Variation in 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Coding Sequences and Glyphosate Response among <i>Cyperus rotundus</i>L. Populations

The gene sequence encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzymatic target site of the herbicide glyphosate, was determined for several purple nutsedge (Cyperus rotundus L.) accessions from geographically distant locations and these were aligned to generate a consensus sequence. The EPSPS sequences each had single nucleotide polymorphisms (SNPs) only a few of which were predicted to cause an amino acid change in the EPSP synthase. None had the proline to serine substitution or other substitutions responsible for glyphosate resistance reported in other species. A dendrogram generated from the cluster analysis of the EPSPS gene sequences indicated similarities between accessions from Tanzania, Indonesia, California-2, Greece, Brazil, Argentina and Iran much like cluster analysis previously reported based on RAPD scores and morphological traits possibly indicating a common genetic background or origin. Considering the differences in EPSPS sequences, the response of these purple nutsedge accessions to 0.84 kg·ae·ha-1 of glyphosate was assessed to determine whether differential tolerance was present. At 7 days after the first application control ranged from 9% for the accession from Greece to 73% for the accession from Tanzania. Control of these accessions increased to 45% and 93% respectively by 14 days after the second application. The I50’s for glyphosate inhibition of growth for four accessions from geographically distant countries (Mississippi, Brazil, Indonesia and Tanzania) were 0.21, 0.10, 0.25 and 0.06 kg·ha-1, respectively, which represented a 4-fold difference. The difference in sensitivity to glyphosate may be a result of a non-target site mechanism such as differences in sequestration, translocation or cuticle thickness rather than alterations in EPSPS.

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.

A gene encoding AtPIP5K2 may be involved in regulating the sensitivity to osmotic stress

One mutant line eto with salt tolerance was screened from a T-DNA insertion mutant collection of Arabidopsis thaliana. In addition to a reduced rate of seed germination, NaCl and ABA also inhibited the growth and the greening of cotyledons of wild-type seedlings, but not the eto mutant. TAIL-PCR analysis showed that T-DNA tag insertion in the eto was located at nucleotide 27,502 in BAC F3M18, upstream （at position -487 relative to the translation initiation codon） of gene At lg77740 （encoding a putative phosphatidylinositol-4-phosphate 5-kinase, AtPIP5K2）. This inserted mutation cosegregated closely with the eto phenotype, Another analysis not only indicated that AtPIP5K2 transcript is expressed predominantly in roots and rosette leaves, but also showed the T-DNA insertion resulted higher accumulation of the AtPIP5K2 in eto mutant plants and did not influenced the expression of the upstream At lg77730 gene. This change may play an essential role in the tolerance of eto mutant plant to the osmotic stress.

生物正交试剂磷酸吡哆醛(PLP)及其类似物的研究进展

近十年来,生物正交试剂作为一种实用工具被广泛用于生物分子的修饰。科研人员也在不断优化现有的生物正交分子结构以获得快速的反应速率、稳定的偶联产物、来提高应用的实用性和广泛性。基于亚胺连接底物醛的独特代表磷酸吡哆醛(PLP)可以通过生物模拟转氨反应间接或利用PLP分子结构中活性正交基团直接参与生物分子的修饰。本文即从生物分子修饰的应用方面介绍生物正交试剂磷酸吡哆醛(PLP)及相关类生物的研究情况。

Fingolimod protects against neurovascular unit injury in a rat model of focal cerebral ischemia/reperfusion injury

Recent research on the underlying mechanisms of cerebral ischemia indicates that the neurovascular unit can be used as a novel subject for general surveys of neuronal damage and protein mechanisms.Fingolimod(FTY-720)is a newly developed immunosuppressant isolated from Cordyceps sinensis that exhibits a wide range of biological activities,and has recently attracted much attention for the treatment of ischemic cerebrovascular diseases.In the current research,the role of FTY-720 and its possible mechanisms were assessed from an neurovascular unit perspective using a rat cerebral ischemia model.Our results revealed that FTY-720 markedly decreased infarct volume,promoted neurological function recovery,and weakened the blood-brain barrier permeability of ischemic rats.The protective roles of FTY-720 in ischemic stroke are ascribed to a combination of sphingosin-1-phosphate receptor-1 and reduced expression of sphingosin-1-phosphate receptor-1 in microvessels and reduction of interleukin-17A protein levels.These findings indicate that FTY-720 has promise as a new therapy for neurovascular protection and functional recovery after ischemic stroke.

