CAROTENÓIDES DE PIMENTÕES AMARELOS (Capsicum annuum, L.). CARACTERIZAÇÃO E VERIFICAÇÃO DE MUDANÇAS COM O COZIMENTO

Três cultivares de pimentões amarelos (Capsicum annuum, L.), Amador Híbrido F1, Zarco Híbrido F1 e Sunboy Híbrido F1, cultivados no Estado de São Paulo, foram analisados com o objetivo de se determinar os teores de carotenóides presentes, avaliar o valor de vitamina A e verificar a perda desta atividade após o cozimento. O procedimento analítico consistiu de saponificação com KOH aquoso 10%, antes da extração com solvente orgânico e separação por coluna cromatográfica. Os três cultivares apresentaram o mesmo perfil de carotenóides, sendo nove identificados: fitoflueno, alfa-caroteno, beta-caroteno, cis-zeta-caroteno, 5,6,5’,6’-diepóxi-beta-caroteno, 5,6,5’,6’-diepóxi-criptoxantina, beta-criptoxantina, luteína e violaxantina. O alfa e o beta-caroteno foram os principais carotenóides que contribuíram com a atividade pró-vitamínica A. Os teores de vitamina A, expressos em equivalentes de retinol/100g variaram entre 23,17 e 48,70 para as amostras cruas. Como os carotenóides são sensíveis ao calor, após o cozimento durante 10 minutos os valores de vitamina A foram menores, variando entre 18,22 e 36,27, com reduções entre 21,08 e 30,80%.

Composição de carotenóides de maracujá-amarelo (Passiflora edulis flavicarpa) in natura

A composição de carotenóides de maracujá-amarelo in natura (Passiflora edulis) comercializado em Campinas, Brasil, foi avaliada utilizando-se cromatografia líquida de alta eficiência. Em todas as amostras foram encontrados os seguintes carotenóides: beta-criptoxantina, prolicopeno, cis-zeta-caroteno, zeta-caroteno, beta-caroteno e 13-cis-beta-caroteno, além de neurosporeno e gama-caroteno em alguns lotes. Em geral, os teores de carotenóides nos diversos lotes foram significativamente diferentes. Em dois dos cinco lotes analisados, o z-caroteno foi o carotenóide principal, contribuindo com 41 e 62% do total de carotenóides, enquanto que em outros dois lotes, o beta-caroteno foi o majoritário, com 55 e 74% do total e em um dos lotes o cis-zeta-caroteno esteve presente em maior concentração, com 29%. Este tipo de inversão entre os carotenóides majoritários não é comumente encontrado em frutas e vegetais.

Pori Sinfoniettan kehitys yhdistyspohjaisesta amatööriorkesterista kunnalliseksi kaupunginorkesteriksi

