让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

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感谢您对 ECIS 的兴趣。

以下是一些条件以及要求演示成功。

应用生物物理学 (ABP) 同意提供 ECIS 仪器，为期 4 周。

电细胞基底阻抗传感所需的所有设备均由 ABP 提供，其中包括：ECIS控制器、阵列站、计算机和阵列启动电源。可以购买其他阵列。

ABP 同意支付从 ABP 到您所在地的运费，并要求研究人员在决定不保留仪器时支付回程运费。

ABP 同意支付从 ABP 到您所在地的运费。 p>

ABP要求至少一名研究人员专门负责仪器的操作和维护。作为专门的运营商，我们要求每周与该研究人员交谈以审查数据和系统操作。

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让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

联系 Applied Biophysicals

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Applied Biophysicals, Inc.185 Jordan RoadTroy, NY 12180

1-866-301-ECIS (3247)

<电话>518-880-6860

518-880-6865

让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

IBCA 会议

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2011 年在雷根斯堡举行的第一届\"基于阻抗的细胞测定会议”开始了新的传统，德国。这些会议的目的是分享所有人使用基于阻抗的方法开发细胞检测的想法和研究发现。

2018201620132011让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

软件

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让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

贸易展览和活动

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有什么比参加我们周围的科学活动更好地了解 ECIS 的力量世界！

以下是 Applied biophysics 将在 2022 年参加的即将举行的展会列表：

目前没有安排任何贸易展会。请尽快回来查看。让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

常见问题解答

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ECIS 代表什么？

ECIS 代表电池-基板阻抗传感。

ECIS 如何发音？

ECIS - (ë\'sis) n。 adj.

ECIS 需要标签吗？

不需要。 ECIS 是一种完全无标记的测量。

细胞能感觉到它们正在受到监控吗？

不能。用于监测电池的交流电流比其感应任何电流的能力低 10 倍。这就好像您在窃听细胞一样。

什么是阻抗？

阻抗是对正弦交流电流的阻力的度量。阻抗将直流电阻的概念扩展到交流电路。它不仅描述了电压和电流的幅值，还描述了电压相对于电流的相位。

ECIS 的作用是什么测量？

ECIS 系统测量细胞覆盖直径 250um 的金电极并阻挡电流时的阻抗变化。

什么是微动？

微动是细胞复杂的运动和独特的波动。

什么是建模？

我们可以应用数学模型由于细胞层的存在而引起的阻抗变化模型，其中阻抗数据可用于计算细胞形态参数，包括细胞层的屏障功能、细胞腹侧与基质之间的间距以及细胞膜电容。在模型中，细胞被表示为具有绝缘膜表面并充满导电电解质的盘状物体。

可以监测哪种细胞？

细胞必须粘附在

阵列表面可以用蛋白质预处理吗？

可以。

让我们谈谈应用程序 866-301-ECIS (3247)

营业时间美国东部时间上午 9 点至下午 5 点

ECIS®，一种无标签、非侵入性的方法，用于电子监测组织培养中生长的细胞。TEER 屏障功能细胞增殖细胞迁移细胞毒性病毒学网络研讨会 2023 年 9 月 5 日

