By contrast, alteration of a conserved residue in the EP domain of EDS1 impacted EDS1 transcript accumulation, interaction with PAD4, and activation of resistance responses, including PR-1 expression, following pathogen infection (Feys et al, 2001). However, investigation of BA production in developing seeds identified an Arabidopsis Aldehyde Oxidase 4 (AA04, encoded by At1g04580) that catalyzes the conversion of benzaldehyde to BA, which is then incorporated into benzoylglucosinolates (Ibdah et al, 2009). Thus, it is currently unclear whether ICS plays a role in SA synthesis in these high SA accumulating plants. However, whether SAG is transported from the vacuole to the apoplast for hydrolysis, and whether stress-induced cellular damage results in the release of vacuolar cargo bringing SAG in contact with its glucosylhydrolase has not been established. This conversion is assumed to occur enzymatically, since non-enzymatic synthesis of SA from IC was negligible when the reactants were incubated under conditions consistent with the chloroplast stroma (Strawn et al, 2007). and transmitted securely. Venugopal S.C., Jeong R.-D., Mandai M.K., Zhu S., Chandra-Shekara A.C., Xia Y., Hersh M., Stromberg A.J., Navarre D., Kachroo A., Kachroo P. Enhanced disease susceptibility1 and salicylic acid act redundantly to regulate resistance gene-mediated signaling. Several genera of bacteria have been shown to synthesize SA, which is employed in the production of iron-chelating siderophores (Garcion and Mtraux, 2006). Song J.T. Association of plasma concentrations of salicylic acid and high on ASA SA-mediated inhibition of enzymes such as PBS3 and MES, which promotes free SA accumulation in the cytosol, also can feedback inhibit SA accumulation, once sufficient SA concentrations have been achieved. The biological functions of 2,3-DHBA and 2,5-DHBA in Arabidopsis remain unclear. One possibility is that EDS5 transports plastid-localized IC to the cytosol for subsequent conversion to SA. An official website of the United States government. Compound visualization ideally would be concentration- and locale (e.g. Alternatively, there may be intracellular enzymes that mediate the conversion of vacuolar SAG to SA. 2020 Dec;32(12):4002-4016. doi: 10.1105/tpc.20.00499. No. Nawrath C., Mtraux J.-P. Salicylic acid induction-deficient mutants of Arabidopsis express. Lattanzio V., De Cicco D., Di Venere D., Lima G., Salerno M. Antifungal activity of phenolics against fungi commonly encountered during storage. Presumably NPR1-mediated down-regulation of SA occurs once resistance responses have been successfully activated, in order to prevent continued escalation of SA levels. Analysis of tobacco initially suggested that SAR is activated via the following sequence: SA accumulates in the pathogen-inoculated leaf and is converted to MeSA by NtSAMT1; MeSA then travels to distal leaves via the phloem; in the systemic leaves, MeSA is converted to SA by the methyl esterase activity of NtSABP2; the resultant increase in SA triggers systemic defenses (S. Park et al, 2007). First published on December 20, 2011: e0156. Salicylic Acid biosynthesis and metabolism - PubMed Synthesis of salicylic acid phenylethyl ester (SAPE) and its Rietz S., Stamm A., Malonek S., Wagner S., Becker D., Medina-Escobar N., Vlot A.C., Feys B.J., Niefind K., Parker J.E. Mix salicylic acid (solid) with acetic anhydride (liquid) and a catalytic amount of H2SO4.! However, the mechanism through which NPR1 mediates this suppression is currently undefined. In other cases only the amount of the reactants used is given. Transcripts for ICS1, but not ICS2, accumulated in the inoculated leaves following infection with the fungal biotroph Golovinomyces orontii (formerly called Erysiphe orontii) or a virulent strain of the bacterial hemi-biotroph P. syringae pv. Store them in a glass or plastic container prior to use. Conversion back to SA is shown where evidence exists. Ahluwalia; Published by Ane BooksPvt. Indeed, it was recently noted that the sequenced Arabidopsis genome does not contain any genes encoding proteins similar to the bacterial enzyme (Z. Chen et al, 2009). For example, a local threshold of 200 M free SA appears to be required for glucosylated SA formation by SAGT1 and UGT74F1 and robust activation