Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

Proton transfer in a reaction catalyzed by onion lachrymatory factor synthase.

We produced a single deuterated lachrymatory factor (propanthial S-oxide, m/z = 91) in a model reaction system comprising purified alliinase, lachrymatory factor synthase (LFS), and (E)-(+)-S-(1-propenyl)-L-cysteine sulfoxide ((E)-PRENCSO) in D(2)O. Onion LFS reacted with the degraded products of (E)-PRENCSO by alliinase, but not with those of (Z)-PRENCSO. These findings indicate that onion LFS is an (E)-1-propenylsulfenic acid isomerase.

Identification of amino acid residues essential for onion lachrymatory factor synthase activity.

Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H(+) substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23-169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23-169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket.

Novel bisthiolane polysulfides from lachrymatory factor synthase-suppressed onion and their in vitro cyclooxygenase-1 inhibitory activity.

Two novel bisthiolane polysulfides (compounds 1 and 2), trivially named thiolanotrisulfide and thiolanotetrasulfide, were isolated from a reaction model of tearless onion (in which lachrymatory factor synthase is suppressed), and the presence of another novel bisthiolane polysulfide (3), trivially named thiolanopentasulfide, was confirmed. On the basis of spectroscopic and mass spectrometric analyses, it was found that these bisthiolane polysulfides were bis(5-hydroxy-3,4-dimethylthiolan-2-yl)-tri/tetra/pentasulfide with the general formulas of C12H22O2S5 (tri-), C12H22O2S6 (tetra-) and C12H22O2S7 (penta-), and they were confirmed to exist in authentic tearless onion juice. Thiolanotrisulfide (1) and thiolanotetrasulfide (2) inhibited cyclooxygenase-1 activity with IC50 values of 720 ± 78 and 464 ± 48 µM respectively, compared with 3282 ± 188 µM for aspirin.

Isolation of dihydrocurcuminoids from cell clumps and their distribution in various parts of turmeric (Curcuma longa).

In addition to well-known curcuminoids, three colored metabolites were isolated from cultured cell clumps that had been induced from buds on turmeric rhizomes. The isolated compounds were identified as dihydro derivatives of curcuminoids, dihydrocurcumin (dihydroCurc), dihydrodesmethoxycurcumin-a (dihydroDMC-a), and dihydrobisdesmethoxycurcumin (dihydroBDMC). The cell clumps did not contain dihydroDMC-b, an isomer of dihydroDMC-a. A comparison of the distribution profiles of curcuminoids and dihydrocurcuminoids in the cell clumps with those in the rhizomes, leaves, and roots revealed the following differences: Unlike rhizomes, the cell clumps, leaves, and roots contained dihydrocurcuminoids as the major colored constituents. Whereas dimethoxy compounds, curcumin and dihydrocurcumin, respectively, were most abundant in the rhizomes and leaves, one of the monomethoxy derivatives, dihydroDMC-a, was found most abundantly in the cell clumps and roots. While both dihydroDMC-a and b were detected in the rhizomes, dihydroDMC-b was not detectable in the cell clumps, leaves, or roots. The occurrence of only one of the two possible isomers of dihydroDMC suggests biosynthetic formation of dihydrocurcuminoids in turmeric.

The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors.

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid >> ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.

Identification of two novel pigment precursors and a reddish-purple pigment involved in the blue-green discoloration of onion and garlic.

By using a model reaction system representing blue-green discoloration that occurs when purees of onion (Allium cepa L.) and garlic (Allium sativum L.) are mixed, we isolated two pigment precursors (PPs) and a reddish-purple pigment (PUR-1) and determined their chemical structures. PPs were isolated from a heat-treated solution containing color developer (CD) and either l-valine or l-alanine, and their structures were determined as 2-(3,4-dimethylpyrrolyl)-3-methylbutanoic acid (PP-Val), and 2-(3,4-dimethyl-1H-pyrrolyl) propanoic acid (PP-Ala), respectively. Next, PUR-1 was isolated from a heat-treated solution containing PP-Val and allicin, and its structure was determined as (1E)-1-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)-prop-1-enylene-3-(1-((1S)-1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-ylidenium). The structure of PUR-1 suggested that PP molecules containing a 3,4-dimethyl pyrrole ring had been cross-linked by an allyl group of allicin to form conjugated pigments. While PUR-1 is a dipyrrole compound exhibiting a reddish-purple color, a color shift toward blue to green can be expected as the cross-linking reaction continues to form, for example, tri- or tetrapyrrole compounds.

