Ruoka-aineista ja sisäilmasta eristettyjen Basillusten ei-proteiini toksiinit : Rakenteet, fysikaalis-kemialliset ominaisuudet ja vaikutusmekanismit eukarioottisissa soluissa

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.Tämän väitöskirjan aiheena on sisäilmasta ja ruoka-aineista eristettyjen itiöitä muodostavien Basillusten tuottamien lämmönkestävien toksisten ei-proteiini yhdisteiden rakenne ja toksisuus nisäkäs soluille Basillusten erittämien toksisten ei-proteiini yhdisteiden esiintymisestä, vaikutuksista ja kulkeutumistavoista kosteusvaurioisten rakennusten sisäilmassa ei ole paljon tutkimustietoa. Basillukset muodostavat ilmassa liikkuvia itiöitä, jotka voivat toimia toksiinien kantajina, kuten sisäilmasta eristetyt toksisten sienien itiöt. Ruoka-aineissa Basillusten itiöt ovat ongelmana, koska ne kestävät ruuanvalmistuksessa käytettäviä lämpötiloja ja ovat valmiita lisääntymään ruoka-aineissa suotuisissa olosuhteissa. Basillusten toksiset yhdisteet löydettiin käyttäen toksisuus indikaattorina sian siittiöiden liikkuvuuden muutosta toksisen altistuksen johdosta. Yhdisteiden puhdistuksessa, detektoinnissa ja rakenteen analysoimisessa käytettiin kaasu- ja nestekromatografiaa, massaspektrometriaa ja ydinmagneettista resonanssispektroskopiaa. Yhdisteiden toksisuutta ja vaikutustapoja tutkittiin käyttäen sian siittiöitä, kissan keuhkosoluja, ihmisen hermosoluja ja rotan maksasta eristettyjä mitokondrioita sekä BLM:ää (Black lipid membrane) ioniforisten ominaisuuksien selvittämiseen. Tämän väitöskirjatyön tuloksena löydettiin uusi K+ > Na+ > Ca2+ ionikanavan muodostava toksiini joka sai nimen amylosiini. Amylosiinia tuottivat kosteusvaurioisen rakennuksen sisäilmasta eristetyt B. amyloliquefaciens kannat. Amylosiini puhdistettiin nestekromatografilla ja monoisotooppinen atomipaino 1197 Da selvitettiin sähkösumutusionisaatio ioniloukku massaspektrillä. Lisäksi sen todettiin olevan peptidi happohydrolyysin ja aminohappojen kaasukromatografisen massaspektrometri analyysin avulla. Sen rakenteen todettiin sisältävän kromoforisen polyeeni ryhmän käyttämällä ydinmagneettista resonanssispektroskopiaa. Amylosiinille kehitettiin nestekromatografinen kvantitointimenetelmä käyttäen makrolaktoni polyeenia amphoterisin B:tä (moolimassa 924 g/mol) referenssiaineena. Ruoka-aine myrkytyksestä eristetty B. licheniformis kanta tuotti solukalvoa rikkovaa lipopeptidiä, likenysiini A:ta, joka puhdistettiin nestekromatografilla. Likenysiini A identifioitiin matriisiavusteisella laserionisaatio lentoaika-massaspektrometrillä sen tuottamien protonoituneiden sekä kationioituneiden ammonium, natrium, kalium molekyylien avulla. Sen tuottamat protonoidut molekyylit olivat m/z 1007, 1021 ja 1035. Likenysiini A:n aminohapot analysoitiin sähkösumutus-ionisaatio kolmoiskvadrupolin massaspektrometrin tuottaman tandem-massaspektrin sekä lipopeptidin happohydrolyysin ja aminohappojen nestekromatografisen analyysin avulla. Sisäilmasta eristetyn B. amyloliquefaciens kannan havaittiin tuottavan amylosiinin lisäksi lipopeptideja: sienille toksista fengysiinia ja solukalvoa hajottavaa surfaktiinia. Ne identifioitiin sähkösumutusionisaatio ioniloukku massaspektrillä niiden tuottamien protonoituneiden ja kationisoituneiden ammonium, natrium, kalium molekyylien sekä analysoimalla niiden tuottamat tandem massaspektrit. Surfaktiinin tuottamat protonoidut molekyylit olivat m/z 1009, 1023 ja 1037 ja fengysiinin m/z 1450, 1463, 1493 ja 1506. Kereulidi (moolimassa 1152 g/mol) puhdistettiin nestekromatografisesti ruokamyrkytys tapaukseen liittyvästä B. cereus kannasta. Kereulidi identifioitiin sen sähkösumutusionisaatio kolmoiskvadrupoli massaspektrometrin tuottamien protonoidun molekyylin m/z 1154, ammonium m/z 1171, natrium m/z 1176 ja kalium m/z 1192 kationisoituneiden molekyylien avulla sekä tulkitsemalla kereulidin protonoidun molekyylin m/z 1154 tandem-massaspektrin tuote-ionit. Kereulidille kehitettiin nestekromatografinen kvantitointimenetelmä käyttäen sitä muistuttavaa valinomysiinia (moolimassa 1110 g/mol) referenssiaineena. Lisäksi osoitettiin empiirisesti, käyttämällä BLM:ää, että B. cereuksen tuottama emeettinen toksiini, kereulidi on tehokas ja spesifinen kalium ionifori, jonka toksinen vaikutus kohdistuu erityisesti mitokondrioiden toimintaan

