Effects of direct-fed microbial supplement on ruminal and plasma metabolome of early-lactation dairy cows: Untargeted metabolomics approach.

We examined the effects of 2 multispecies direct-fed microbial (DFM) supplements on ruminal and plasma metabolome of early-lactation dairy cows using a high-coverage untargeted metabolomics approach. A total of 45 multiparous Holstein cows (41 ± 7 DIM) were enrolled for the 14-d pre-experimental and 91-d experimental period and were a subset from a lactation performance study, which used 114 cows. Cows were blocked using pre-experimental energy-corrected milk yield and randomly assigned within each block to 1 of 3 treatments: (1) corn silage-based diet with no DFM supplement (control; CON), (2) basal diet top-dressed with a mixture of Lactobacillus animalis and Propionibacterium freudenreichii at 3 × 109 cfu/d (PRO-A), or (3) basal diet top-dressed with a mixture of L. animalis, P. freudenreichii, Bacillus subtilis, and Bacillus licheniformis at 11.8 × 109 cfu/d (PRO-B). The basal diet was fed ad libitum daily as a TMR at 0600 and 1200 h for a duration of 91 d. Rumen fluid and blood samples were taken on d -3, 28, 49, 70, and 91 and immediately stored at -80°C. Before analysis, ruminal and plasma samples from d 28, 49, 70, and 91 were composited. An in-depth, untargeted metabolome profile of the composite rumen and plasma samples and the d -3 samples was developed by using a chemical isotope labeling/liquid chromatography-mass spectrometry (LC-MS)-based technique. Differentially abundant metabolites (taking into account fold change [FC] values and false discovery rates [FDR]) were identified with a volcano plot. In the rumen, compared with the CON diet, supplemental PRO-A increased (FC ≥1.2; FDR ≤0.05) the relative concentrations of 9 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, quinolinic acid, and shikimic acid, and PRO-B increased relative concentrations of 16 metabolites, including 2-hydroxy-2,4-pentadienoic acid, glutaric acid, 16-hydroxypalmitic acid, and 2 propionate precursors (succinic and methylsuccinic acids). Relative to PRO-A, supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative rumen concentrations of 3 metabolites, 16-hydroxypalmitic acid, indole-3-carboxylic acid, and 5-aminopentanoic acid, but reduced relative rumen concentrations of 13 metabolites, including carnitine, threonic acid, and shikimic acid. Compared with the CON diet, relative concentrations of 13 plasma metabolites, including myxochelin A and glyceraldehyde, were increased (FC ≥1.2; FDR ≤0.05) by PRO-A supplementation, whereas those of 9 plasma metabolites, including 4-(2-aminophenyl)-2,4-dioxobutanoic acid, N-acetylornithine, and S-norlaudanosolin, were reduced (FC ≤0.83; FDR ≤0.05). Supplemental PRO-B increased (FC ≥1.2; FDR ≤0.05) relative concentrations of 9 plasma metabolites, including trans-o-hydroxybenzylidenepyruvic acid and 3-methylsalicylaldehyde, and reduced relative concentrations of 4 plasma metabolites, including ß-ethynylserine and kynurenine. Pathway analysis of the differentially abundant metabolites in both rumen and plasma revealed that these metabolites are involved in AA and fatty acid metabolism and have antimicrobial and immune-stimulating properties. The results of this study demonstrated that dietary supplementation with either PRO-A or PRO-B altered the plasma and ruminal metabolome. Notably, ruminal and plasma metabolites involved in the metabolism of AA and fatty acids and those with immunomodulatory properties were altered by either or both of the 2 microbial additives.

Synergism with Shikimic Acid Restores ß-Lactam Antibiotic Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved into a dangerous pathogen resistant to beta-lactam antibiotics (BLAs) and has become a worrisome superbug. In this study, a strategy in which shikimic acid (SA), which has anti-inflammatory and antibacterial activity, is combined with BLAs to restart BLA activity was proposed for MRSA treatment. The synergistic effects of oxacillin combined with SA against oxacillin resistance in vitro and in vivo were investigated. The excellent synergistic effect of the oxacillin and SA combination was confirmed by performing the checkerboard assay, time-killing assay, live/dead bacterial cell viability assay, and assessing protein leakage. SEM showed that the cells in the control group had a regular, smooth, and intact surface. In contrast, oxacillin and SA or the combination treatment group exhibited different degrees of surface collapse. q-PCR indicated that the combination treatment group significantly inhibited the expression of the mecA gene. In vivo, we showed that the combination treatment increased the survival rate and decreased the bacterial load in mice. These results suggest that the combination of oxacillin with SA is considered an effective treatment option for MRSA, and the combination of SA with oxacillin in the treatment of MRSA is a novel strategy.