The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes

Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.

Arginine methylation of ribose-5-phosphate isomerase A senses glucose to promote human colorectal cancer cell survival

Cancer cells remodel their metabolic network to adapt to variable nutrient availability. Pentose phosphate pathway(PPP) plays protective and biosynthetic roles by oxidizing glucose to generate reducing power and ribose. How cancer cells modulate PPP activity in response to glucose supply remains unclear. Here we show that ribose-5-phosphate isomerase A(RPIA), an enzyme in PPP, directly interacts with co-activator associated arginine methyltransferase 1(CARM1) and is methylated at arginine 42(R42). R42 methylation up-regulates the catalytic activity of RPIA. Furthermore, glucose deprivation strengthens the binding of CARM1 with RPIA to induce R42 hypermethylation. Insufficient glucose supply links to RPIA hypermethylation at R42, which increases oxidative PPP flux. RPIA methylation supports ROS clearance by enhancing NADPH production and fuels nucleic acid synthesis by increasing ribose supply. Importantly, RPIA methylation at R42 significantly potentiates colorectal cancer cell survival under glucose starvation. Collectively, RPIA methylation connects glucose availability to nucleotide synthesis and redox homeostasis.

Homology modeling and docking studies of IscS from extremophile Acidithiobacillus ferrooxidans

The gene iscS-3 from ,4cidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional （3D） molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5＇-phosphate（PLP） and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS（P-I） and cysteine-PLP-IscS（C-P-I） complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glul05, Glu79, Ser203 and Hisl80 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.

磷酸吡哆醛对H22肝癌细胞DNA拓扑异构酶Ⅰ活性的影响

为建立DNA拓扑异构酶活性测定方法和探讨磷酸吡哆醛(PLP)抑制肿瘤细胞增殖的可能机制,本实验以DNA琼脂糖凝胶电泳EB荧光法测定了H22肝癌细胞DNA拓扑异构酶I(TOPOI)的活性。结果表明维生素B6的活性形式PLP能有效地抑制TOPOI松弛负超螺旋的作用,且抑制效应与PLP剂量呈正相关。从而证实PLP能明显抑制TOPOI的活性并在分子水平上调节细胞周期活动和肿瘤细胞增殖活动。

氧化亚铁钩端螺旋菌的固氮供硫蛋白与底物的相互作用

氧化亚铁钩端螺旋菌的nifS基因可能在它的固氮酶成熟中发挥重要的作用.为了考察由这个基因编码的蛋白质,建立了一个关于它的可靠的完整三维分子结构.建立的结构被进一步进行绑定位点搜索,跟辅因子磷酸吡哆醛(PLP)和底物半胱氨酸进行柔性分子对接,并据此识别其关键残基.在跟半胱氨酸对接中,半胱氨酸中的氨基非常靠近His102,Lys204和PLP,半胱氨酸跟它们的相互作用能也很大,这些情况跟那些来自其他生物的固氮供硫蛋白(NifS)类似物的实验事实一致.根据这些结果,这个基因编码NifS;底物半胱氨酸能被有效地募集到激活位点.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

吡哆醛激酶研究进展

吡哆醛激酶(PLK)属于核糖激酶超家族成员,广泛存在于生物有机体中,它是维生素B6的关键代谢酶,PLK能够催化维生素B6的磷酸化,使之转化为有活性的吡哆醛磷酸(PLP),而PLP在氨基酸合成分解代谢中发挥着重要的作用。本文从PLK结构、分类、生物学功能及其与某些疾病的联系等方面,进行详细的综述,为了解这一激酶、开展相关研究提供便利。

Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa

It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.