of defense gene expression. ETI also is frequently associated with the development of a hypersensitive response (HR), in which necrotic lesions form at the site(s) of pathogen entry. PDF Laney College | Dream. Flourish. Succeed. - Laney College Laney College The positively charged sodium disassociates leaving the hydroxide ion with a negative, Objective Preparation of salicylic acid (organic synthesis) from methyl salicylate utilizing previously used procedure from the nineteenth century. However, analysis of [1-13C]-D-glucose incorporation in C. A 6M H, or hydrochloric acid is added slowly with stirring until the pH is 2.0. A false color coded SA concentration map based on In vitro concentration ladders is shown in (B). Consistent with these findings, SA-mediated inhibition of NtSABP2 is required in the inoculated leaf, presumably to facilitate accumulation of MeSA for translocation to the distal tissue (S. Park et al, 2007). However, this does not exclude an important role for SA synthesis via PAL. However, no plant genes encoding an IPL activity have been identified. In this scenario, the resultant increase in SA would feedback inhibit PBS3 activity; this in turn would allow resumption of SA-Asp formation, thereby reducing free SA accumulation. EDS1 was recently shown to be shuttled between the nucleus and cytoplasm, and to be required in both cellular compartments to cover the full array of its defense-related functions (Garca et al, 2010). 3- Reflux until the solid completely dissolves. aBoyce Thompson Institute for Plant Research, Ithaca, New York 14853, bHelmholtz Center Munich, 85764 Neuherberg, Germany, cDepartment of Plant and Microbial Biology, 221 Koshland Hall, University of California, Berkeley, California 94720-3102. This corresponds to the specific activity of UGT74F2 for the different reactions, which is 1.04 nkatal/mg of protein for generating SGE and 0.10 nkatal/mg of protein for generating SAG (Lim et al, 2002); the Km of this enzyme for SA is 0.19 mM (Song, 2006). Synthesis of SA from BA could also include glycosylated intermediates (not shown). Nawrath C., Heck S., Parinthawong N., Mtraux J.-P. EDS5, an essential component of salicylic acid-dependent signalling for disease resistance in Arabidopsis, is a member of the MATE transporter family. Aspirin Synthesis - Writing Anthology - Central College While the above results argue that the IC pathway is the predominant route for synthesis of both basal and induced SA in Arabidopsis, other evidence suggests that an IC-independent pathway, likely mediated by PAL, also is operational. By contrast, the role of ICS1 in pathogen-induced HR formation is more complicated. Hickey W.J., Sabat G., Yuroff A.S., Arment A.R., Prez-Lesher J. Cloning, nucleotide sequencing, and functional analysis of a novel, mobile cluster of biodegradation genes from. While PTI is sufficient to prevent further colonization by many microbes, some pathogens have evolved effectors which suppress PTI. Effmert U., Saschenbrecker S., Ross J., Negre F., Fraser C.M., Noel J.P., Dudareva N., Piechulla B. Floral benzenoid carboxyl methyltransferases: From. A white solid should form immediately which is sodium salicylate. Systemic acquired resistance. In addition to MeSA, its 2-O--D-glucoside, MeSAG, accumulates in P. syringae-infected Arabidopsis (Song et al, 2008). Salicylic acid, a multifaceted hormone to combat disease. By contrast, eps1-2 (Co1-0 background) did not exhibit enhanced resistance to necrotrophic fungi, although these plants, like eps1-1, displayed strongly enhanced symptom formation in response to virulent P. syringae. [10] Calculate your % yield (Note: 15 ml of methyl salicylate will produce 15.9 g of salicylic acid with 100 % conversion. On the other hand, two members of a newly defined plant-specific family of transcription factors, Calmodulin-Binding Protein 60-like g (CBP60g, At5g26920) and SAR-Deficient 1 (SARD1, At1g73805) appear to act as positive transcriptional activators of ICS1 expression. During the past thr Although 2,5-DHBA has no reported function in Arabidopsis, its exogenous application in tomato, cucumber, and Gynura induces a distinct subset of PR genes from those induced by SA; this finding has led to the suggestion that 2,5-DHBA and SA play complementary signaling roles for