Model studies on precursor system generating blue pigment in onion and garlic.

Reactions involved in blue-green discoloration in a mixture of onion (Allium cepa L.) and garlic (Allium sativum L.) were investigated. Vivid-blue color was successfully reproduced by using a defined model reaction system comprising only trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (1-PeCSO) from onion, S-allyl-L-cysteine sulfoxide (2-PeCSO) from garlic, purified alliinase (EC 4.4.1.4), and glycine (or some other amino acids). Four reaction steps identified and factors affecting the blue color formation were in good agreement with those suggested by earlier investigators. When crude onion alliinase was used in place of garlic alliinase, less pigment was formed. This result was explained by a difference in the amount of thiosulfinates, colorless intermediates termed color developers, yielded from 1-PeCSO by these enzymes.

A novel PCR method for quantification of buckwheat by using a unique internal standard material.

A novel quantitative and specific method for detection of buckwheat, a known food allergen, in diverse food materials was developed by using a unique internal standard to compensate for the variability in DNA extraction and amplification efficiencies. The method was based on a real-time PCR targeting the internal transcribed spacer region of Fagopyrum spp. and was designed to detect both cultivated and wild buckwheat, because wild buckwheat might be potentially allergenic. As the internal standard material, ground seeds of statice (Limonium sinuatum) were added to food samples prior to DNA extraction, and the amount of statice DNA measured by real-time PCR was used to standardize the buckwheat content. Statice, an ornamental plant, was chosen as the internal standard material because it was readily available and was inferred to be least likely to be commingled in foods. The specificity of the PCR system was tested against commonly used food materials of plant origin. Quantitative results expressed in buckwheat protein concentrations (mean +/- standard deviation) for various food samples prepared to contain 10 ppm (wt/wt) of buckwheat flour (corresponding to 1.2-microg/g [ppm] buckwheat protein) ranged from 0.7 +/- 0.2 (rice) to 0.9 +/- 0.4 (wheat) and for 100-ppm (wt/wt) samples (12-microg/g [ppm] buckwheat protein) from 7.7 +/- 1.0 (pepper) to 9.8 +/- 0.5 (wheat) microg/g (ppm). The method's accuracy, sensitivity, and specificity were considered sufficient for detection of buckwheat contamination at the level required for compliance with the Japanese Food Allergen Labeling Regulation.

[A case report of a patient with advanced rectal cancer who was administered chemotherapy in collaboration with a clinical pathway and visiting nursing care].

Recently, chemotherapy for the treatment of colorectal cancer was widely administered in Japan. Many various ideas were necessary to perform safe and efficient chemotherapy at our hospital. We experienced that even a patient with intellectual disability could have chemotherapy carry out smoothly in collaboration with a clinical pathway and visiting nursing care. The patient was a 63-year-old man who underwent low anterior resection of the rectum for advanced rectal cancer on April 2004. Multiple liver metastases had appeared in November 2004. Chemotherapy was administered because of the liver metastases. However, it was difficult to manage the side effect of the chemotherapy and we could not get cooperation from his family. A visiting nursing care was employed in cooperation with outpatient nursing for managing of the side effect. IFL regimen was carried out using a clinical pathway. The response became PD after 5 courses of an IFL regimen. The regimen was changed to FOLFOX4 on April 2005. No adverse events were seen beyond grade 2 during 10 courses of FOLFOX4 regimen.

Production and characterization of tearless and non-pungent onion.

