Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species

BACKGROUND: Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS: Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS: Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study

A method for the extraction and quantitation of phycoerythrin from algae

A summary of a new technique for the extraction and quantitation of phycoerythrin (PHE) from algal samples is described. Results of analysis of four extracts representing three PHE types from algae including cryptomonad and cyanophyte types are presented. The method of extraction and an equation for quantitation are given. A graph showing the relationship of concentration and fluorescence units that may be used with samples fluorescing around 575-580 nm (probably dominated by cryptophytes in estuarine waters) and 560 nm (dominated by cyanophytes characteristics of the open ocean) is provided

A potential cyanobacterial ancestor of Viridiplantae chloroplasts

The theory envisaging the origin of plastids from endosymbiotic cyanobacteria is well-established but it is difficult to explain the evolution (spread) of plastids in phylogenetically diverse plant groups. It is widely believed that primordial endosymbiosis occurred in the last common ancestor of all algae^1^, which then diverged into the three primary photosynthetic eukaryotic lineages, viz. the Rhodophyta (red algae), Glaucocystophyta (cyanelle-containing algae) and Viridiplantae (green algae plus all land plants)^2^. Members of these three groups invariably have double membrane-bound plastids^3^, a property that endorses the primary endosymbiotic origin of the organelles. On the other hand, the three or four membrane-bound plastids of the evolutionary complicated Chromalveolates [chromista (cryptophytes, haptophytes, and stramenopiles) and alveolata (dinoflagellates, apicomplexans, and ciliates)] are inexplicable in the light of a single endosymbiosis event, thereby necessitating the postulation of the secondary^4,5^ and tertiary^6^ endosymbiosis theories where a nonphotosynthetic protist supposedly engulfed a red or a green alga^7^ and an alga containing a secondary plastid itself was engulfed^8^ respectively. In the current state of understanding, however, there is no clue about the taxonomic identity of the cyanobacterial ancestor of chloroplasts, even though there is a wide consensus on a single primordial endosymbiosis event. During our metagenomic investigation of a photosynthetic geothermal microbial mat community we discovered a novel order-level lineage of Cyanobacteria that - in 16S rRNA gene sequence-based phylogeny - forms a robust monophyletic clade with chloroplast-derived sequences from diverse divisions of Viridiplantae. This cluster diverged deeply from the other major clade encompassing all hitherto known groups of Cyanobacteria plus the chloroplasts of Rhodophyta, Glaucocystophyceae and Chromalveolates. Since this fundamental dichotomy preceded the origin of all chloroplasts, it appears that two early-diverging cyanobacterial lineages had possibly given rise to two discrete chloroplast descents via two separate engulfment events

The fundamental role of quantized vibrations in coherent light harvesting by cryptophyte algae

The influence of fast vibrations on energy transfer and conversion in natural molecular aggregates is an issue of central interest. This article shows the important role of high-energy quantized vibrations and their non-equilibrium dynamics for energy transfer in photosynthetic systems with highly localized excitonic states. We consider the cryptophyte antennae protein phycoerythrin 545 and show that coupling to quantized vibrations which are quasi-resonant with excitonic transitions is fundamental for biological function as it generates non-cascaded transport with rapid and wider spatial distribution of excitation energy. Our work also indicates that the non-equilibrium dynamics of such vibrations can manifest itself in ultrafast beating of both excitonic populations and coherences at room temperature, with time scales in agreement with those reported in experiments. Moreover, we show that mechanisms supporting coherent excitonic dynamics assist coupling to selected modes that channel energy to preferential sites in the complex. We therefore argue that, in the presence of strong coupling between electronic excitations and quantized vibrations, a concrete and important advantage of quantum coherent dynamics is precisely to tune resonances that promote fast and effective energy distribution.Comment: 16 Pages, 10 figures. Version to appear in The Journal of Chemical Physic

PHYCOBILISOMES AND ISOLATED PHYCOBILIPROTEINS. EFFECT OF GLUTARDIALDEHYDE AND BENZOQUINONE ON FLUORESCENCE

The fluorescence of the biliproteins C-phycocyanin from Spirulina platensis, B-phycoerythrin from Porphyridium cruentum and of isolated whole P. cruentum phycobilisomes is quenched in the presence of glutardialdehyde (GA) or benzoquinone (BQ). The kinetics of fluorescence decrease thus induced is biphasic. If GA is used as a quencher, the fluorescence can be recovered at 77 K. Contrary to the GA-effect, only a minor recovery takes place with BQ at 77K, thus demonstrating a different mechanism of action of GA and BQ on biliprotein

Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria

Understanding the functional diversity of specific microbial groups at the global scale is critical yet poorly developed. By combining the considerable knowledge accumulated through recent years on the molecular bases of photosynthetic pigment diversity in marine Synechococcus, a major phytoplanktonic organism, with the wealth of metagenomic data provided by the Tara Oceans expedition, we have been able to reliably quantify all known pigment types along its transect and provide a global distribution map. Unexpectedly, cells able to dynamically change their pigment content to match the ambient light color were ubiquitous and predominated in many environments. Altogether, our results unveiled the role of adaptation to light quality on niche partitioning in a key primary producer

Karakteristik Fikoeritrin sebagai Pigmen Asesoris pada Rumput Laut Merah, Serta Manfaatnya

