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17.4E: Jestive alge - biologija

17.4E: Jestive alge - biologija


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Jestive alge stoljećima se koriste kao hrana u mnogim obalnim regijama diljem svijeta.

ciljevi učenja

  • Opišite hranjivu vrijednost algi

Ključne točke

  • Alge su vrlo raznolika skupina općenito jednostavnih jednostaničnih ili višestaničnih eukariotskih organizama.
  • Alge su izvrsne hranjive vrijednosti jer sadrže potpune proteine, vlakna, a ponekad i visoku razinu omega-3 masnih kiselina, mnoge vitamine i minerale.
  • Neki spojevi koji se koriste kao dodaci u prehrambenoj industriji izolirani su iz algi.

Ključni uvjeti

  • potpuni protein: Potpuni protein (cijeli protein) je protein koji sadrži svih devet esencijalnih aminokiselina.

Alge su vrlo raznolika skupina općenito jednostavnih jednostaničnih ili višestaničnih eukariotskih organizama. Većina njih su autotrofne što znači da mogu iz atmosfere pokupiti ugljikov dioksid i pretvoriti ga u organsku tvar. Svoj fotosintetski aparat naslijedili su od cijanobakterija. Cijanobakterije se ponekad nazivaju i plavo-zelene alge, ali su prokariotski organizmi i nisu prave alge. Neke vrste cijanobakterija koriste se i kao hrana.

Morske alge su jestive alge koje se stoljećima koriste kao hrana u mnogim obalnim regijama diljem svijeta. Mogu pripadati jednoj od tri skupine višestaničnih algi: crvenoj, zelenoj ili smeđoj. U zemljama poput Kine, Japana, Koreje i donekle Islanda, Irske, Čilea i Novog Zelanda alge su dio svakodnevne prehrane ljudi. Obično su morskog podrijetla budući da su slatkovodne alge često otrovne.

Alge su izvrsne hranjive vrijednosti jer sadrže potpune proteine ​​(za razliku od biljne hrane ubrane na kopnu), vlakna, a ponekad i visoku razinu omega-3 masnih kiselina. Zapravo, omega-3 kiseline u ribi potječu iz mikroalgi koje se konzumiraju na dnu piramida hrane i postupno prelaze do ribe na vrhu. Alge su također bogate mnogim vitaminima, poput A, C, B1, B2, B3 i B6, kao i minerali, poput joda, kalcija, kalija, magnezija i željeza. Konzumiraju se kuhani, sušeni i sirovi.

Uzgojene mikroalge i cijanobakterije kao npr Spirulina i Chlorella prodaju se kao dodaci prehrani. Hidrokoloidi kao što su agar, alginat i karagenan izolirani su iz divljih i uzgojenih algi i koriste se kao dodaci u prehrambenoj industriji zbog svojih svojstava emulgiranja i zgušnjavanja. Neki od složenih polisaharida koji se nalaze u algama mogu biti probavljeni bakterijama u crijevima budući da su potrebni enzimi za probavu obilno prisutni kod Japanaca, ali ih nema u Sjevernoj Americi.


Interferon

Interferoni (IFNs, / ˌ ɪ n t ər ˈ f ɪər ɒ n / [1]) su skupina signalnih bjelančevina [2] koje stvaraju i oslobađaju stanice domaćini kao odgovor na prisutnost nekoliko virusa. U tipičnom scenariju, stanica zaražena virusom oslobađa interferone uzrokujući da stanice u blizini pojačaju svoju protuvirusnu obranu.

IFN -i pripadaju velikoj klasi proteina poznatih kao citokini, molekule koje se koriste za komunikaciju između stanica kako bi pokrenule zaštitnu obranu imunološkog sustava koja pomaže u iskorjenjivanju patogena. [3] Interferoni su dobili ime po sposobnosti da "ometaju" replikaciju virusa [3] štiteći stanice od virusnih infekcija. IFN također imaju razne druge funkcije: aktiviraju imunološke stanice, poput prirodnih stanica ubojica i makrofaga, povećavaju obranu domaćina tako što reguliraju prezentaciju antigena zahvaljujući povećanju ekspresije glavnih antigena kompleksa histokompatibilnosti (MHC). Određeni simptomi infekcija, poput vrućice, bolova u mišićima i "simptoma sličnih gripi", također su uzrokovani proizvodnjom IFN-a i drugih citokina.

U životinja, uključujući ljude, identificirano je više od dvadeset različitih IFN gena i proteina. Obično su podijeljeni u tri klase: IFN tipa I, IFN tipa II i IFN tipa III. IFN -i koji pripadaju sve tri klase važni su za borbu protiv virusnih infekcija i za regulaciju imunološkog sustava.


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Metabolomsko profiliranje različitih vrsta morskih algi razlikuje smeđe, crvene i zelene alge

Među skupinama algi, smeđe alge imale su karakteristično visoku koncentraciju manitola, a zelene alge fruktozu. Kod crvenih algi potrebno je ocijeniti profile metabolita pojedinih vrsta.

Morske alge metabolički se razlikuju od kopnenih biljaka. Međutim, opći profili metabolita tri glavne skupine morskih algi, smeđih, crvenih i zelenih algi, te učinak različitih metoda ekstrakcije na rezultate profiliranja metabolita nisu svestrano istraženi. U ovom smo istraživanju ocijenili metabolite topive u vodi u četiri smeđe, pet crvenih i dvije zelene alge prikupljene s dva mjesta u sjevernom Japanu, smještena u Japanskom moru i Tihom oceanu. Liofilizirani uzorci algi obrađeni su ekstrakcijom metanol-voda sa ili bez kloroforma i analizirani kapilarnom elektroforezom i tekućinskom kromatografijom-masenom spektrometrijom za karakterizaciju metabolita. Profili koncentracije metabolita pokazali su karakteristične karakteristike koje ovise o vrstama i taksonomskim skupinama, dok metode ekstrakcije nisu imale značajan učinak. Taksonomske razlike između različitih profila metabolita morskih algi bile su dobro definirane korištenjem samo metabolita šećera, ali bez drugih glavnih vrsta spojeva. Manitol je bio glavni metabolit šećera u smeđim algama, dok su fruktoza, saharoza i glukoza pronađeni u visokim koncentracijama u zelenim algama. U crvenim algama pojedine vrste imale su neke karakteristične metabolite, poput sorbitola u Pyropia pseudolinearis i panose u Dasya sessilis. Profili metabolita generirani u ovoj studiji bit će resurs i pružit će smjernice za nutraceutska istraživanja jer su podaci o metabolitima u morskim algama još uvijek vrlo ograničeni u usporedbi s podacima o kopnenim biljkama.

Ovo je pregled sadržaja pretplate, pristup putem vaše ustanove.


Materijali i metode

Kemikalije i reagensi

Sve kemikalije su nabavljene od tvrtke Sigma (St. Louis, MO, USA), osim ako nije drugačije naznačeno.

Materijali od dijatoma i priprema uzoraka

C. weissflogii ND-8 izoliran je iz obalne vode Zhoushana, provincije Zhejian u Kini. Uzgajan je u Guillardovom mediju f/2 pripremljenom od filtrirane, sterilizirane prirodne morske vode, s omjerom inokulacije 1: 8, pod 12 sati svjetlosti (intenzitet svjetlosti 75 μmol/m 2/s) i 12 sati tamno vrijeme u 1 danu na 20 ଌ � ଌ.

Karakterizacija C. weissflogii ND-8

Fotomikrografije C. weissflogii ND-8 snimljeni su optičkim mikroskopom (FM 10 fotoaparat Nikon, Tokio, Japan) i skenirajućim elektronskim mikroskopom (JSM-6380LV, JEOL, Tokio, Japan). Molekularna identifikacija izvedena je kako je prethodno opisano (Su i Yang, 2015). Temeljni premazi ITS5-F (5 ’-TCACCTACGGAAACCTTGT-3 ’)/ITS5-R (5 ’-TTCAGCGGGTAGTCTTGCCTC-3 ’) i 18S-F (5 ’-ACCTCGGT) (5 ’-TCACCTACGGAAACCTTGT-3 ’) korišteni su za pojačavanje ND-8 ITS i 18S fragmenata. Njihovi ITS i 18S nizovi uspoređeni su s onima dostupnim u NCBI bazama podataka pomoću BLAST -a. Rezultati pretraživanja obrađeni su softverom MEGA5.2 (Tamura i sur., 2011.). Filogenetsko stablo izgrađeno je metodom spajanja susjeda, provedeno je 1.000 ponavljanja slučajnog pretraživanja kako bi se procijenila pouzdana razina stabla (Saitou i Nei, 1987.).