Vitamin B6 and colorectal cancer:Current evidence and future directions

Colorectal cancer remains the third most common cancer in both women and men worldwide.Identifying modifiable dietary factors is crucial in developing primary prevention strategies.Vitamin B6 is involved in more than 100 coenzyme reactions,and may influence colorectal cancer risk in multiple ways including through its role in one-carbon metabolism related DNA synthesis and methylation and by reducing inflammation,cell proliferation,and oxidative stress.Observational studies of dietary or dietary plus supplementary intake of vitamin B6 and colorectal cancer risk have been inconsistent with most studies reporting nonsignificant positive or inverse associations.However,published studies of plasma pyridoxal 5'-phosphate(the active form of vitamin B6) levels consistently support an approximately 30%-50% reduction in risk of colorectal cancer comparing high with low concentrations.The reasons for the discrepancy in the results between dietary-based and plasma-based studies remain unresolved.Other unresolved questions include the effects of vitamin B6 intake in early life(i.e.,childhood or adolescence) and of suboptimal vitamin B6 status on colorectal cancer risk,whether the associations with vitamin B6 differ across molecular subtypes of colorectal cancer,and whether the vitamin B6-colorectal cancer association is modified by genetic variants of one-carbon metabolism.

p38 mitogen-activated protein kinase regulates type-Ⅰ vs type-Ⅱ phenotyping of human vascular endothelial cells

AIM: To identify kinases involved in phenotype regulation of vascular endothelial cells(VECs): Proproliferative G-protein signaling 5(RGS5)^(high)(typeⅠ) vs anti-proliferative RGS5^(low)(typeⅡ) VECs.METHODS: Proteomic kinase assays were performed to identify the crucial kinase involved in the phenotype regulation of human VECs using typeⅠ VECs, which promotes the proliferation of human vascular smooth muscle cells(VSMCs), and typeⅡ VECs, which suppress the proliferation of human VSMCs. The assays were performed using multiple pairs of typeⅠ and typeⅡ VECs to obtain the least number of candidates. The involvement of the candidate kinases was verified by evaluating the effects of their specific inhibitors on the phenotype regulation of human VECs as well as the expression levels of regulator of RGS5, which is the causative gene for the "typeⅡ to typeⅠ" phenotype conversion of human VECs. RESULTS: p38α mitogen-activated protein kinase(p38α MAPK) was the only kinase that showed distinctive activities between typeⅠ and typeⅡ VECs: p38α MAPK activities were low and high in type-Ⅰand typeⅡ VECs, respectively. We found that an enforced expression of RGS5 indeed lowered p38α MAPK activitiesin typeⅡ VECs. Furthermore, treatments with a p38α MAPK inhibitor nullified the anti-proliferative potential in typeⅡ VECs. Interestingly, MAPK inhibitor treatments enhanced the induction of RGS5 gene. Thus, there is a vicious cycle between "RGS5 induction" and "p38α MAPK inhibition", which can explain the unidirectional process in the stress-induced "typeⅡ to typeⅠ" conversions of human VECs. To understand the upstream signaling of RGS5, which is known as an inhibitory molecule against the G protein-coupled receptor(GPCR)-mediated signaling, we examined the effects of RGS5 overexpression on the signaling events from sphingosine-1-phosphate(S1P) to N-cadherin, because S1 P receptors belong to the GPCR family gene and N-cadherin, one of their downstream effectors, is reportedly involved in the regulation of VEC-VSMC interactions. We found that RGS5 specifically bound with S1P1. Moreover, N-cadherin localization at intercellular junctions in typeⅡ VECs was abolished by "RGS5 overexpression" and "p38α MAPK inhibition".CONCLUSION: p38α MAPK plays crucial roles in "type-Ⅰ vs type-Ⅱ" phenotype regulations of human VECs at the downstream of RGS5.