Tutkielmassa tarkastellaan Porin kaupunginorkesterin, nykyisen Pori Sinfoniettan kehitystä yhdistyspohjaisesta amatööriorkesterista kunnalliseksi ammattiorkesteriksi. Arkistoaineisto muodostaa historiantutkimukseen kuuluvan tutkimuksen primäärilähdeaineiston. Tutkimuksellinen ote syntyy ennen kaikkea orkesterin kehitysvaiheiden kontekstoimisesta suomalaisen orkesterilaitoksen ja hyvinvointivaltion historiaan, sekä porilaiseen kulttuuripolitiikkaan. Porin ammattiorkesterihistoria alkaa jo 1800-luvulta Porin Soitannollisen Seuran perustamisesta. Yhteiskunnallisten muutosten vaikutuksesta sen toiminta loppui kokonaan 1910-luvulla. Orkesteritoiminta alkoi uudelleen 1920-1930-luvulla mutta tällä kertaa amatööripohjalla. Kaupungissa toimivan kahden eri orkesteriyhdistyksen toimintaa pyrittiin yhdistämään, mutta poliittiset erimielisyydet olivat esteenä. Porin kaupungin palkatessa kaupunginkapellimestarin orkesterin johtajaksi vuonna 1938, yhdistysten yhteistoiminta pääsi viimein alkamaan. Orkesterin yhteistoiminnasta alettiin nopeasti käyttää nimitystä Porin kaupunginorkesteri. 1950-1960-luvut olivat orkesteritoiminnan vakiintumisen aikaa. Orkesteria ei enää uhannut jatkuva pelko toiminnan loppumisesta. Vuonna 1955 orkesteri kunnallistettiin ja se sai ensimmäisen ammattilaisvakanssinsa. 1960-luvun lopulta alkaen orkesterin ammattivakanssit alkoivat lisääntyä.Orkesterista tuli ensin runko-orkesteri, jonka ammattilaiset saattoivat aluksi esiintyä pieninä yhtyeinä, sitten kamariorkesterina. 1970-luvun lopulla kaikki soittajat olivat päätoimisia ammattisoittajia. Ammattiorkesterin myötä orkesteri sai paljon uusia toimintamuotoja, kuten alueorkesteritoiminta, lastenkonsertit, ooppera jne. Orkesterilla oli kuitenkin yhä monia haasteita. Orkesterin toimintakulttuuri ei ollut ammattimaisella tasolla, mikä johti moniin orkesterin sisäisiin ja orkesterihallinnon välisiin ristiriitoihin. Monista yrityksistä huolimatta kaupunkiin ei tahdottu saada konserttitaloa ja kapellimestarin valinta jakoi kaupunkia. Kaupungin henkiset ja musiikilliset resurssit eivät ole aina olleet optimaaliset orkesterin tasapainoiselle kehitykselle. 1990-luvulta alkaen Pori Sinfonietta on yhä enemmän alkanut muistuttaa kansainvälisesti vertailukelpoista orkesteria. Orkesterin kapellimestarit hankitaan kutsumenettelyllä, sillä on oma konserttisali, konserttipäivä, omia levytyksiä ja sen toiminta edellytykset ovat toimistohenkilökunnan ja jousiston kasvun myötä parantuneet.

Qualidade microbiológica do leite determinada por características de produção

A atividade leiteira vem se tornando cada vez mais importante para a economia brasileira, apesar de ainda ser caracterizada como típica de pequenos produtores com baixa produtividade. Devido a esse perfil ocorre pouco investimento na atividade resultando em uma produção leiteira com baixa qualidade, identificada por altas contagens microbianas. O objetivo deste trabalho foi estabelecer uma relação entre práticas higiênicas de ordenha e a qualidade microbiológica do leite. Assim, um questionário sobre características de produção, ordenha e cuidados sanitários foi aplicado em 60 propriedades leiteiras localizadas na região de Viçosa, MG - Brasil, das quais foram coletadas amostras de leite para enumeração de aeróbios mesófilos (AM) e psicrotróficos (PSI). A maioria das propriedades foi caracterizada como pequena, com menos que 15 animais em lactação e produção diária menor que 50 L. De forma geral, verificou-se que os produtores adotam várias práticas higiênicas, mais frequentes em propriedades com produção diária superior a 100 L de leite (significativa em relação a pré e pós-dipping e uso do CMT). O reflexo dessas práticas foram as baixas contagens de AM e PSI nas amostras analisadas, com médias menores de forma significativa em propriedades que refrigeram a produção (Tukey, p < 0,05).

Molho cremoso à base de extrato de soja: estabilidade, propriedades reológicas, valor nutricional e aceitabilidade do consumidor