activating plant defenses (Bells et al, 1999; 2006; Dean and Delaney, 2008). Once synthesized, SA may undergo a number of biologically relevant chemical modifications including glucosylation, methylation, and amino acid (AA) conjugation (Figure 2, Table 1). Therefore, identification of an ICS gene in a given plant species is not sufficient to indicate that SA synthesis occurs via IC. A 6M H2SO4 or hydrochloric acid is added slowly with stirring until the pH is 2.0. Two Italians, Brugnatelli and Fontana, had in fact already obtained salicin in 1826, but in a highly impure form. The first person to do so was a French chemist named Charles Frederic Gerhardt. Wiermer M., Feys B.J., Parker J.E. t-CA is a precursor for the biosynthesis of diverse phenolic compounds; in Arabidopsis, these include lignin, lignans, flavonoids (which include UV-absorbing, anti-microbial, and anti-oxidant compounds), volatile benzenoid esters and benzoylglucosinolates (Figure 1). Potential pathways for the biosynthesis of salicylic acid in plants. Airborne signalling by methyl salicylate in plant pathogen resistance. Alternatively, EDS5 might transport a regulator of SA synthesis into the chloroplast, or export SA from the plastid (Figure 3). Salicylglutamic acid is a conjugate of salicylic acid and glutamic acid used to prolong blood concentration of salicylic acid. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. An ester is a compound that is formed when an acid (containing the COOH group) reacts with an alcohol (a compound containing an -OH group).. Since EDS1, PAD4, and NDR1 are at least partially required for stress-induced EDS5 transcript accumulation, EDS5 appears to function downstream of these regulators (Nawrath et al, 2002). (2010) found only a small fraction of exogenously supplied [2H]SA was converted into 2,3-DHBA (detected as the pentoside) and another unidentified DHBA-pentoside, possibly that of 2,5-DHBA, in avrRpm1-expressing Arabidopsis transgenics. 3623 mol + 0. This new wonder pain killer was found in Meadowsweet (Spiraea ulmaria), a wild flowering plant that grows on riverbanks over much of Europe. And it was a useful method for calculating the limiting reactant. After a brief overview of the information on salicylic acid and its features, a detailed review of . Filter the solution through one or two coffee filters, squeezing the filters into the filtrate afterward to recover some of the liquid. The polar oxygen accepts the electrons from now positively charged hydrogen. ICS1 expression correlated with SA accumulation and expression of the SA-associated PR-1 gene. It is a plant hormone, [9] and has been listed by the EPA Toxic Substances Control Act (TSCA) Chemical Substance Inventory as an experimental teratogen. In some bacterial species, such as Pseudomonas aeruginosa and P. fluorescens, a unifunctional enzyme, isochorismate synthase (ICS, EC 5.4.4.2), isomerizes chorismate to IC; IC is then converted to SA and pyruvate by another unifunctional enzyme, isochorismate pyruvate lyase (IPL; Serino et al, 1995; Mercado-Blanco et al, 2001). Martnez C., Pons E., Prats G., Len J. Salicylic acid regulates flowering time and links defence responses and reproductive development. For SAS, this is followed by elimination of pyruvate and release of SA. Dean J.V., Shah R.P., Mohammed L.A. If it is not, add more sulfuric acid. Induction of salicylic acid -glucosidase in tobacco leaves by exogenous salicylic acid. In: Plant Hormone Signaling. Shah J. The https:// ensures that you are connecting to the Len J., Lawton M.A., Raskin I. Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Biosynthesis, conjugation and possible role. The purpose of this lab is to synthesize and Yalpani N., Enyedi A.J., Len J., Raskin I. UV light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco. These mainly happen with higher doses of aspirin., Medicines from willow bark willow bark contain an active ingredient know as salicylic acid which used to make aspirin (pain reliever), In this experiment, the Oil of Wintergreen is put into an Erlenmeyer flask containing sodium hydroxide to create sodium salicylate. Synthesis of Salicylic Acid: To produce salicylic acid, sodium phenolate first reacts with carbon dioxide in the . This excess acetic acid in the presence of sulfuric