Exploiting the synergistic antibacterial activity of shikimic acid and ceftiofur against methicillin-resistant Staphylococcus aureus.

Efforts to curtail the escalating health threat posed by methicillin-resistant Staphylococcus aureus (MRSA), a formidable superbug, necessitate the development of innovative treatment strategies. Leveraging potential compounds from natural sources in tandem with antibiotics has emerged as a promising approach against MRSA. These strategies should enhance the antibiotic efficacy, reduce dosage and toxicity, and bypass MRSA resistance. In this study, we used a checkerboard assay to illustrate the significant synergistic anti-MRSA effect of shikimic acid (SA), a naturally occurring compound, and ceftiofur (CF). Time-kill curves further revealed that a combination of 1/4 of the minimum inhibitory concentration (MIC) of SA and 1/8 MIC of the sodium CF eradicated MRSA within 2 h, with no noticeable toxicity observed with these concentrations. In vivo experiments confirmed that this combination therapy demonstrated robust antimicrobial activity against MRSA-induced bacteremia in mice, significantly reducing bacterial loads in the kidneys, liver, and spleen, attenuating inflammatory cell infiltration, and alleviating pathological damage. This study not only offers a compelling strategy, capitalizing on the synergistic potential of SA and CF, to rapidly address antibiotic resistance but also contributes significantly to the refinement of antimicrobial therapeutic strategies.

Natural Xanthine Oxidase Inhibitor 5-O-Caffeoylshikimic Acid Ameliorates Kidney Injury Caused by Hyperuricemia in Mice.

Xanthine oxidase (XOD) inhibition has long been considered an effective anti-hyperuricemia strategy. To identify effective natural XOD inhibitors with little side effects, we performed a XOD inhibitory assay-coupled isolation of compounds from Smilacis Glabrae Rhizoma (SGR), a traditional Chinese medicine frequently prescribed as anti-hyperuricemia agent for centuries. Through the in vitro XOD inhibitory assay, we obtained a novel XOD inhibitor, 5-O-caffeoylshikimic acid (#1, 5OCSA) with IC50 of 13.96 µM, as well as two known XOD inhibitors, quercetin (#3) and astilbin (#6). Meanwhile, we performed in silico molecular docking and found 5OCSA could interact with the active sites of XOD (PDB ID: 3NVY) with a binding energy of -8.6 kcal/mol, suggesting 5OCSA inhibits XOD by binding with its active site. To evaluate the in vivo effects on XOD, we generated a hyperuricemia mice model by intraperitoneal injection of potassium oxonate (300 mg/kg) and oral gavage of hypoxanthine (500 mg/kg) for 7 days. 5OCSA could inhibit both hepatic and serum XOD in vivo, together with an improvement of histological and multiple serological parameters in kidney injury and HUA. Collectively, our results suggested that 5OCSA may be developed into a safe and effective XOD inhibitor based on in vitro, in silico and in vivo evidence.

The regulatory role of shikimate in plant phenylalanine metabolism.