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b–LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨm), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨm. Fusaricidins A plus B, LI-F05a, and LI-F05b–LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies

The Barnacle Goose (Branta leucopsis) in the archipelago of southern Finland - population growth and nesting dispersal

We studied the population growth and expansion of Barnacle Goose (Branta leucopsis) in Helsinki archipelago, southern Finland. Barnacle Goose breeding was first recorded in Helsinki in 1989. During our study 1996-2013 the number of nesting geese increased from 24 to 740 pairs. We analyzed the role of protected islands in the population growth, and the factors behind differences in growth rates. Our study data consisted of 104 islands. Of these, 29 are protected from private recreational activity (nature reserve ormilitary areas) and were established prior to the start of our study. We predicted that protected areas would have a positive impact on Barnacle Goose population growth. In part of the study period (2002-2013) the population growth in our study area was much steeper in protected islands compared to islands with open access. However, breeding densities in those unprotected islands were higher than in protected islands in the early years of the study. We found that the most important factors affecting pair numbers in islands are island size and the time it has been inhabited, in addition to island distance from the islands southeast of Helsinki, where breeding expansion started. Island protection had no effect on the breeding geese numbers or current densities on the islands. Results indicate that early breeders like Barnacle Geese do not benefit from island protection probably because the recreational use of the islands is scant early in the spring.Peer reviewe

Endotoxin levels and contribution factors of endotoxins in resident, school, and office environments - A review

As endotoxin exposure has known effects on human health, it is important to know the generally existing levels of endotoxins as well as their contributing factors. This work reviews current knowledge on the endotoxin loads in settled floor dust, concentrations of endotoxins in indoor air, and different environmental factors potentially affecting endotoxin levels. The literature review consists of peer-reviewed manuscripts located using Google and PubMed, with search terms based on individual words and combinations. References from relevant articles have also been searched. Analysis of the data showed that in residential, school, and office environments, the mean endotoxin loads in settled floor dust varied between 660 and 107,000 EU/m(2), 2180 and 48,000 EU/m(2), and 2700 and 12,890 EU/m(2), respectively. Correspondingly, the mean endotoxin concentrations in indoor air varied between 0.04 and 1610 EU/m(3) in residences, and 0.07 and 9.30 EU/m(3) in schools and offices. There is strong scientific evidence indicating that age of houses (or housing unit year category), cleaning, farm or rural living, flooring materials (the presence of carpets), number of occupants, the presence of dogs or cats indoors, and relative humidity affect endotoxin loads in settled floor dust. The presence of pets (especially dogs) was extremely strongly associated with endotoxin concentrations in indoor air. However, as reviewed articles show inconsistency, additional studies on these and other possible predicting factors are needed. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Acrebol, a novel toxic peptaibol produced by an Acremonium exuviarum indoor isolate

Aims: To identify a toxin and its producer isolated from woody material in a building where the occupants experienced serious ill health symptoms. Methods and Results: Hyphal extracts of an indoor fungus, identified as the cycloheximide-tolerant species Acremonium exuviarum, inhibited motility of boar spermatozoa (EC50 5 ± 2 lg of crude solids ml)1) and caused cytolysis of murine neuroblastoma cells (MNA) and feline fetal lung cells (FL). The responsible substances were purified and identified as two structurally similar, heatstable, novel, toxic peptaibols, 1726 Da and 1740 Da, respectively, with amino acid sequences of Acetyl-Phe-Iva ⁄ Val-Gln-Aib-Ile-Thr-Leu-Aib-Pro-Aib-Gln- Pro-Aib-(X-X-X)-SerOH and Acetyl-Phe-Iva ⁄ Val-Gln-Aib-Ile-Thr-Leu-Val-Pro- Aib-Gln-Pro-Aib-(X-X-X)-SerOH. Purified acrebol inhibited motility of boar sperm, depleted ATP half-content in 1 day (EC50 of 0Æ1 lg ml)1, 60 nmol l)1) depolarised the mitochondria after 2 days, but did not affect the cellular content in NADH. This indicates mitochondrial toxicity. Plate-grown biomass of A. exuviarum BMB4 contained 0Æ1–1% (w ⁄ w) of acrebol, depending on the culture medium. Conclusions: Acrebol paralysed the energy generation of mammalian cells suggesting that mitochondria were its target of action. Significance and Impact of the Study: Acremonium exuviarum, as an indoor fungus, is potentially hazardous to health because of the toxic peptaibols that it produces.Peer reviewe

Sadonkorjuun jälkeisen kuivauksen ja siilo-olosuhteiden vaikutus mallasohran elävyyteen ja mikrobiologiseen laatuun

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