Ekstrakcija i izolacija deviza

C. weissflogii ND-8 uzgajan je u Guillardovoj sredini f/2 na 20 ଌ � ଌ 5 dana, nakon čega je uslijedilo centrifugiranje na 4000 × g 15 min. Mulj od algi je sakupljen, sušen liofilizacijom na � ଌ 2 dana. Pročišćavanje FX -a provedeno je kako je prethodno opisano (Xia et al., 2013). Osim toga, kako bi se izbjegle smetnje svjetlosti, svi su pokusi izvedeni u mraku. Aktivne frakcije su sakupljene pomoću TLC u sistemu otapala koji sadrži naftni eter/etil acetat, 1: 1 (v/v). Faktor zadržavanja (R.f) izračunato je na sljedeći način:

Aktivne frakcije su sakupljene i koncentrirane u vakuumu. Koncentrati su konačno osušeni duhanikom za daljnje odvajanje i analizu.

HPLC-MS

HPLC-MS analiza provedena je na Termo HPLC-MS sustav (Thermo Scientific, Waltham, MA, SAD) pomoću Thermo Hypersil GOLD C18 stupac (veličina čestica 1,9-μm, 2,1 mm × 100  mm) s metanolom i vodom kao eluentom. Eksperimentalni uvjeti bili su sljedeći: volumen ubrizgavanja: 5   μM mobilna faza: 0 𠄰.2 min, 95% B 0.2 𠄳.5 min, 95% 𠄲% B 3.5 𠄵 min, 2% B 5 𠄷,5 min, 2% �% B 7,5 � min, protok 95% B: 0,3 ml ·min 𠄱. HPLC eluat je davan u MS sustav s naponom raspršivanja od 1,0 kV. MS vrhovi su zabilježeni i uspoređeni s onima u FX standardu.

Izolirani ciljni uzorak (2,0 mg) i standardni FX (2,0 mg) otopljeni su u 0,5 ml deuterokloroforma (CDCl3) i 1H nuklearne magnetske rezonancije (NMR) mjerena je pomoću Bruker 400 MHz NMR spektrometra (MA, SAD).

Životinje i tretmani

Odrasli miševi C57BL/6 bez specifičnih patogena u dobi od 8 � tjedana stari 20 ± 1 g kupljeni su u Medicinskom sveučilištu Fujian Medical Center (Fujian, Kina). Uzorak je uključivao 126 životinja, pola mužjaka i pola ženke. Svi pokusi na životinjama provedeni su u skladu s Vodičem za njegu i uporabu laboratorijskih životinja koji je odobrio Pokrajinski ured za upravljanje laboratorijskim životinjama u Fujianu, a vodio ih je Odbor za njegu i uporabu životinja Sveučilišta Fujian (Odobrenje br. 201800013).

Nakon aklimatizacije tijekom 1 tjedna, 90 miševa nasumično je podijeljeno u devet skupina (n = 10/skupini) s pola mužjaka i pola ženke, uključujući lažnu skupinu (miš je primio intraperitonealnu injekciju normalne fiziološke otopine 1,0 ml/kg), liječen deviznim lijekom (0,1 �,0 mg/kg), liječen LPS-om (20 mg/kg), tretirane FX+LPS (0,1 i#x201310,0 mg/kg odnosno 20 mg/kg) i grupe urinastatina (10 4 U/kg). LPS je dobiven iz Escherichia coli 0111: B4 stanice (Cell Signaling Technology, Beverley, MA, USA). Ulinastatin je korišten kao pozitivna kontrola. Lažna skupina je ubrizgana s istom količinom fiziološke otopine puferirane fosfatom (PBS) (0,0067M pH 7,4, HyClone, GE Healthcare Life Sciences, UT, SAD). Miševima je intraperitonealno (ip) ubrizgan FX 30 minuta prije ip primjene smrtonosne doze LPS (20 mg/kg) ili PBS. Svi su miševi postili 12 sati prije operacije, ali su mogli slobodno piti vodu. Stopa preživljavanja miševa zabilježena je svakih 6 sati tijekom 120 sati, a Kaplan –Meier krivulje preživljavanja generirane su pomoću softvera GraphPad Prism 6 (v.5.01 za Windows GraphPad Software, San Diego, Kalifornija, SAD) i analizirane pomoću log- rang test. Na temelju gornjih pokusa, ostalih 36 miševa nasumično je podijeljeno u šest skupina (n = 6/skupina) s pola muškarca i pola žene. Nakon anestezije pomoću natrijeve soli Pentobarbital, uzeta je mišja krv preko retro-orbitalna punkcija 6 sati nakon izazivanja i ostavljena da se zgruša na 28 ଌ 30 minuta. Serum je zatim prikupljen centrifugiranjem na 2000 ×g 30 minuta i pohranjeno na � ଌ radi daljnje analize. Tkiva miševa prikupljena su za daljnje analize.

Histopatološki pregled

Mišja tkiva su fiksirana paraformaldehidom (4%) u PBS za histološku analizu. Tkiva su isprana vodom, dehidrirana etanolom i stavljena u parafin, nakon čega je uslijedilo presijecanje kriostata (𢏄 μm) i postavljanje na staklene pločice. Presjeci su zatim deparafificirani pomoću ksilena i etanola i obojeni općim hematoksilinom i eozinom (H & ampE) kako bi se otkrila hemoragična nekroza u tkivima. Histološke promjene opažene su pod svjetlosnim mikroskopom (Olympus, Japan) pri povećanju od 100 × i 200 ×. Prema Eriksson i sur., Ocjena oštećenja jetre mjerena je na presjecima obojenim H & ampEE-om koristeći ocjene od 0 do 4, kako slijedi: rezultat 0 ne predstavlja upalne infiltrate 1 predstavlja male upalne stanice između hepatocita 2 predstavlja veća žarišta od> 100 upalnih stanica 3 predstavljaju> 10% poprečnog presjeka, 4 predstavljaju> 30% uključenog presjeka (Eriksson i sur., 2003.).

Kultura stanica

Mišji makrofag RAW 264.7 kupljen je od American Type Culture Collection (Manassas, VA, USA) i korišten kao in vitro model za istraživanje protuupalnih svojstava FX-a. Stanice su kultivirane u Dulbecco -ovom modificiranom mediju Eagle koji sadrži 10% (v/v) fetalnog goveđeg seruma (Gibco, Kalifornija, SAD) i 1% (v/v) penicilina/streptomicina (Gibco, Kalifornija, SAD) pri 37 &# xb0C u vlažnom inkubatoru s 5% ugljičnog dioksida (CO2).

Test održivosti stanica

Stabilnost stanica procijenjena je u RAW 264.7 stanicama pomoću Cell Counting Kit-8 (CCK-8 Beyotime Biotechnology, Peking, Kina). Apsorbancija uzorka na 450 nm mjerena je pomoću čitača mikroploča (Synergy HT BioTek, Winooski, VT, SAD), a ucrtan je postotak preživjelih stanica u svakoj tretiranoj skupini.

Kvantitativni PCR u stvarnom vremenu

Ekspresija i sekrecija citokinske mRNA mjerena je kvantitativnom lančanom reakcijom polimeraze u stvarnom vremenu nakon reverzne transkripcije (RT-qPCR). Ukupna RNA je odvojena pomoću TRIZOL -a (Invitrogen, Carlsbad, CA, USA). RT-qPCR je izveden korištenjem SYBR Green (Applied Biosystems, Foster City, CA, USA) kako je prethodno opisano (Fan et al., 2018). Primeri korišteni za RT-qPCR bili su sljedeći: IL-1 β-F (5 ’-ACAGGCTCCGAGATGAACAA-3 ’)/IL-1 β-R (5 ’-TGGGAGTAGACAAGGTACAACCC-3 &#-2019) 6-F (5 ’-TAGTCCTTCCTACCCCAATTTCC-3 ’)/IL-6-R (5 ’-CCTCTCGGCAGTGGATAAAG-3 ’) i TNF-α-F (5 ’-CATCTA & CATCTA )/TNF-α-R (5 ’-TGGGAGTAGACAAGGTACAACCC-3 ’).