In higher plants, the amino acid phenylalanine is a substrate of both primary and secondary metabolic pathways. The primary pathway that consumes phenylalanine, protein biosynthesis, is essential for the viability of all cells. Meanwhile, the secondary pathways are not necessary for the survival of individual cells, but benefit of the plant as a whole. Here we focus on the monolignol pathway, a secondary metabolic pathway in the cytosol that rapidly consumes phenylalanine to produce the precursors of lignin during wood formation. In planta monolignol biosynthesis involves a series of seemingly redundant steps wherein shikimate, a precursor of phenylalanine synthesized in the plastid, is transiently ligated to the main substrate of the pathway. However, shikimate is not catalytically involved in the reactions of the monolignol pathway, and is only needed for pathway enzymes to recognize their main substrates. After some steps the shikimate moiety is removed unaltered, and the main substrate continues along the pathway. It has been suggested that this portion of the monolignol pathway fulfills a regulatory role in the following way. Low phenylalanine concentrations (viz. availability) correlate with low shikimate concentrations. When shikimate concentratios are low, flux into the monolignol pathway will be limited by means of the steps requiring shikimate. Thus, when the concentration of phenylalanine is low it will be reserved for protein biosynthesis. Here we employ a theoretical approach to test this hypothesis. Simplified versions of plant phenylalanine metabolism are modelled as systems of ordinary differential equations. Our analysis shows that the seemingly redundant steps can be sufficient for the prioritization of protein biosynthesis over the monolignol pathway when the availability of phenylalanine is low, depending on system parameters. Thus, the phenylalanine precursor shikimate may signal low phenylalanine availability to secondary pathways. Because our models have been abstracted from plant phenylalanine metabolism, this mechanism of metabolic signalling, which we call the Precursor Shutoff Valve (PSV), may also be present in other biochemical networks comprised of two pathways that share a common substrate.

Hypolipogenic Effect of Shikimic Acid Via Inhibition of MID1IP1 and Phosphorylation of AMPK/ACC.

Although shikimic acid from Illicium verum has antioxidant, antibacterial, anti-inflammatory, and analgesic effects, the effect of shikimic acid on lipogenesis has not yet been explored. Thus, in the present study, hypolipogenic mechanism of shikimic acid was examined in HepG2, Huh7 and 3T3-L1 adipocyte cells. Shikimic acid showed weak cytotoxicity in HepG2, Huh7 and 3T3-L1 cells, but suppressed lipid accumulation in HepG2, Huh7 and 3T3-L1 cells by Oil Red O staining. Also, shikimic acid attenuated the mRNA expression of de novo lipogenesis related genes such as FAS, SREBP-1c, and LXR-α in HepG2 cells by RT-PCR analysis and suppressed the protein expression of SREBP-1c and LXR-α in HepG2 and 3T3-L1 cells. It should be noted that shikimic acid activated phosphorylation of AMP-activated protein kinase (AMPK)/Aacetyl-coenzyme A carboxylase (ACC) and reduced the expression of MID1 Interacting Protein 1 (MID1IP1) in HepG2, Huh7 and 3T3-L1 cells. Conversely, depletion of MID1IP1 activated phosphorylation of AMPK, while overexpression of MID1IP1 suppressed phosphorylation of AMPK in HepG2 cells. However, AMPK inhibitor compound c did not affect the expression of MID1IP1, indicating MID1IP1 as an upstream of AMPK. Taken together, our findings suggest that shikimic acid has hypolipogenic effect in HepG2 and 3T3-L1 cells via phosphorylation of AMPK/ACC and inhibition of MID1IP1 as a potent candidate for prevention or treatment of fatty liver and hyperlipidemia.

Shikimic Acid Inhibits Osteoclastogenesis in Vivo and in Vitro by Blocking RANK/TRAF6 Association and Suppressing NF-κB and MAPK Signaling Pathways.

BACKGROUND/AIMS: Bone homeostasis is associated with the balance between bone-resorbing osteoclasts and bone-forming osteoblasts. Unbalanced bone homeostasis as a result of reduced osteogenesis or excessive osteoclastogenesis can lead to disorders such as osteoporosis, Paget's disease, and rheumatoid arthritis. Shikimic acid is a cyclohexanecarboxylic acid, reported to exhibit pharmacological properties including anti-inflammatory and antioxidant activities. However, its effects on bone homeostasis remain unknown. METHODS: First, the in vitro MTT cell viability assay was performed. Tartrate-resistant acid phosphatase (TRAP) and actin ring formation assays, as well as immunofluorescence staining were then performed to evaluate osteoclastogenesis. Potential signaling pathways were characterized by western blotting and verified in overexpression experiments. Related factors were examined by western blotting, reverse transcription polymerase chain reaction, electrophoretic mobility shift assay, and co-immunoprecipitation. Ovariectomized mice were used for the in vivo study. RESULTS: TRAP staining showed that shikimic acid significantly inhibited osteoclastogenesis and pit resorption in bone marrow monocytes and RAW264.7 cells, and actin ring formation assays showed that shikimic acid suppressed the bone resorption function of osteoclasts. Furthermore, shikimic acid inhibited the receptor activator of nuclear factor-κB RANK/tumor necrosis factor receptor-associated factor 6 (TRAF6) association, suppressed nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and downregulated nuclear factor of activated T-cell cytoplasmic 1. The expression of osteoclastogenesis biomarkers, including TRAF6, calcitonin receptor, TRAP, cathepsin K, and matrix metalloproteinase-9, was inhibited. In vivo, shikimic acid also significantly ameliorated bone loss and prevented osteoclastogenesis in ovariectomized mice. CONCLUSION: Shikimic acid inhibited osteoclastogenesis and osteoclast function by blocking RANK ligand-induced recruitment of TRAF6, as well as downstream signaling pathways in vitro. Shikimic acid also reduced ovariectomy-induced osteoclastogenesis and bone loss in vivo.

