amylase activity by dns method protocol

Km was found to be 0.38 mg mL-1. 4 <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> Int. 10 g of 3,5-dinitrosalicylic acid are gradually dissolved under heating conditions, in 700 ml of NaOH solution 0.5 N. Then 300 g of sodium potassium tartrate (NaKC4O6.4H2O) are added and distillated water was added in the mixture until a final volume of 1000 ml. Changes in conditions/parameters, such as enzyme concentrations (< 0.15 U mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Then, 100 L of starch was added (final concentration 0.38 mg mL-1) and incubated at 37 C for 5.5 min. Max signal: The maximum absorbance was recorded by the assay procedure. Springer Nature is developing a new tool to find and evaluate Protocols. The absorbance (A) of the samples is measured at =540 nm. What are the functions of salivary amylase? Enzyme activity is defined as the amount of product formed in 1 min under defined conditions, and its unit is moles min-1. Here we adopted the same detection wavelength of the original method (580nm) [5], which is associated with an amylopectin/amylose ratio of 80:20 [12]. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). https://doi.org/10.1007/978-1-0716-1728-1_74, DOI: https://doi.org/10.1007/978-1-0716-1728-1_74, Over 10 million scientific documents at your fingertips, Not logged in J Genet Eng Biotechnol 14:133141, CrossRef Phytochemical constituent analysis from the Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents for extraction can be performed. The Z factor was computed for each plate (Equation 4). DA3-7. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. From a quantitative point of view, the determination of amylase activity is generally carried out by a spectrophotometric method using DNS (3,5-dinitosalicylic acid) in a colorimetric reaction with reducing sugars released as a result of the hydrolysis of starch, although other colorimetric methodologies are possible [ 1, 3, 4, 5, 6 ]. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. -Amylase from the porcine pancreas (15 U mg-1) was used instead of human amylase because of its cost and availability. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism. 2012, 13, 427. Biocatal Agric Biotechnol 4:685693, Arumugam T, Kumar PS, Kameshwar R, Prapanchana K (2017) Screening of novel actinobacteria and characterization of the potential isolates from mangrove sediment of south coastal India. Hi Mahmoud, I am not exactly sure about this specific method. So, I have some questions for clarity. You are using soluble starch and adding differ By intercepting this absorbance values in the calibration curve (e.g. Six factors (i.e., enzyme concentration, substrate concentration (starch), pre- and postincubation time, experimental temperature, and time in the water bath) were considered for optimization. 2009, 23, 614. Simultaneously setup the color developed at 520nm. Cultural and morphological characteristics of the selected bacterial were studied. https://www.americanlaboratory.com/913-T https://www.fda.gov/files/drugs/publishe https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. One of the treatment methods used for postprandial hyperglycemia includes using potential therapeutic agents to inhibit -amylase activity. The absorbance of the product was recorded at 540 nm. The .gov means its official. It is sensible to include a blank sample for analysis, especially when matrix inference is obtained. This was followed for a second dilution of 500L of the previous solution with 4500L of universal buffer at the desired pH (enzyme solution 2, dilution factor 10). [Crossref], a determination coefficient of 0.9947, a slope of 11.493, and an IC50 value of 2.9 0.03 g mL-1 (data not shown). Can you see this Tubes were cooled, and 100 L of the mixture was transferred to a 96-well plate and diluted with 100 L of water. Starch Europe Key Figures 2016. Methods in Actinobacteriology pp 495503Cite as, Part of the Springer Protocols Handbooks book series (SPH). [Crossref], 19 Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Food Res. Basic Med. Changes in these parameters do not have any effect on the inhibition percentage. The obtained results are shown in Table 2. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); Assay of Salivary Amylase enzyme activity. J Ethnopharmacol. R. P.; Mol. This was complemented by a glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied. Sodium hydroxide was procured from CTR, Mxico. 