The onion lachrymatory factor (LF) is produced from trans-S-1-propenyl-L-cysteine sulfoxide (PRENCSO) through successive reactions catalyzed by alliinase (EC 4.4.1.4) and lachrymatory factor synthase (LFS), and is responsible for the tear inducing-property and the pungency of fresh onions. We developed tearless, non-pungent onions non-transgenically by irradiating seeds with neon-ion at 20 Gy. The bulbs obtained from the irradiated seeds and their offspring bulbs produced by selfing were screened by organoleptic assessment of tear-inducing property or HPLC analysis of LF production. After repeated screening and seed production by selfing, two tearless, non-pungent bulbs were identified in the third generation (M3) bulbs. Twenty M4 bulbs obtained from each of them showed no tear-inducing property or pungency when evaluated by 20 sensory panelists. The LF production levels in these bulbs were approximately 7.5-fold lower than those of the normal onion. The low LF production levels were due to reduction in alliinase activity, which was a result of low alliinase mRNA expression (less than 1% of that in the normal onion) and consequent low amounts of the alliinase protein. These tearless, non-pungent onions should be welcomed by all who tear while chopping onions and those who work in facilities where fresh onions are processed.

Changes in trans-S-1-Propenyl-l-cysteine Sulfoxide and Related Sulfur-Containing Amino Acids during Onion Storage.

trans-S-1-Propenyl-l-cysteine sulfoxide (PRENCSO) is the most abundant flavor precursor in onions. The changes in PRENCSO, its biosynthetic precursors, and cycloalliin contents in bulbs stored at 0 °C [i.e., recommended by the Food and Agriculture Organization of the United Nations (FAO)] are investigated. Ion-exchange high-performance liquid chromatography analysis revealed larger amounts of PRENCSO and cycloalliin, which were stoichiometrically balanced by the decrease in the combined amounts of the three biosynthetic precursors. The molar balance of the PRENCSO biosynthesis process during onion storage was explained well by the changes of these five compounds. High-throughput integrated liquid chromatography-tandem mass spectrometry analysis was employed to analyze multiple samples at 5 and 20 °C. The molar concentration data, converted using linear regression equations, showed a similar molar balance for the PRENCSO biosynthesis process comprising these five compounds during storage at all temperatures tested. Furthermore, the content of cycloalliin significantly increased at the expense of that of PRENCSO during storage at 20 °C.

Progressive improvement of prognosis for patients with gastric cancer (dynamic stage grouping) with increasing survival interval from initial staging: how much longer can a given survivor expect to live?

BACKGROUND: The prognosis in gastric cancer is usually defined at the time of presentation. Prognosis, however, is a variable that alters with time. Once a patient has survived for 1 year, the prognosis will change. This study examines this variable in patients who have survived from 1 to 5 years after operation. METHODS: Of 1468 patients with gastric cancer, the next 5-year survival (5YS) of patients who already survived for 1, 2, 3, 4, and 5 years after the initial operation was examined, ie, for patients who have survived 1 year, the 5YS (the 6-year survival from operation) was calculated. Similar survival was determined for patients who had survived more than 2, 3, 4, or 5 years and reported according to stage. RESULTS: In patients who survived 2 years, the next 5YS from that time of patients in stage III a stage was similar to that in stage II, and was similar to the 5YS of patients in stage II at the time of diagnosis. In patients who survived 3, 4, or 5 years, the next 5YS from that time of patients in stage III b and stage III a was similar to that of stage II, and subsequently improved. Similarly, the relationship between stage and survival was disordered subsequently with the passage of time. Similar improvement in survival such that 5YS from the 3rd, 4th, and 5th postoperative year approximated that of the survival of patients at an earlier stage at the time of diagnosis. CONCLUSIONS: Survival is not only stage-dependent at the time of diagnosis but dependent on the length of survival from the initial operation. Progressive improvement in subsequent 5YS appears to approximate survival of lower-stage disease with the passage of time.

Does the surgical stress associated with palliative resection for patients with incurable gastric cancer with distant metastasis shorten their survival?