Synthesis, biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli.

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC50 588 and 589 µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.

Preformulation study and initial determination of biological Properties of isopropylidene shikimic acid.

Isopropylidene shikimic acid (ISA), a new drug derviatived from Shikimic Acid, had been proved to be effective in the cerebral protection after cerebral ischemia and reperfusion. But there was little research on the physical pharmacy and biopharmaceutical properties about the drug. In order to provide some useful data for the pharmaceutical development of ISA, the solubility, stability and Oil/Water partition coefficient (LogP) were determined by the classic preformulation study method, and the transmembrane performance of ISA was studied by Franz -diffusion cell method in vitro. The results showed that ISA was water-soluble with a solubility 32.52mg/ml, which could be improved to 44.32 mg/ml by 1% (w/v) sodium dodecylsulfate; the LogP was -0.63; ISA was less stable in water but it was stable when pH greater than 6.0 and unstable when pH less than 6.0; the accumulated permeation rates at 1h were about 50% and more than 80% at 6h. Data obtained by the study indicated that the medium selection and pH control were important for liquid preparation of ISA, and avoiding dissolution and absorption in stomach was critical for the oral solid dosage forms. Mucosal drug delivery systems would be considered, according to the certain hydrophilic-lipophilic characters and good transmembrane capability.

[Lipid-lowering effect of seven traditional Chinese medicine monomers in zebrafish system].

The present study aimed to study lipid-lowering effect of seven traditional Chinese medicine monomers in zebrafish system. Zebrafish were fed with high fat diet to establish a hyperlipemia model, then fasted and bathed with seven traditional Chinese medicine monomers stigmasterol, triacontanol, chrysophanol, vanillic acid, shikimic acid, polydatin and oleanolic acid respectively. The oil red O staining was used to detect the blood lipids of zebrafish. Serum total cholesterol and triglyceride levels were detected to validate the lipid-lowering effect. The result showed that a zebrafish model of hyperlipemia could be established by feeding larvae zebrafish with high fat diet. Among the seven traditional Chinese medicine monomers, chrysophanol had lipid-lowering effect. Chrysophanol significantly reduced serum total cholesterol and triglyceride levels in adult zebrafish fed with high fat diet. Chrysophanol accelerated peristalsis frequency of zebrafish intestine and the excretion of high fat food. It is concluded that chrysophanol has lipid- lowering effect in zebrafish, and the mechanism of the effect may be due to the roles of chrysophanol in reducing lipid absorption from gastrointestinal tract and accelerating the excretion of food.

Insights into the metabolic mechanism of rapamycin overproduction in the shikimate-resistant Streptomyces hygroscopicus strain UV-II using comparative metabolomics.

Rapamycin is a polyketide with a 31-membered macrolide ring that possesses powerful immunosuppressant activity. In this study, we firstly obtained a mutant, shikimate-resistant Streptomyces hygroscopicus strain UV-II, which displayed about 3.20-fold higher rapamycin production (305.9 mg/L) than the wild-type S. hygroscopicus ATCC29253 (95.5 mg/L). Under optimal conditions, with the addition of 2 g/L shikimic acid, the strain's rapamycin production was further increased by approximately 34.9%, to 412.6 mg/L. To gain deeper insights into the effects of shikimic acid resistance and supplementation, the fermentation properties, metabolite concentrations, and transcriptional levels of relevant genes were analyzed and evaluated for differences between this improved mutant and its parental strain. The results showed that most of the metabolic modules involved in rapamycin biosynthesis were upregulated in the mutant strain. Analysis of metabolic pathways and gene expression levels further revealed that shikimic acid metabolism plays a crucial role in the synthesis of rapamycin, and identified the rapK gene as a potential target for genetic manipulation to obtain rapamycin-producing strains with improved product yield. Consequently, the rapK gene was overexpressed in the UV-II strain, which to our delight further improved rapamycin production to 457.3 mg/L. These findings thus provide a theoretical basis for further improvements in the production of not only rapamycin, but also of other, analogous macrolide compounds.