Na preparação de molhos de salada substitutos da maionese, para reduzir a quantidade de ovos e de óleo são utilizados emulsificantes e espessantes associados ou não à proteína de soja. Neste trabalho, o extrato de soja integral homogeneizado foi utilizado como base para a preparação de emulsão cremosa para substituir a maionese. O extrato de soja foi elaborado com teor de sólidos totais de 6 a 12%, após cozimento em água com bicarbonato, trituração e homogeneização a alta pressão. A emulsão preparada com diversas proporções de extrato de soja e óleos de soja e girassol foi também submetida à homogeneização a alta pressão variando de 1000 a 4000 psi. Na formulação do molho cremoso foram utilizados: açúcar, sal, ácido cítrico e diferentes hidrocolóides como estabilizantes. Os produtos foram avaliados quanto às características nutricionais, sensoriais, reológicas e de estabilidade. As condições mais favoráveis quanto à estabilidade e comportamento reológico para obtenção de molho cremoso foram atingidas quando se utilizou extrato de soja com 10% de sólidos, pressão de homogeneização de 3000 psi e relação extrato:óleo de 70:30, condição que apresentou redução de calorias de até 60%, em relação à maionese. Não foi observada diferença significativa (p < 0,05) quanto à preferência em relação ao sabor e consistência, bem como considerando a intenção de compra para as amostras de molho cremoso preparadas com os óleos de soja e girassol, utilizando-se como estabilizantes a goma xantana, maltodextrina ou carboximetilcelulose. O produto final é livre de colesterol, apresenta a vantagem do maior conteúdo proteico e teor reduzido de óleo e de calorias, além de adequada aceitação pelo consumidor, em relação à maionese tradicional e molhos para saladas.

Acetic acid as an intervention strategy to decontaminate beef carcasses in mexican commercial slaughterhouse

Beef can be contaminated during the slaughter process, thus other methods, besides the traditional water washing, must be adopted to preserve meat safety. The objective of this study was to evaluate the effect of 2% acetic acid interventions on the reduction of indicator bacteria on beef carcasses at a commercial slaughterhouse in Mexico. Reduction was measured by the count of mesophilic aerobic bacteria (TPC), total coliform (TC), and fecal coliform (FC) (log CFU/ cm²). Among the different interventions tested, treatments combining acetic acid solution sprayed following carcass water washing had greater microbial reduction level. Acetic acid solution sprayed at low pressure and longer time (10-30 psi/ 60 s) reached higher TPC, TC, and FC reductions than that obtained under high pressure/ shorter time (1,700 psi/ 15 s; P<0.05). Exposure time significantly affected microbial reduction on carcasses. Acetic acid solution sprayed after carcass washing can be successfully used to control sources of indicator bacteria on beef carcasses under commercial conditions.

Characterization, physicochemical stability, and evaluation of in vitro digestibility of solid lipid microparticles produced with palm kernel oil and tristearin

Solid lipid particles have been investigated by food researchers due to their ability to enhance the incorporation and bioavailability of lipophilic bioactives in aqueous formulations. The objectives of this study were to evaluate the physicochemical stability and digestibility of lipid microparticles produced with tristearin and palm kernel oil. The motivation for conducting this study was the fact that mixing lipids can prevent the expulsion of the bioactive from the lipid core and enhance the digestibility of lipid structures. The lipid microparticles containing different palm kernel oil contents were stable after 60 days of storage according to the particle size and zeta potential data. Their calorimetric behavior indicated that they were composed of a very heterogeneous lipid matrix. Lipid microparticles were stable under various conditions of ionic strength, sugar concentration, temperature, and pH. Digestibility assays indicated no differences in the release of free fatty acids, which was approximately 30% in all analises. The in vitro digestibility tests showed that the amount of palm kernel in the particles did not affect the percentage of lipolysis, probably due to the high amount of surfactants used and/or the solid state of the microparticles.

Pori Sinfoniettan kehitys yhdistyspohjaisesta amatööriorkesterista kunnalliseksi kaupunginorkesteriksi

