RE-ESTABLISHMENT OF SYMBIOSIS TO Anabaena-FREE Azolla

Symbiosis to Anabaena-free Azolla has been re-established by placing indusium containing Anabaena onto the Anabaena-free megasporocarps. Anabaena-free megasporocarps were obtained by removing the indusium and the apical membrane of the megaspore apparatus of normal Azolla. The recovery of symbiosis in artificially reconstituted Azolla-Anabaena association was confirmed using the scanning electron microscope, monoclonal antibody test (McAb-C_(16)), and nitrogen fixation capacity (ARA).Laboratory-grown Anabaena azollae cells inoculated subsequently to the decapitated megaspore apparatus were observed to have entered the leaf cavities of sporophyte, although some portions of Anabaena filaments were found outside the leaf cavity. Plants invaded by artificially inoculated Anabaena did not have sufficient N_2-fixing activity to allow the growth in the N-free medium.     

A Comparative Kinetic Analysis of the Flavin-Photosensitized Oxidation and Reduction of Plastocyanin and Cytochrome c6 from Different Organisms

Abstract— Laser flash photolysis spectroscopy has been used to investigate the kinetics of oxidation and reduction of pho-tosynthetic plastocyanin (Pc) and cytochrome c₆ (Cyt) from the cyanobacteria Anabaena PCC 7119 and Syne-chocystis PCC 6803 by lumiflavin, riboflavin and flavin mononucleotide (FMN). For both redox reactions similar steric and electrostatic effects were observed with Syne-chocystis proteins and Anabaena Cyt, thus suggesting that the same (or closely adjacent) sites are being used for electron entry and removal. In the case of Anabaena Pc, however, both the steric and electrostatic effects suggest that FMN-dependent protein oxidation and reduction may occur at different sites, that is oxidation at the hydrophobic patch and reduction at the hydrophilic one. Kinetic pK_a values of 5.8 and 6.2 have been determined for the lumiflavin-dependent reduction of Anabaena and Monoraphidium Pc, respectively, whereas the oxidation reactions appear to be pH independent. Not only the reduction kinetics but also protein tyrosine fluorescence quenching by iodide ions suggest the occurrence of pH-induced conformational changes in Pc. In conclusion, kinetic and fluorescent studies indicate that there are considerable quantitative similarities between Cyt and Pc when isolated from the same organism, consistent with the identical physiological role that these two proteins play inside the cells.     

A critical role for dermal mast cells in cis-urocanic acid-induced systemic suppression of contact hypersensitivity responses in mice

Many studies have implicated cis-urocanic acid (cis-UCA) in UVB-induced immunomodulation. The strongest evidence came from studies in mice whereby a cis-UCA antibody blocked UVB-induced suppression of delayed-type hypersensitivity responses. Furthermore, in several studies, the cis-UCA antibody at least partially reversed UVB suppression of contact hypersensitivity responses. Previous reports suggested that cis-UCA was immunomodulatory through its effects on keratinocytes, Langerhans cells, fibroblasts, T lymphocytes, natural killer cells and monocytes/macrophages. As dermal mast cells were recently demonstrated to be critical to UVB-induced systemic suppression of certain delayed-type and contact hypersensitivity responses, we investigated whether they were involved in the processes by which cis-UCA was immunomodulatory. Not only was there a correlation between dermal mast cell prevalence and the degree of susceptibility of different strains of mice to the immunomodulatory effects of cis-UCA, there was also a functional link. Mast cell-depleted Wf/Wf mice were rendered susceptible to immunomodulation by cis-UCA injected subcutaneously only after their dorsal skin had been reconstituted with bone marrow-derived mast cell precursors. These studies suggest that mast cells are critical to the processes by which cis-UCA suppresses systemic contact hypersensitivity responses to the hapten, trinitrochlorobenzene, in mice.     

INCREASED ENERGY TRANSFER FROM PHYCOBILISOMES TO PHOTOSYSTEM II IN HIGH LIGHT ADAPTED Anabaena cylindrica

Excitation energy transfer from phycobilisomes to photosystem II in high-light adapted cells of Anabaena cylindrica was studied by fluorescence spectroscopy and compared to that of low-light adapted cells. Measurements were made on membrane fragments containing phycobilisomes, photosystem I and II, isolated in 0.75 M K-phosphate. Relative efficiency of 430 to 590 nm light in the excitation of F₆₈₀ chlorophyll fluorescence was compared in low and high light adapted cells, respectively. The values indicate that light energy absorbed by phycobilisomes is transferred to photosystem II antenna chlorophylls with higher efficiency in high-light adapted cells than in low-light adapted cells. Partial dissociation and uncoupling of energy transfer caused by low ion concentration were different in the membrane fragments isolated from the two kinds of cells and indicated a higher aggregation state of pigment-protein complexes of phycobilisomes in high-light adapted A. cylindrica cells.     