Anti-inflammatory, analgesic and antioxidant activities of 3,4-oxo-isopropylidene-shikimic acid.

Context 3,4-Oxo-isopropylidene-shikimic acid (ISA) is an analog of shikimic acid (SA). SA is extracted from the dry fruit of Illicium verum Hook. f. (Magnoliaceae), which has been used for treating stomachaches, skin inflammation and rheumatic pain. Objective To investigate the anti-inflammatory, analgesic and antioxidant activities of ISA. Materials and methods Analgesic and anti-inflammatory activities of ISA were evaluated using writhing, hot plate, xylene-induced ear oedema, carrageenan-induced paw oedema and cotton pellets-induced granuloma test, meanwhile the prostaglandin E2 (PGE2) and malondialdehyde (MDA) levels were assessed in the oedema paw tissue. ISA (60, 120 and 240 mg/kg in mice model and 50, 120 and 200 mg/kg in rat model) was administered orally, 30 min before induction of inflammation/pain. Additionally, ISA was administered for 12 d in rats from the day of cotton pellet implantation. The active oxygen species scavenging potencies of ISA (10(-3)-10(-5) M) were evaluated by the electron spin resonance spin-trapping technique. Results ISA caused a reduction of inflammation induced by xylene (18.1-31.4%), carrageenan (7.8-51.0%) and cotton pellets (11.4-24.0%). Furthermore, ISA decreased the production of PGE2 and MDA in the rat paw tissue by 1.0-15.6% and 6.3-27.6%, respectively. ISA also reduced pain induced by acetic acid (15.6-48.9%) and hot plate (10.5-28.5%). Finally, ISA exhibited moderate antioxidant activity by scavenging the superoxide radical and hydroxyl radical with IC50 values of 0.214 and 0.450 µg/mL, respectively. Discussion and conclusion Our findings confirmed the anti-inflammatory, analgesic and antioxidant activities of ISA.

Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

Shikimic acid, a base compound for the formulation of swine/avian flu drug: statistical optimization, fed-batch and scale up studies along with its application as an antibacterial agent.

The sudden outbreak of swine flu has increased the global demand of shikimic acid which is an industrially interesting compound, as it is used as a key starting material for the synthesis of a neuraminidase inhibitor Tamiflu(®), for the treatment of antiviral infections such as swine flu. Statistical optimization and evaluation of medium components for the production of shikimic acid by Citrobacter freundii is addressed in the present investigation. Plackett-Burman design was applied for the screening of the most significant variables affecting shikimic acid production, where glucose, asparagine, KH2PO4, CaCO3 and agitation rate were the most significant factors. Response surface methodology was also employed to study the interaction among the most significant variables through which shikimic acid production increased to 12.76 g/L. Further, fed-batch studies resulted in the production of 22.32 g/L of shikimic acid. The scalability of the process was also confirmed by running 14 L bioreactor (7.5 L production medium) where 20.12 g/L of shikimic acid was produced. In addition the antibacterial activity of the shikimic acid produced was analysed against four Gram positive and four Gram negative bacteria and it was found to have a greater inhibition effect against the Gram negative bacteria.

Antibacterial Activity of Shikimic Acid from Pine Needles of Cedrus deodara against Staphylococcus aureus through Damage to Cell Membrane.