Mjerenje razine proupalnih citokina pomoću ELISA-e

RAW 264.7 stanice prethodno su obrađene FX-om u naznačenim dozama tijekom 6 sati, nakon čega je uslijedilo 3-satno liječenje LPS-om (1.0 mg/L), te su uzgajane u pločama s 24 jažice (1 × 10 6 stanica/jažici) tijekom 24 sata h Sakupljeni su supernatanti kultiviranih RAW stanica 264.7.

Razine TNF-α, IL-1 β i IL-6 u supernatantima i uzorcima seruma (vidi gore) kvantificirane su pomoću ELISA kompleta (TNF-α, R & ampD Systems, Minneapolis, MN, SAD , kataloški broj SMTA00B IL-1 β, R & ampD Systems, Minneapolis, MN, SAD, kataloški broj SMLB00C IL-6, R & ampD Systems, Minneapolis, MN, SAD, kataloški broj SM6000B) prema protokolima proizvođača ’s.

Western blot analiza

Western blotting izveden je kako je prethodno opisano (Chen i sur., 2002. Xiao i sur., 2019.). Antitijela gen protiv primarne reakcije na mijeloidnu diferencijaciju 88 (MyD88) (D80F5#4283), anti-fosfo-IKK α/β (Ser176/180) (16A6#2697), anti-IKK α (#2682), Phospho-IKK α/β (Ser176/180) (16A6,#2697), anti-I 㮫 α (44D4,#4812), anti-Phospho-I 㮫 α (Ser32/36) ( 5A5, #9246), anti-NF-& #x3baB p65 (D14E12, #8242) i anti-fosfo-NF-& #x3baB p65 (Ser536) (93H1, #3033) kupljeni su od Cell Signaling Technology.

NF-㮫 Test aktivnosti luciferaze

Humane stanice THP1-Lucia TM NF-㮫 izvedene su iz stanične linije humanog monocita THP-1 stabilnom integracijom NF-㮫-inducibilne reporterske konstrukcije Lucia TM. Reporterske stanice THP-1 Lucia NF 㮫 kupljene su od tvrtke InvivoGen (San Diego, Kalifornija, SAD). THP1-Lucia TM NF-㮫 stanice posebno su dizajnirane za praćenje puta transdukcije signala NF-㮫 u fiziološki relevantnoj staničnoj liniji. Stanice THP1-Lucia TM NF-㮫 uzgojene su na ploči sa 96 jažica (1   × 10 5 /jažici) 18 h u prisutnosti različitih koncentracija FX nakon čega slijedi LPS (1,0 mg /L) za stimulaciju . Za određivanje aktivnosti luciferaze, 20   μl alikvota medija stanične kulture premješteno je u crne ploče sa 96 jažica (Corning, NY, SAD), a zatim otopina za ispitivanje QUANTI-Luc TM (InvivoGen). Ploče su odmah izmjerene za aktivnost luciferaze pomoću čitača Victor 2 s više ploča (PerkinElmer) prema uputama proizvođača.

Imunofluorescentno bojenje

Stanice su uzgojene i fiksirane s 4% paraformaldehidom 10   minuta na sobnoj temperaturi, nakon čega je slijedila obrada otopinom za penetraciju membrane (0,3% Triton-100) 10 minuta na sobnoj temperaturi. Stanice su isprane s 1 × PBS pet puta, zatim inkubirane s anti-NF-㮫 (p65) primarnim antitijelima (razrjeđenje 1: 200) (Cell Signaling Technology, USA) preko noći na 4 ଌ, nakon čega je uslijedila inkubacija sa AlexaFluor 488 kozje anti -zečje sekundarno antitijelo na 37 ଌ u mraku 30 min. Jezgre su obojene s 0,5 μg/ml 4 ′,6-diamidino-2-fenilindola (DAPI) (1: 800, Santa Cruz) u PBS 2 minute. Negativne kontrole pripremljene su izostavljanjem primarnih antitijela. Nakon tri ispiranja PBS-om, uzorci su montirani u nosač za montažu (M1289, Sigma-Aldrich), promatrani pod fluorescentnim mikroskopom Zeiss (Carl Zeiss, Oberkochen, Njemačka), a analize slika provedene su pomoću softvera Zeiss LSM 510.

Statističke analize

Podaci su izraženi kao srednja ± standardna devijacija (SD). Statistička značajnost utvrđena je jednosmjernim ANOVA i Tukeyjevim testom za post hoc višestruka usporedba s 5 softvera. The P vrijednost

17.4E: Jestive alge - biologija

Proučavana je primjena LC-NMR/MS za izravnu identifikaciju ugljikohidrata u pivu. Ugljikohidrati su glavne komponente piva, a njihovo strukturno obilježavanje samo pomoću NMR -a ozbiljno je ometeno snažnim spektroskopskim preklapanjem. Izravna analiza piva pomoću LC-NMR/MS omogućuje brzu (1-2 h) identifikaciju dekstrina sa stupnjem polimerizacije (DP) do devet monomera, pri čemu je jedino potrebno otpuštanje. Iako NMR lako ukazuje na prisutnost točaka grananja α (1 → 6) za svaku podfrakciju odvojenu LC, javljaju se poteškoće zbog nedvosmislenog dodjeljivanja linearnih ili razgranatih oblika dekstrina visokog DP. Za dva uzorka piva koja su ispitana u ovom radu utvrđeno je da imaju značajno različite sastave oligosaharida, što odražava različite uvjete proizvodnje. Korištenje NMR -a sa crticama za brzu karakterizaciju ugljikohidratnog sastava piva može biti temelj korisnog alata za kontrolu kvalitete piva.

Ključne riječi: Sastav ugljikohidratnih dekstrina piva NMR LC-NMR/MS

Ekstrakcija čilija, crnog papra i đumbira s gotovo kritičnim CO2, Propan i dimetil eter: analiza ekstrakata kvantitativnom nuklearnom magnetskom rezonancijom
  • Owen J. Catchpole,
  • John B. Grey,
  • Nigel B. Perry,
  • Elaine J. Burgess,
  • Wayne A. Redmond, i
  • Noel G. Porter

Đumbir, crni papar i čili u prahu ekstrahirani su pomoću gotovo kritičnog ugljičnog dioksida, propana i dimetil etera u laboratorijskim razmjerima kako bi se odredio ukupni prinos i učinkovitost ekstrakcije za odabrane ljute komponente. Ovisnost prinosa i učinkovitosti ekstrakcije o temperaturi također je utvrđena za crni papar i čili upotrebom propana i dimetil etera. Oštrina ekstrakata određena je pomoću NMR tehnike razvijene za ovaj rad. Također je utvrđen sadržaj hlapljivih sastojaka ekstrakta đumbira i crnog papra. Ekstrakcija svih vrsta začina izvedena je acetonom radi usporedbe ukupnih prinosa. Podkritični dimetil eter bio je jednako učinkovit u ekstrakciji oštrih načela iz začina kao i natkritični ugljikov dioksid, iako je također ekstrahirana znatna količina vode. Subkritični propan bio je najmanje učinkovito otapalo. Sva otapala kvantitativno su ekstrahirala gingerole iz đumbira. Prinosi kapsaicina dobivenih nadkritičnim CO2 i dimetil eterom bili su slični i približno dvostruko veći od ekstrahiranog propanom. Prinos piperina dobivenih ekstrakcijom propana crnog papra bio je nizak i iznosio je ∼10% od onog postignutog s dimetil eterom i CO2, ali se povećavao s povećanjem temperature ekstrakcije.

Ključne riječi: začini đumbir crni papar ekstrakt čilija gotovo kritičan ljut gingerol piperin kapsaicin kvantitativna NMR

Istodobno tekućinsko kromatografsko određivanje kreatinina i pseudouridina u goveđem urinu i utjecaj pH uzorka na analizu

Opisana je brza i pouzdana metoda za istodobno određivanje kreatinina i pseudouridina. Oba su analita otkrivena pri optimalnoj valnoj duljini detekcije (262 nm), uzimajući u obzir relativne razine prisutne u goveđem urinu. Cimetidin je korišten kao interni standard i detektiran pri maksimalnoj valnoj duljini apsorpcije (220 nm) na zasebnom kanalu. Sva tri spoja eluirana su u roku od 15 minuta, koristeći gradijent metanola od 10 mmol/L fosfata (pH 6,8) na stupcu C18. Utvrđeno je da podaci o kreatininu značajno ovise o pH uzorka. Oporavak obaju analita bio je iznad 96%. Najniže razine kreatinina i pseudouridina koje su se mogle otkriti bile su 0,28 nmol odnosno 9,0 pmol. Korištenje internog standarda rezultiralo je metodom s visokom preciznošću (standardna devijacija od 1,42 mmol/L i 0,027 mmol/L za kreatinin i pseudouridin), no ipak jednostavnom i brzom.

