A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.Peer reviewe

Structural Studies of Membrane-Bound Pyrophosphatases

Membrane-bound pyrophosphatases (M-PPases) catalyze a reaction where the free energy released from pyrophoshate (PPi) hydrolysis is converted into a sodium and/or proton gradient by pumping these ions across the membrane [1]. They are found in plants, protozoans and prokaryotes and are important for survival in abiotic stress conditions such as cold, hypoxia, salt stress and low-light intensity [2, 3][4]. In plants, M-PPases are the main hydrolysers of cytoplasmic pyrophosphate, which source in plants, and in other organisms, are the various anabolic reactions such as DNA, RNA and protein synthesis in which PPi is released as a by-product of ATP hydrolysis [5]. This hydrolytic activity is important for plant maturation [6], as build up of pyrophosphate inhibits the gluconeogenesis and cellulose synthesis and the above mentioned anabolic reactions [5]. Based on their function, M-PPases can be divided into four groups: K+-dependent Na+-pumps, K+-dependent H+-pumps, K+-dependent Na+, H+-pumps and K+-independent H+-pumps (Luoto et al., 2013). The K+-dependent pumps require potassium for full activity, but have reduced activity without it [7]. M-PPases are dimeric [4] and have 14 to 17 transmembrane helices [8]. Most of the conserved residues necessary for M-PPase activity occur in the cytoplasmic part of the protein [7] [9][4]. These residues take part in binding Mg2PPi, the physiological substrate of M-PPases, and Mg2+, a necessary cofactor for catalysis. The aim of this study was to solve the structure of a membrane-bound pyrophosphatase. To find a suitable target protein for X-ray crystallography, eight M-PPases were expressed in Saccharomyces cerevisiae. Three expressed at levels of 0.5 mg/l or higher: the K+-dependent Na+-pump of Thermotoga maritima (TmPPase) and the K+-independent H+-pumps of Pyrobaculum aerophilum (PaPPase) and Thiobacillus denitrificans (TdPPase). TmPPase and PaPPase were purified by the hot-solve -protocol [10, 11] and I showed that both proteins were purified in their native oligomeric, dimeric, form [10]. Both TmPPase and PaPPase were crystallised, and the activity and crystallisability of both of these proteins were tested in a range of different detergents [10, 12]. TmPPase crystals diffracting to 2.6 Å could be grown [13] [13] in the presence of a novel octyl neopentyl glycol detergent (OGNPG, [14], and these crystals allowed the protein structure to be solved. Phasing the TmPPase structure was done by multiple isomorphous replacement with anomalous scattering (MIRAS) using Na2WO4 and tri-metyl lead acetate (TMLA) derivatised crystals and molecular replacement with Rosetta [15]. This work yielded a 2.6 Å structure of TmPPase in the metal-bound, resting state (TmPPase:Mg:Ca). Molecular replacement using this structure as a model was used to solve a 4 Å structure of TmPPase in the product bound conformation (TmPPae:Mg4:K:Pi2). The solved, high-resolution TmPPase structure is very similar (r.m.s.d 1.57 Å for 517/618 aligned Cas) to that of Vigna radiata proton pumping M-PPase, which was also solved in 2012 (VrPPase:PNP, [16]. Both structures show a dimeric protein where the monomer consists of 16 α-helix containing subunits. The membrane spanning helices extend up to 27 Å into the cytoplasm and six of them (helices 5, 6, 11, 12, 15 and 16) enclose the active site cavity. Below the active site is a series of small cavities surrounded by helices 5, 6, 12 and 16 that leads to the periplasm/lumen and seems to form the exit channel for ion transfer. The cytoplasmic active site cavity is lined by conserved residues and has a three part structure consisting of a hydrolytic centre where the substrate binds, a coupling funnel that couples pyrophosphate hydrolysis to ion-pumping, and a gate that connects the cytoplasmic and periplamic/vacuolar cavities. Comparison of the TmPPase:Mg4Pi2 and TmPPase:Mg:Ca structures with VrPPase:PNP allowed