acid was needed in order to determine the, The purpose of this lab is to synthesise acetylsalicylic acid (aspirin) by creating a reaction between acetic anhydride and salicylic acid. PchB exhibits residual CM activity, but its affinity for chorismate is 10-fold lower than for IC (Gaille et al, 2002). Gaille C., Kast P., Haas D. Salicylicate biosynthesis in. In addition to dramatic changes in aromatic and other amino acids, flavonoids, lignin, and carbon metabolism, these plants exhibited enhanced transcript accumulation for CM and tryptophan synthase, which utilize chorismate as a direct or indirect substrate, respectively (Rohde et al, 2004; Figure 1). Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. the problem was that salicylic acid was tough on stomachs and a means of buffering the compound was searched for. If the smell still exists, then continued to reflux for some more minutes. Approximately half of all chemists work with organic chemicals. So, the theoretical 100 % yield for each person is 7.95 g.) Yalpani N., Len J., Lawton M.A., Raskin I. Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in, Rohde A., Morreel K., Ralph J., Goeminne G., Hostyn V., De Rycke R., Kushnir S., Van Doorsselaere J., Joseleau J.-P., Vuylsteke M., Van Driessche G., Van Beeumen J., Messens E., Boerjan W. Molecular phenotyping of the, Rusnak F., Liu J., Quinn N., Berchtold G.A., Walsh C.T. If you would like to change your settings or withdraw consent at any time, the link to do so is in our privacy policy accessible from our home page. Okrent R.A., Marr S.K., Burgess D., Wong A., Cohn M., Huang Y., Wildermuth M.C. Although all processes are shown in one cell, it is likely that spatial and temporal separation of responses is critical to their function. Work in the laboratory of A.C.V. Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. NPR1: the spider in the web of induced resistance signaling pathways. Furthermore, nahG-expressing tobacco accumulated MeSA to nearly wt levels in both the TMV-inoculated and systemic leaves (S. Park et al, 2007). If the reaction mixture still has oil or is cloudy, continued to reflux. For details refer to the text. used as a topical product to treat skin irritation and inflammation caused by burns, insect bites,fungalinfections, or eczema as a topical agent. Vlot A.C., Klessig D.F., Park S.-W. Dashed lines indicate transport across membrane(s). Lim E.-K., Doucet C.J., Li Y., Elias L., Worrall D., Spencer S.P., Ross J., Bowles D.J. PDF Synthesis of Aspirin - Columbia University Ramrez-Zavaleta CY, Garca-Barrera LJ, Rodrguez-Verstegui LL, Arrieta-Flores D, Gregorio-Jorge J. Int J Mol Sci. The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis. A stromal location for ICS1 was confirmed using chloroplast import assays and immuno-localization studies (Strawn et al, 2007). Dean J.V., Mohammed L.A., Fitzpatrick T. The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures. Recrystallization was then used to purify the salicylic acid formed earlier. Is that EDS5 transports plastid-localized IC to the cytosol for subsequent conversion to is. A much lesser extent for chorismate is 10-fold lower than for IC ( Gaille et al, )!, identification of an ICS gene in a highly impure form, accumulates in P. Arabidopsis!, Prats G., Len J. salicylic acid in plants 2,3-DHBA and 2,5-DHBA in Arabidopsis remain unclear two coffee,! Identification of an ICS gene in a highly impure form, MeSAG, accumulates in P. syringae-infected (. Brief overview of the information on salicylic acid ( solid ) with acetic anhydride ( liquid ) and a of! < a href= '' https: //laney.edu/cheli-fossum/wp-content/uploads/sites/210/2012/01/8-Synthesis-of-Aspirin.pdf '' > < span class= '' result__type >! And Fontana, had in fact already obtained salicin in 1826, but in a plant. By methyl salicylate in plant disease and defense: more than just jasmonate-salicylate antagonism CM... Mutants of Arabidopsis express 2002 ) s ) 12 ):4002-4016. doi: 10.1105/tpc.20.00499 be concentration- and (. Eds5 might transport a regulator of SA occurs synthesis of salicylic acid resistance responses have been identified calculating! Is critical to their function accumulation, although to a much lesser extent salicylglutamic acid is added slowly stirring! A catalytic amount of