Ključne riječi: kreatinin pseudouridin HPLC diodni niz unutarnjih standarda cimetidin

Diferencijacija prirodnog i sintetičkog fenilalanina i tirozina kroz prirodno obilje 2H nuklearna magnetska rezonancija
  • Elisabetta Brenna,
  • Giovanni Fronza,
  • Claudio Fuganti i
  • Matteo Pinciroli

NMR karakterizacija prirodnog obilja deuterija na uzorcima uzoraka aminokiselina tirozina (1) i fenilalanina (2), ispitanih kao acetilirani metilni esteri 4 i 6, provedena je s ciljem da se ovim sredstvima utvrdi doprinos hidroksilacije u životinjama ishrane l-fenilalaninom (2) do stvaranja l-tirozina (1), što je prethodno otkriveno na istim uzorcima utvrđivanjem fenolnih vrijednosti δ18O. Studija, koja također uključuje NMR ispitivanje benzojeve kiseline (5) iz 2 i tirosola (7) iz 1, bitno ne uspijeva pružiti tražene podatke jer je način označavanja deuterija uzorcima tirozina različitog podrijetla prilično sličan, ali ukazuje na dramatična razlika u obrascu označavanja deuterija dvije aminokiseline 1 i 2. Najvažnija varijacija je u pogledu obogaćivanja deuterija na položajima CH2 i CH, koji su obrnuti u dvije aminokiseline prirodnog izvođenja. Štoviše, dok se čini da su atomi diastereotopskih benzilnih vodika l-tirozina (1) jednako obogaćeni deuterijem, u l-fenilalaninu (2) (D/H) 3R> (D/H) 3S. Slično, benzojeva kiselina (5) pokazuje zasebne signale za jezgre aromatičnog deuterija, koji prilično ukazuju na prirodnu ili sintetsku derivaciju. Način označavanja deuterija bočnim lancem 1 i 2 okvirno je povezan s različitim podrijetlom dviju aminokiselina, prirodnim iz životinjskih izvora za l-tirozin i biotehnološkim vjerojatno iz genetski modificiranih mikroorganizama za l-fenilalanin.

Ključne riječi: tirozin fenilalanin tirosol životinjsko biljno podrijetlo aspartam prirodno obilje deuterij NMR

Identifikacija hlapivih spojeva u soji u različitim razvojnim fazama pomoću mikroekstrakcije u čvrstim fazama
  • Stephen M. Boué,
  • Betty Y. Shih,
  • Carol H. Carter-Wientjes, i
  • Thomas E. Cleveland

Hlapljive tvari iz sjemena soje (Glycine max) analizirane su metodom mikroekstrakcije u krutoj fazi (SPME) u kombinaciji s plinskom kromatografijom-masenom spektrometrijom (GC-MS). Trideset hlapljivih spojeva već prijavljenih za soju je pronađeno, a identificirano je ili uslovno identificirano dodatnih 19 spojeva koji nisu prethodno prijavljeni. Metoda SPME upotrijebljena je za usporedbu hlapljivog profila sjemena soje u tri različite faze razvoja. Većina novo prijavljenih spojeva u sjemenu soje bili su aldehidi i ketoni. Tijekom ranih razdoblja razvoja u fazi zrelosti R6, nekoliko hlapivih tvari bilo je prisutno u relativno visokim koncentracijama, uključujući 3-heksanon, (E) -2-heksenal, 1-heksanol i 3-oktanon. U fazi zrelosti R7 i R8, opažene su smanjene količine 3-heksanona, (E) -2-heksenala, 1-heksanola i 3-oktanona. U fazi zrelosti R8 heksanal, (E) -2-heptenal, (E) -2-oktenal, etanol, 1-heksanol i 1-okten-3-ol detektirani su u relativno visokim koncentracijama. SPME nudi mogućnost razlikovanja između tri razvojne faze soje koje daju temeljne i praktične informacije.

Ključne riječi: isparljive tvari soje zrelost plinska kromatografija masena spektrometrija mikroekstrakcija u čvrstom stanju SPME

Izotopsko označavanje i LC-APCI-MS kvantifikacija za ispitivanje apsorpcije karotenoida i filokinona iz kelja (Brassica oleracea)
  • Anne C. Kurilich,
  • Steven J. Britz,
  • Beverly A. Clevidence, i
  • Janet A. Novotny

Sposobnost proučavanja bioraspoloživosti hranjivih tvari iz hrane važan je korak u određivanju utjecaja tih nutrijenata na zdravlje. Ovaj rad opisuje metodu za proučavanje bioraspoloživosti hranjivih tvari iz kelja (Brassica oleracea var. Acephala) označavanjem hranjivih tvari ugljikom-13, hranjenjem kelja odraslim dobrovoljcem i analizom uzoraka plazme za označene hranjive tvari. Rezultati su pokazali da uvjeti za proizvodnju atmosfersko svojstveno obilježenog kelja nisu imali štetan učinak na rast biljaka. Lutein, β-karoten, retinol i filokinon analizirani su pomoću masene spektrometrije s kemijskom ionizacijom s atmosferskim tlakom s tekućinskom kromatografijom. Analiza uzoraka plazme pokazala je da je označeni lutein dosegao vrhunac u plazmi 11 sati (0,23 μM), β-karoten svoj vrhunac 8 (0,058 μM) i 24 sata (0,062 μM), retinol vrhunac 24 sata (0,10 μM), a filokinon vrhunac pri 7 h (3,0 nM). Ova metoda označavanja kelja s 13C bila je uspješna za stvaranje jasno definiranih kinetičkih krivulja za 13C-lutein, 13C-β-karoten, 13C-retinol i 13C-filokinon.

Ključne riječi: karotenoid β-karoten lutein retinol vitamin A filokinon oznaka izotopa vitamina K masena spektrometrija LC-MS kelj Brassica oleracea

BIOAKTIVNI Sastav
Ovicidni i adulticidni učinci Eugenia caryophyllata Spojevi ulja pupoljaka i lišća na Pediculus capitis
  • Young-Cheol Yang,
  • Si-Hyeock Lee,
  • Won-Ja Lee,
  • Don-Ha Choi, i
  • Young-Joon Ahn

Toksičnost Eugenia caryophyllata pupoljaka i spojeva dobivenih iz ulja iz lista (acetileugenol, β-kariofilen, eugenol, α-humulen i metil salicilat) i srodnika eugenola (izoeugenol i metileugenol) na jaja i ženke Pediculus capitis ispitana je metode primjene i fumigacije te se uspoređuju s onima široko rasprostranjenih δ-fenotrina i piretruma. U difuzijskom biološkom testu na filtriranom papiru sa ženskim P. capitisom, pedikulicidno djelovanje ulja pupoljka i lista Eugenia bilo je usporedivo s djelovanjem δ-fenotrina i piretruma na temelju vrijednosti LT50 pri 0,25 mg/cm2. Pri 0,25 mg/cm2, najotrovniji spoj za ženski P. capitis bio je eugenol, a zatim metil salicilat. Acetileugenol, β-kariofilen, α-humulen, izoeugenol i metileugenol nisu bili učinkoviti. Eugenol pri 0,25 mg/cm2 bio je snažan poput δ-fenotrina i piretruma, ali je bio nešto manje učinkovit od piretroida pri 0,125 mg/cm2. Protiv jaja P. capitis, metil salicilat i eugenol bili su visoko učinkoviti pri 0,25 odnosno 1,0 mg/cm2, dok je mala ili nikakva aktivnost pri 5 mg/cm2 primijećena kod ostalih ispitivanih spojeva, kao i kod δ-fenotrina i piretruma. U testovima fumigacije sa ženskim P. capitisom pri 0,25 mg/cm2, eugenol i metil salicilat bili su učinkovitiji u zatvorenim čašama nego u otvorenim, što ukazuje da je učinak spojeva uvelike posljedica djelovanja u parnoj fazi. Ni δ-fenotrin ni piretrum nisu pokazali toksičnost fumiganata. Eterična ulja pupoljka i lista Eugenia, osobito eugenol i metil salicilat, zaslužuju daljnje proučavanje kao potencijalna sredstva za kontrolu P. capitis ili spojevi olova.