me to analyse the catalytic cycle of M-PPases. The three M-PPase structures show that binding of the substrate induces both the ordering and movement of the loop between helices 5 and 6, which in turn closes the active site, and the movement of helix 12 towards the periplasmic part of the protein. Also, in the VrPPase:PNP structure, a conserved arginine is close to the cluster of conserved residues forming the gate. Based on these observations, I proposed a model of the catalytic cycle of M-PPases in which binding of the substrate leads to the formation of a transitory intermediate in which movement of helix 12 and the conserved arginine leads to opening of the gate and exit channel and to ion pumping. The molecular structures of TmPPase have also shed light on the evolution of M-PPases: superposition of a structural motif containing four α-helices shows that M-PPases arose through gene triplication. 1. Malinen, AM, GA Belogurov, AA Baykov, R Lahti: Na+-pyrophosphatase: a novel primary sodium pump. Biochemistry 2007, 468872-8878. 2. Garcia-Contreras, R, H Celis, I Romero: Importance of Rhodospirillum rubrum H+-pyrophosphatase under low-energy conditions. J Bacteriol 2004, 1866651-6655. 3. Lopéz-Marqués, RL, JR Péréz-Castineira, M Losada, A Serrano: Differential regulation of soluble and membrane-bound inorganic pyrophosphatases in the photosynthetic bacterium Rhodospirillum rubrum provides insights into pyrophosphate-based stress bioenergetics. J Bacteriol 2004, 1865418-5426. 4. Serrano, A, JR Pérez-Castineira, M Baltscheffsky, H Baltscheffsky: H+-PPases: yesterday, today and tomorrow. IUBMB Life 2007, 5976-83. 5. Baykov, AA, Cooperman, BS, Goldman, A, Lahti, R: Cytoplasmic Inorganic Pyrophosphatase. In Inorganic polyphosphates(23). Edited by HC Schröder. Springer Verlag; 1999:127-150. 6. Ferjani, A, S Segami, G Horiguchi, Y Muto, M Maeshima, H Tsukaya: Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 2011, 232895-2908. 7. Maeshima, M: Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 2000, 146537-51. 8. Mimura, H, Y Nakanishi, M Hirono, M Maeshima: Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis. J Biol Chem 2004, 27935106-35112. 9. McIntosh, MT, AB Vaidya: Vacuolar type H+-pumping pyrophosphatases of parasitic protozoa. Int J Parasitol 2002, 321-14. 10. Kellosalo, J, T Kajander, MG Palmgren, RL Lopéz-Marqués, A Goldman: Heterologous expression and purification of membrane-bound pyrophosphatases. Protein Express Purif 2011, 7925-34. 11. López-Marqués, RL, JR Pérez-Castiñeira, MJ Buch-Pedersen, S Marco, JL Rigaud, MG Palmgren, A Serrano: Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: a "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins. Biochim Biophys Acta 2005, 171669-76. 12. Kellosalo, J, T Kajander, R Honkanen, A Goldman: Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases. Mol Membr Biol 2013, 3064-74. 13. Kellosalo, J, T Kajander, K Kogan, K Pokharel, A Goldman: The structure and catalytic cycle of a sodium pumping pyrophosphatase. Science 2012, 337473-476. 14. Chae, PS, SGF Rasmussen, RR Rana, K Gotfryd, R Chandra, MA Goren, AC Kruse, S Nurva, CJ Loland, Y Pierre: Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. nAture methods 2010, 71003-1008. 15. Cowtan, K: Fast Fourier feature recognition. Acta Crystallogr D 2001, 571435-1444. 16. Lin, SM, JY Tsai, CD Hsiao, YT Huang, CL Chiu, MH Liu, JY Tung, TH Liu, RL Pan, YJ Sun: Crystals structure of membrane-embedded H+-translocating pyrophosphatase. Nature 2012, 484399-403.Kalvoon sitoutuneet pyrofosfataasit (M-PPaasit) katalysoivat kemiallista reaktioita, jossa pyrofosfaatin (PPi) hydrolyysistä vapautunut vapaa energia ajaa natriumionien ja/tai protonien pumppausta solukalvon yli. Kyseisiä proteiineja esiintyy kasveissa, alkueläimissä ja prokaryooteissa ja ne ovat tärkeitä abioottisen stressin, kuten kylmän, kuivuuden, vähävaloisuuden, hypoxian ja suolastressin siedon kannalta [1, 2][3]. Kasveissa M-PPaasit huolehtivat sytoplasmisen PPi:n hydrolyysistä, jonka kertyminen estää