The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

Amerika Birleşik Devletleri&#39;nde düzenlenmiş değildir, burada yapılan omurgasız hayvan çalışmaları; Bu nedenle Kurumsal Hayvan Bakım ve Kullanım Komitesi, protokollerin incelenmesi için yetkisi yoktur. Bu ABD&#39;de düzenlemenin yetki alanına giren değil yerine, yazarlar burada bu çalışmalar samimi etik kullanımı konusunda çaba, özen ve bu hayvanların tedavi, birey sayısı minimize edilmiş ve bu çabaların ile yapılmıştır devlet olduğunu Basel Bildirgesi ve Laboratuar Hayvan Uluslararası Bilim Konseyi (ICLAS) etik kurallarına uyum içindedir. 1. Doryteuthis pealeii (D pealeii) Diseksiyon <ol><li> Satın alma decapitated kalamar D. Woods Hole, MA Marine Biological Laboratory at Deniz Kaynakları Bölümü veya herhangi bir taze balık pazarından pealeii. yuvarlanabilir veya daha önce bu prosedürü uygulamak için değiştirilmiş olan örnekleri kullanmayın. Özellikle, kromatophore tabakası hala örnek üzerinde bozulmamış olmalıdır. <ol><li> Hayvanlar hemen kullanılmazsa, kullanılana kadar -20 ° C&#39;de saklayın. örnekleri defrost için, diseksiyon önce 1 saat boyunca oda sıcaklığında su koyun. </li></ol></li><li> hayvanın arka tarafı boyunca kesilmiş paslanmaz çelik düz ince nokta diseksiyon makas, kullanma. ventral manto üssünde başlayın ve yüzgeç bölgede biter. </li><li> forseps diseksiyon ve diseksiyon makas kullanarak bağ dokusu kesilmesi ile kaldırarak iç organların (mide, solungaç, mürekkep bezi, üreme organları, sistemik kalp ve brakiyal kalp) tüm çıkarın. Bir örnek torbaya organları atın. </li><li> Standart 11-1 / 2&#39;de (fin tarafı) manto pin T-işaretçilerine diseksiyon kullan &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;esnek bir diseksiyon pedi içeren alüminyum diseksiyon tava. doku Batmak bir tek kullanımlık polistiren vakum filtrasyon sistemi ile filtre edilmiş deniz suyu 250 ml0.2 um gözenek boyutlarına ihtiva etmektedir. </li><li> Dikkatle bozulmamış chromatophore içeren cilt tabakası tutarak, diseksiyon forseps ile epidermal deri tabakası (opak renk) çıkartın. epidermal tabaka kaldırıldıktan sonra, bir numune torbasına atın. </li><li> forseps ve diseksiyon makas ile chromatophore tabakasının küçük (1 cm x 1 cm) bölümleri parçalara ayır. 1.5 ml mikro santrifüj tüplerine numune toplamak (disseke doku ile her boş tüpün yarısını doldurun). </li><li> tüpler içeren dokuya süzülmüş deniz suyu 500 ul ekle. Numuneler 4 ° C&#39;de gece boyunca saklanır. </li></ol> 2. Ayırma chromatophore Pigment Granül <ol><li> Doku sindirimi <ol><li> Papain / kolajenaz hazırlanması <ol><li> Kağıt tartmak kollajenaz 30 mg ve 4 &quot;x 4&quot; düşük azot kullanarak papain 5 mg tartın. bir vida