Compartmentalisation of nitrogenase in a non-heterocystous cyanobacterium: Trichodesmium contortum

Abstract In Trichodesmium contortum , nitrogenase was detected in only a limited number (about 10%) of microscopically distinguishable, consecutively arranged cells in central regions of the trichomes. Cells with nitrogenase also contained the photosystem II associated pigment phycoerythrin. These cells were not distinguishable from other cells on a structural basis, but were clearly visible at low magnification microscopy as all in the zone were more compact and shorter than those on either side. The compartmentalisation of nitrogenase into a chain of cells and in a possibly photosynthetic environment represents a previously undescribed phenomenon. The nitrogenase containing cells apparently perform the O₂ protective function of heterocysts yet are different in several aspects.     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

The marine picoplankter Synechococcus sp. WH 7803 exhibits an adaptive response to restricted iron availability

Abstract: Laboratory cultures of marine Synechococcus sp. WH 7803 were grown under conditions of restricted iron availability. The culture medium was adjusted to restrict iron availability: (i) by adding the iron chelator EDDA; (ii) by omitting iron; and (iii) by omitting both iron and EDTA. An adaptive response was observed to these iron-restricted conditions, including a decrease in cellular phycoerythrin and synthesis of a 36 kDa polypeptide in [³⁵S]methionine radiolabelled whole cell lysates separated by SDS-PAGE. The polypeptide was synthesized within 48 h of transferring exponential phase cells to the iron-restricted medium. The protein was localized to the cell membranes and not the cytoplasmic fraction.     

Multiple forms of phycoerythrin-545 from Cryptomonas maculata

Three multiple phycoerythrin-545 forms were purified from crude extracts of Cryptomonas maculata by preparative isoelectric focusing. The phycoerythrin forms are charge isomers with isoelectric points at 7.83, 5.05 and 4.84. The multiple pigment forms have similar molecular weights of 44500 daltons and are composed of subunits of unequal size in a 1:1 stoichiometry with molecular weights of () 9900 and () 15700 daltons, twice. The proposed quarternary structure of the native pigments is ()₂()₂.The charge differences of the phycoerthrins are caused by a charge heterogeneity of the light subunits, as revealed by urea gel electrophoresis. The chains of pigment form pI 7.83 had a greater electrophoretic mobility than those subunits of the acidic pigment forms pI 5.05 and pI 4.84.The phycoerythrin forms have an absorption spectrum with similar absorption maxima at 544 nm, but differ in the position of the long wavelength shoulders lying at 555 and 557 nm in the negatively charged pigment forms and at 560 nm for the phycoerythrin form with a pI at 7.83.The fluorescence emission spectra coincide in their asymmetrical shape with shoulders at about 620 nm; they slightly differ int he position of the emission maxima at 586 nm for the phycoerythrins with pIs at 4.84 and 5.05 and at 584 nm for phycoerythrin with pI at 7.83.Abbreviations PC phycocyanin - PE phycoerythrin - pI isoelectric point - SDS sodium dodecyl sulphate     

Freeze fracture immunocytochemistry of light-harvesting pigment complexes in a cryptophyte

Summary Immunocytochemical techniques using colloidal gold as the marker have been used to examine the location of the two light harvesting pigment-protein complexes in cryptophyte chloroplasts. A comparison of post-embedding thin section labelling and freeze fracture labelling has been carried out onRhodomonas salina using polyclonal antibodies to a chlorophylla/c ₂ light-harvesting complex, phycoerythrin and the -subunit of phycoerythrin. The effect of different fixation procedures on the intensity of labelling and ac curacy of antigen location have been examined and the effectiveness of uranyl acetate and tannic acid in improving both the preservation of thylakoid structure and labelling density of phycoerythrin has been demonstrated. Freeze fracture labelling gives better spatial res olution of the different antigens than post-embedding labelling, as well as better definition of thylakoid membranes. It confirms the location of phycoerythrin in the thylakoid lumen and the location of the chlorophylla/c ₂ LHC in both appressed and unappressed thylakoid membranes.Abbreviations PE phycoerythrin - chl chlorophyll - LHC light-har-vesting complex     