毒理学ECIS

11:00AM EDT

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请尽快回来查看。

ECIS 学校日期：待定

2 天课程

纽约州特洛伊

Brachypodium distachyon triphosphate tunnel metalloenzyme 3 is both a triphosphatase and an adenylyl cyclase upregulated by mechanical wounding - Świeżawska - 2020 - FEBS Letters - Wiley Online Library orcid.org/0000-0001-8155-8716 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, Poland Correspondence B. Świeżawska, Nicolaus Copernicus University, Chair of Plant Physiology and Biotechnology, Lwowska St. 1, PL 87-100 Torun, Poland Tel: +48-56-611-44-56 E-mail: swiezawska@umk.plSearch for more papers by this authorMaria Duszyn, orcid.org/0000-0003-3144-9516 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorMateusz Kwiatkowski, orcid.org/0000-0002-1473-4545 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorKrzysztof Jaworski, orcid.org/0000-0002-8597-268X Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorAgnieszka Pawełek, orcid.org/0000-0003-3951-1193 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorAdriana Szmidt-Jaworska, orcid.org/0000-0002-8139-4763 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this author orcid.org/0000-0001-8155-8716 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, Poland Correspondence B. Świeżawska, Nicolaus Copernicus University, Chair of Plant Physiology and Biotechnology, Lwowska St. 1, PL 87-100 Torun, Poland Tel: +48-56-611-44-56 E-mail: swiezawska@umk.plSearch for more papers by this authorMaria Duszyn, orcid.org/0000-0003-3144-9516 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorMateusz Kwiatkowski, orcid.org/0000-0002-1473-4545 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorKrzysztof Jaworski, orcid.org/0000-0002-8597-268X Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorAgnieszka Pawełek, orcid.org/0000-0003-3951-1193 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this authorAdriana Szmidt-Jaworska, orcid.org/0000-0002-8139-4763 Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, PolandSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Proteins with a CyaB, thiamine triphosphatase domain (CYTH domain) may play a central role at the interface between nucleotide and polyphosphate metabolism. One of the plant CYTH domain-containing proteins from Brachypodiumdistachyon, BdTTM3, is annotated in NCBI databases as an ‘adenylyl cyclase (AC)’ or a ‘triphosphate tunnel metalloenzyme’. The divergent nomenclature and the search for plant ACs induced us to experimentally confirm the enzymatic activity of BdTTM3. Based on invitro analysis, we have shown that the recombinant form of BdTTM3 is a protein with high triphosphatase activity (binding both tripolyphosphate and ATP) and low AC activity. Furthermore, the analysis of BdTTM3 transcriptional activity indicates its involvement in the mechanism underlying responses to wounding stress in B.distachyon leaves. Abbreviations AC, adenylyl cyclase ATPase, adenosine triphosphatase CYTH domain, CyaB, thiamine triphosphatase domain PPPase, tripolyphosphatase ThTPase, thiamine triphosphatase TTM, triphosphate tunnel metalloenzyme The CyaB, thiamine triphosphatase domain (CYTH domain) is a conserved amino acid motif found in some groups of proteins such as prokaryotic adenylyl cyclases (AC), mammalian thiamine triphosphatases (ThTPase), RNA triphosphatases, membrane-associated polyphosphate polymerases, tripolyphosphatases (PPPase), nucleoside triphosphatases, and nucleoside tetraphosphatases. The ‘CYTH’ abbreviation is derived from the presence of this domain in the CyaB adenylate cyclase from Aeromonashydrophila and the mammalian ThTPase. Bioinformatics tools enable the identification of many proteins belonging to the CYTH superfamily. This motif is widespread in proteins among bacteria, archeons, fungi, plants, and animals (excluding birds). The common and basic feature of all these proteins is their ability to hydrolyze triphosphate-containing substrates in the presence of divalent metal cations (Mg2+ or Mn2+) [1]. In addition, the spatial conformations of the β-barrel structure of these proteins show great similarity, although the degree of the similarity of the amino acid sequences among the CYTH domain superfamily members is very different (excluding Nitrosomonaseuropaea PPPase and Musmusculus ThTPase) [1-4]. The CYTH superfamily members meeting both criteria are called triphosphate tunnel metalloenzymes (TTMs) [5]. According to previous reports, the CYTH domain could be a fundamental cross-point between the nucleotide and phosphate metabolic pathways [6]. This assumption is derived from the fact that both bacterial CyaB and YpAC2 proteins from A.hydrophila and Yersiniapestis, respectively, belonging to the CYTH superfamily have adenylate cyclase activity [2, 4, 7]. To date, analysis of the biochemical properties of plant AtTTM1-AtTTM3 from Arabidopsisthaliana and mammalian ThTPases [5, 8-11] did not show adenylate cyclase activity. The physiological function of many CYTH superfamily members is still unknown. It was experimentally confirmed that yeast RNA triphosphatases are involved in the first step of eukaryotic mRNA capping [1, 12-14]. TTMs in many bacteria, for example, Clostridiumthermocellum (CthTTM), N.europaea (NeuTTM), or Escherichiacoli (Ygif), are known to be PPPases with various affinities for different substrates [5, 15-18]. A similar situation for organophosphate substrate specificity applies to three plant TTM proteins identified in A.thaliana [8-10]. Biochemical analyses revealed that AtTTM1 and AtTTM2 have the highest affinity for pyrophosphate (PPi), whereas AtTTM3 has a strong PPPase activity similar to that of TTMs from C.thermocellum and N.europaea. In addition, the biological function of these three plant TTMs is very diverse. Analysis of TTM-knockout A.thaliana transgenic plants revealed the possible involvement of AtTTM3 in root development [8], that of AtTTM2 in pathogen resistance, and that of AtTTM1 in leaf