kapaklı 50 ml&#39;lik bir polipropilen santrifüj tüpü içine bu aktarın. </li><li> deioniz 10 ml ölçünBir dereceli pipet kullanarak ed su. papain / kollajenaz içeren flakon doğrudan ekleyin. çözelti kadar yüksek ayarda Vortex açıktır. </li></ol></li><li> Hücre dışı Doku Çıkarma <ol><li> x g 5 dakika 14,000 Aşama 1.7 toplanan chromatophore doku santrifüjleyin. Bir plastik atık kovaya deniz suyunun üst tabaka atın. </li><li> P1000 değişken hacimli tek kanallı mikropipet kullanarak doku papain / kollajenaz çözüm 500 ul ekleyin. En yüksek ayarda 1-2 dakika boyunca her bir örnek Vortex. </li><li> çevreleyen doku hücreleri ayırmak için 40 kHz&#39;lik bir frekansta 5 dakika boyunca numune sonikasyon. </li><li> 14.000 x g&#39;de 5 dakika boyunca santrifüje. Bir plastik atık kovaya süpernatant atın. </li><li> x g 5 dakika 14,000 toplanan chromatophore doku santrifüjleyin. Bir plastik atık kovaya deniz suyunun üst tabaka atın. bir kez daha tekrarlayın. </li><li> El ile kalan geniş doku sn kaldırmakBir sonraki adıma geçmeden önce forseps ile bu süreçte sindirimi yoktu leri. </li></ol></li></ol></li><li> Pigment granüller Yalıtımlı <ol><li> Homojenizasyon tamponu hazırlanması <ol><li> 2.38 g tartılır (4- (2-hidroksietil) -1-piperazinetansülfonik asit) (HEPES, 100 mM), magnezyum klorür (10 mM), potasyum-aspartat 0.856 g (50 mM) 0.095 gr, 0.015 g ditiyotireitol (1 mM). bir vida kapaklı 150 ml&#39;lik bir polistiren kap içine aktarın. Ticari bir küçük tablet proteaz önleyici. </li><li> Bir dereceli silindir kullanarak, iyonu giderilmiş su, 100 ml ölçün. homojenizasyon tuzlar içeren kabın doğrudan ekleyin. çözelti kadar Vortex açıktır. </li></ol></li><li> Pigment Granül homojenizasyon <ol><li> 5 dakika boyunca 14.000 xg&#39;de adım 2.1 her bir örnek santrifüj. Bir plastik atık kovaya süpernatant atın. Bu örnek dışı doku geçersiz olmalıdır. </li><li> Ekle 500# 181; 1-2 dakika her biri için her bir tüp ve vorteks homojenizasyon tamponu iyice örnekleri karıştırın. 30 dakika (~ 40 kHz) numuneler sonikasyon ve 14,000 x g &#39;de santrifüj 5 dakika ile takip edin. </li><li> Bir plastik atık kovaya supernatant atmak ve chromatophore sakkuluslar ve protein hayvan iplerini tam sindirim sağlamak için önceki adımı 2-3 kez tekrarlayın. Geri kalan çözelti, açık sarı bir süpernatan ve izole pigment granülleri içeren kırmızı renkli bir pelet içermelidir. </li></ol></li></ol></li></ol> 3. Pigment Ekstraksiyon <ol><li> Asidik metanol preparasyonu (HCl-MeOH) <ol><li> Dikkatle 5 ml metanol (MeOH) konsantre (36,5-38% (w / w)), hidroklorik asit (HCI) 25 ul ekle. bir vida kapaklı bir cam numune şişesine HCl-MeOH çözeltisi saklayın. eşit çözüm karıştırmak için hafifçe döndürün. NOT: Daha fazla asidik çözelti, daha pigment fi sırasında çıkartılacaktırilk çıkarma. </li></ol></li><li> Pigment Ekstraksiyon <ol><li> 14.000 xg&#39;de 5 dakika boyunca (2.2.2) pigment granül süspansiyonu Santrifüj ve bir plastik atık kovaya süpernatant atın. </li><li> HCl-MeOH çözeltisi 500 ul ilave edin ve 3-4 dakika ~ için her bir örnek girdap. 