BACKGROUND/AIMS: In cases of incurable stage IV gastric cancer with distant metastases, surgical treatment has usually consisted merely of palliation. The effect of palliative resection in these highly advanced cases remains controversial. Palliative resection may be prohibited by the potential disadvantages of surgical stress. METHODOLOGY: Over the past 23 years, 382 stage IV incurable gastric cancer patients with distant metastases were classified into a resection group (group R) whose subjects underwent a palliative resection of the primary tumor and the non-resection group (group N) who were treated without resection of primary tumor. In order to exclude patients with very poor prognosis due to irresectability even if trying to resect, we restricted the subjects to patients who survived more than 30 and 60 days and some months and estimated the mean survival. Cumulative survival rates were calculated by using the Kaplan-Meier method, and the mean survivals of groups R and N were compared. RESULTS: A significantly longer mean survival was observed in group R than in group N (381 vs. 181 days, P<0.0001). Restricting the subjects to patients who survived more than 30 and 60 days, there is also a significant difference between the mean survival of group R and that of group N. However, restricting the subjects to patients who survived more than 300 days, no significant difference was seen between the two groups. The rate of hospital death was higher in group N than in group R (15.9% vs. 3.4%) CONCLUSIONS: Palliative resection of the primary tumor in stage IV gastric cancer is meaningful in view of hospital stay, long-term survival, and satisfaction with the treatment. We should resect the primary tumor in cases in which it is resectable.

Does body mass index (BMI) influence morbidity and long-term survival in gastric cancer patients after gastrectomy?

BACKGROUND/AIMS: The long-term survival of patients with gastric cancer is governed by various factors, such as the clinical stage of the cancer, the patient's nutritional state, and the treatment and may be governed by the volume of intraperitoneal adipose tissue. The aim of this study is to clarify the relationship between the degree of the patients' body mass index and their long-term survival. METHODOLOGY: Gastric cancer patients who had undergone a gastrectomy with D2-lymphadenectomy and with resection A and B according to the criteria of the Japanese Research Society for Gastric Cancer Rules were subgrouped into those patients with a body mass index < 0.185 (the lower body mass index group) and those patients with a body mass index > 0.210 (the higher body mass index group). The patient's morbidity and long-term survival rate was retrospectively compared between the 2 groups. RESULTS: A significantly longer mean survival rate was observed for the lower body mass index group in stage 2 (1667 vs. 1322 days, P = 0.0240). Also, a significantly longer mean survival rate was observed for the higher BMI group in stage 3a (1431 vs. 943, P = 0.0071). CONCLUSIONS: The body mass index is one of the prognostic factors of stage 2 and stage 3a gastric cancer. However, it does not appear to be useful for determining the prognosis of stage 1a, 1b, 3b, and 4a gastric cancers.

Structures and reactions of compounds involved in pink discolouration of onion.

In "pinking" of onion, E-(+)-S-(1-propenyl)-L-cysteine sulfoxide is first cleaved by alliinase to yield colour developers (CDs), which react with amino acids, such as valine, to form pigment precursors (PPs). The PPs react with naturally occurring carbonyls (NOCs) to form pigments. By inducing a PP from previously isolated cepathiolanes and L-valine, it was confirmed that cepathiolanes constitute at least a part of the CDs. From the PP and formaldehyde as a NOC, two colourless and two pink compounds were derived. The structure of one of the colourless compounds was established as 2-(2-(1-(1-carboxy-2-methylpropyl)-3,4-dimethyl-1H-pyrrol-2-yl)methyl-3,4-dimethyl-1H-pyrrol-1-yl)-3-methylbutanoic acid. The structures of the other colourless compound and the pink pigments were predicted based on their molecular formula and the MS(n) spectral data. A trimeric pigment structure was predicted for one of the pink pigments, which was believed to be the first to be reported in the literature. With these, a new reaction scheme for "pinking" of onion is proposed.

Structure and bioactivity of thiosulfinates resulting from suppression of lachrymatory factor synthase in onion.