Shikimic acid (SA) has been reported to possess antibacterial activity against Staphylococcus aureus, whereas the mode of action of SA is still elusive. In this study, the antibacterial activity and mechanism of SA toward S. aureus by cell membrane damage was investigated. After SA treatment, massive K+ and nucleotide leakage from S. aureus, and a significant change in the membrane potential was observed, suggesting SA may act on the membrane by destroying the cell membrane permeability. Through transmission electron microscopic observations we further confirmed that SA can disrupt the cell membrane and membrane integrity. Meanwhile, SA was found to be capable of reducing the membrane fluidity of the S. aureus cell. Moreover, the fluorescence experiments indicated that SA could quench fluorescence of Phe residues of the membrane proteins, thus demonstrating that SA can bind to S. aureus membrane proteins. Therefore, these results showed the antibacterial activity of SA against S. aureus could be caused by the interactions of SA with S. aureus membrane proteins and lipids, resulting in causing cell membrane dysfunction and bacterial damage or even death. This study reveals the potential use of SA as an antibacterial agent.

Involvement of the cell-specific pigment genes pks and sult in bacterial defense response of sea urchins Strongylocentrotus intermedius.

Bacterial infections are one of the most important problems in mass aquaculture, causing the loss of millions of juvenile organisms. We isolated 22 bacterial strains from the cavity fluid of the sea urchin Strongylocentrotus pallidus and used phylogenetic analysis based on 16S rRNA gene sequences to separate the bacterial strains into 9 genera (Aliivibrio, Bizionia, Colwellia, Olleya, Paenibacillus, Photobacterium, Pseudoalteromonas, Shewanella, and Vibrio). Incubating Strongylocentrotus intermedius larvae with a strain from each of the 9 bacterial genera, we investigated the viability of the larvae, the amount of pigment cells, and the level of polyketide synthase (pks) and sulfotransferase (sult) gene expression. Results of the assay on sea urchin development showed that all bacterial strains, except Pseudoalteromonas and Bizionia, suppressed sea urchin development (resulting in retardation of the embryos' development with cellular disorders) and reduced cell viability. We found that pks expression in the sea urchin larvae after incubation with the bacteria of 9 tested genera was significantly increased, while the sult expression was increased only after the treatment with Pseudoalteromonas and Shewanella. Shikimic acid, which is known to activate the biosynthesis of naphthoquinone pigments, increased the tolerance of the sea urchin embryos to the bacteria. In conclusion, we show that the cell-specific pigment genes pks and sult are involved in the bacterial defense response of sea urchins.

An overview of mechanism and chemical inhibitors of shikimate kinase.

Tuberculosis is a highly infectious disease other than HIV/AIDS and it is one of the top ten causes of death worldwide. Resistance development in the bacteria occurs because of genetic alterations, and the molecular insights suggest that the accumulation of mutation in the individual drug target genes is the primary mechanism of multi-drug resistant tuberculosis. Chorismate is an essential structural fragment for the synthesis of aromatic amino acids and synthesized biochemically by a number of bacteria, including Mycobacterium tuberculosis, utilizing the shikimate pathway. This shikimate kinase is the newer possible target for the generation of novel antitubercular drug because this pathway is expressed only in mycobacterium and not in Mammals. The discovery and development of shikimate kinase inhibitors provide an opportunity for the development of novel selective medications. Multiple shikimate kinase inhibitors have been identified via insilico virtual screening and related protein-ligand interactions along with their in-vitro studies. These inhibitors bind to the active site in a similar fashion to shikimate. In the current review, we present an overview of the biology and chemistry of the shikimate kinase protein and its inhibitors, with special emphasis on the various active scaffold against the enzyme. A variety of chemically diversified synthetic scaffolds including Benzothiazoles, Oxadiazoles, Thiobarbiturates, Naphthoquinones, Thiazoleacetonitriles, Hybridized Pyrazolone derivatives, Orthologous biological macromolecule derivatives, Manzamine Alkaloids derivatives, Dipeptide inhibitor, and Chalcones are discussed in detail. These derivatives bind to the specific target appropriately proving their potential ability through different binding interactions and effectively explored as an effective and selective Sk inhibitor.Communicated by Ramaswamy H. Sarma.

AhpC is required for optimal production of enterobactin by Escherichia coli.