10 dakika boyunca (~ 40 kHz) numuneler sonikasyon, daha sonra 14,000 x g&#39;de 5 dakika boyunca santrifüj. </li><li> Bir transfer pipeti kullanarak, bir 1 dramlık vidalı kapaklı cam şişede supernatant toplamak. süpernatant rengi koyu kırmızı olmalıdır. </li><li> başka bir renk (~ 4-5 yıkar) ekstre kadar çıkarma adımı 3.2.2 tekrarlayın. NOT: Her çıkarma ~ pigment 1.5 ml verecektir. Geri kalan renksiz bir pelet pigmenti ekstre granüller olup. Not: renksiz granül ve ekstre pigmentler iki sonraki kullanıma kadar 4 ° C sıcaklıkta su içinde saklanabilir. </li></ol></li></ol> Ekstraksiyon Süreci boyunca 4. Karakterizasyonu <ol><li> Tarama ElECTRON Mikroskopi <ol><li> ekstraksiyon işlemi saflığını doğrulamak için taramalı elektron mikroskobu (SEM) kullanılarak (adım 2.2.1 ve pigment ekstre adım 3.2.3 dan girmemiş) Görüntü granülleri. </li><li> 14.000 xg&#39;de 5 dakika boyunca görüntüleme, ilk santrifüj hem reaksiyona girmemiş granül ve pigment çıkarılan granüller granül örnekleri hazırlamak ve bir plastik atık kovaya süpernatant atmak için. 1 ml etanol ve mevduat bir transfer pipet kullanarak alüminyum SEM taslakları üzerine granül süspansiyonu 2-3 damla bu yeniden askıya. </li><li> gece boyunca örnekleri kurutulduktan sonra 15 mil bir kaplama elde etmek 3 dakika için altın, platin kaplamalı kat püskürtmeyle çökeltilmesi. </li><li> Bir Schottky verici ve bir ikincil elektron dedektörü ile bir SEM ile görüntü. 5-7 kV ışını gerilim ve görüntüye 8 ve 9 mm, bu malzemeler arasında bir çalışma mesafesi kullanın. </li></ol></li><li> Ultraviyole-Görünür (UV-Vis) Spektrofotometrik Ölçümler <ol><li> absorbans pro Monitör200-800 nm bir çift ışın spektrofotometre kullanılarak 3 koşulları için dosyaları (2.2.2 girmemiş granül, 3.2.3 çıkarılan pigment ve 3.2.4 pigment çıkarılan granül). </li><li> reaksiyona girmemiş granüller ve pigment ekstre granüller için 1 ml&#39;lik bir küvete deiyonize su kullanılarak boş bir ölçüm toplayın. Her iki numune için UV-Vis absorpsiyon spektrumları toplayın. </li><li> ekstre pigment için 1 ml&#39;lik bir küvete asidik metanol çözeltisi kullanılarak boş bir ölçüm toplayın. numune için UV-Vis absorpsiyon spektrumu toplayın. </li><li> Soğurma tepe noktasının maksimum görece yüksekliğe ulaşır dalgaboyunu tanımlayarak Her numunenin maksimum bir dalga boyuna belirler. </li></ol></li></ol>

