanese hospitals when ready-made formulations to meet patients' needs are unavailable. Although the quality of hospital preparations have to be ensured for efficacy and safety, such quality evaluation tends to be insufficient mainly due to lack of manpower and experimental environments in hospitals. In this paper, we investigated the applicability of quantitative (q)NMR spectroscopy to the quality control of diclofenac gargles as examples of hospital preparations, as it has various merits for the quantitative analysis of mixtures in solutions. Diclofenac gargles are composed of diclofenac, tranexamic acid, and lidocaine, and are used for the pain relief of stomatitis induced by cancer chemotherapy. Aliquots of the gargles, which were prepared five times, were mixed with dimethylsulfone as an internal standard, followed by qNMR measurements. Water signal suppression was achieved using a pulse program, water suppression enhanced through T1 effects, because the pulse program was superior to other ones such as presaturation and one-dimensional nuclear Overhauser effect spectroscopy in terms of quantitativeness. Concentrations of the three medicinal ingredients were simultaneously determined based on the signals selected by considering the spectral separation and the quantitativeness. Consequently, the gargles were found to be prepared with constant quality, and were stable at room temperature for at least four weeks. qNMR is considered to be potentially useful for the quality control of various hospital preparations because of minimal sample pretreatments, lack of need of calibration curves, and its comprehensive detection abilities.

Amitorines A and B, Nitrogenous Diterpene Metabolites of Theonella swinhoei: Isolation, Structure Elucidation, and Asymmetric Synthesis.

Two new nitrogenous prenylbisabolanes never before found in Lithistid sponges have been isolated from Theonella swinhoei. These new diterpenes, named amitorine A (1) and amitorine B (2), containing a prenylbisabolane skeleton have been characterized by spectroscopic analyses, and the relative and absolute configurations of 1 and 2 were determined by asymmetric synthesis of both diastereomers via the common bicyclic lactone 6 intermediate.

Synthesis of ¹³C-lidocaine as a probe of breath test for the evaluation of cytochrome P450 activity.

(13)C-Labeled lidocaine, 2-di[1-(13)C]ethylamino-N-(2,6-dimethylphenyl)acetamide (1), was synthesized from [1-(13)C]acetic acid in six steps, as a probe for a breath test to evaluate in vivo cytochrome P450 activity. The measurement of (13)CO2 in breath was successfully performed following oral administration of (13)C-lidocaine 1 to mice.

An efficient laboratory-scale preparative method for [1-(13)C]glycocholic acid.

A breath test using [1-(13)C]glycocholic acid as a substrate is a potential diagnostic method for small intestinal bacterial overgrowth syndrome. [1-(13)C]Glycocholic acid has been thus synthesized in an excellent yield from ethyl [1-(13)C]glycinate hydrochloride in a one-pot reaction. This method is suitable for the preparation of the labeled compound on a laboratory scale, which helps to perform extensive clinical studies of the breath test.

A study on the chemical stability of cholesterol-lowering drugs in concomitant simple suspensions with magnesium oxide.

BACKGROUND: Difficulty in taking solid medicines is a common issue particularly for the elderly because of a decline in swallowing function, also known as dysphagia. For patients with such a dysfunction, a simple suspension method, in which solid medicines are disintegrated and suspended using warm water, has been developed and widely used in Japanese clinical settings. However, there is little information on drug stability in the simple co-suspension of multiple formulations especially including acidic or alkaline ones. In this study, the chemical stability of typical cholesterol-lowering drugs was investigated in a simple co-suspension with alkaline magnesium oxide (MgO) which is frequently used as a laxative or antacid in Japan. METHODS: A cholesterol-lowering drug (one tablet) was soaked with or without MgO in warm water (55°C), and the vessel was left at room temperature for 10 min or 5 h. The suspensions prepared were then analyzed by high-performance liquid chromatography. Degradation products were analyzed by nuclear magnetic resonance spectroscopy and mass spectrometry for the structural elucidation. RESULTS: In the simple co-suspension with MgO, no significant degradation was observed for atorvastatin or pravastatin, while a significant decrease of the recovery from the co-suspension was observed for rosuvastatin after 5 h. On the other hand, simvastatin and ezetimibe co-suspended with MgO were partially degraded to simvastatin acid and a pyran compound, respectively. CONCLUSIONS: A simple co-suspension with MgO is feasible for atorvastatin, pravastatin, and rosuvastatin, although the rosuvastatin tablet should not be left soaking for a long time. Further it is inadvisable to suspend simvastatin or ezetimibe together with MgO because of their partial degradation.