Escherichia coli alkyl hydroperoxide reductase subunit C (AhpC) is a peroxiredoxin that detoxifies peroxides. Here we show an additional role for AhpC in cellular iron metabolism of E. coli. Deletion of ahpC resulted in reduced growth and reduced accumulation of iron by cells grown in low-iron media. Liquid chromatography-mass spectroscopy (LC-MS) analysis of culture supernatants showed that the ahpC mutant secreted much less enterobactin, the siderophore that chelates and transports ferric iron under iron-limiting conditions, than wild-type E. coli did. The ahpC mutant produced less 2,3-dihydroxybenzoate, the intermediate in the enterobactin biosynthesis pathway, and providing 2,3-dihydroxybenzoate restored wild-type growth of the ahpC mutant. These data indicated that the defect was in an early step in enterobactin biosynthesis. Providing additional copies of entC, which functions in the first dedicated step of enterobactin biosynthesis, but not of other enterobactin biosynthesis genes, suppressed the mutant phenotype. Additionally, providing either shikimate or a mixture of para-aminobenzoate, tryptophan, tyrosine, and phenylalanine, which, like enterobactin, are synthesized from the precursor chorismate, also suppressed the mutant phenotype. These data suggested that AhpC affected the activity of EntC or the availability of the chorismate substrate.

Protective effects of 3,4-oxo-isopropylidene-shikimic acid on experimental colitis induced by trinitrobenzenesulfonic acid in rats.

BACKGROUND: 3,4-Oxo-isopropylidene-shikimic acid (ISA) is a derivative of shikimic acid (SA). SA is extracted from Illicium verum Hook.fil., which has been used in traditional Chinese medicine and used for treating vomiting, stomach aches, insomnia, skin inflammation, and rheumatic pain. AIMS: To investigate the effects and the protective mechanism of 3,4-oxo-isopropylidene-shikimic acid on experimental colitis model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. METHODS: Colitis in rats was induced by colonic administration with TNBS. ISA (50, 100, and 200 mg/kg) was administered for 12 days to experimental colitis rats. The inflammatory degree was assessed by macroscopic damage score, colon weight/length ratios (mg/cm), and myeloperoxidase (MPO) activity. Malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) activities were measured with biochemical methods. RESULTS: ISA significantly ameliorated macroscopic damage, reduced colon weight/length ratios and the activity of MPO, depressed MDA and NO levels and iNOS activity, and enhanced GSH level, and GSH-Px and SOD activities in the colon tissues of experimental colitis in a dose-dependent manner. Moreover, the effect of ISA (200 mg/kg) was as effective as sulfasalazine (500 mg/kg). CONCLUSIONS: The findings of this study demonstrate the protective effect of ISA on experimental colitis, probably due to an antioxidant action.

Inhibition of Biofilm Formation and Virulence Factors of Cariogenic Oral Pathogen Streptococcus mutans by Shikimic Acid.

Streptococcus mutans is known as an important oral pathogen causing dental caries, a widespread oral infectious disease. S. mutans synthesize exopolysaccharide (EPS) using glucosyltransferases (Gtfs), resulting in biofilm formation on the tooth surface. Bacterial cells in the biofilms become strongly resistant to a harsh environment, such as antibiotics and host defense mechanisms, making biofilm-based infections difficult to eliminate. Discovering novel antibiofilm agents, especially from natural products, helps to develop effective strategies against this kind of diseases. The present study investigated the inhibitory effect of shikimic acid (SA), one abundant compound derived from Illicium verum extract, on the biofilm formation of S. mutans. We found SA can reduce the EPS synthesized by this oral pathogen and modulate the transcription of biofilm formation related genes, leading to fewer bacterial cells in its biofilm. SA also interacted with cell membrane and membrane proteins, causing damage to bacterial cells. Ex vivo testing of biofilm formation on bovine teeth showed SA strongly decreased the number of S. mutans cells and the number of EPS accumulated on dental enamel surfaces. Moreover, SA exhibits almost no toxicity to human oral cells evaluated by in vitro biocompatibility assay. In conclusion, shikimic acid exhibits remarkable antibiofilm activity against S. mutans and has the potential to be further developed as a novel anticaries agent. IMPORTANCE Natural products are an important and cost-effective source for screening antimicrobial agents. Here, we identified one compound, shikimic acid, from Illicium verum extract, exhibiting antimicrobial activity against S. mutans proliferation. It also inhibits biofilm formation of this bacteria through decreasing Gtf expression and EPS synthesis. Furthermore, this compound exhibits no significant cytotoxicity at its MIC against S. mutans, providing evidence for its clinical application.