senescence [9, 10]. However, recent studies verified the role of AtTTM3 in root growth because different studied ttm3 mutant alleles show inconsistent phenotypes related to hypocotyl growth [19]. The ttm3-1 mutant has reduced hypocotyl and root growth, but the ttm3-3 and ttm3-4 mutants showed a wild-type A.thaliana phenotype. It is intriguing that structurally similar proteins belonging to the same CYTH superfamily can play a role in very different processes and require different substrates. In this study, we investigated TTM (BdTTM3) from Brachypodiumdistachyon, which belongs to the CYTH protein family. Based on invitro analysis, it was shown that the recombinant form of BdTTM3 is a protein with triphosphatase and AC activity; however, the triphosphatase activity is absolutely predominant. Additionally, our assay revealed that the BdTTM3 gene is significantly upregulated by mechanical wounding, suggesting that BdTTM3 may be involved in the response to injury in B.distachyon leaves. The investigations were carried out on B.distachyon Bd21, belonging to Poaceae (Gramineae), a large family of monocotyledonous flowering plants. Seedlings were grown in growth chambers at 23°C with a photoperiod consisting of 16h of light (300µmol·m−2·s−1) and 8h of darkness. Mechanical wounding stress was induced in fully developed leaves of 3-week-old B.distachyon seedlings. A technique involving the ‘pinch/cut’ of leaves, described in the literature as a form of mechanical wounding, was used [20]. Leaves were collected 0.5, 1, 2, 3, 4, 6, 8, 24, and 48h postwounding and just after pinching (0h). Three independent biological replicates of the experiment were performed. The ORF of the BdTTM3 cDNA (NCBI accession number: XM_010236256.3) was amplified by PCR using specific primers (Table S1). The PCR product was introduced into the linearized pGEX-6P-2 expression vector using In-Fusion cloning technology (In-Fusion HD Cloning Kit; Takara Bio USA, Inc.,Mountain View, CA, USA). The E.coli BL21 strain, which was transformed with the resulting plasmid, was used to produce the glutathione S-transferase (GST)-tagged protein. Cells were grown in LB medium supplemented with 2% glucose at 37°C. Fusion protein production was induced by adding IPTG at a final concentration of 0.5mm and incubating the cells at 22°C for 4h in glass vessels connected to a BioFlo 120 bioprocess control station (Eppendorf,Hauppauge, NY, USA). The pH was controlled at 6.5 (±0.2), the dissolved oxygen parameter was set to 30%, and the agitation speed was 300r.p.m. Bacteria were collected by centrifugation, and BdTTM3 was purified as previously described [21]. For the control expression vector, pGEX-6-P2 was used, or GST alone was purified as described above. The homogeneity and purity of the protein fractions were analyzed with 10% (v/v) SDS/PAGE [22], and the gels were stained with Coomassie Blue. For western blotting analysis, the proteins separated by SDS/PAGE were transferred to polyvinylidene fluoride membranes (Hybond-P) by semidry system (Bio-Rad, Hercules, CA, USA) in 25mm Tris/HCl buffer with 192mm glycine (pH 8.3) and 20% (v/v) methanol. After blocking in TBS buffer (20mm Tris/HCl and 100mm NaCl) containing 3% (w/v) nonfat dry milk, the membranes were incubated with polyclonal anti-GST antibodies (1:15000). The western blots were visualized with horseradish peroxidase-conjugated secondary anti-goat IgG (1:50000) for GST and detected by chemiluminescence. Confirmation of the amino acid sequence of BdTTM3 was performed using a Thermo Scientific™ Q Exactive™ Mass Spectrometer(Thermo Fisher Scientific,Waltham, MA, USA). Verification of the molecular mass of BdTTM3 was performed using a Waters Q-TOF Premier™ Mass Spectrometer (Institute of Biochemistry and Biophysics of the Polish Academy of Sciences). The AC activity of BdTTM3 was determined by estimating the rate of cAMP formation. For the BdTTM3 assay, the reaction mixture contained 50mm Tris/HCl buffer (pH 7.5), 5mm MgCl2, 5mm MnCl2, 0.5mm IBMX (3-isobutyl-1-methylxanthine), 0.5mm dipyridamole, 1mm DTT, ATP as a substrate with a concentration between 0.1 and 2mm, and 7µg of the purified enzyme in a final volume of 100µL. After incubation at 30°C for 30min, the reaction was stopped by the addition of 900µL of 75% EtOH, and the samples were centrifuged at 16000g for 10min. Each sample was lyophilized, and the total cAMP concentration was determined in triplicate using a cAMP Biotrak EIA Kit (GE Healthcare, Uppsala, Sweden) according to the acetylation protocol. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used as an alternative method for the AC activity assay. LC-MS/MS experiments were performed using the Nexera UHPLC and LCMS-8045 integrated system (Shimadzu Corporation, Canby, OR, USA). The conditions of the experiments are detailed in Table S1. The enzyme activity was defined as the amount of cAMP produced by 1mg of protein per minute. Free phosphate released by BdTTM3 was measured with the Malachite Green Phosphate Assay Kit (Echelon Biosciences Inc.,Salt Lake City, UT, USA). The reaction mixture contained 50mm Tris/HCl, 5mm MgCl2, 5mm MnCl2, pentasodium tripolyphosphate hexahydrate (PPPi) or ATP as substrates at concentrations between 0.1 and 2mm, and 7µg of the purified enzyme in a final volume of 25µL. After incubation at 30°C for 30min, the reaction was stopped by the addition of 100µL of Malachite Green solution. After incubation at room temperature for 20min, the absorbance at 620nm was recorded (Epoch2 Microplate Reader; BioTek, Winooski, VT, USA). The enzyme activity was defined as the amount of free phosphate produced by 1mg of protein per minute. Two E.coli strains deficient in AC, the E.coli SP850 strain (lam-, el4-, relA1, spoT1, cyaA1400(::kan), thi-1; accession number 7200) and the E.coli CA8306 strain (lam-, el4-, relA1, spoT1, cya854(del), thiE1; accession number 6027), were obtained from the E.coli Genetic Stock Center (Yale University, New Haven, CT, USA). The