Study on the Use of Ozone Water as a Chemical Decontamination Agent for Antineoplastic Drugs in Clinical Settings.

The exposure of healthcare workers to antineoplastic drugs in hospitals has been recognized to be harmful. To minimize the risk of exposure, the removal of these drugs from work environments, such as compounding facilities, has been recommended. In our previous paper, the degradation and inactivation efficacy of ozone water, which is being introduced into Japanese hospitals as a chemical decontamination agent, was reported for its effects on typical antineoplastic drugs (gemcitabine, irinotecan, paclitaxel). This article aims to further investigate the efficacy of ozone water for eight antineoplastic drugs to clarify its application limitations. A small amount (medicinal ingredient: typically ca. 1.5 µmol) of formulation containing 5-fluorouracil, pemetrexed, cisplatin, oxaliplatin, cyclophosphamide, ifosfamide, doxorubicin, or docetaxel was mixed with 50 mL of ozone water (~8 mg/L), and the resulting solutions were analyzed by high-performance liquid chromatography over time to observe the degradation. Consequently, the ozonation was overall effective for the degradation of the drugs, however this varied depending on the chemical structures of the drugs and additives in their formulations. In addition, after the parent drugs were completely degraded by the ozonation, the degradation mixtures were subjected to 1H nuclear magnetic resonance spectroscopy and evaluated for mutagenicity against Salmonella typhimurium strains and cytotoxicity against human cancer cells. The degradation mixtures of cisplatin and ifosfamide were mutagenic while those of the other drugs were non-mutagenic. Further, the ozonation resulted in clear decreases of cytotoxicity for 5-fluorouracil, oxaliplatin, and doxorubicin, but increases of cytotoxicity for pemetrexed, cisplatin, cyclophosphamide, and ifosfamide. These results suggest that the ozone water should be restrictedly used according to the situation of contamination in clinical settings because the ozonation enhances toxicity depending on the drug even if degradation is achieved.

1H NMR-based metabonomic analysis of urine from young spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) and their substrains are a useful model for studying essential hypertension which is a complex, polygenic, and multifactorial disorder. Their genetic and metabolic features are of great interest because they may provide insights into the mechanism of blood pressure regulation. We have compared urinary metabolic profiles of young SHR with those of their age-matched normotensive controls, Wistar Kyoto rats, using (1)H NMR-based metabonomics. Principal components analysis was applied to the NMR spectral data after data-reduced and normalized by the total integral or the creatinine integral. Consequently, a clear separation of urine samples between the two strains was observed in the principal components scores plot. The loadings plot from the data normalized by the creatinine integral showed that many metabolites such as citrate, alpha-ketoglutarate, and hippurate contributed to the separation, and the urinary levels of most metabolites used in this study, including these three, were lower in SHR than in Wistar Kyoto rats. These metabolic changes may be concerned with blood pressure regulation in SHR, although a relation to other strain differences cannot be ruled out. The present study suggests the usefulness of a (1)H NMR-based metabonomic approach using SHR in the field of hypertension research.

New polyhydroxylated sterols stylisterols A-C and a novel 5,19-cyclosterol hatomasterol from the Okinawan marine sponge Stylissa sp.

New polyhydroxylated sterols, stylisterol A-C (1-3), and a novel 5,19-cyclosterol, hatomasterol (4) were isolated from the Okinawan sponge Stylissa sp. Structural determinations of these compounds were made by spectroscopic analysis and chemical conversion. Assessment of cytotoxicity toward HeLa cells was also determined.