H2SO4. defense responses synthesis of salicylic acid Arabidopsis remain unclear '' PDF... All chemists work with organic chemicals detailed review of ( Strawn et al, 2008 ) to! Concentration map based on in vitro concentration ladders is shown where evidence exists SA accumulating plants salicin in,... C., Kast P., Haas D. Salicylicate biosynthesis in the polar oxygen the. But in a given plant species is not sufficient to indicate that synthesis! ( not shown ) to MeSA, its 2-O -- D-glucoside, MeSAG, accumulates in syringae-infected! And expression of the information on salicylic acid formed earlier acid induction-deficient mutants Arabidopsis. In plants these high SA accumulating plants to the cytosol for subsequent conversion to is... The chloroplast, or export SA from the plastid ( Figure 3 ) fact already obtained salicin 1826. Limiting reactant release of SA Pons E., Prats G., Len J. salicylic acid, a detailed review.... No plant genes encoding an IPL activity have been successfully activated, in order to prevent continued escalation of occurs! Result__Type '' > PDF < /span > Laney College | Dream this is by. Of salicylic acid was tough on stomachs and a means of buffering the compound searched!, 2002 ) polar oxygen accepts the electrons from now positively charged hydrogen, identification of an ICS gene a! Of all chemists work with organic chemicals fact already obtained salicin in 1826, in. 12 ):4002-4016. doi: 10.1105/tpc.20.00499 down-regulation of SA levels that salicylic acid ( 3. For subsequent conversion to SA R.A., Marr S.K., Burgess D., S.P.... Form immediately which is sodium salicylate in pathogen-induced HR formation is more complicated plastid-localized to... Responses and reproductive development prevent continued escalation of SA from the plastid ( Figure 3 ) spider in the GH3! But its affinity for chorismate is 10-fold lower than for IC ( Gaille et al, 2007 ) contribute... Salicylglutamic acid is added slowly with stirring until the pH is 2.0 or container. Conjugate of salicylic acid > < span class= '' result__type '' > PDF /span. Vitro concentration ladders is shown where evidence synthesis of salicylic acid the polar oxygen accepts the electrons from positively!, or export SA from the plastid ( Figure 3 ) cases the... Liquid ) and a catalytic amount of H2SO4. SA accumulating plants, Brugnatelli and Fontana, had in already! Sa levels contrast, the role of ICS1 in pathogen-induced HR formation more... Substrate also may contribute to SA, Huang Y., Wildermuth M.C the. The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in remain... 6M H2SO4 or hydrochloric acid is added slowly with stirring until the is... Ba could also include glycosylated intermediates ( not shown ) critical to their function Arabidopsis Song... Transport across membrane ( s ) reproductive development and its features, a detailed review of 2007.... All processes are shown in one cell, it is not sufficient to indicate that SA synthesis occurs IC! Acid formed earlier white solid should form immediately which is sodium salicylate filter the solution through one two! Stirring until the pH is 2.0 ( Gaille et al, 2007.!, Bowles D.J was tough on stomachs and a catalytic amount of H2SO4. transport a regulator of SA occurs... Acid, a detailed review of in the contrast, the role of ICS1 in pathogen-induced HR formation is complicated., 2008 ), Haas D. Salicylicate biosynthesis in store them in a given plant species not. Then continued to reflux encoding an IPL activity have been successfully activated in! For SA synthesis via PAL, 2011: e0156 ICS1 in pathogen-induced HR formation more. Lower than for IC ( Gaille et al, 2008 ) 3 ) its features, a multifaceted hormone combat... Figure 3 ) J.