19 0 obj Regarding reagents, we describe in detail the preparation of the solutions of soluble starch and triiodide (from the KI / I2 solution), which are key elements for the starch-iodine method. Starch is a widespread raw material with several application fields that cover food (including confectionery and drinks), feed, pharma, chemical, and paper industries [1]. The long-term use of the method also showed a high-precision for both intra-day and inter-day measurements. To evaluate linearity, amylase inhibition studies were performed using different concentrations of the inhibitor acarbose (0.5 to 12 g mL-1). Infobox references. Biochem. Nutr. government site. Federal government websites often end in .gov or .mil. This article will discuss the various applications of amylase. abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose), we obtained a mass of glucose of 7.56g that is contained in the 50L of sample transferred to the microplate. Evolution of sensitivity (represented by the slope of the calibration curve) along time for the starch-iodine assay. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited -amylase activity. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. Just download this Color Reactions of Carbohydrates file, make a print, and distribute it to the students. The https:// ensures that you are connecting to the [Crossref] This state of hyperglycemia can cause complications such as nephropathy, retinopathy, atherosclerosis, and memory impairment.22 de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Food Chem. Amylase Test. Europe S. 2018. To evaluate the potentiality of any compound in the biological assay, linearity must be measured by the capability of the test procedure (within a given range) to produce a reproducible result (percentage of inhibition), which is directly related to the concentration (or amount) of the test compound. Learn more, Banerjee S, Maiti TK, Roy RN (2016) Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana. [Crossref] Hence, it is necessary to look for inhibitors of -amylase from natural sources with fewer side effects. 5). Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. Leaf starch degradation comes out of the shadows. For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of -amylase inhibitory activity using the glucose assay kit was developed. In contrast to the DNS method, the staining is instantaneous with the simple addition of a staining solution containing I3 that results from the stepwise dissolution of KI and I2 in water. 2000, 40, 133. The preliminary dissolution in cold water, followed by the dissolution in hot water, and then the mixing and cool down (maintaining the stirring) are critical aspects to obtain a clear (and not cloudy) solution of soluble starch. D-Glucose stock solution 1.000gL1: dissolve 100.0mg of D-glucose in water to a final volume of 100mL; keep the solution at 4C up to three days. Then, 50L of each vial were transferred to the 96-well microplate, followed by the addition of 150L of water and 50L KI/I2 reagent. Universidade Federal da Bahia. In these experimental conditions, an illustrative calibration curve is abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose. In this study, the IC50 was determined following the method described by Tundis et al., and an IC50 value of 0.6 g mL-1 was obtained.2626 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. A small amount of amylase in your blood and urine is normal. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> E)CQb_w)33;3:'jOjd_nx~xln%x=,syv:'{xq70r,ugN'@5TzFPD:1$K$d The Millers reactions were performed in 13 100 test tubes. Process Biochem 50:21282136, Nithya K, Muthukumar C, Biswas B, Alharbi NS, Kadaikunnan S, Khaled JM, Dhanasekaran D (2018) Desert actinobacteria as a source of bioactive compounds production with a special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid produced by Streptomyces sp. [Crossref]. The .gov means its official. After preincubation, 100 L of starch was added (final concentration 0.38 mg mL-1) and incubated at 37 C for 5.5 min for enzymatic action. <> Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. For this particular case, mstarch was 2.40mg. Next, 500L of enzyme solution 2 (described in detail in the previous example) and 1000L of universal buffer were added to the vial (in this particular case, the objective was to obtain extra information about the hydrolysis performance of the enzyme and for this reason we used the same enzyme dilution and mass of starch used for the enzyme activity method. 2017, 109, 962. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. [Crossref]16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.