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We have nothing to disclose.

Bu kalamar chromatophore granüllerden pigmentlerin elde etmek için bir yöntem gösterilmiştir. Özellikle granülleri hedefleyerek, hedefimiz adaptif renklenmeye aracılık rollerini tespit etmektir. Bu yöntem toplu doku örnekleri 14 veya dondurularak kurutulmuş bir cilt 15 kullanılarak cephalopod pigmentler karakterize etmek için tasarlanan önceki raporlarda farklıdır. Bu protokol, chromatophore pigmentler ekstre etkili olsa da, küçük reaksiyon ölçekle...

Bu tür kalamar, mürekkepbalığı ve ahtapot gibi kafadan bacaklılar dinamik kamufle ve sinyalizasyon için kendi görünümünü değiştirmek için yeteneği var. 1-6 Bu özellik kromatoforların olarak bilinen pigmentli organların seçici alansal genişleme kısmen destekleniyor. 4,7-9 kromatoforların vardır onlar harekete geçirilir gibi kas lifleri tarafından radyal demirlemiş bir cytoelastic sacculus içinde sınırlı nano yapılı pigment granüllerinin bir ağ ihtiva yumuşak aktüatörler. 1,3, kromatoforlar organı boyunca granülleri dağıtmak sunulan yüzey alanı% 500 genişletin. 3,7 Bu işlem, kromatoforların bir dizi üzerinden birbiriyle uyumlu olduğunda, 10,11, hayvanın genel rengi değiştirilir. Bu pigment granülleri bu renk değişikliğine neden olduğu bilinmekle birlikte, bunların bileşimi bilinmemektedir. Bu izole edilmesi ve gelecekteki kompozisyon çalışmalar için uyarlanabilir chromatophore pigmentler saflaştırmak için bir prosedür açıklanmaktadır. 3,12 chromatophore içeren doku kafadanbacaklı itibaren dikkatli çıkarıldıktan ile hasat edilir. Bir sindirim ve homojenizasyon işlem daha sonra chromatophore hücreleri çevreleyen doku ayırmak ve ayırmak için kullanılır. nano yapılı granüller tekrar sonifikasyon ve santrifüjlemeden kullanılarak geri kalan kromatoforların den daha sonra izole edilir ve saflaştırılır. Saflaştırma üzerine, pigmentler, asidik metanol solüsyonları kullanılarak kelebek kanatları görünür renk ekstre uyarlanmış olan bir işlemde granüller elde edilir. 13 tarama elektron mikroskobu (SEM) ve spektrofotometri chromatophore pigmentler kullanarak başarılı şekilde ekstre onaylamak için kullanılan bu süreç. Bu yöntem, C moleküler katılım keşfetmek için kullanılan chromatophore granüller izolasyonunu anlatmaktadıroloration Kafadanbacaklılarda içinde. Bütün hayvanlardan 12 Küçük molekül ekstraksiyon genellikle uzun ve sıkıcı bir süreç olabilir. Buradaki amaç Kafadanbacaklılarda içinde nanoyapılı granüllerden pigmentlerin edinimi için etkili ve kolay protokolün gelecek araştırmacıları bilgilendirmektir.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Kromatoforlar D. disseke edilir pealeii dorsal manto (Şekil 1A, 1B). Bunlar kaldırıldıktan sonra, kromatoforlar lize edildi ve pigment granülleri (Şekil 2A, 2B) üzerinden izole etmek için santrifüj ve yıkama döngüleri kullanılarak saflaştırılır. Asidik metanol solüsyonları (HCl-MeOH) çözülebilir pigment özü ve çözünmeyen, renksiz bir pigment ekstre granüller hasıl granüller...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Kalamar Doryteuthis pealeii kromatoforlardaki nano yapılı granüllerden pigmentlerin çıkarılması için bir protokol verilmektedir.

Squid pigmentlerin çıkarılması için bir yöntem<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

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A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K Görüntüleme.  University of New Hampshire.  Kalamar Doryteuthis pealeii kromatoforlardaki nano yapılı granüllerden pigmentlerin çıkarılması için bir protokol verilmektedir. 

Video: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Bir Balık-besleme Laboratuvar biyodeneyinde Deniz Organizmaların Dokular Sekonder Metabolitlerin Antipredatory Aktivite değerlendirmek için

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Çevre

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Karın Pigmentasyonunun Karaciğer Miktarında Nicelleştirilmesi<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Genetik

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Petrol Emülsiyonlar Su: Hidrofobik Pigmentler ile Suda çözünür Klorofil bağlayıcı Proteinler Montaj için Yeni Bir Sistem

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Kimya

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

İzole ve ışık-hasat antenler Diatom Cyclotella Meneghiniana lipozomlar Thylakoid lipidler ile gelen ekleme

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Biyokimya

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

İmmünoloji ve Enfeksiyon

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Mühendislik

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

Bitkiler ve Yeşil Alg Işık hasat Kompleksleri İn Vitro Sulandırma içinde

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Biyoloji

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Nörobilim

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Zebra Balığı Kullanarak Pigmentasyon Düzenleyicilerini Tanımlamak için Ters Genetik Yaklaşım

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

Squid pigmentlerin çıkarılması için bir yöntem

Özet

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Bir Balık-besleme Laboratuvar biyodeneyinde Deniz Organizmaların Dokular Sekonder Metabolitlerin Antipredatory Aktivite değerlendirmek için

Karın Pigmentasyonunun Karaciğer Miktarında Nicelleştirilmesi

Petrol Emülsiyonlar Su: Hidrofobik Pigmentler ile Suda çözünür Klorofil bağlayıcı Proteinler Montaj için Yeni Bir Sistem

İzole ve ışık-hasat antenler Diatom Cyclotella Meneghiniana lipozomlar Thylakoid lipidler ile gelen ekleme

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

Bitkiler ve Yeşil Alg Işık hasat Kompleksleri İn Vitro Sulandırma içinde

Drosophila Fotoreseptörlerinde Fotopigment Düzeylerini Ölçmek için Elektrofizyolojik Yöntemler

Zebra Balığı Kullanarak Pigmentasyon Düzenleyicilerini Tanımlamak için Ters Genetik Yaklaşım