glukoneogeneesiä ja selluloosan, DNA:n, RNA:n ja proteiinien synteesiä ja haittaa täten kasvien kehitystä [4]. Pyrofosfaattia muodostuu edellä mainituissa anabolisissa reaktioissa, joissa se vapautuu ATP:n hydrolyysissä [5]. Toimintansa perusteella M-PPaasit voidaan luokitella neljään ryhmään: K+-riippuvaisiin Na+-pumppuihin, K+-riippuvaisiin H+-pumppuihin, K+-riippuvaisiin Na+, H+ -pumppuihin ja K+-riippumattomiin H+-pumppuihin (Luoto et al., 2013). K+-riippuivaiset pumput ovat aktiivisimmillaan sitoessaan kaliumia, mutta niillä on kuitenkin mitattava, vaikkakin alhaisempi, aktiivisuus ilman kaliumia [6]. M-PPaasit ovat dimeerisiä proteiineja ja niillä on proteiinista riippuen 14-17 transmembraanihelixiä (TMH) [7]. Suurin osa M-PPaasien aktiivisuuteen tarvittavista konservoituneista aminohapoista sijaitsee proteiinin sytoplasmisella puolella [6] [8][3]. Nämä aminohapot ottavat osaa Mg2PPi:n, entsyymin fysiologisen substraatin, ja Mg2+:n, aktiivisuuden kannalta välttämättömän ligandin, sitomiseen. Tämän työn tavoitteena oli M-PPaasin molekyylirakenteen ratkaiseminen. Kahdeksasta tähän tarkoitukseen valitusta kohdeproteiinista, yksi, Thermotoga maritiman K+-riippuvainen Na+-pumppu (TmPPase), tuotti kiteitä joita pystyttiin käyttämään rakenteen ratkaisemiseen (Kellosalo et al., 2012). TmPPaasin rakenne, ensimmäinen Suomessa ratkaistu kalvoproteiinin rakenne, paljasti dimeerisen proteiinin, jonka monomeeri koostuu 16:sta TMH:sta (Kellosalo et al., 2012). Nämä TMH:t jatkuvat n. 27 Å sytoplasman puolelle ja ne muodostavat hydrolyyttisesta keskuksesta , kytkentäsuppilosta , portista ja ulostuloskanavasta koostuvan, neliosaisen rakenteen (Kellosalo et al., 2012). Pyrofosfaatti sitoutuu proteiinin sytoplasmisessa päässä olevaan hydrolyyttiseen keskukseen (Lin et al., 2012), jossa tapahtuvat muutokset välittyvät konservoituneista, varautuneista aminohapoista muodostunutta kytkentäsuppiloa pitkin portille (Kellosalo et al., 2012). Muutokset portin rakenteessa mahdollistavat ionin pumppaamisen ulostulokanavan kautta periplasmiseen tilaan (Kellosalo et al., 2012). TmPPaasi-rakenteiden, joissa proteiini on lepotilassa tai sitoo lopputuoteena olevaa fosfaattia (Kellosalo et al., 2012), vertailu Vigna radiatan protoneja pumppaavan M-PPaasin substraattia sitovan rakenteen (Lin et al., 2012) kanssa paljasti TMH12:sta ja R191:n liikkuvan katalyyttisen syklin aikana (Kellosalo et al., 2012). Uskomme, että nämä muutokset proteiinirakenteessa ajavat portin ja ulostulokanavan avautumista ja täten ionin pummpausta. TmPPaasin molekyylirakenne on myös auttanut M-PPaasien evolution selvittämisessä: proteiinirakenteessa toistuu neljästä alfa-heliksistä koostuva motiivi, joka osoittaa että M-PPaasit ovat kehittyneet geenitriplikaation myötä (Kellosalo et al., 2012). 1. Garcia-Contreras, R, H Celis, I Romero: Importance of Rhodospirillum rubrum H+-pyrophosphatase under low-energy conditions. J Bacteriol 2004, 1866651-6655. 2. Lopéz-Marqués, RL, JR Péréz-Castineira, M Losada, A Serrano: Differential regulation of soluble and membrane-bound inorganic pyrophosphatases in the photosynthetic bacterium Rhodospirillum rubrum provides insights into pyrophosphate-based stress bioenergetics. J Bacteriol 2004, 1865418-5426. 3. Serrano, A, JR Pérez-Castineira, M Baltscheffsky, H Baltscheffsky: H+-PPases: yesterday, today and tomorrow. IUBMB Life 2007, 5976-83. 4. Ferjani, A, S Segami, G Horiguchi, Y Muto, M Maeshima, H Tsukaya: Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 2011, 232895-2908. 5. Baykov, AA, Cooperman, BS, Goldman, A, Lahti, R: Cytoplasmic Inorganic Pyrophosphatase. In Inorganic polyphosphates(23). Edited by HC Schröder. Springer Verlag; 1999:127-150. 6. Maeshima, M: Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 2000, 146537-51. 7. Mimura, H, Y Nakanishi, M Hirono, M Maeshima: Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis. J Biol Chem 2004, 27935106-35112. 8. McIntosh, MT, AB Vaidya: Vacuolar type H+-pumping pyrophosphatases of parasitic protozoa. Int J Parasitol 2002, 321-14