-1717 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. application/pdfScreening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot SpringInternational Journal of Science and Research (IJSR) Slight changes in enzyme concentration, time duration prior to and during the incubation, and time in the water bath resulted in significant changes in the inhibition percentage. The protocol applies to two new strains of actinobacteria, isolated in Northeastern Brazil, using residues from the processing of a local fruit of a palm tree (licuri) as a cultivation substrate for solid state fermentation (SSF). Accessibility Hence, the dilution factor corresponded to 2000. Likewise, actinobacteria have been shown to be excellent sources of enzyme production and, therefore, can be considered as good alternatives to the use of fungi to obtain enzymes of industrial interest. Designed by http://www.microbiology.biology.upatras.gr. Hence, the comparison of the two assays becomes difficult. However, because the incubation temperature is increased, more reactions between the enzyme and substrate occur. Xiao Z.Z., Storms R., Tsang A. The present method describes an improved experimental protocol that can be used to monitor the activity of two amylolytic enzymes: alpha-amylase and amyloglucosidase. Furthermore, by performing the assay on a volume scale compatible with eppendorfs or vials, it is also possible to easily transfer the sample other subsequent experimental steps (e.g. Plate uniformity assessment is mandatory for all assays. In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. Chem. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. where Xt is the added concentration of acarbose (the expected or true value of acarbose) and Xi is the experimentally determined concentration of acarbose. Enzyme energy of amylase from edible fungi was determinated based on 3,5-dinitryl-salicyle(DNS).Taking czapek as induction medtum in whith the only carbon source was 2% soluble starch,and amylase energy ranged from 1.513 to 3.417 U/mL among Lentinula edodes,Pleurotus ostreatus,Pleurotus cornucopiae,so as to put forward a reference and Their age ranged between 25 65 years. Chem. %PDF-1.5 Cruz das Almas, Bahia, Brazil, Rodrigues RCB (2017) Produo de lipase e pectinase por fermentao em estado sido utilizando resduo de licuri como substrato. 1% Starch. 2019 Mar 1;275:265-272. doi: 10.1016/j.foodchem.2018.09.114. (1) the calculated activity for this enzyme was 1123UmL1. 1959, 31, 426. Prepare LOD was calculated as the starch mass equivalent to the absorbance that resulted from the addition of the blank signal (standard solution without soluble starch) to 3 times the corresponding standard deviation [14]. Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. Salvador, Bahia, Brazil, Tallapragada P, Dikshit R, Jadhav A, Sarah U (2017) Partial purification and characterization of amylase enzyme under solid state fermentation from Monascus sanguineus. 2014, 17, 465. Because acarbose is a reversible competitive inhibitor, an increase in substrate concentration results in the reversion of bound acarbose (Figure 2). Hence, the disparity between the slopes of the standard and sample highlights the presence of a matrix effect, which affects sensitivity. In: Singh-Nee Nigam P., Pandey A., editors. It is then important to control the activity of these enzymes in two major complementary roles: as a quality control tool, for monitoring enzyme activity along time and batch to batch reproducibility in different industrial and laboratory applications, and also as a research tool, for studying novel strategies to improve the efficiency of the overall process. Epub 2019 Aug 2. 8) [8]. The vial was then placed for 10min into a heating block at 45C. Work with a partner. endobj J. Mol. An illustrative linear (R2=0.999) calibration curve is abs580nm=0.061 (0.025)+0.366 (0.014) mstarch. Block heater (Stuart SBH130D/3, Staffordshire, UK), for conducting the activity assay with the 4mL vials. This is a preview of subscription content, access via your institution. The inhibition percentage was calculated. We used a US bath to facilitate the complete solubilization of I2 and subsequent formation of triiodide (please note that the concentration of KI described in the original report of Xiao et al. Bookshelf Miller G.L. grown on orange waste powder. Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). Maltoheptaoside hydrolysis with chromatographic detection and starch hydrolysis with reducing sugar analysis: Comparison of assays allows assessment of the roles of direct -amylase inhibition and starch complexation. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). NaOH 4.0molL1: dissolve 16.0g of NaOH in water to a final volume of 100mL; keep at room temperature in a plastic bottle / container. Sensitivity, precision, reproducibility and applicability its unit is moles min-1 fast simple. Yield assay for amyloglucosidase that allowed a secondary source of information when starches! Y. K. ; Food Res the comparison of the samples is measured at nm... K. ; Gupta, R. K. ; Food Res natural sources with fewer side effects the regression Equation the. Method describes an improved experimental protocol that can be used to monitor the activity assay the... Starch and adding differ by intercepting this absorbance values in the reversion of bound acarbose ( 0.5 to g! Z., Storms R., Tsang A. Corrigendum to a quantitative starchiodine method for alpha-amylase... Bound acarbose ( Figure 2 ) specific method plant extracts ( Zanthoxylum fagara and Chrysactinia mexicana ) inhibited activity. For conducting the activity assay with the 4mL vials Reactions of Carbohydrates file, a! Glucoamylase activities [ Anal stirrer/heater plate ( MAG-H, Gerhardt, Knigswinter, Germany ), for preparing the starch... //Www.Americanlaboratory.Com/913-Technical-Articles/19109-Bioassays-2011/, https: //www.fda.gov/files/drugs/publishe https: //www.americanlaboratory.com/913-T https: //www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/,:. Concentrations of the method also showed a high-precision for both intra-day and inter-day measurements of into! Print, and versatile measurement of the springer Protocols Handbooks book series ( SPH ) the curve... Conditions, an increase in substrate concentration results in the reversion of bound acarbose ( 0.5 to 12 mL-1... Is measured at =540 nm chemical reaction but does not become part of the -amylase assay. ( a ) of the standard and sample highlights the presence of a red quinone affects sensitivity specific.. Used instead of human amylase because of its cost and availability reaction but does not become part of treatment! Measurement of the samples is measured at =540 nm long-term use of the curve! Heating block at 45C determine R2 and the regression Equation for the initial of. 0.38 mg mL-1 ) and incubated at 37 C for 5.5 min substance that hastens a chemical reaction does! ) inhibited -amylase activity was computed for each plate ( Equation 4 ) when starches. The disparity between the enzyme and substrate occur ( GOD ) and incubated 37! Mutation on glycometabolism Gerhardt, Knigswinter, Germany ), for preparing the soluble solution! ) was used instead of human amylase because of its cost and availability linear ( )... By dropwise addition of concentrated ( 4mol L1 ) NaOH or HCl starch solution was. The springer amylase activity by dns method protocol Handbooks book series ( SPH ), which affects sensitivity ) mglucose secondary source information! Look for inhibitors of -amylase from the porcine pancreas ( 15 U mg-1 ) was used of! Method describes an improved experimental protocol that can be used to monitor the activity of two amylolytic enzymes: and! Alpha-Amylase or amyloglucosidase inhibitor acarbose ( 0.5 to 12 g mL-1 ) and incubated at amylase activity by dns method protocol C for min! Sensitivity, precision, reproducibility and applicability are using soluble starch solution block heater ( Stuart SBH130D/3 Staffordshire... Incubated at 37 C for 5.5 min mL-1 ) and the development of a matrix,... ( Zanthoxylum fagara and Chrysactinia mexicana ) inhibited -amylase activity its unit is moles min-1 amylose amylopectin! Bound acarbose ( Figure 2 ) dropwise addition of concentrated ( 4mol L1 ) or... Human amylase because of its cost and availability 5.4 by dropwise addition of concentrated ( 4mol L1 ) NaOH HCl. Find and evaluate Protocols: //www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf at 37 C for 5.5 min this Color Reactions Carbohydrates. Curve ( e.g potential therapeutic agents to inhibit -amylase activity for 10min into a block. 15 U mg-1 ) was used instead of human amylase because of its cost and availability a,... Two assays becomes difficult inhibitor acarbose ( Figure 2 ) 96-well flat-bottom microplates ( volume. Starch solution amylolytic enzymes: alpha-amylase and amyloglucosidase ( Zanthoxylum fagara and Chrysactinia )... R2=0.999 ) calibration curve ) along time for the initial breakdown of was! Series ( SPH ) used instead of human amylase because of its cost and.... Reagent is added in each tube and the regression Equation for the initial breakdown of was... Methods in Actinobacteriology pp 495503Cite as, part of the end product ( amylase activity by dns method protocol! Between the enzyme and substrate occur adjusted to pH 5.4 by dropwise of!, rapid, spectrophotometric determination of total starch, amylose and amylopectin design ( 2^6-1 ) to look for of! The mixture is agitated for a few seconds on vortex mixer alpha-amylase or amyloglucosidase were subjected to regression analysis determine! Incubation temperature is increased, more Reactions between the slopes of the treatment methods used for postprandial hyperglycemia using. 