Tutkielmassa tarkastellaan Porin kaupunginorkesterin, nykyisen Pori Sinfoniettan kehitystä yhdistyspohjaisesta amatööriorkesterista kunnalliseksi ammattiorkesteriksi. Arkistoaineisto muodostaa historiantutkimukseen kuuluvan tutkimuksen primäärilähdeaineiston. Tutkimuksellinen ote syntyy ennen kaikkea orkesterin kehitysvaiheiden kontekstoimisesta suomalaisen orkesterilaitoksen ja hyvinvointivaltion historiaan, sekä porilaiseen kulttuuripolitiikkaan. Porin ammattiorkesterihistoria alkaa jo 1800-luvulta Porin Soitannollisen Seuran perustamisesta. Yhteiskunnallisten muutosten vaikutuksesta sen toiminta loppui kokonaan 1910-luvulla. Orkesteritoiminta alkoi uudelleen 1920-1930-luvulla mutta tällä kertaa amatööripohjalla. Kaupungissa toimivan kahden eri orkesteriyhdistyksen toimintaa pyrittiin yhdistämään, mutta poliittiset erimielisyydet olivat esteenä. Porin kaupungin palkatessa kaupunginkapellimestarin orkesterin johtajaksi vuonna 1938, yhdistysten yhteistoiminta pääsi viimein alkamaan. Orkesterin yhteistoiminnasta alettiin nopeasti käyttää nimitystä Porin kaupunginorkesteri. 1950-1960-luvut olivat orkesteritoiminnan vakiintumisen aikaa. Orkesteria ei enää uhannut jatkuva pelko toiminnan loppumisesta. Vuonna 1955 orkesteri kunnallistettiin ja se sai ensimmäisen ammattilaisvakanssinsa. 1960-luvun lopulta alkaen orkesterin ammattivakanssit alkoivat lisääntyä. Orkesterista tuli ensin runko-orkesteri, jonka ammattilaiset saattoivat aluksi esiintyä pieninä yhtyeinä, sitten kamariorkesterina. 1970-luvun lopulla kaikki soittajat olivat päätoimisia ammattisoittajia. Ammattiorkesterin myötä orkesteri sai paljon uusia toimintamuotoja, kuten alueorkesteritoiminta, lastenkonsertit, ooppera jne. Orkesterilla oli kuitenkin yhä monia haasteita. Orkesterin toimintakulttuuri ei ollut ammattimaisella tasolla, mikä johti moniin orkesterin sisäisiin ja orkesterihallinnon välisiin ristiriitoihin. Monista yrityksistä huolimatta kaupunkiin ei tahdottu saada konserttitaloa ja kapellimestarin valinta jakoi kaupunkia. Kaupungin henkiset ja musiikilliset resurssit eivät ole aina olleet optimaaliset orkesterin tasapainoiselle kehitykselle. 1990-luvulta alkaen Pori Sinfonietta on yhä enemmän alkanut muistuttaa kansainvälisesti vertailukelpoista orkesteria. Orkesterin kapellimestarit hankitaan kutsumenettelyllä, sillä on oma konserttisali, konserttipäivä, omia levytyksiä ja sen toiminta edellytykset ovat toimistohenkilökunnan ja jousiston kasvun myötä parantuneet. Asiasanat: Porin musiikkielämä, kaupunginorkesteri, yhdistyshistoria, kulttuurihistoria, orkesterihistoria, sinfoniaorkesteri

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Metal oxides prepared through the nanocasting approach-mechanistic study, surface interactions and applications in separation