Ključne riječi: Prirodni insekticid pedikulicid ovicid fumigant Pediculus capitis eterično ulje Eugenia caryophyllata GC-MS eugenol metil salicilat način djelovanja

Sadržaj antocijanina i proantocijanidina u odabranim bijelim i crnim vinima. Usporedba radikalno apsorbirajućeg kapaciteta kisika s netradicionalnim vinima dobivenim od borovnice Highbush

Antioksidacijski kapacitet, mjeren kapacitetom apsorpcije radikala kisika (ORACPE), ukupnim fenolnim, ukupnim i pojedinačnim antocijaninima te sadržajem frakcija proantocijanidina ocijenjen je u crnim i bijelim vinima iz grožđa. Usporedba s obzirom na antioksidacijski kapacitet napravljena je s netradicionalnim vinima od borovnice. Borovnice su među voćem koje je najbolje prepoznato po svojim potencijalnim zdravstvenim prednostima. U crnim vinima ukupni sadržaj oligomernih proantocijanidina, uključujući katehine, bio je znatno veći (177,18 ± 96,06 mg/L) nego u bijelim vinima (8,75 ± 4,53 mg/L). Relativno visoka korelacija u crnim vinima pronađena je između vrijednosti ORACPE -a i spojeva malvidina (r = 0,75, P <0,10) i proantocijanidina (r = 0,87, P <0,05). U bijelim vinima pronađena je značajna korelacija između trimerne frakcije proantocijanidina i vrijednosti uklanjanja peroksilnih radikala (r = 0,86, P <0,10). Umjereno piće (1 napitak dnevno, oko 140 ml) crnog vina ili bijelog vina ili vina od borovnice visokog ugla odgovara unosu 2,04 ± 0,81 mmol TE, 0,47 ± 0,15 mmol TE i 2,42 ± 0,88 mmol TE ORACPE -a dnevno.

Ključne riječi: antocijani proantocijanidini vinski vinski grožđe borovnice fenoli ORAC

Izolacija i antihipertenzivni učinak inhibitornih peptida angiotenzin I-konvertirajućeg enzima (ACE) iz špinata Rubisco
  • Yanjun Yang,
  • Ewa D. Marczak,
  • Megumi Yokoo,
  • Hachiro Usui i
  • Masaaki Yoshikawa

Četiri nova inhibitorna peptida za enzim koji pretvara angiotenzin I (ACE), to jest MRWRD, MRW, LRIPVA i IAYKPAG, izolirana su iz digestije pepsin-pankreatin špinata Rubisco uz upotrebu HPLC. IC50 vrijednosti pojedinih peptida bile su 2,1, 0,6, 0,38 i 4,2 μM, respektivno. MRW i MRWRD imali su antihipertenzivni učinak nakon oralne primjene na spontano hipertenzivne štakore. Maksimalno smanjenje dogodilo se 2 sata nakon oralne primjene MRW -a, dok je MRWRD pokazalo maksimalno smanjenje 4 sata nakon oralne primjene u dozama od 20 odnosno 30 mg/kg. IAYKPAG je također pokazao antihipertenzivno djelovanje nakon oralne primjene u dozi od 100 mg/kg, dajući maksimalno smanjenje 4 sata nakon oralne primjene. IAYKP, IAY i KP, fragmentni peptidi IAYKPAG -a, također su imali antihipertenzivno djelovanje. LRPVIA nije pokazao nikakav antihipertenzivni učinak u dozi od 100 mg/kg unatoč snažnom inhibitornom djelovanju ACE.

Ključne riječi: Rubisco špinat ACE-inhibitorni peptidi antihipertenzivni učinak spontano hipertenzivni štakori (SHR)

Kemijske karakteristike i biološki učinci sicilijanaca Opuntia ficus indica (L.) Mlin. Voćni sok: antioksidativno i antiulcerogeno djelovanje
  • Enza Maria Galati,
  • Maria Rita Mondello,
  • Daniele Giuffrida,
  • Giacomo Dugo,
  • Natalizia Miceli,
  • Simona Pergolizzi, i
  • Maria Fernanda Taviano

Istražen je sok cijelih plodova sicilijanskih sorti opuncije (Opuntia ficus indica (L.) Mill.) I utvrđen sadržaj askorbinske kiseline, ukupnih polifenola i flavonoida. U soku je ferulinska kiselina bila glavni derivat hidroksicimetne kiseline, a srednja koncentracija ukupnih fenolnih spojeva bila je 746 μg/mL. The flavonoid fraction, analyzed by high-performance liquid chromatography−diode array detection, consisted of rutin and isorhamnetin derivatives. The juice showed antioxidant activity in the DPPH• test, probably due to the phenolic compounds that are effective radical scavengers. The preventive administration of the juice inhibited the ulcerogenic activity of ethanol in rat. Light microscopy observations showed an increase in mucus production and the restoration of the normal mucosal architecture. The juice is nutritionally interesting, and its dietary intake could provide protection against oxidative damage.

Keywords: Opuntia ficus indica fruit juice antioxidant flavonols antiulcer activity mucosa structural changes

Enhancing Volatile Phenol Concentrations in Wine by Expressing Various Phenolic Acid Decarboxylase Genes in Saccharomyces cerevisiae
  • Annél Smit ,
  • Ricardo R. Cordero Otero ,
  • Marius G. Lambrechts ,
  • Isak S. Pretorius , and
  • Pierre van Rensburg

Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1P) and terminator (PGK1T) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Σ1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Σ1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future.

Keywords: Phenolic acid decarboxylation volatile phenols wine yeast wine aroma

Molluscicidal Saponins from Sapindus mukorossi, Inhibitory Agents of Golden Apple Snails, Pomacea canaliculata
  • Hui-Chi Huang ,
  • Sin-Chung Liao ,
  • Fang-Rong Chang ,
  • Yao-Haur Kuo , and
  • Yang-Chang Wu

Extracts of soapnut, Sapindus mukorossi Gaertn. (Sapindaceae) showed molluscicidal effects against the golden apple snail, Pomacea canaliculata Lamarck. (Ampullariidae) with LC50 values of 85, 22, and 17 ppm after treating 24, 48, and 72 h, respectively. Bioassay-directed fractionation of S. mukorossi resulted in the isolation of one new hederagenin-based acetylated saponin, hederagenin 3-O-(2,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (1), along with six known hederagenin saponins, hederagenin 3-O-(3,4-O-di-acetyl-α-l-arabinopyranoside)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (2), hederagenin 3-O-(3-O-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (3), hederagenin 3-O-(4-O-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (4), hederagenin 3-O-(3,4-O-di-acetyl-β-d-xylopyranosyl)-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (5), hederagenin 3-O-β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (6), and hederagenin 3-O-α-l-arabinopyranoside (7). The bioassay data revealed that 1−7 were molluscicidal, causing 70−100% mortality at 10 ppm against the golden apple snail.

Keywords: Sapindus mukorossi Sapindaceae molluscicidal effects Pomacea canaliculata hederagenin saponins


5. Related Species of Malus domestica

As mentioned earlier, the number of species in the genus Malus varies widely, with different taxonomic treatments recognizing anywhere from 8 to 78 primary species (see Section 2.1). Many of the crabapple species can be difficult to differentiate due to the lack of distinguishing characters (Dickson et al. 1991).

Table 2: Provincial distribution of Malus species present in Canada outside of cultivation (from: Brouillet et al. 2010+ CFIA and NRCan/CFS 2011+ Kartesz 1999 Scoggan 1979).
Descriptive text:

The purpose of the table is to illustrate the provincial distribution of Malus species present in Canada outside of cultivation. The table describes whether the species is native to Canada or introduced and the species distribution by province.

Distribution collated from reports of synonyms Malus pumila Mlin. i Malus sylvestris (L. ) Mill. in the literature.

5.1 Inter-species/genus hybridization

Table 3: Reports of experimental interspecific hybrid crosses reported for Malus species present in Canada.
Descriptive text:

The purpose of the table is to highlight reports of experimental interspecific hybrid crosses reported for the Malus species present in Canada. It describes the cross, the number of pollinations and matured fruits, as well as provides references for each cross.

5.2 Potential for introgression of genetic information from Malus domestica into relatives

5.3 Summary of the ecology of relatives of Malus domestica


Materijal i metode

In this study, a total of 34 PEPC protein sequences of 14 different families in C4 and CAM plants were collected from National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov).

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING 10) database (http://string-db.org/) was used to foresee the interacting proteins (Szklarczyk et al. 2014 ). The database contains information from numerous sources, including experimental repositories, computational prediction methods and public text collections.