Kendomycin Cytotoxicity against Bacterial, Fungal, and Mammalian Cells Is Due to Cation Chelation

Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.Peer reviewe

Relation of gallbladder function and Helicobacter pylori infection to gastric mucosa inflammation in patients with symptomatic cholecystolithiasis

Background. Inflammatory alterations of the gastric mucosa are commonly caused by Helicobacter pylori (Hp) infection in patients with symptomatic gallstone disease. However, the additional pathogenetic role of an impaired gallbladder function leading to an increased alkaline duodenogastric reflux is controversially discussed. Aim:To investigate the relation of gallbladder function and Hp infection to gastric mucosa inflammation in patients with symptomatic gallstones prior to cholecystectomy. Patients: Seventy-three patients with symptomatic gallstones were studied by endoscopy and Hp testing. Methods: Gastritis classification was performed according to the updated Sydney System and gallbladder function was determined by total lipid concentration of gallbladder bile collected during mainly laparoscopic cholecystectomy. Results: Fifteen patients revealed no, 39 patients mild, and 19 moderate to marked gastritis. No significant differences for bile salts, phospholipids, cholesterol, or total lipids in gallbladder bile were found between these three groups of patients. However, while only 1 out of 54 (< 2%) patients with mild or no gastritis was found histologically positive for Hp, this infection could be detected in 14 (74%) out of 19 patients with moderate to marked gastritis. Conclusion: Moderate to marked gastric mucosa inflammation in gallstone patients is mainly caused by Hp infection, whereas gallbladder function is not related to the degree of gastritis. Thus, an increased alkaline duodenogastric reflux in gallstone patients seems to be of limited pathophysiological relevance. Copyright (c) 2006 S. Karger AG, Basel

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61 alpha subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substratenonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61 alpha mutations identified from human HCT116 cells su ests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61 alpha mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61 alpha resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved druglike properties that are based on the coibamide pharmacophore.Peer reviewe

Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5-6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a 'molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.Peer reviewe

Overcoming bottlenecks in the membrane protein structural biology pipeline

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future

A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins

VIRTUALISOINTIA HYÖDYNTÄVÄ LINUX-POHJAINEN CNA-LABORATORIOYMPÄRISTÖ

Työn tavoitteena on toteuttaa virtualisointia hyödyntävä Linux-pohjainen CNA-laboratorioympäristö, jossa opettajalla on etäyhteys mahdollisuus oppilaiden virtuaalikoneisiin ja niihin kytkettyihin CNA-laitteisiin. Työssä asennetaan myös uudet Cisco-laitteet CNA-luokan kytkinkaappiin ja järjestellä vanhat uudelleen siten, että kunkin laboratoriotyöryhmän olisi helpompi hahmottaa heille kuuluva laiteko-koonpano. Opinnäytetyössä tutustutaan Lubuntu-käyttöjärjestelmään, live levykuvan luomiseen Pinguybuilder-ohjelman avulla ja virtualisoinnin toteuttamiseen Vmware-ohjelmalla. Lisäksi opettajalle tehdään mah-dollisuus muodostaa etäyhteys SSH:n avulla oppilas koneisiin ja sieltä telnet-yhteyden avulla Cisco-laitteisiin. Tarvittavat toiminnot toteutetaan käyttämällä BASH-skriptausta ja JAVA-ohjelmointikieltä.The objective of this thesis was to implement virtualization using Linux based operating system in CNA laboratory environment. The instructor has remote access to students’ virtual machines and Cis-co equipment that is connected. The thesis also includes information on the installation of new equip-ment to the switch rack in the CNA classroom and reorganization of the old equipment for better view of machine groups. The thesis introduce Lubuntu operating system, creating of live image using PinguyBuilder program and utilizing virtualization using Vmware. Additionally, the instructor will have remote access to stu-dent machine using SSH and from there via telnet to Cisco equipment. The necessary functions were implemented by using BASH scripting and JAVA language