complementation test was performed as previously described [23]. Quantitative RT–PCR was performed to analyze the dynamic changes in the transcript level of BdTTM3 after mechanical wounding of B.distachyon leaves. Total RNA was isolated from plant tissues with the GeneMATRIX Universal RNA Purification Kit (EURx) and used as a template for first-strand cDNA synthesis (Transcriptor High Fidelity cDNA Synthesis Kit; Roche,Mannheim, Germany). For the qRT–PCR analysis of BdTTM3 transcript levels, gene-specific primers and a specific hydrolysis probe were used. The actin gene, BdACT1, from B.distachyon (Genebank no. XP_003560589), was used as an internal control (Table S1). Expression analyses were performed using the LightCycler TaqMan Master Ready-to-use Hot Start Reaction Mix and the LightCycler 2.0 Carousel-Based System (Roche) in controlled conditions (Table S1). The software used for the relative mRNA-level analyses was lightcycler Software 4.0 (Roche). The relative expression was calculated using the standard curves obtained from serial dilution of cDNAs for both the studied and reference genes. The final values were determined by an automated method (light cycler Software 4.0; Roche), which is recommended for all applications. All measurements were performed with three replicates. All data are presented as the mean±standard error (SE). Statistical differences were determined with the t-test at the level of P 0.05, and P 0.01 was considered significant. The amino acid sequence of BdTTM3, which is referred to in the NCBI database as a putative ‘TTM 3’ (NCBI: XM_010236256.3), indicates that the studied protein belongs to the CYTH-like phosphatase superfamily. A.thaliana CYTH superfamily members (AtTTM1-AtTTM3) were previously characterized in the context of their biochemical properties and physiological function [8-10, 19]. To date, these three proteins have been the only experimentally studied TTMs in the entire plant kingdom. Analysis of the amino acid sequence of BdTTM3 from B.distachyon revealed only 43% amino acid identity with AtTTM3 from A.thaliana [8]. B.distachyon is a monocotyledonous plant, whereas A.thaliana a dicotyledonous plant, so discrepancies in the amino acid sequences of these proteins may result from their separate evolutionary development. The evolutionary relationships between the amino acid sequences in these proteins identified by the BlastP program were established by constructing a dendrogram (Fig. 1). The results show that the highest amino acid identity was observed between ‘AC’ and ‘TTMs 3’ belonging to monocotyledons such as Panicumhallii (88%), Setariaitalica (86%), Oryzasativa (85%), Hordeumvulgare (85%), Sorghumbicolor (85%), Aegilopstauschii (85%), and Zeamays (83%), and the lowest homology ( 30%) was observed between BdTTM3 and prokaryote CYTH domain-containing proteins. Figure 1Open in figure viewerPowerPoint Species-specific relationships between known plant TTM s. A rooted phylogenetic tree was constructed using the CLC Main Workbench 5 software with the neighbor-joining method. The EMBL database accession numbers are as follows: TTM 3 B.distachyon (XM_010236256.3), adenylate cyclase Z.mays (NP_001136955.1), TTM 3 O.sativa (XP_015648702.1), predicted protein H.vulgare (BAJ88761.1), TTM 3-like A.tauschii (XP_020152618.1), hypothetical protein P.hallii (PUZ62805.1), TTM 3 S.italica (XP_004978471.1), TTM 3-like Panicummiliaceum (RLM48708.1), TTM 3 S.bicolor (XP_002463412.1), hypothetical protein Dichantheliumoligosanthes (OEL29426.1), protein product Triticumaestivum (CDM81715.1), adenylate cyclase Saccharumhybrid cultivar (AGT17183.1), TTM 3-like Musaacuminata (XP_009392140.1), TTM 3 Ananascomosus (OAY78534.1), TTM 3 Solanumlycopersicum (XP_004241687.1), TTM 3-like, TTM 3-like Solanumtuberosum (XP_006356230.1), adenylate cyclase Ipomoeanil (ADM83596.1), TTM 3-like Quercussuber (XP_023887999.1), and TTM 3 A.thaliana (sp|Q9SIY3.1). The CYTH domain in BdTTM3 includes the characteristic EXEXK signature, which contains highly conserved glutamate residues important for catalytic activity [6] (Fig. S1). According to DIALIGN program analysis (http://www.genomatix.de/cgi-bin//dialign/dialign.pl), the most highly conserved region of BdTTM3 is a 38-aa-long motif that includes the EXEXK signature located at the N terminus of the protein. Alignment of the BdTTM3 aa sequence with that of proteins annotated as ‘ACs’ or ‘TTMs’ from the seven abovementioned plant species revealed 89% identity within this 38-aa-long region. It was discovered that the four glutamate residues (Glu-2, Glu-4, Glu-90, and Glu-171) form an acidic patch in the vicinity of the AtTTM3 tripolyphosphate substrate [5]. These aa are conserved among many TTMs with absolutely different catalytic activities, including the RNA triphosphatase Cet1p [3], the mouse ThTPase, and E.coli ygiF [5] (Fig. S1). Many members of the TTM family contain two metal ion-binding sites and bind Mg2+/Mn2+ cofactors [3, 5, 24, 25]. The first binding site is formed by the triphosphate substrate with a divalent metal ion complex binding to the tunnel center. In this location, the binding of the ion is facilitated by a glutamate residue (Glu-169 in AtTTM3). A second site containing three glutamate residues originating from the conserved acidic patch coordinates the second metal ion binding. The authors suggest that site no. 1 is essential for proper substrate coordination, whereas no. 2 is involved in the nucleophilic attack of the triphosphate substrate [5]. The proposed TTM reaction mechanism is similar to that found in mammalian AC type V [26]. To assess the properties of the TTM3 protein encoded by the B.distachyon 639bp ORF, the gene was cloned into a pGEX-6P-2 expression vector in frame with a GST tag and then expressed in E.coli BL21 as a GST-BdTTM3 fusion protein. The fusion protein GST-BdTTM3 emerged as a clear main band with a molecular mass of ~55kDa (Fig. 2A). The molecular weight of the 213-aa-long BdTTM3 polypeptide