Various online web services and software were used for analyses of PEPC proteins in C4 and CAM plants. Comparative and bioinformatic analyses were carried out online at the website ExPASy (http://expasy.org/tools). Functional domains in PEPC proteins were identified by ProDom server (http://prodom.prabi.fr/prodom/current/html/form.php) (Altschul et al. 1997 ). Physico-chemical parameters of PEPC proteins were analyzed by ProtParam (http://web.expasy.org/protparam) (Gasteiger et al. 2005 ) and the secondary structure prediction was analyzed by SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) (Geourjon and Deleage 1995 ).

Prediction of mitochondrial and plastid targeting sequences was accomplished by Predotar 1.03 server (https://urgi.versailles.inra.fr/predotar/predotar.html) while prediction of signal peptide cleavage sites was achieved by SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP) (Petersen et al. 2011 ). Identification of subcellular locations and transmembrane helices in proteins was performed by TargetP 1.1 and (www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al. 2000 ) and TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (Moller et al. 2001 ) servers, respectively.

The tertiary structure prediction analysis of PEPC proteins was performed by the Phyre2 server using profile–profile matching and secondary structure (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (Kelley and Sternberg 2009 ). Chimera 1.10.1 was used for 3D structure visualization of Zea mays as the model of plants (https://www.cgl.ucsf.edu/chimera/). Backbone similarities and differences of obtained models were estimated by TM-score server (http://zhanglab.ccmb.med.umich.edu/TM-score/) (Zhang and Skolnick 2004 Xu and Zhang 2010 ). Additionally, the SuperPose web server was used to find the most and the least similarities of PEPC sequences in C4 and CAM plants (http://wishart.biology.ualberta.ca/SuperPose/) (Maiti et al. 2004 ). Finally, stereochemical quality and accuracy of the model was evaluated with PROCHECK 3.5 (http://www.ebi.ac.uk/thornton-srv/software/PROCHECK) by Ramachandran plot analysis (Laskowski et al. 1993 ). Z-score was calculated using interactive ProSA-web service (https://prosa.services.came.sbg.ac.at/prosa.php) for the recognition of errors in three-dimensional structure which indicated model quality and total energy deviation of the structure with respect to energy distribution derived from random conformations (Wiederstein and Sippl 2007 ).

The motifs of protein sequences were discovered using the program of Multiple Em for Motif Elicitation (MEME version 4.10.2) (Bailey et al. 2009 ) and Motif Alignment and Search Tool (MAST version 4.9.1) (Bailey and Gribskov 1998 ) at the website http://meme.nbcr.net/meme. The parameters of MEME analyses were applied as follows: distribution of motif occurrences, zero or one per sequence number of different motifs, 10 minimum motif width, six and maximum motif width, 50.

The višestruka sequence alignment of PEPC proteins was performed with ClustalW algorithm implemented in Molecular Evolutionary Genetic Analysis (MEGA 6.06) (http://www.megasoftware.net) (Tamura et al. 2013 ) with default parameters. The phylogenetic tree was constructed using the neighbor-joining (NJ) method and the bootstrap test carried out with 1000 replicates.


RASPRAVA

This study expanded our understanding of the anatomy and function of the EO in two species of planktivorous fishes of special importance, H. molitrix i H. nobilis, by demonstrating that the EO is an important chemosensory organ in these species. We suggest that it uses this sense to accumulate tiny food particles. In addition, this study provides the first detailed description of the morphology of the gill rakers and EO in these important species and shows that their extremely fine gill rakers and EO canals are closely associated and capable of functioning as an integrated unit. While differences in gill raker morphology and spacing were noted between species, their EOs seem much the same (if not identical). We also present histological evidence suggesting that the EO functions as a sophisticated pharyngeal chemosensory organ with both taste buds and SCCs. Our electrophysiological recordings, the first reported from an EO, demonstrate that the EO detects food-related chemicals including l -amino acids and likely other yet unknown chemicals. Together, these data suggest that the EO via pump filtering identifies and packages planktonic food that bigheaded carp have become specialized to consume. This ability might explain the extreme efficiency with which these carp species feed and thus their invasiveness in eutrophic waters.

Histological staining with nuclear red/light green/orange of 14 μm cryosections of the EO. Cartilage and bone were stained green, the brain, nerves and nerve fiber bundles were stained orange, and epithelia including taste buds were stained purple. These stainings were done for a general overview on sections adjacent to the sections used for immunohistochemistry. Images A, B and C show H. molitrix images D, E, F and G show H. nobilis. A, B and C depict cross-sections through the epibranchial organ and its tubes (T). Image B shows the supporting cartilaginous structures (*) and the ridges at the outside of the epibranchial organ (arrowheads). (D) Horizontal section: the tubes are lined with epithelium that contains taste buds (arrows in C). In some areas modified gill rakers face the areas with taste buds. (E) Higher magnification of the modified gill rakers. (F) Some areas of the tubes contain small flaps as shown in Fig. 4C. (G) Higher magnification of the small flaps, which are lined with abundant mucus cells (arrows).

Histological staining with nuclear red/light green/orange of 14 μm cryosections of the EO. Cartilage and bone were stained green, the brain, nerves and nerve fiber bundles were stained orange, and epithelia including taste buds were stained purple. These stainings were done for a general overview on sections adjacent to the sections used for immunohistochemistry. Images A, B and C show H. molitrix images D, E, F and G show H. nobilis. A, B and C depict cross-sections through the epibranchial organ and its tubes (T). Image B shows the supporting cartilaginous structures (*) and the ridges at the outside of the epibranchial organ (arrowheads). (D) Horizontal section: the tubes are lined with epithelium that contains taste buds (arrows in C). In some areas modified gill rakers face the areas with taste buds. (E) Higher magnification of the modified gill rakers. (F) Some areas of the tubes contain small flaps as shown in Fig. 4C. (G) Higher magnification of the small flaps, which are lined with abundant mucus cells (arrows).

The primary finding of this study is that the EO of bigheaded carps functions as a chemoreceptive organ. It has large numbers of taste buds and SCCs and is innervated by vagal nerve fibers that respond to relevant chemical stimuli. Specifically, we found large numbers of both taste buds and SCCs both within and outside the EO in both species. The taste buds stained with calretinin, typical of taste buds described in other fish (Reutter et al., 1974). In contrast, we found few taste buds and no SCCs either on the lips or within the buccal cavity of either species, consistent with our inability to record neural activity to chemical stimuli applied to these areas. Together, these data strongly suggest that the EO is the primary taste organ in these species. Although rather small, taste buds of the bigheaded carps resembled those of other fish species (Reutter et al., 1974). For comparison, taste buds in catfish of sizes comparable to the size of our specimens are about 50–80 μm high (Kirino et al., 2013) and in other fish species reach heights even up to 80 μm and widths 40–60 μm (Hansen and Reutter, 2004). The taste buds in the bigheaded carps were only 22–25 μm high and 10–18 μm wide. That these taste buds responded to AAs is typical of those on other organs in other fishes (Sorensen and Caprio, 1998) (see below). Our experiments also described the presence of SCCs on the EO as well as on the internal flaps within the EO and the gill rakers. SCCs have been observed on gill rakers in other teleosts (Hansen, 2005) as well as other organs of other vertebrates (Finger et al., 2003), but their function is not well understood. SCC morphology varies with respect to the apical endings of the cells and may vary even in the same fish species (Kotrschal et al., 1997), as seen here in the bigheaded carps. Only in the sea robin, Prinotus carolinus (Silver and Finger, 1984), is there direct evidence for the types of chemical stimuli detected by SCCs in fish, and l -amino acids have been implicated. It is possible, but unknown, whether our electrophysiological recordings included responses from SCCs.

In addition to presenting clear histological results that the EO serves as a specialized pharyngeal taste organ, we present electrophysiological evidence that it is responsive to the chemical stimuli found in their planktonic foods. It is notable that the detection threshold of the EO was about 1% that of stock concentration because this concentration would be relevant within the buccal cavity, which lacks other chemosensory structures: the EO appears to be the primary taste organ in these species. The mixture of l -amino acids found in their algal food was only partly responsible for the responsiveness, strongly suggesting that additional unidentified stimuli exist. This is notable because it is commonly thought that l -amino acids are the primary feeding cues in fishes, although most work has focused on carnivores (Sorensen and Caprio, 1998) likely some not yet tested chemostimulatory metabolites are present in their specialized planktonic diet which includes cyanobacteria. Although future studies should examine the physiological function of the EO in greater detail, we believe our work establishes the EO as a new type of internal, pharyngeal taste organ.