Hoitotyön opiskelijan valmistautuminen ensimmäiseen ohjattuun harjoitteluun-opetusvideo

Opinnäytetyömme tarkoituksena oli tehdä opetusvideo Centria-ammattikorkeakoulun hoitotyön opiskelijoille ennen ensimmäiseen ohjattuun harjoitteluun menoa tuotekehitysprojektin muodossa. Opettajat voivat hyödyntää opetusvideota tulevien hoitotyön opiskelijoiden opetuksessa. Opinnäytetyömme tavoitteena on auttaa uusia hoitotyön opiskelijoita oppimaan sekä omaksumaan ohjeet ja toimimaan ohjatussa harjoittelussa niiden mukaan, jolloin ohjattuun harjoitteluun on helpompi mennä. Toivomme että ensimmäisen vuoden hoitotyön opiskelijat saavat opetusvideomme kautta varmuutta ja tietoa, miten toimia, kun he aloittavat ensimmäisen ohjatun harjoittelunsa. Opinnäytetyö tuotettiin tuotekehittelyprojektina, jonka lopputuloksena syntyi opetusvideo Centria-ammattikorkeakoulun käyttöön. Centria-ammattikorkeakoulu voi hyödyntää opetusvideota uusien hoitotyön opiskelijoiden opetuksessa sekä ohjattuun harjoitteluun menossa. Hoitotyön opiskelijoiden ja työelämän yhteyshenkilöiden sekä teoriatiedon avulla tuotimme kattavan sekä katsojaa palvelevan asiakokonaisuuden opetusvideon muodossa. Opinnäytetyön tietoperusta sisältää tietoa oppimismenetelmistä, opetusvideon merkityksestä, hoitotyön koulutuksesta ja ohjatusta harjoittelusta. Tietoperusta sisältää laajemmin kaikki aiheet, joita opetusvideossa käsitellään. Opetusvideo tuotettiin projektin muodossa kevään 2017 aikana. Mukana oli kaksi hoitotyön ammattilaista Kokkolan terveyskeskussairaalasta sekä hoitotyön opiskelijoita. Teimme tiiviisti yhteistyötä toisen opinnäytetyöryhmän kanssa, jotka tekivät samasta aiheesta verkkotehtävän. Opetusvideon jätämme vapaaseen käyttöön Centria-ammattikorkeakoululle. Jatkotutkimusaiheena nousi esille opinnäytetyönä tehtävä ohjelehtinen kaikista ohjatuista harjoitteluista. Se sisältäisi asioita, mistä pitää ottaa selvää ennen harjoittelujakson alkamista, ohjatun harjoittelun tavoitteet ja kaikki tarvittavat asiakirjat, jotka harjoittelujaksoon kuuluvat.The purpose of this thesis was to do a teaching video for nursing students of Centria University of Applied Sciences before they go to their first guided practical training in the form of a product development project. Teachers can take advantage of the video in the nursing students education. The aim of the thesis is to help new nursing students to learn and to adopt the instructions and act in guided practical training according to them, so it will be easier to go to the guided practical training. We hope that our teaching video gives confidence and knowledge to the first year nursing students how to act when they start their first guided practical training. The thesis was produced as the product development project the result of which was the teaching video for Centria University of Applied Sciences. Centria University of Applied Sciences can utilize the teaching video in the teaching of new nursing students as well as in guided practical training. With the help of nursing students, contacts of working life and theoretical knowledge we produced comprehensive and user-oriented complex of issues in the form of the teaching video. The thesis data base contains knowledge of learning methods, significance of the teaching video, education of nursing and guided practical training. The data base contains more broadly all topics discussed in the teaching video. The teaching video was produced in the form of the project during the spring of 2017. There were involved two nursing professionals from Kokkola Health Centre Hospital and nursing students from Centria University of Applied Sciences. We worked closely with another thesis group who did an online task on the same subject. We leave the teaching video to free use for Centria University of Applied Sciences. As a further study subject came up a thesis to be made of a data sheet on all guided practical trainings, which contains things that you have to know before starting practical training, practical training objectives and all documents needed and included in practical training