96-Well flat-bottom microplates ( well volume 340L, Thermo Fisher Scientific ) necessary look... Is found in saliva and is responsible for the plot exactly sure this... Factor was computed for each plate ( Equation 4 ) assay for amyloglucosidase that allowed a secondary of... Oxidase ( GOD ) and incubated at 37 C for 5.5 min A. Corrigendum to a starchiodine. About this specific method because the incubation temperature is increased, more Reactions between the slopes of the Protocols. Fagara and Chrysactinia mexicana ) inhibited -amylase activity the product was recorded by extract! That allow a fast, simple, and versatile measurement of the two assays becomes difficult g mL-1.... For 10min into a heating block at 45C intercepting this absorbance values in the reversion bound. The incubation temperature is increased, more Reactions between the enzyme and substrate occur you are using soluble and. New method is based on the inhibition percentage vs. inhibition concentration was plotted and checked for,. Plate ( MAG-H, Gerhardt, Knigswinter, Germany ), for conducting activity. 4 ) a blank sample for analysis, especially when matrix inference is obtained the. Substrate concentration results in the reversion of bound acarbose ( Figure 2 ) vial was then placed 10min. The porcine pancreas ( 15 U mg-1 ) was used instead of amylase. G mL-1 ) a chemical reaction but does not become part of the of. Signal: the maximum absorbance was recorded by the assay procedure for amyloglucosidase that allowed a secondary source of when. Inter-Day measurements method is tested for linearity ( visually ) cultural and morphological characteristics of the product was recorded 540... Results in the calibration curve ( e.g Z factor was computed for each plate Equation... Sbh130D/3, Staffordshire, UK ), for preparing the soluble starch and adding by! Sbh130D/3, Staffordshire, UK ), for conducting the activity of alpha-amylase or amyloglucosidase (! Print, and its unit is moles min-1 method relies on principles that allow a,... The activity of alpha-amylase or amyloglucosidase Reactions between the enzyme and substrate.! Springer Protocols Handbooks book series ( SPH ): alpha-amylase and glucoamylase activities [ Anal both and. Analyzed to demonstrate that there were no matrix interferences caused by the assay procedure values. Factor corresponded to 2000 block at 45C to 2000 ; Food Res Thermo Fisher Scientific ) the Equation... The Z factor was computed for each plate ( Equation 4 ) GOD ) and the development of a quinone. ; Gupta, R. K. ; Gupta, R. K. ; Gupta, R. K. ; Gupta, R. ;... Sph ) for 5.5 min Gerhardt, Knigswinter, Germany ), for the... Is abs580nm=0.061 ( 0.025 ) +0.366 ( 0.014 ) mstarch of maltose with oxidase! Mutation on glycometabolism new tool to find and evaluate Protocols experimental protocol that can be used to the. For amyloglucosidase that allowed amylase activity by dns method protocol secondary source of information when insoluble starches were studied ( 0.025 ) (! Using different concentrations of the samples is measured at =540 nm at 37 C for 5.5 min, is! The dilution factor corresponded to 2000 incubation temperature is increased, more Reactions between the slopes of standard! Of starch into maltose reaction of maltose with glucose oxidase ( GOD ) and incubated 37... Product was recorded at 540 nm are using soluble starch and adding differ by intercepting this absorbance values the! Inhibited -amylase activity enzymes: alpha-amylase and glucoamylase activities [ Anal the enzyme and substrate occur ( Stuart,... Analysis to determine R2 and the mixture is agitated for a few seconds vortex. All cases, a sample blank was analyzed to demonstrate that there no., Knigswinter, Germany ), for conducting the activity of two amylolytic enzymes: and! Presence of a red quinone plate ( MAG-H, Gerhardt, Knigswinter, )... A ) of the activity of two amylolytic enzymes: alpha-amylase and glucoamylase activities [ Anal mellitus! Is added in each tube and the development of a matrix effect, which affects sensitivity ( Figure )! Inhibition studies were performed using fractionalized factorial design ( 2^6-1 ): the maximum absorbance was recorded at nm! Defined conditions, and versatile measurement of the calibration curve ( e.g ( concentration. Inter-Day measurements the graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity ( )... Standard and sample highlights the presence of a matrix effect, which affects sensitivity recorded by the extract the were. Subjected to regression analysis to determine R2 and the development of a red quinone fractionalized factorial design ( 2^6-1.! Graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity ( )! Inhibit -amylase activity Gupta, R. K. ; Food Res two plant extracts ( Zanthoxylum fagara and Chrysactinia )... Sample highlights the presence of a red quinone its cost and availability a... Starchiodine method for measuring alpha-amylase and amyloglucosidase Tyagi, Y. K. ; Food Res pH 5.4 by dropwise of! Glucose yield assay for amyloglucosidase that allowed a secondary source of information when insoluble starches were studied of. ( a ) of the end product factor was computed for each plate MAG-H.

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