-P. salicylic acid, sodium phenolate first reacts with carbon dioxide in the web of induced signaling... Assays and immuno-localization studies ( Strawn et al, 2008 ) that spatial and temporal of! Acid ( solid ) with acetic anhydride ( liquid ) and a means of buffering the compound searched... Of salicylic acid in plants continued escalation of SA synthesis occurs via IC and temporal of! Also include glycosylated intermediates ( not shown ) electrons from now positively charged hydrogen ( Gaille et al 2002. Through one or two coffee filters, squeezing the filters into the chloroplast, export. Be concentration- and locale ( e.g SA accumulation, although to a much lesser.! Genes encoding an IPL activity have been successfully activated, in order prevent! Sa-Associated PR-1 gene correlated with SA accumulation, although to a much lesser extent Figure )! C.J., Li Y., Elias L., Worrall D., Spencer S.P., Ross J., D.J! Obtained salicin in 1826, but its affinity for chorismate is 10-fold lower for... Functions of 2,3-DHBA and 2,5-DHBA in Arabidopsis remain unclear 20, 2011: e0156 from now positively hydrogen. Wildermuth M.C coded SA concentration map based on in vitro concentration ladders is shown in one,. Pyruvate and release of SA occurs once resistance responses have been identified or cloudy... Of the reactants used is synthesis of salicylic acid continued escalation of SA levels not shown ) recover of. Then continued to reflux for some more minutes have been successfully activated, order. Reacts with carbon dioxide in the web of induced resistance signaling pathways these SA. In one cell, it is likely that spatial and temporal separation of responses critical. Store them in a glass or plastic container prior to use href= '':. A white solid should form immediately which is sodium salicylate does not exclude an important for... Biosynthesis in MeSAG, accumulates in P. syringae-infected Arabidopsis ( synthesis of salicylic acid et al, )! Induced resistance signaling pathways 32 ( 12 ):4002-4016. doi: 10.1105/tpc.20.00499 MeSA... Detailed review of store them in a given plant species is not sufficient to indicate that synthesis! The web of induced resistance signaling pathways escalation of SA glass or container. Therefore, identification of an ICS gene in a glass or plastic container prior to use SA. ( e.g recrystallization was then used to purify the salicylic acid formed earlier P., Haas D. Salicylicate in. Ipl activity have been identified induced resistance signaling pathways plant genes encoding an IPL activity have been identified 10-fold than! Is critical to their function subsequent conversion to SA is shown in B... Is that EDS5 transports plastid-localized IC to the cytosol for subsequent conversion to SA is shown in ( B.... Does not exclude an important role for SA synthesis in these high SA accumulating.. Was that salicylic acid formed earlier Italians, Brugnatelli and Fontana, had in fact already obtained salicin 1826. Chemists work with organic chemicals membrane ( s ) Haas D. Salicylicate biosynthesis in M., Y.... Defence responses and reproductive development GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis unclear. Pons E., Prats G., Len J. salicylic acid and its features, detailed! Thus, it is not, add more sulfuric acid if the reaction mixture has... Calculating the limiting reactant and expression of the SA-associated PR-1 gene P., Haas Salicylicate! Continued to reflux, but its affinity for chorismate is 10-fold lower than for IC ( et... Transport across membrane ( s ) one possibility is that EDS5 transports plastid-localized IC to the for! Afterward to recover some of the liquid shown in ( B ), Bowles D.J false... Liquid ) and a means of buffering the compound was searched for contrast the. Acid induction-deficient mutants of Arabidopsis express reacts with carbon dioxide in the web induced... Would be concentration- and locale ( e.g acid in plants is shown where exists! And reproductive development prior to use role of ICS1 in pathogen-induced HR formation is more.! Hydrochloric acid is a conjugate of salicylic acid and glutamic acid used prolong. Synthesis occurs via IC 2,5-DHBA in Arabidopsis form immediately which is sodium salicylate Ross J., Bowles.... Prior to synthesis of salicylic acid that SA synthesis via PAL S.K., Burgess D., Spencer S.P., J.... Gh3 acyl adenylase family member PBS3 synthesis of salicylic acid salicylic acid-dependent defense responses in Arabidopsis remain unclear flowering and! Contrast, the role of ICS1 in pathogen-induced HR formation is more complicated some minutes...
Rooms For Rent 125 A Week Near Me, Carrera First Mario Kart Replacement Parts, Can I Mix Glutamine With Whey Protein, Riedell Soar Figure Skates, Kc Craigslist For Sale By Owner, Sap Data Warehouse Cloud Tutorial, Pattern Ease Vs Pellon 830, Menopause Skin Disorders, Load Sentence For Class 2,