Varying Tolerance to Glyphosate in a Population of Palmer Amaranth with Low EPSPS Gene Copy Number

A Palmer amaranth population (seeds collected in the year 2000;Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha?1) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD50 of 0.75 kg·ha?1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.

Full structure building and docking of NifS from extremophile Acidithiobacillus ferrooxidans

The gene iscS-2 from extremophile Acidithiobacillus ferrooxidans may play a crucial role in nitrogenase maturation. To investigate the protein encoded by this gene, a reliable integral three-dimensional molecular structure was built. The obtained structure was further used to search binding sites, carry out the flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine, and identify its key residues. The docking results of PLP reveal that the residues of Lys203, His100, Thr73, Ser200, His202, Asp177 and Gln180 have large interaction energies and/or hydrogen bonds fixation with PLP. The docking results of cysteine show that the amino group in cysteine is very near His100, Lys203 and PLP, and the interaction energies for cysteine with them are very big. These identified residues are in line with the experimental facts of NifS from other sources. Moreover, the four residues of Asn152, Val179, Ala102 and Met148 in the PLP docking and the two residues of Lys208 and Ala102 in the cysteine docking also have large interaction energies, which are fitly conserved in NifS from all kinds of sources but have not been identified before. According to these results, this gene encodes NifS protein, and the substrate cysteine can be effectively recruited into the active site. Furthermore, all of the above detected key residues are directly responsible for the binding and/or catalysis of PLP and cysteine.

Vitamin B6 and Lipid Contents in Engraulisjaponica Specifically Caught for Production of Japanese Soup Stock

The purpose of this study was to elucidate the relationship between percentage of body lipid and individual vitamin B6 contents in the anchovy used for production of Japanese soup stock. The contents of individual vitamin B6 compound in foods should be determined to estimate the functionality of foods. Anchovies （Engraulisjaponica） for use as production of iriko, which is a raw material for preparation of Japanese soup stock, were caught using a medium-sized purse seine on May 26th, June 1st, and July 6th 2011 in Tachibana Bay, Nagasaki, Japan. Vitamin B6 contents were analyzed by 4-pyridoxolacone-coversion （all-enzymatic） HPLC. The relationship between body length, lipid content and the individual contents of vitamin B6 compounds in the anchovies was determined. The propriety of anchovies for iriko preparation could not be estimated based on the appearance of length of fishes. The anchovies were rich in vitamin B6, especially pyridoxamine 5＇-phosphate, which may prevent diabetic complications. The percentage of body lipid significantly showed a negative correlation with the content of pyridoxamine 5＇-phosphate. The contents of the other vitamin B6 compounds did not show the significant correlation with the percentage of body lipid.

Stabilization of Alliinase from Garlic by Osmolytes and the Mannose-Specific Lectin ASAI

Objectives： Alliinase is a pyridoxal-5＇-phosphate （PLP）-dependent enzyme responsible for the production of diallyl thiosulfinate （allicin）, the biologically active component of garlic, from alliin. The use of allicin for treatment of various diseases has been proposed but it is very unstable in the blood stream. This difficulty can be overcome by administration of alliin, together with a conjugate of alliinase directed towards the target cells. This, in turn requires a stable and active form of the enzyme. In this study we evaluate the stability of alliinase itself, in the presence mad absence of osmolytes, as well as that of its catalytically active complex with a mannose-specific lectin, ASAI （Allium sativum agglutinin I）, also presents in garlic. Methods： Alliinase, mad ASAI were both purified from garlic cloves. Thermal stability of alliinase itself, mad of its complexes with PLP and ASAI, in the presence mad absence of osmolytes, was analyzed by monitoring enzymic activity, and using DSC （differential scanning calorimetry）. Key findings： PLP exerts only a minor influence on alliinase structure and stablity. But both osmolytes and ASAI stabilize the enzyme considerably. Conclusions： The principle finding is that ASAI greatly stabilizes alliinase. Thus, the lectin-enzyme complex, which can be lyophilized and stored until used, provides an effective formulation of alliinase for generation of allicin from alliin in vivo.