Rumput laut di Indonesia telah banyak dimanfaatkan sebagai obat-obatan, bahan makanan, bahan dasar kosmetik, dan senyawa bioaktif serta nutrisi. Salah satu senyawa bioaktif yang dominan terkandung pada rumput laut merah adalah fikobilin, terdiri dari fikoeritrin dan fikosianin. Fikobilin terbentuk oleh reduksi biliverdin mealalui fitokromobilin. Pigmen tersebut berperan penting sebagai pigmen pelengkap pada proses fotosintesis rumput laut merah dengan membantu klorofil-a dalam menyerap cahaya, fikoeritrin menyerap cahaya hijau yang dapat menutupi warna hijau dari klorofil dan biru dari fikosianin. Struktur subunit fikoeritrin (PE) adalah (αβ)6γ dengan nilai absorbansi maksimal sekitar 580 nm. Jenis-jenis fikoeritrin berdasarkan serapan spektranya dibagi menjadi beberapa macam, yaitu B-fikoeritrin (B-PE), R-fikoeritrin (R-PE) dan C-fikoeritrin (C-PE), R-PE jenis fikobiliprotein yang mendominasi algae merah. Beberapa penelitian telah menunjukkan banyaknya manfaat dari pigmen tersebut. PE telah digunakan secara luas dalam industri dan laboratorium penelian immunologi, contoh sebagai label antibodi, reseptor antigen dan molekul biologi yang lain. Selain itu PE digunakan dalam aplikasi histokimia, digunakan sebagai fotosensitizer untuk pengobatan tumor dan berpotensi sebagai antioksidan

Potensi Rumput Laut Gracilaria SP sebagai Bahan Alternatif Dye Sensitized Solar Cell (Dssc)

Rumput laut Gracilaria sp adalah salah satu komoditas di daerah perairan bagian selatan Porong, Sidoarjo. Telah dilakukan studi untuk mengetahui potensi rumput laut gracilaria sp sebagai materialalternatif pelapis DSSC, dengan memanfaatkan fikoeritrin atau pigmen berwarna merah yangterkandung dalam tumbuhan tersebut. Nilai penyerapan cahaya diidentifikasi sebagai parameter untukmenentukan potensial dari fikoeritrin menggunakan metode regresi. Hasil yang diperoleh menunjukkanbahwa fikoeritrin yang terkandung dalam Gracilaria sp memiliki potensial penyerapan cahaya antara489-545 nm yang nilainya mendekati panjang gelombang sinar matahari dengan rentang antara 300-800 nm. Dengan perolehan rentang tersebut disimpulkan bahwa Gracilaria sp dapat digunakan sebagai material alternatif DSSC

An improved crystal structure of C-phycoerythrin from the marine cyanobacterium Phormidium sp. A09DM

C-Phycoerythrin (PE) from Phormidium sp. A09DM has been crystallized using different conditions and its structure determined to atomic resolution (1.14 Å). In order for the pigment present, phycoerythrobilin (PEB), to function as an efficient light-harvesting molecule it must be held rigidly (Kupka and Scheer in Biochim Biophys Acta 1777:94–103, 2008) and, moreover, the different PEB molecules in PE must be arranged, relative to each other, so as to promote efficient energy transfer between them. This improved structure has allowed us to define in great detail the structure of the PEBs and their binding sites. These precise structural details will facilitate theoretical calculations of each PEB’s spectroscopic properties. It was possible, however, to suggest a model for which chromophores contribute to the different regions of absorption spectrum and propose a tentative scheme for energy transfer. We show that some subtle differences in one of these PEB binding sites in two of the 12 subunits are caused by crystal contacts between neighboring hexamers in the crystal lattice. This explains some of the differences seen in previous lower resolution structures determined at two different pH values (Kumar et al. in Photosyn Res 129:17–28, 2016)

Inhibition of tumor necrosis factor α–stimulated monocyte adhesion to human aortic endothelial cells by AMP-activated protein kinase

<b>Objective</b>— Proatherosclerotic adhesion of leukocytes to the endothelium is attenuated by NO. As AMP-activated protein kinase (AMPK) regulates endothelial NO synthesis, we investigated the modulation of adhesion to cultured human aortic endothelial cells (HAECs) by AMPK. <b>Methods and Results</b>— HAECs incubated with the AMPK activator, AICAR, or expressing constitutively active AMPK demonstrated reduced TNF α-stimulated adhesion of promonocytic U-937 cells. Rapid inhibition of TNF α-stimulated U-937 cell adhesion by AICAR was NO-dependent, associated with unaltered cell surface adhesion molecule expression, and reduced MCP-1 secretion by HAECs. In contrast, inhibition of TNF α-stimulated U-937 cell adhesion by prolonged AMPK activation was NO-independent and associated with reduced cell surface adhesion molecule expression. <b>Conclusions</b>— AMPK activation in HAECs inhibits TNF α-stimulated leukocyte adhesion by a rapid NO-dependent mechanism associated with reduced MCP-1 secretion and a late NO-independent mechanism whereby adhesion molecule expression, in particular E-selectin, is suppressed. We investigated the functional effects of AMPK activation in cultured human endothelial cells. Stimulation of AMPK inhibited TNF α-stimulated monocyte adhesion by two distinct mechanisms: a rapid NO-dependent mechanism associated with a reduction in chemokine release and a late NO-independent mechanism whereby adhesion molecule expression is suppressed