Metabonomic study on the biochemical response of spontaneously hypertensive rats to chronic taurine supplementation using (1)H NMR spectroscopic urinalysis.

There is a wealth of experimental information and some clinical evidence available in the literature suggesting that taurine exerts preventive effects on cardiovascular diseases. In particular, taurine has been shown to reduce blood pressure in not only hypertensive patients but also in a number of hypertensive rodent models such as spontaneously hypertensive rats (SHR). However, the molecular basis of the efficacy and toxicity of the compound has not been fully characterized. We have investigated the effects of taurine supplementation to urinary low-molecular-weight endogenous metabolites in SHR using a (1)H NMR-based urinary metabonomic approach. The SHR were chronically treated with 3% taurine in drinking water from four to 14 weeks of age, and 24-h urine samples were analyzed using (1)H NMR spectroscopy. Metabolic information was extracted from the NMR data by principal components analysis as well as visual inspection. Consequently, the metabolite profile started to change with considerable interindividual variation from six weeks of age. The extent of change became increasingly remarkable with the duration of treatment, with the concurrent observation of the hypotensive effect. The metabolic changes included a decreased urinary output of tricarboxylic acid cycle intermediates (citrate, α-ketoglutarate, and succinate) and an increased output of phenylacetylglycine and p-cresol sulfate. The results suggest that chronic taurine supplementation to the SHR resulted in an acceleration of metabolic acidosis with perturbation in the tricarboxylic acid cycle and the modulation of the intestinal microbial metabolism.

Sample preparation method to minimize chemical shift variability for NMR-based urinary metabonomics of genetically hypertensive rats.

Urinary metabonomics based on proton nuclear magnetic resonance ((1)H NMR) has been widely employed to study metabolic differences associated with gene function and pathophysiological and toxicological stimuli. However, the chemical shift variability of (1)H NMR signals, which is due to differences in pH and ionic strength among urine samples, remains an outstanding problem for efficient data mining. Thus, we have proposed an improved sample preparation method where urine samples are lyophilized and reconstituted in a buffer solution (pH 7.40) so that the extent of urine concentration becomes constant based on creatinine concentration. In order to examine the usefulness of the proposed method, urine samples taken from spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) were treated not only by the proposed method but also by the usual method where urine with various concentrations is mixed with an equivalent volume of buffer solution (pH 7.40). Consequently, the pH of the urine samples prepared by the proposed method was precisely controlled to 6.89-7.01, whereas the pH of samples by the usual method was in the range of 6.81-7.18. The chemical shift variations of various metabolites having ionizable groups such as succinate, α-ketoglutarate, cis-aconitate, taurine, and glycine were significantly reduced with decreases in pH variability. A preliminary multivariate statistical analysis was carried out for the (1)H NMR spectral data obtained by the proposed method, where the metabolic profiles were distinguished between the SHR and SHRSP. The proposed sample preparation method will be particularly useful to closely inspect NMR-based urinary metabonomic data for the exploration of metabolic changes.

A metabonomic study of biochemical changes characteristic of genetically hypertensive rats based on (1)H NMR spectroscopic urinalysis.