predicted insilico was 23.04kDa, and the isoelectric point was 5.36 (https://web.expasy.org/compute_pi/). When the purified fusion protein was digested, one main 30kDa band appeared (Fig. 2A). Because the predicted molecular mass was not consistent with the protein band observed on SDS/PAGE, the molecular weight of recombinant protein was additionally verified by mass spectrometry. The obtained results confirmed that the assayed protein was 24.05kDa, and the amino acid composition corresponds to that of BdTTM3 (Fig. 2C). Western blot analysis showed that the anti-GST antibody reacted with the 55kDa protein as well as with pure GST (26kDa), demonstrating that the produced protein was GST-BdTTM3 (Fig. 2B). Figure 2Open in figure viewerPowerPoint Purification of the GST-BdTTM3 fusion protein and BdTTM3 pure protein. Panel A (stained gel after protein separation with 10% SDS/PAGE) and Panel B (western blot analysis with anti-GST antibodies) show the steps of purifying the recombinant form of BdTTM3. Lane 1, protein fraction from IPTG-induced E.coli BL21 containing the construct pGEX-6P2-BdTTM3. Lane 2, supernatant of non-IPTG-induced E.coli. Lane 3, purified GST-BdTTM3 fusion protein. Lane 4, recombinant GST-BdTTM3 after protease digestion (in tube). Lane 5, classical protease digestion of fusion protein (directly on resin). Lane 6, GST protein alone. Panel C shows the analysis of the molecular mass of BdTTM3 by mass spectrometry. Based on the available literature data concerning A.thaliana and the divergent nomenclature used in the NCBI database to describe CYTH domain-containing proteins, we decided to experimentally confirm the enzymatic activity of BdTTM3. Thus, the resulting pure recombinant BdTTM3 protein was used to estimate both triphosphatase activity and AC enzymatic activity. Based on previous reports describing the strong affinity of AtTTM3 for tripolyphosphate (PPPi), we tested this compound as an expected substrate for BdTTM3 [8]. The highest PPPase activity was observed in the presence of 0.5mm PPPi, manganese, and magnesium ions when free phosphate reached a maximum concentration of ~11.5nm Pi mg protein−1·min−1 at 30°C (Fig. 3A). Figure 3Open in figure viewerPowerPoint Enzymatic activity of BdTTM3 in response to various concentrations of PPPi (Panel A) and ATP (Panels B, C). The values are the mean of three replicates. Bars represent the SE. Panel D shows complementation of the SP850 and CA8306 E.coli cyaA strains by BdTTM3. Colonies of host cells transformed with the vector containing the BdTTM3 insert developed a strong purple color (I—CA8306 strain with pGEX-BdTTM3 construct, II—SP850 strain with pGEX-BdTTM3 construct), whereas cell transformed with the empty pGEX vector remained colorless (III—SP850 strain with pGEX vector). The ATP substrate for BdTTM3 was also tested in the context of ADP and Pi release and cyclic AMP biosynthesis. The ability of BdTTM3 to hydrolyze ATP was studied in an ATP concentration range between 0.1 and 2mm in the presence of Mg2+ and Mn2+. Free phosphate reached a maximum concentration of ~7.0nm Pi mg protein−1·min−1 in the presence of 0.25mm ATP in the reaction mixture (Fig. 3B). It was revealed that BdTTM3 is approximately two times more active as a PPPase than as an adenosine triphosphatase (ATPase). Interestingly, BdTTM3 was also able to convert the ATP substrate to cyclic AMP in the presence of divalent ions. The maximal BdTTM3 activity was reached at 2mm ATP, and the level of cAMP was ~6pmol·mg protein−1·min−1 (as determined by an ELISA method; Fig. 3C) and 4.5pmol·mg protein−1·min−1 (as determined by an LC/MS/MS method; Fig. 4). It is worth pointing out that the AC activity of BdTTM3 is ~1100 times lower than its activity in catalyzing the decomposition of ATP into ADP and a free phosphate. The cAMP level produced by other proteins with AC activity from A.thaliana was 2.2pmol protein-1·min-1 for AtKUP7 [27] and 7.3pmol protein-1·min-1 for AtClAP [28]. However, for AtTTM1, AtTTM2, and AtTTM3, no cyclase activity was determined [8]. Prokaryotic TTMs function as ACs in the case of Aeromonas and Yersinia, but such enzymatic activity was not detected for C.thermocellum and Nitrosomonaseuropeae [2, 7, 15, 16]. It should be noted here that the level of cAMP synthesized by bacterial and animal ACs is higher than that synthesized by plant enzymes. The average human AC activity was ∼40pmol cAMP mg−1 protein min−1 [29], while kidney-specific rat AC activity was 145pmol cAMP mg−1 protein min−1 [30]; bacterial photoactivated cyclase bPAC from Beggiatoa sp produced ~10nm cAMP mg−1 protein min−1 [31]. Based on the obtained results and the available literature, it can be concluded that cAMP synthesis is not the main function of plant proteins with AC domains [27, 28]. Figure 4Open in figure viewerPowerPoint (A) Determination of AC activity of the BdTTM3 protein by LC-MS/MS. Ion chromatogram of cAMP was generated from a reaction mixture containing 7μg of purified protein and ATP as a substrate in the presence of 4mm Mn2+ and Mg2+. The maximum adenylate cyclase activity of BdTTM3 was reached in the presence of 2mm ATP, generating 4.5pmol cAMP·mg protein−1·min−1. (B) Inset showing the parent cAMP ion at m/z 330.20 [M+H]+ and the corresponding fragmented daughter ion at m/z 136.30 [M+H]+. The fragmented product ion was used for quantitation. Additionally, to confirm the AC activity of BdTTM3, the complementation of the cyaA mutation in two E.coli AC-deficient strains (E.coli SP850 and E.coli CA8306) was performed. The complementation test using E.coli strains lacking the AC (cyaA) has already been used to confirm the activity of some potential plant ACs [23, 27, 28]. It is a fast and valuable screening tool for candidates for the AC nomenclature. Neither mutant was able to induce the expression of the lactose operon or utilize lactose, which resulted in the production of colorless colonies on MacConkey agar. Bacterial mutants were transformed with the