Mesoporous metal oxides are nowadays widely used in various technological applications, for instance in catalysis, biomolecular separations and drug delivery. A popular technique used to synthesize mesoporous metal oxides is the nanocasting process. Mesoporous metal oxide replicas are obtained from the impregnation of a porous template with a metal oxide precursor followed by thermal treatment and removal of the template by etching in NaOH or HF solutions. In a similar manner to the traditional casting wherein the product inherits the features of the mold, the metal oxide replicas are supposed to have an inverse structure of the starting porous template. This is however not the case, as broken or deformed particles and other structural defects have all been experienced during nanocasting experiments. Although the nanocasting technique is widely used, not all the processing steps are well understood. Questions over the fidelity of replication and morphology control are yet to be adequately answered. This work therefore attempts to answer some of these questions by elucidating the nanocasting process, pin pointing the crucial steps involved and how to harness this knowledge in making wholesome replicas which are a true replication of the starting templates. The rich surface chemistry of mesoporous metal oxides is an important reason why they are widely used in applications such as catalysis, biomolecular separation, etc. At times the surface is modified or functionalized with organic species for stability or for a particular application. In this work, nanocast metal oxides (TiO2, ZrO2 and SnO2) and SiO2 were modified with amino-containing molecules using four different approaches, namely (a) covalent bonding of 3-aminopropyltriethoxysilane (APTES), (b) adsorption of 2-aminoethyl dihydrogen phosphate (AEDP), (c) surface polymerization of aziridine and (d) adsorption of poly(ethylenimine) (PEI) through electrostatic interactions. Afterwards, the hydrolytic stability of each functionalization was investigated at pH 2 and 10 by zeta potential measurements. The modifications were successful except for the AEDP approach which was unable to produce efficient amino-modification on any of the metal oxides used. The APTES, aziridine and PEI amino-modifications were fairly stable at pH 10 for all the metal oxides tested while only AZ and PEI modified-SnO2 were stable at pH 2 after 40 h. Furthermore, the functionalized metal oxides (SiO2, Mn2O3, ZrO2 and SnO2) were packed into columns for capillary liquid chromatography (CLC) and capillary electrochromatography (CEC). Among the functionalized metal oxides, aziridinefunctionalized SiO2, (SiO2-AZ) showed good chemical stability, and was the most useful packing material in both CLC and CEC. Lastly, nanocast metal oxides were synthesized for phosphopeptide enrichment which is a technique used to enrich phosphorylated proteins in biological samples prior to mass spectrometry analysis. By using the nanocasting technique to prepare the metal oxides, the surface area was controlled within a range of 42-75 m2/g thereby enabling an objective comparison of the metal oxides. The binding characteristics of these metal oxides were compared by using samples with different levels of complexity such as synthetic peptides and cell lysates. The results show that nanocast TiO2, ZrO2, Fe2O3 and In2O3 have comparable binding characteristics. Furthermore, In2O3 which is a novel material in phosphopeptide enrichment applications performed comparably with standard TiO2 which is the benchmark for such phosphopeptide enrichment procedures. The performance of the metal oxides was explained by ranking the metal oxides according to their isoelectric points and acidity. Overall, the clarification of the nanocasting process provided in this work will aid the synthesis of metal oxides with true fidelity of replication. Also, the different applications of the metal oxides based on their surface interactions and binding characteristics show the versatility of metal oxide materials. Some of these results can form the basis from which further applications and protocols can be developed.

Pissakoulu lasten toiminnallisen kastelun hoidossa: Intervention vaikuttavuuden arviointi kastelun määrän ja lasten kokemuksien avulla