Spontaneously hypertensive rats (SHR) provide a simple model for studying essential hypertension. Their genetic and metabolic features are of great interest because they may provide insights into the pathophysiological processes underlying essential hypertension. We have thus investigated the metabolic characteristics of SHR at various ages, covering the prehypertensive stage and the developmental phase of hypertension, using a (1)H nuclear magnetic resonance (NMR)-based metabonomic approach. Twenty-four-hour urine samples from the SHR and their age-matched normotensive control, Wistar-Kyoto rats, were analyzed using (1)H NMR spectroscopy, and the spectral data were subjected to principal components analysis (PCA) to find metabolic differences between the two strains. Consequently, it was possible to separate the urine samples between the two strains at any age ranging from 4 to 20 weeks in the principal component scores plots. The major spectral regions and signals (metabolites) contributing to the separation were picked up based on the loadings. Subsequently, the urinary excreted levels of metabolites highlighted by the PCA were compared based on the signal intensities corrected by urine volume and body weight. These investigations revealed the major metabolic changes characteristic of the SHR, which included differences in citrate, α-ketoglutarate, succinate, hippurate, phenylacetylglycine, p-cresol glucuronide, creatine, taurine, medium-chain dicarboxylates (tentative), unknown (δ 3.11), and the regions at 3.60, 3.64, 3.68 and 3.88 p.p.m. The results supported the occurrence of metabolic acidosis in the SHR in the period of prehypertension as well as rapidly rising blood pressure. In addition, the intestinal microfloral populations in the SHR were suggested to be altered in the developmental phase of hypertension.

Total synthesis of antimalarial diterpenoid (+)-kalihinol A.

Total synthesis of antimalarial diterpenoid (+)-kalihinol A, isolated from marine sponge Acanthella sp., is achieved. This total synthesis involves regioselective alkylation of an epoxide, construction of a tetrahydropyran ring by iodo-etherification, construction of a cis-decalin ring by intramolecular Diels-Alder reaction, isomerization of cis-decalin to trans-decalin, and subsequent functionalization of the trans-decalin ring.

LC-NMR identification of a novel taurine-related metabolite observed in (1)H NMR-based metabonomics of genetically hypertensive rats.

This paper describes the LC-NMR spectroscopic identification of a novel urinary endogenous metabolite responsible for the signals, which were found as major contributors to the separation between genetically hypertensive rats (SHRSP) and normotensive control rats (WKY) in previous NMR-based metabonomic studies. Urine samples from 26-week-old normotensive rats were analyzed by an LC-NMR system equipped with a reversed-phase column having high retention ability for polar compounds. (1)H NMR spectra were continuously obtained in the on-flow mode, and the retention times of the unassigned signals in question were determined. Various two-dimensional spectra were subsequently measured for the fraction containing the unassigned signals under the stop-flow mode, which enables for a long accumulation resulting in the enhancement of signal-to-noise ratios. The candidate compound obtained from these LC-NMR data was synthesized, and the NMR and mass spectra were compared with those of the LC-NMR fraction. The unknown metabolite was identified as succinyltaurine from these experiments together with standard addition experiments. This novel metabolite, which is characteristic of the normotensive rats, is very interesting because it is structurally related to hypotensive taurine, and not substantially detected in the genetically hypertensive rats, which excreted more taurine than the normotensive rats. The biological and pathophysiological significances of succinyltaurine remain to be investigated.

Practical synthesis of a key intermediate for lactacystin from (R)-4-hydroxymethyl-2-phenyl-4,5-dihydrooxazol-4-ylmethyl acetate.

A practical synthesis of a key intermediate for the proteasome inhibitor lactacystin from (R)-4-hydroxymethyl-2-phenyl-4,5-dihydrooxazol-4-ylmethyl acetate was established. (R)-4-Hydroxymethyl-2-phenyl-4,5-dihydrooxazol-4-ylmethyl acetate is a useful chiral building block for the synthesis of biologically active compounds containing alpha-substituted alpha-amino acid moieties.

PLA2 inhibitory activity of marine sesterterpenoids cladocorans, their diastereomers and analogues.

Inhibition of secretory phospholipase A(2) (sPLA(2)) by cladocorans A and B and their diastereomers almost equaled that of manoalide.

8,17-Epoxybriarane diterpenoids, briaranolides A-J, from an Okinawan Gorgonian Briareum sp.

Ten new 8,17-epoxybriarane diterpenoids, briaranolides A-J (1-10), were isolated from an Okinawan gorgonian Briareum sp. The structure of the diterpenoids was determined on the basis of spectroscopic analysis, chemical conversions, and X-ray analysis.