pGEX-6P-2-BdTTM3 construct containing the BdTTM3 ORF and empty pGEX vector (negative control) and then streaked on MacConkey agar containing ampicillin, IPTG, and maltose. It was noted that both E.coli cyaA-deficient mutants containing the BdTTM3 insert formed red-colored colonies, indicating acid production during lactose fermentation, which confirmed that BdTTM3 can act as an AC (Fig. 3D). Bacterial colonies transformed with the empty pGEX vector remained colorless on MacConkey plates. In summary, the results indicated BdTTM3 had the strongest affinity for PPPi and weaker affinity for ATP as a substrate. The ATP substrate was mainly used to release ADP and Pi; however, the production of cyclic AMP was confirmed by three independent methods. It is worth pointing out that a large difference in the ATPase and adenylate cyclase activity of BdTTM3 was observed, which suggests that the predominant function of the studied protein is as an ATPase and that AC activity can occur occasionally. It was previously confirmed that proteins belonging to the TTM family show many different catalytic activities and substrate preferences [1, 5]. As an example, we can site three known plant TTMs from A.thaliana that differ in their affinity for the substrate. AtTTM3 had a strong preference for tripolyphoshpate (PPPi), whereas AtTTM1 and AtTTM2 had a high affinity for PPi [9, 10]. Differences in the substrate preferences of individual TTM family members from E.coli, mice, yeast, and some fungi have also been studied and published previously [5, 32, 33]. The authors discovered how some TTMs specifically bind different triphosphate-containing substrates and the kind of catalysis that occurs in the tunnel center. They proposed a model of directional substrate binding for the tunnel center of TTM proteins [5]. It was shown that the triphosphate parts of all ligands are closely aligned in the tunnel center, but the ‘tail’ moieties of the substrates can enter the tunnel domain from opposite sites in different TTMs. This special method of substrate binding allows the proteins from the TTM family to carry out different reactions and produce absolutely different products, while the cleavage site of the substrate is maintained in the vicinity of metal-binding site no. 2. Based on this, it is obvious that the substrate specificity of each individual TTM protein must be experimentally verified because despite the similar structure of all TTMs, the existence of differences in the regulation mechanism has been proven. For example, despite the strong structural homology between TTM polyphosphatases such as AtTTM3 and ygiF and polyphosphate polymerases such as Vtc4p from Saccharomycescerevisiae, they show very significant mechanistic differences [5, 33]. It was proposed that the binding of PPPi could be the most basic role of CYTH superfamily members [1]. PPPase activity may be a starting point for multifunctional enzymes, which subsequently have evolved several other types of metal-dependent catalytic mechanisms and acquired other enzymatic activities and functions. It can be concluded that the CYTH superfamily proteins have a highly conserved three-dimensional structure accompanied by vast functional diversity [1]. BdTTM3 gene expression profile in Brachypodiumdistachyon leaves subjected to mechanical wounding The substrate plasticity of TTMs probably allows them to act in a wide spectrum of biological processes. However, knowledge about their physiological function is still limited, especially in plants [8-10, 19]. As a consequence of mechanical wounding, plants initiate a complex cascade of reactions leading to activation of both local and systemic defense mechanisms. It is known that the activity of 8% of all A.thaliana genes is modulated in response to wounding, which demonstrates the extraordinary complexity of these processes [34]. Our studies revealed that the mRNA level of BdTTM3 is significantly upregulated after mechanical wounding of B.distachyon leaves (Fig. 5). The relative expression of BdTTM3 was measured 48h following injury and compared with the BdTTM3 mRNA profile in unwounded B.distachyon leaves. The transcriptional activity of the studied gene in unwounded leaves was consistently low. Mechanical injury stimulated changes in the BdTTM3 expression level both at the beginning (a rapid sixfold increase was observed after 30min postwounding) and in the later stages of the stress response (6–48h). Figure 5Open in figure viewerPowerPoint Expression analysis of BdTTM3 in wounded and unwounded B.distachyon leaves. The data represent three independent experiments showing similar results. Bars represent the SE. White boxes show the calculated values of the BdTTM3 mRNA level in unwounded (control) tissues, and black boxes depict changes in the transcript level of BdTTM3 in mechanically wounded tissues during the 48-h test cycle. Bars represent the SE. Statistical differences between the mean values were determined with the t-test at the level of *P 0.05, and **P 0.01 was considered significant. It is intriguing that BdTTM3 is transcribed as a bicistronic transcript with the anaphase-promoting complex subunit called CDC26 (Fig. S2). Recently, such bicistronic transcripts in A.thaliana AtTTM3 were characterized [19]. It is known that upstream ORFs are coding sequences located in the 5′ untranslated region of mRNAs that may encode small functional peptides regulating the expression of the main ORF. The authors revealed that AtTTM3 and AtCDC26 are translated from a single transcript conserved across the plant lineage. The ORFs of CDC26 and TTM3 are always in close proximity (~150 base pairs in the case of Marchantiapolymorpha and Chlamydomonasreinhardtii and only eight base pairs in A.thaliana) [19]. The protein sequence for BdCDC26 is located 20bp upstream (not in frame) of the BdTTM3 sequence and encodes a 77-aa-long peptide. According to the genetic analysis, the transcription and translation of both genes can be