Kastelu on merkittävästi lapsen arkielämää haittaava kiusallinen oire. Koulun aloittavista perusterveistä lapsista 10 – 25 % kastelee viikoittain joko päivällä ja/tai yöllä. Keskeistä kastelevan lapsen hoidossa on kastelua vähentävien ja virtsarakon hallintaa lisäävien tapa-asioiden ohjaus ja uusien toimintamallien vakiinnuttaminen lapsen arkeen. International Children’s Continence Societyn (ICCS) suositusten mukaan arjen tapa-asioiden ohjaukseen perustuvan uroterapian tulisi olla kastelevan lapsen ensisijainen hoitomuoto. Tutkimuksen tarkoituksena oli arvioida pissakoulu-intervention vaikuttavuutta kastelun hoidossa sekä kuvata lasten kokemuksia pissakoulusta. Tutkimusasetelmassa käytettiin toisiaan täydentävinä sekä kvantatiivisia että kvalitatiivisia aineistonkeruumenetelmiä. Kuusikymmentäyhdeksän hoitoresistentiksi kastelijaksi luokiteltua 6-12-vuotiasta lasta osallistui polikliiniseen pissakouluun 4-6 lapsen ryhmissä kahtena erillisenä päivänä kahden kuukauden välein. Otoskooksi määriteltiin voima-analyysin perusteella 52. Ohjauksen sisältö perustui ICCS:n suosituksiin ja toteutuksessa painotettiin lapsilähtöisyyttä sekä yhdessä tekemistä ja keskustelua. Kvantitatiivisen osan aineistonkeruumenetelmänä käytettiin viikon WC-päiväkirjaa. Kastelun määrän muutoksia mitattiin ennen ja jälkeen molempien pissakoulupäivien ja tulokset analysoitiin SPSS tilasto-ohjelmalla. Kvalitatiivinen aineisto kerättiin pissakoulun toisen päivän päätteeksi haastattelemalla viisi ensimmäistä pissakouluryhmää, jonka jälkeen aineiston todettiin saturoituneen. Ryhmäkeskustelut analysoitiin aineistolähtöisellä sisällönanalyysillä. Tutkimukseen osallistuneista 69 lapsesta 58:lta saatiin tiedot kaikilta neljältä mittauskerralta. Lapset olivat iältään keskimäärin 8,3 vuotiaita, 34 oli tyttöä ja 24 poikaa. Verrattaessa ensimmäisen ja viimeisen mittauksen keskiarvojen välisiä eroja kuivat päivät (3,5 vrt. 5,3, p<0.001) ja kuivat yöt (2,4 vrt. 3,9, p<0.001) lisääntyivät tilastollisesti merkitsevästi. Päiväkastelun osalta 24/40 (60 %) ja yökastelun osalta 23/46 (50 %) lapsella kastelu väheni ≥50 %. Kuivaksi tuli 13/58 (22 %) lasta. Ryhmähaastatteluihin osallistui 19 lasta (12 tyttöä ja 7 poikaa, keski-ikä 8,9 vuotta). Lapset kokivat kastelun monella tapaa arkea haittaavaksi noloksi pulmaksi. Pissakoulu auttoi heitä tulemaan pissarakon pomoksi, jolloin pulmasta tuli hallittava tai siitä pääsi kokonaan eroon. Erityisen tärkeää lapsille oli tavata toisia kastelevia lapsia ja jakaa kokemuksia heidän kanssaan sekä oppia tekemällä lapsilähtöisin menetelmin. Pissakoulu on lapsilähtöinen ja vaikuttava hoitotyön interventio, joka arjen tapa-asioiden muutoksilla auttaa lapsia saavuttamaan paremman virtsarakon hallinnan ja vähentämään kastelua tarjoten myös arvokasta vertaistukea.

Characterization of the phosphorylation of thylakoid membrane proteins from cyanobacterium synechococcus sp. PCC 6301 in light state 1 and light state 2

The distribution of excitation energy between the two photosystems (PSII and PSI) of photosynthesis is regulated by the light state transition. Three models have been proposed for the mechanism of the state transition in phycobilisome (PBS) containing organisms, two involving protein phosphorylation. A procedure for the rapid isolation of thylakoid membranes and PBS fractions from the cyanobacterium Synechococcus m. PCC 6301 in light state 1 and light state 2 was developed. The phosphorylation of thylakoid and soluble proteins rapidly isolated from intact cells in state 1 and state 2 was investigated. 77 K fluorescence emission spectra revealed that rapidly isolated thylakoid membranes retained the excitation energy distribution characteristic of intact cells in state 1 and state 2. Phosphoproteins were identified by gel electrophoresis of both thylakoid membrane and phycobilisome fractions isolated from cells labelled with 32p orthophosphate. The results showed very close phosphoprotein patterns for either thylakoid membrane or PBS fractions in state 1 and state 2. These results do not support proposed models for the state transition which required phosphorylation of PBS or thylakoid membrane proteins.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

The measurement of the optical absorption cross section of photosystem 1 and photosystem 2 from whole live cells of porphyridium cruentum, in light state 1 and light state 2