Multifaceted applications of nanomaterials in cell engineering and therapy

Nanomaterials with superior physiochemical properties have been rapidly developed and integrated in every aspect of cell engineering and therapy for translating their great promise to clinical success. Here we demonstrate the multifaceted roles played by innovatively-designed nanomaterials in addressing key challenges in cell engineering and therapy such as cell isolation from heterogeneous cell population, cell instruction in vitro to enable desired functionalities, and targeted cell delivery to therapeutic sites for prompting tissue repair. The emerging trends in this interdisciplinary and dynamic field are also highlighted, where the nanomaterial-engineered cells constitute the basis for establishing in vitro disease model; and nanomaterial-based in situ cell engineering are accomplished directly within the native tissue in vivo. We will witness the increasing importance of nanomaterials in revolutionizing the concept and toolset of cell engineering and therapy which will enrich our scientific understanding of diseases and ultimately fulfill the therapeutic demand in clinical medicine.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.     

High-performance liquid chromatography with on-line post-column immunoreaction detection of digoxin and its metabolites based on fluorescence energy transfer in the far-red spectral region

The combination of immunoassays with separation techniques such as chromatography can result in enhanced selectivity and sensitivity. This paper describes an on-line chromatography with immunochemical post-column fluorescence energy transfer detection for digoxin and its metabolites. R-phycoerythrin (PE) was used as the donor and an indodicarbocyanine dye (Cy5) as the acceptor label. These labels allow the detection in the far-red spectral region, which is more selective for biological samples. Hence, digoxin was labeled with PE using the activated digoxigenin-NHS-ester and monoclonal anti-digoxin antibody was labeled with Cy5. Digoxin and its metabolites was injected into the HPLC system followed by post-column injection of R-phycoerythrin labeled digoxin and by Cy5 labeled anti-digoxin antibody. Incubation time was provided using an open tubular reactor coil at room temperature. The detection was performed by measurement of the sensitized emission of Cy5 at 670 nm due to fluorescence energy transfer from PE labeled with digoxin. The system was optimized with regard to the concentrations of the used post-column reagents as well as incubation time and temperature. The dynamic range of digoxin spiked in 0.01 M phosphate buffer (pH 7.4) was 0.05 to 10 ng/ml with a correlation coefficient of 0.989. The limit of detection was 33 pg/ml. The precision of two controls, 0.4 and 4 ng/ml, was found to be 2.2 and 8.7% RSD, respectively, accuracy was 10.7 and 20.3% (n=6 in each case).     

Energy transfers from Photosystem II to Photosystem I in Cryptomonas rufescens (Cryptophyceae)

In Cryptomonas rufescens (Cryptophyceae), phycoerythrin located in the thylakoid lumen is the major accessory pigment. Oxygen action spectra prove phycoerythrin to be efficient in trapping light energy.The fluorescence excitation spectra at ?196°C obtained by the method of Butler and Kitajima (Butler, W.L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85) indicate that like in Rhodophycease, chlorophyll a is the exclusive light-harvesting pigment for Photosystem I.For Photosystem II we can observe two types of antennae: (1) a light-harvesting chlorophyll complex connected to Photosystem II reaction centers, which transfers excitation energy to Photosystem I reaction centers when all the Photosystem II traps are closed. (2) A light-harvesting phycoerythrin complex, which transfers excitation energy exclusively to the Photosystem II reaction complexes responsible for fluorescence at 690 nm.We conclude that in Cryptophyceae, phycoerythrin is an efficient light-harvesting pigment, organized as an antenna connected to Photosystem II centers, antenna situated in the lumen of the thylakoid. However, we cannot afford to exclude that a few parts of phycobilin pigments could be connected to inactive chlorophylls fluorescing at 690 nm.