induced upon wounding; however, the mechanism of CDC26 and TTM translation and the functional connection between them remains unknown. Based on the biochemical analysis of BdTTM3, indicating its triphosphatase activity, it can be assumed that BdTTM3 protein synthesized as a consequence of mechanical damage probably acts mainly as a PPPase or an ATPase, and additionally as an AC in B.distachyon cells. In contrast to the previously revealed role of ATP and cyclic AMP in plant stress-induced signaling [21, 35], the function of polyphosphates in these processes was not considered. Most data concerning the PPPi role come from microbial systems. Kohn et al. [18] proposed that PPPases can prevent the accumulation of high levels of PPPi in cells, which is a very strong chelator of divalent cations, including Ca2+ ions. As shown previously, calcium elevation is activated upon wounding in plants [36], so we can assume that the high PPPase activity of BdTTM3 could indirectly regulate the Ca2+ level by hydrolysis of its chelator—PPPi. However, as noted, a high level of ATP is released as a consequence of wounding and mechanical stress in plants and can act as a DAMP (damage-associated molecular pattern) signal [35]. The damage-induced changes are mediated by signaling networks, which include receptors, calcium (Ca2+) influx, kinase cascades, reactive oxygen species (ROS), reactive nitrogen species, and ATP release [37]. Then, ATP is recognized by downstream elements. In light of the biochemical analysis of BdTTM3, it can be suggested that the high expression of BdTTM3 is important for maintaining a balance between ATP, ADP, and cyclic AMP after the injury reaction. In summary, in databases, many proteins belonging to the CYTH family can be found. The phyletic distribution of the CYTH domain suggests that it is an ancient enzymatic domain that is involved in both nucleotide and organic phosphate metabolism [6]. The actual biological functions of proteins with such domains are not clearly understood. The discrepancies in the terminology of ‘ACs’ or ‘TTMs’ with respect to plant proteins with the CYTH domain led us to address this problem and to analyze one of these proteins, BdTTM3, from B.distachyon. Our studies revealed that BdTTM3 acts on nucleotides and has strong triphosphatase and weak AC activity; however, the PPPase and ATPase activities are absolutely predominant. At the moment, it is not possible to explain why PPPase activity prevails. However, the BdTTM3 gene is significantly upregulated by mechanical wounding, suggesting that it may be involved in responses to injury as an element of energy transformation or signal transduction in plant repair programs. This work was supported by grant from the National Science Center (Poland; Grant No. 2018/02/X/NZ1/00103, MINIATURA2) and funds provided by Nicolaus Copernicus University (Torun, Poland) for the Research Program of the Chair of Plant Physiology and Biotechnology. BŚ performed molecular biology and biochemical experiments, analyzed data and wrote the manuscript, MD performed biochemical experiments, MK performed a LC/MS/MS experiments, AP performed qPCR analysis, KJ and AS-J supervised the research design, revised and critically evaluated the manuscript. Table S1. Sequence of the primers employed for PCR amplification in this study. Fig. S1. (A): The colour scheme of the alignment for plant triphosphate tunnel metalloenzymes amino acid conservation. The conservation scores were determined by the program PRALINE. The amino acid alignment sequences are from Brachypodiumdistachyon (XM_010236256.3), Zeamays (NP_001136955.1), Oryzasativa (XP_015648702.1), Aegilopstauschii (XP_020152618.1), Panicumhallii (PUZ62805.1), Setariaitalica (XP_004978471.1), Hordeumvulgare (BAJ88761.1) and Sorghumbicolor (XP_002463412.1). (B) The alignment of BdTTM3 and AtTTM3 sequences with other animal and bacterial described TTMs. The amino acid alignment includes: BdTTM3 from Brachypodiumdistachyon (XM_010236256.3), AtTTM3 from Arabidopsisthaliana (5A5Y_A), thiaminetriphosphatase from Musmusculus (NP_694723.1), ygiF from Escherichiacoli (NP_417526.1), NeuTTM from Nitrosomonaseuropaea (WP_011112064.1). The scoring scheme works from 0 to the last conserved alignment position, up to 10 for the most conserved alignment position. Red colour represents high conservation of amino acids, blue colour low conservation. Fig. S2. (A): A scheme of bicistronic CDC26-TTM3 transcript in Brachypodiumdistachyon. (B) The nucleotide and amino acid sequences BdCDC26 and BdTTM3. BdCDC26 is located 20bp upstream of BdTTM3 sequence and encodes a 77 aa-long peptide. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 1Bettendorff L and Wins P (2013) Thiamine triphosphatase and the CYTH superfamily of proteins. FEBS J 280, 6443– 6455. Wiley Online Library 2Sismeiro O, Trotot P, Biville F, Vivares C and Danchin A (1998) Aeromonas hydrophila adenylyl cyclase 2: a new class of adenylyl cyclases with thermophilic properties and sequence similarities to proteins from hyperthermophilic archaebacteria. J Bacteriol 180, 3339– 3344. 3Gong C, Smith P and Shuman S (2006) Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins. RNA 12, 1468– 1474. 4Gallagher DT, Smith NN, Kim SK, Heroux A, Robinson H and Reddy PT (2006) Structure of the class IV adenylyl cyclase reveals a novel fold. J Mol Biol 362, 114– 122. 5Martinez J, Truffault V and Hothorn M (2015) Structural determinants for substrate binding and catalysis in triphosphate tunnel metalloenzymes. J Biol Chem 290, 23348– 23360. 6Iyer LM and Aravind L (2002) The catalytic domains of thiamine triphosphatase and CyaB-like adenylyl cyclase define a novel superfamily of domains that bind organic phosphates. BMC Genom 3, 33. 