Histology of the epibranchial organ. Sections adjacent to those of Fig. 5 were treated with antibodies against calretinin (red), a marker for taste buds and solitary chemosensory cells, and acetylated tubulin (green), a marker for nerve fibers. Images A, B, D and E show H. molitrix, images C, F, G and H show H. nobilis. (A) As seen in Fig. 5, parts of the tubes are lined with epithelium containing taste buds whereas taste buds in other areas are scarce. (B) The epithelium outside the EO has ridges that contain taste buds (cf. Fig. 5B). (C) Taste buds innervated by small tubulin-positive nerve fibers lie opposite modified gill rakers (*), which usually do not have taste buds. (D) The modified gill rakers (*) contain few solitary chemosensory cells (arrow). (E) Higher magnification of the modified gill rakers. The red dots depict a few calretinin-positive solitary chemosensory cells. (F) The ridges on the outside of the EO have small protrusions that contain several taste buds. (G) Higher magnification of a taste bud (red) contacted by small nerve fibers (green). (H) Higher magnification of a solitary chemosensory cell also contacted by small nerve fibers.

Histology of the epibranchial organ. Sections adjacent to those of Fig. 5 were treated with antibodies against calretinin (red), a marker for taste buds and solitary chemosensory cells, and acetylated tubulin (green), a marker for nerve fibers. Images A, B, D and E show H. molitrix, images C, F, G and H show H. nobilis. (A) As seen in Fig. 5, parts of the tubes are lined with epithelium containing taste buds whereas taste buds in other areas are scarce. (B) The epithelium outside the EO has ridges that contain taste buds (cf. Fig. 5B). (C) Taste buds innervated by small tubulin-positive nerve fibers lie opposite modified gill rakers (*), which usually do not have taste buds. (D) The modified gill rakers (*) contain few solitary chemosensory cells (arrow). (E) Higher magnification of the modified gill rakers. The red dots depict a few calretinin-positive solitary chemosensory cells. (F) The ridges on the outside of the EO have small protrusions that contain several taste buds. (G) Higher magnification of a taste bud (red) contacted by small nerve fibers (green). (H) Higher magnification of a solitary chemosensory cell also contacted by small nerve fibers.

Our study extends our understanding of the gross morphology of the EO in bigheaded carps while elaborating on its remarkable anatomical specialization. In particular, we confirmed Boulenger's (Boulenger, 1901) century-old observation that the EO in bigheaded carp contains four blind tubes and demonstrate that the gill rakers of these species directly continue into these tubes through a series of specialized protrusions, probably allowing it to serve as an integrated feeding system. These protrusions, which have not been noted before, may keep larger undesirable particles from entering the EO. As long suspected, but not previously demonstrated, our histological results show that the EO contains large numbers of mucus cells and food boli inside the EO, suggesting that it does indeed aggregate food particles (Wilamovski, 1972). No other specialized secretory cell types were found in the EO, adding no support to a previous conjecture that the organ may also have a digestive function (Bertmar et al., 1969). While confirming an earlier report that the EO is muscular (Wilamovski, 1972 Bauchot et al., 1993), we found new evidence that the EO is reinforced with cartilage, which probably facilitates its ability to forcefully intake and expel water and food particles. Additionally, we illustrated gill raker morphology in both species in a detail not previously shown (Boulenger, 1901 Fang, 1928). Their fine structure is consistent with the likelihood that the gill rakers function with the EO to direct food for aggregation at the entrance of the alimentary canal via cross-flow filtration (Sandersen et al., 2001).

Finally, our study adds new insight into the function of the EO. We show that the EO contains numerous mucus and chemosensory cells, its canals are continuous with the gill rakers, and it contracts strongly when exposed to chemical stimuli. These findings directly support conjecture by Wilamovski (Wilamovski, 1972) that the EO in the bigheaded carps aggregate food from the gill rakers by secreting mucous and pumping and expelling it as boli, to the floor of the pharynx near the tiny alimentary canal for consumption (see Fig. 8 for schematic detail). From this study it now appears that the EO detects the presence of accumulating, desirable food particles in its canals using food chemicals detected by its taste buds and SCCs. Given the huge mass of fine particles that frequently exist in eutrophic (and dimly lit) waters, many of which would not be expected to be edible, but which would tend to accumulate in the gill rakers, the presence of chemosensory cells to discern food consumption would be highly adaptive. The fact that EO detects compounds other than AAs is intriguing given that many phytoplankton species (i.e. cyanobacteria) contain toxins (Beveridge et al., 1993 Leflaive and Ten-Hage, 2007), which might also be detected as part of a possible role of the EO in food selection. Whether mucus production in the EO might be directly stimulated by appropriate food chemicals is unknown. The precise connection between the presence of food particles and their chemicals, and EO pumping will be critical to unravel. Analogies may exist between the EO and the palatal organ, a specialized internal food recognition and sorting system in Eurasian carps including the goldfish, Carassius auratus (Finger, 2008), and common carp (Sibbing, 1982). It is interesting that taste buds and SCCs both occur on the EO, but any functional consequences of this association are unknown at present.

Electrophysiological responses of a branch of the vagus nerve which innervates the epibranchial organ of bighead carp to chemical feeding stimuli. (A) Integrated gustatory electrophysiological responses from a branch of the vagus nerve in bighead carp to increasing concentrations of the filtrate of the algal food (AF) and the l -amino acids this food contains (AA). Mean responses (± s.e.m.) are expressed relative to those elicited by the AA mixture (STD). Data represent three preparations. (B) Representative integrated traces from one of the carp whose data are represented in panel A. Responses are shown to 0.1, 1.0, 10.0, 33.3 and 100% AF (Control: well water control STD: AA mixture at full strength). The responses elicited to 100% AA and AF differed (P<0.10, paired t-test N=3).

Electrophysiological responses of a branch of the vagus nerve which innervates the epibranchial organ of bighead carp to chemical feeding stimuli. (A) Integrated gustatory electrophysiological responses from a branch of the vagus nerve in bighead carp to increasing concentrations of the filtrate of the algal food (AF) and the l -amino acids this food contains (AA). Mean responses (± s.e.m.) are expressed relative to those elicited by the AA mixture (STD). Data represent three preparations. (B) Representative integrated traces from one of the carp whose data are represented in panel A. Responses are shown to 0.1, 1.0, 10.0, 33.3 and 100% AF (Control: well water control STD: AA mixture at full strength). The responses elicited to 100% AA and AF differed (P<0.10, paired t-test N=3).

In conclusion, this study demonstrates new aspects of the function of the EO in fishes, and in bigheaded carps in particular. Our results show that the EO is chemosensitive, and suggest that it plays a role in ingestion and food selection. It is possible that the chemosensitivity of the EO function might be exploited using flavored nanoparticles that are now being considered as means to selectively deliver toxins to these species for control (Hinterthuer, 2012). Further studies will need to determine the full range of chemical classes detected by the EO and its precise role in food ingestion in these species and other species that possess this intriguing organ. How this system might work together with the sense of smell (which is seemingly well developed) to locate, select and ingest novel planktonic food will also be interesting to determine.


Advanced BioFuels USA

Teachers will find many useful links and information on the Education Resources page and on the Grants page. In addition, Advanced Biofuels USA has prepared PowerPoint presentations available in the Biofuels Basics section on the PowerPoint Presentations page.

This page provides links and information about a sample of programs and activities in schools stories about teachers and students who “learned by doing” and news reports about educational activities.

For more examples, click on categories such as Education and Teacher Resources along the right margin of the web site. And, subscribe to our free monthly newsletter.

Network, Share, Collaborate

Advanced Biofuels USA is gathering contact information for educators around the world who are working on curriculum-based educational materials to teach about advanced biofuels. If you or someone you know is working on such materials–or wants to, please provide us with contact information and the reason you/they want to be a part of this network. Let us know what sorts of materials or services this collaborative network might provide that would be useful for developing quality, effective, up-to-date educational materials. Email us at [email protected] and put Educational Network in the subject line.