The optical cross section of PS I in whole cells of Porphyridium cruentum (UTEX 161), held in either state 1 or state 2, was determined by measuring the change in absorbance at 820nm, an indication of P700+; the X-section of PS2 was determined by measuring the variable fluorescence, (Fv-Fo)/Fo, from PS2. Both cross-sections were 7 determined by fitting Poisson distribution equations to the light saturation curves obtained with single turnover laser flashes which varied in intensity from zero to a level where maximum yield occurred. Flash wavelengths of 574nm, 626nm, and 668nm were used, energy absorbed by PBS, by PBS and chla, and by chla respectively. There were two populations of both PSi and PS2. A fraction of PSi is associated with PBS, and a fraction of PS2 is free from PBS. On the transition S1->S2, only with PBS-absorbed energy (574nm) did the average X-section of PSi increase (27%), and that of PS2 decrease (40%). The fraction of PSi associated with PBS decreased, from 0.65 to 0.35, and the Xsection of this associated PS 1 increased, from 135±65 A2 to 400±300A2. The cross section of PS2 associated with PBS decreased from 150±50 A2 to 85±45 A2, but the fraction of PS2 associated with PBS, approximately 0.75, did not change significantly. The increase in PSi cross section could not be completely accounted for by postulating that several PSi are associated with a single PBS and that in the transition to state2, fewer PSi share the same number of PBS, resulting in a larger X-section. It is postulated that small changes occur in the attachment of PS2 to PBS causing energy to be diverted to the attached PSi. These experiments support neither the mobile-PBS model of state transitions nor that of spillover. From cross section changes there was no evidence of energy transfer from PS2 to PSi with 668nm light. The decrease in PS2 fluorescence which occurred at this wavelength cannot be explained by energy transfer; another explanation must be sought. No explanation was found for an observed decrease in PSi yield at high flash intensities.

Pigment orientation changes detected by low temperature linear dichroism spectroscopy: cold-hardening transition in phycobilisome-containing organisms

Low temperature (77K) linear dichroism spectroscopy was used to characterize pigment orientation changes accompanying the light state transition in the cyanobacterium, Synechococcus sp. pee 6301, and cold-hardening in winter rye (Secale cereale L. cv. Puma). Samples were oriented for spectroscopy using the gel squeezing method (Abdourakhmanov et aI., 1979) and brought to 77K in liquid nitrogen. The linear dichroism (LD) spectra of Synechococcus 6301 phycobilisome/thylakoid membrane fragments cross-linked in light state 1 and light state 2 with glutaraldehyde showed differences in both chlorophyll a and phycobilin orientation. A decrease in the relative amplitude of the 681nm chlorophyll a positive LD peak was observed in membrane fragments in state 2. Reorientation of the phycobilisome (PBS) during the transition to state 2 resulted in an increase in core allophycocyanin absorption parallel to the membrane, and a decrease in rod phycocyanin parallel absorption. This result supports the "spillover" and "PBS detachment" models of the light state transition in PBS-containing organisms, but not the "mobile PBS" model. A model was proposed for PBS reorientation upon transition to state 2, consisting of a tilt in the antenna complex with respect to the membrane plane. Linear dichroism spectra of PBS/thylakoid fragments from the red alga, Porphyridium cruentum, grown in green light (containing relatively more PSI) and red light (containing relatively more PSll) were compared to identify chlorophyll a absorption bands associated with each photosystem. Spectra from red light - grown samples had a larger positive LD signal on the short wavelength side of the 686nm chlorophyll a peak than those from green light - grown fragments. These results support the identification of the difference in linear dichroism seen at 681nm in Synechococcus spectra as a reorientation of PSll chromophores. Linear dichroism spectra were taken of thylakoid membranes isolated from winter rye grown at 20°C (non-hardened) and 5°C (cold-hardened). Differences were seen in the orientation of chlorophyll b relative to chlorophyll a. An increase in parallel absorption was identified at the long-wavelength chlorophyll a absorption peak, along with a decrease in parallel absorption from chlorophyll b chromophores. The same changes in relative pigment orientation were seen in the LD of isolated hardened and non-hardened light-harvesting antenna complexes (LHCII). It was concluded that orientational differences in LHCII pigments were responsible for thylakoid LD differences. Changes in pigment orientation, along with differences observed in long-wavelength absorption and in the overall magnitude of LD in hardened and non-hardened complexes, could be explained by the higher LHCII monomer:oligomer ratio in hardened rye (Huner et ai., 1987) if differences in this ratio affect differential light scattering properties, or fluctuation of chromophore orientation in the isolated LHCII sample.