7Smith N, Kim SK, Reddy PT and Gallagher DT (2006) Crystallization of the class IV adenylyl cyclase from Yersinia pestis. Acta Crystallogr F Struct Biol Cryst Commun 62, 200– 204. Wiley Online Library 8Moeder W, Garcia-Petit C, Ung H, Fucile G, Samuel MA, Christendat D and Yoshioka K (2013) Crystal structure and biochemical analyses reveal that the Arabidopsis triphosphate tunnel metalloenzyme AtTTM3 is a tripolyphosphatase involved in root development. Plant J 76, 615– 626. Wiley Online Library 9Ung H, Moeder W and Yoshioka K (2014) Arabidopsis triphosphate tunnel metalloenzyme2 is a negative regulator of the salicylic acid-mediated feedback amplification loop for defense responses. Plant Physiol 166, 1009– 1021. 10Ung H, Karia P, Ebine K, Ueda T, Yoshioka K and Moeder W (2017) Triphosphate tunnel metalloenzyme function in senescence highlights a biological diversification of this protein superfamily. Plant Physiol 175, 473– 485. 11Makarchikov AF, Lakaye B, Gulyai IE, Czerniecki J, Coumans B, Wins P, Grisar T and Bettendorff L (2003) Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals. Cell Mol Life Sci 60, 1477– 1488. 12Tsukamoto T, Shibagaki Y, Imajoh-Ohmi S, Murakoshi T, Suzuki M, Nakamura A, Gotoh H and Mizumoto K (1997) Isolation and characterization of the yeast mRNA capping enzyme beta subunit gene encoding RNA 5’-triphosphatase, which is essential for cell viability. Biochem Biophys Res Commun 239, 116– 122. 13Pei Y, Schwer B, Saiz J, Fisher RP and Shuman S (2001) RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans. BMC Microbiol 1, 29. 14Bisaillon M and Shuman S (2001) Structure–function analysis of the active site tunnel of yeast RNA triphosphatase. J Biol Chem 276, 17261– 17266. 15Keppetipola N, Jain R and Shuman S (2007) Novel triphosphate phosphohydrolase activity of Clostridium thermocellum TTM, a member of the triphosphate tunnel metalloenzyme superfamily. J Biol Chem 282, 11941– 11949. 16Delvaux D, Murty MR, Gabelica V, Lakaye B, Lunin VV, Skarina T, Onopriyenko O, Kohn G, Wins P, De Pauw E et al. (2011) A specific inorganic triphosphatase from Nitrosomonas europaea: structure and catalytic mechanism. J Biol Chem 286, 34023– 34035. 17Jain R and Shuman S (2008) Polyphosphatase activity of CthTTM, a bacterial triphosphate tunnel metalloenzyme. J Biol Chem 283, 31047– 31057. 18Kohn G, Delvaux D, Lakaye B, Servais AC, Scholer G, Fillet M, Elias B, Derochette JM, Crommen J, Wins P et al. (2012) High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved. PLoS One 7, e43879. 19Lorenzo-Orts L, Witthoeft J, Deforges J, Martinez J, Loubéry S, Placzek A, Poirier Y, Hothorn LA, Jaillais Y and Hothorn M (2019) Concerted expression of a cell cycle regulator and a metabolic enzyme from a bicistronic transcript in plants. Nat Plants 5, 184– 193. 20Cui F, Brosche M, Sipari N, Tang S and Overmyer K (2013) Regulation of ABA dependent wound induced spreading cell death by MYB108. New Phytol 200, 634– 640. Wiley Online Library 21Świeżawska B, Jaworski K, Duszyn M, Pawełek A and Szmidt-Jaworska A (2017) The Hippeastrum hybridum PepR1 gene (HpPepR1) encodes a functional guanylyl cyclase and is involved in early response to fungal infection. J Plant Physiol 216, 100– 107. 22Laemmli UK (1970) Cleavage of structural proteins during the assembly of the heat of bacteriophage T4. Nature 227, 680– 685. 23Świeżawska B, Jaworski K, Pawełek A, Grzegorzewska W, Szewczuk P and Szmidt-Jaworska A (2014) Molecular cloning and characterization of a novel adenylyl cyclase gene, HpAC1, involved in stress signaling in Hippeastrum x hybridum. Plant Physiol Biochem 80, 41– 52. 24Martins A and Shuman S (2003) Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism. Nucleic Acids Res 31, 1455– 1463. 25Gong C, Martins A and Shuman S (2003) Structure-function analysis of Trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism. J Biol Chem 278, 50843– 50852. 26Tesmer JJ, Sunahara RK, Johnson RA, Gosselin G, Gilman AG and Sprang SR (1999) Two-metal-Ion catalysis in adenylyl cyclase. Science 285, 756– 760. 27Al-Younis I, Wong A and Gehring Ch (2015) The Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre. FEBS Lett 589 (Part B), 3848– 3852. Wiley Online Library 28Chatukuta P, Dikobe TB, Kawadza DT, Sehlabane KS, Takundwa MM, Wong A, Gehring C and Ruzvidzo O (2018) An Arabidopsis clathrin assembly protein with a predicted role in plant defense can function as an adenylate cyclase. Biomolecules 8, 15. 29Hope BT, Nagarkar D, Leonard S and Wise RA (2007) Long-term upregulation of protein kinase A and adenylate cyclase levels in human smokers. J Neurosci 27, 1964– 1972. 30Khan J and Backer W (2003) Distribution of adenylyl cyclase and guanylyl cyclase in rat tissues. J King Abdulaziz Univ Sci 15, 115– 128. https://doi.org/10.4197/Sci.15-1.9. 31Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gärtner W, Petereit L, Efetova M, Schwarzel M et al. (2011) Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. J Biol Chem 286, 1181– 1188. 32Hothorn M, Neumann H, Lenherr ED, Wehner M, Rybin V, Hassa PO, Uttenweiler A, Reinhardt M, Schmidt A, Seiler J et al. (2009) Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science 324, 513– 516. 33Pei Y, Schwer B, Hausmann S and Shuman S (2001) Characterization of Schizosaccharomyces pombe RNA triphosphatase. Nucleic Acids Res 29, 387– 396. 34Cheong YH, Chang HS, Gupta R, Wang X, Zhu T and Luan S (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129, 661– 677. 35Tanaka K, Choi J, Cao Y and Stacey G (2014) Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants. Front Plant Sci 5, 446. 36Kiep V, Vadassery J, Lattke J, Maaß JP, Boland W, Peiter E and Mithöfer A (2012) Systemic cytosolic Ca(2+) elevation is activated upon wounding and herbivory in Arabidopsis. New Phytol 207, 996– 1004. Wiley Online Library 37Prasad A, Sedlářová M, Kale RS and Pospíšil P (2017) Lipoxygenase in singlet oxygen generation as a response to wounding: in vivo imaging in Arabidopsis thaliana. Sci Rep 7, 9831. Please check your email for instructions on resetting your password. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. Can\'t sign in? Forgot your username? 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