The Bioenergy and Bioproducts Education Programs (BBEP) (formerly Northeast Bioenergy & Bioproducts (NBB) Programs)

Based at Cornell University, Bioenergy & Bioproducts Education Programs provide professional development and hands-on teaching tools for educators (grades 6 – 16 in service and pre-service teachers and extension educators) who want to learn and teach about the Bioenergy and Bioproducts systems currently in use and under development in the United States. Through the collaborative efforts and expertise of six institutions of research and higher learning, this program aims to inspire today’s students to pursue careers in math and science by aligning concern for the natural environment with the emerging bioenergy and bioproducts industries. ČITAJ VIŠE

Smithsonian Science Education Academies for Teachers

SSEAT Academies provide teachers with an opportunity to take part in a week-long professional development course behind-the-scenes at Smithsonian museums and other world class research facilities throughout the greater Washington, DC area.

Since 2005, the Smithsonian Science Education Center (SSEC) has held summer academies, Smithsonian Science Education Academies for Teachers (SSEATs). The SSEAT Academies provide teachers with an opportunity to take part in a week-long professional development course behind-the-scenes at Smithsonian museums and other world class research facilities throughout the greater Washington, DC area. The academies help to bridge the gap between the formal science education programs of the SSEC and the informal science education that exists throughout the Smithsonian, and combine training in science pedagogy with content presented by scientists and researchers who are experts in their fields.

Recent topics of the SSEATs include Biodiversity, Energy’s Innovations and Implications, Earth’s History and Global Change, and Space Science. Each SSEAT academy engages 20-30 teachers from grades K–12 over a week. Each day, participants engage in carefully selected science experiences directly related to concepts addressed in the content and pedagogical training, and guided by scientists, curators, and educators from a variety of facilities including the Smithsonian. The goals and design of the academy align with the Smithsonian’s goals and the Smithsonian’s Strategic Plan for Science. The Smithsonian Institution strongly supports the initiative, which it sees as complementing its education outreach work of promoting and providing a framework for the participation of their staff in the professional development of teachers.

  • Learn from distinguished guests such as world-renowned NASA scientific illustrator Sally Bensusen
  • Participate in hands-on activities that translate directly into the classroom using a variety of resources, including Smithsonian Science for Global Goals curriculum
  • Tour state of the art facilities such as the Harvard College observatory or get a behind-the-scenes look at Smithsonian museum

The Smithsonian Science Education Center is proud to offer Continuing Education Units (CEUs) for our Academies through Virginia Commonwealth University. The VCU Office of Continuing and Professional Education offers opportunities for personal and professional development as well as custom training solutions, logistical support and skills training in negotiation and mediation. Take the next step at: ocpe.vcu.edu.

* Not all academies are offered every year. Please see here to see which academies will be offered this year. ČITAJ VIŠE

National Council for Science and the Environment

NCSE has established a range of programs to increase the number, quality and diversity of people capable of bringing science to bear on the critical environmental challenges facing our society. These include the:

    EnvironMentors is a science education and national college access program with a mission to mentor and motivate high school students from communities underrepresented in the sciences as they plan and conduct environmental research and acquire skills that will allow them to build careers and become more active stewards of their communities and the environment. EnvironMentors chapters are located around the country and are hosted through partnerships with universities and educational based nonprofit organizations. Since 1992, the program has paired over 2,000 high school students with mentors through its network of chapters throughout the country.

NCSE Alliance of Sustainability and Environmental Leaders The NCSE Alliance of Sustainability and Environmental Academic Leaders (NCSE Leaders’ Alliance) brings together over 100 deans, directors, and academic leaders of environmental and sustainability departments, programs, and schools from all NCSE Members. Participation in the NCSE Leaders’ Alliance provides a valuable peer network for academic leaders to improve the quality and effectiveness of environmental and sustainability higher education programs, research, curricula, and workforce development.

Formerly known as the NCSE Council of Environmental Deans and Directors (CEDD) and the NCSE Community College Alliance for Sustainability Education (CCASE), the NCSE Leaders’ Alliance now captures both CEDD and CCASE and includes the expansion of academic leaders beyond deans and directors as the landscape of sustainability education has continued to evolve and grow. Each NCSE Member is invited to select two deans, directors, and academic leaders to represent their institution on the NCSE Leaders’ Alliance. The NCSE Leaders’ Alliance provides input and perspective on priorities of NCSE Members and advances the quality and effectiveness of interdisciplinary environmental and sustainability education and scholarship.

Current Membership Benefits

  • Participate in the NCSE Alliance of Sustainability and Environmental Academic Leaders (NCSE Leaders’ Alliance), a group for deans, directors, and academic leaders to connect with peers on a range of issues in higher education.
  • Attend the NCSE Annual Conference with select complimentary registrations and unlimited discounted registrations.
  • Engage with local governments and support students to navigate the science-policy interface.
  • Lead and participate in Communities of Practice, technical working groups that support and amplify education, research, and analysis from higher education institutions.
  • Access to NCSE Higher Education Research Reports for analysis of national trends in environmental and sustainability education and research to help institutions innovate.
  • Receive exclusive communications that shares tangible tools for science to engage in environmental decision-making, such as NCSE Pathways and a members-only email list.
  • Attend in-person and virtual events and webinars that convene scientists, thought leaders, and decision-makers.

National Energy Education Development (NEED) Project

Started in 1980, The National Energy Education Development (NEED) Project began as a one-day celebration of energy education when National Energy Education Day was recognized by a Joint Congressional Resolution. In the same year, President Jimmy Carter issued a Presidential Proclamation stressing the need for comprehensive energy education in our schools, a reduction in our dependence of fossil fuels, and increasing energy efficiency and the use of renewable energy technologies. Since its founding 40 years ago, NEED has kept its Kids Teaching Kids philosophy as a fundamental principle of NEED programming – encouraging students to explore, experiment, engage, and encouraging teachers to embrace student leadership in the classroom. NEED trains and assists teachers in harnessing the energy of the classroom – the energy of students.

NEED is expanding and evolving to best meet the needs of both teachers and students – in the classroom and beyond. In just the last decade The NEED Project has grown to encompass a curriculum portfolio of 100+ teacher and student guides designed to engage and teach teachers and students about energy. At the same time, the training opportunities offered by NEED expanded to include a variety of teacher professional development and training for educators and school district energy personnel as well. NEED’s work in after school programs, student clubs, scouting groups, and home school networks also continues to grow.

Growth Energy and National Association of Agricultural Educators (NAAE) High School Biofuels Curriculum

The curriculum is the first industry-supported biofuels curriculum that provides students a guided in-classroom experience and will offer agricultural educators the tools needed to provide students with an array of technical skills and historical knowledge in biofuels.

Agricultural educators considering including this curriculum into their lesson plan for the semester will have access to a number of resources to help supplement the activities provided within the curriculum. These include helpful presentation slides on the history, technology, and policy that make biofuels important for the rural economy, and assessment tools that educators can use to assess and track student progress. Additionally, each activity concludes with a short self-assessment meant to encourage discussion and identify key takeaways that students can reflect on. The curriculum can be downloaded here.

ExploraVision Science Competition K-12

ExploraVision is a science competition that encourages K-12 students of all interest, skill and ability levels to create and explore a vision of future technology by combining their imaginations with the tools of science. All inventions and innovations result from creative thinking and problem solving. That’s what ExploraVision is all about. ČITAJ VIŠE

Ethanol 101

Basic information about ethanol from “Let’s Clear the Air.” READ MORE

University of Idaho 4-H

The University of Idaho’s Biodiesel Education Program has released curriculum designed to help students between the ages of eight and 12 understand the concepts of energy and renewable energy. While the free seven-lesson program was written for 4-H clubs, the Biodiesel Education Program stresses it is also appropriate for use by elementary school teachers.

The curriculum features several hand-on activities, including a matching game, a fossil fuels timeline and a renewable energy model. Other components of the program include an energy tour and viscosity wands.

The program includes two parts, a student workbook and an instructor’s manual. The student workbook contains lessons titled “What is Energy,” “Liquid Fuels as Energy Sources,” “Fossil Fuels,” “Renewable Energy,” “Vegetable Oil and Animal Fat as Sources of Energy,” “How is Biodiesel Made and Used,” and “Scientists and Engineers.” READ MORE and MORE



Komentari:

  1. Zoloktilar

    Konačan sam, žao mi je, ali ova varijanta mi ne prilazi.

  2. Tolrajas

    Very curious:)

  3. Chevalier

    potpuno se slažem

  4. Mora

    Off your shoulders! From the tablecloth the way! To je već bolje!

  5. Migal

    To je laž.

  6. Tokala

    Vjerojatno ste u krivu?

  7. Etlelooaat

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