In normal onion (Allium cepa), trans-S-1-propenyl-L-cysteine sulfoxide is transformed via 1-propenesulfenic acid into propanethial S-oxide, a lachrymatory factor, through successive reactions catalyzed by alliinase and lachrymatory factor synthase (LFS). A recent report showed that suppression of the LFS activity caused a dramatic increase in thiosulfinates previously reported as "zwiebelane isomers". After purification by recycle high-performance liquid chromatography and subsequent analyses, we established the planar structure of the putative "zwiebelane isomers" as S-3,4-dimethyl-5-hydroxythiolane-2-yl 1-propenethiosulfinate, in which two of the three molecules of 1-propenesulfenic acid involved in the formation gave the thiolane backbone, and the third molecule gave the thiosulfinate structure. Of at least three stereoisomers observed, one in the (2'R,3'R,4'R,5'R)-configuration was collected as an isolated fraction, and the other isomers were collected as a combined fraction because spontaneous tautomerization prevented further purification. Both fractions showed inhibitory activities against cyclooxygenase-1 and α-glucosidase in vitro.

Using synthesized onion lachrymatory factor to measure age-related decreases in reflex-tear secretion and ocular-surface sensation.

PURPOSE: To use synthesized onion lachrymatory factor (SOLF) to investigate age-related changes in reflex-tear secretion and ocular-surface sensation. METHODS: We separated 91 healthy volunteers into four groups: groups A, age 20-29 years; B, 30-39; C, 40-49; and D, older than 50 years. We exposed one eye of each subject to SOLF and measured the elapsed time until the subject's limit of irritation tolerance (TLI) was reached and an increase in the tear meniscus radius (DeltaR). After the SOLF stimulus, corneal sensitivity was examined by Cochet-Bonnet esthesiometry (CB), and reflex-tear secretion was examined by the Schirmer I-test (ST). RESULTS: TLI was significantly shorter in group A than in the other groups (P < 0.0001), and the groups B and D also differed significantly from each other (P = 0.0013). The increase in DeltaR was significantly greater in group A than in group C (P = 0.0306) or D (P < 0.0001), and groups B (P = 0.0002) and C (P = 0.0308) also differed significantly from group D. There were no significant intergroup differences in the CB and ST results. CONCLUSIONS: An age-related decrease in reflex-tear secretion and ocular-surface sensation was revealed by the SOLF test but could not be detected by either CB or the ST.

Silencing onion lachrymatory factor synthase causes a significant change in the sulfur secondary metabolite profile.

Through a single genetic transformation in onion (Allium cepa), a crop recalcitrant to genetic transformation, we suppressed the lachrymatory factor synthase gene using RNA interference silencing in six plants. This reduced lachrymatory synthase activity by up to 1,544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatory factor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-l-cysteine sulfoxide breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of this raised thiosulfinate level was a marked increase in the downstream production of a nonenzymatically produced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported in trace amounts or had not been detected in onion. The consequences of this dramatic simultaneous down- and up-regulation of secondary sulfur products on the health and flavor attributes of the onion are discussed.

PCR method for detecting trace amounts of buckwheat (Fagopyrum spp.) in food.

Buckwheat often causes severe allergic reactions, even when its ingestion level is extremely low. Therefore, buckwheat is listed in several countries as a common food allergen. In addition to common buckwheat and Tartarian buckwheat that are cultivated and consumed widely, wild buckwheat may be potentially allergenic. Food containing undeclared buckwheat poses a risk to patients with the buckwheat allergy. We describe in this report a PCR method to detect buckwheat DNA by using primers corresponding to the internal transcribed spacer region and the 5.8S rRNA gene. The method is buckwheat-specific and compatible with both cultivated and wild buckwheat of the Fagopyrum spp. Its sensitivity was sufficient to detect 1 ppm (w/w) of buckwheat DNA spiked in wheat DNA. This method should benefit food manufacturers, clinical doctors, and allergic patients by providing information on the presence of buckwheat contamination in food.