Intra‐strain Variability in the Effects of Temperature on UV‐B Sensitivity of Cyanobacteria

Stratospheric ozone depletion is mostly marked over the Antarctic and to a lesser extent over the Arctic, though recent reports have revealed that this also occurs at lower latitudes. Continued depletion of ozone in the lower stratosphere allows more UVR to reach the Earth's surface. Furthermore, it is projected that surface water temperatures will increase by between 0.2 and 2.0°C by the year 2060 and this will directly or indirectly influence algal growth. The interactions between environmental factors are complicated by the existence of different strains (ecotypes) of the same species that may respond differently. To understand the interactive effects of temperature and UV‐B on two strains of Anabaena circinalis, we investigated the damaging effects of UV‐B on cell numbers and photosynthetic characteristics and also examined the effect of temperature on the capacity of cells to recover from such stress. Both strains of A. circinalis responded differently in terms of survival, photosynthetic characteristics and recovery with interactions between temperature and UV‐B. This could be due to the variations in strain‐specific photoreactive mechanisms. This needs to be explored further including more strains and species before definitive conclusions can be reached about effects of global change on cyanobacteria generally.     

Reconstitution of the Torpedo californica nicotinic acetylcholine receptor into planar lipid bilayers

Detergent-solubilized acetylcholine receptor (nAcChR) proteins can be purified by affinity chromatography and reconstituted into lipid vesicles and afterwards into planar lipid bilayer membranes via, in principle, two methods: fusion or assembly from two vesicle-spread monolayers. In the presence of agonists (carbamoylcholine, suberoyldicholine) different kinds of channel openings are recorded: fast single channels, bursts and long openings with short closures in between. Similar results have been obtained with reconstituted membrane fragments rich in nAcChR. In addition, Torpedo californica nAcChR proteins give rise to fuzzy channels and less defined events of conductivity, which “reemerge” again all the time. Frequently the channel events have conductance levels of about 200 to 300 pS, obviously simultaneous openings of several aggregated receptors. Under these conditions 40 pS conductance events occur also. It appears that the conductance of the channels measured is a multiple of 6.3 pS. Often, with the same sample, no channel openings are seen. Contrary to patch-clamp investigations on whole cells, AcChR-channel openings in reconstituted systems occur only several minutes after agonist application and not immediately. It is not clear whether the “reconstituted channels” reflect rapid activation or whether they result from desensitized receptor states only. Although a clear-cut correlation of channel event and channel protein unit is only possible by reconstitution of the biochemically characterized protein, e.g. monomer, dimer or higher oligomers, the reconstitution technique is still in its infancy.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

Analysis of the interaction of a hybrid system consisting of bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119

The mitochondrial steroid-hydroxylating system in vertebrates and the NADPH producing electron transfer chain in photosynthetic organisms contain structurally and functionally similar components. Examination of a potential hybrid reconstitution of the electron transfer chain between different components of both systems could help to improve our knowledge on protein-protein interaction and subsequent electron transfer. Here we analyzed the interaction between bovine adrenodoxin reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119. Optical biosensor as well as steady state and fast kinetic experiments showed their ability to form distinct productive complexes. Compared with the corresponding physiological systems the electron transfer is rather slow, probably due to the lack of specificity at the interaction surface.     

Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems

A one-step immunomagnetic separation technique was performed on a microfluidic platform for the isolation of specific cells from blood samples. The cell isolation and purification studies targeted T cells, as a model for low abundance cells (about 1:10,000 cells), with more dilute cells as the ultimate goal. T cells were successfully separated on-chip from human blood and from reconstituted blood samples. Quantitative polymerase chain reaction analysis of the captured cells was used to characterize the efficiency of T cell capture in a variety of flow path designs. Employing many (4-8), 50 microm deep narrow channels, with the same overall cross section as a single, 3 mm wide channel, was much more effective in structuring dense enough magnetic bead beds to trap cells in a flowing stream. The use of 8-multiple bifurcated flow paths increased capture efficiencies from approximately 20 up to 37%, when compared to a straight 8-way split design, indicating the value of ensuring uniform flow distribution into each channel in a flow manifold for effective cell capture. Sample flow rates of up to 3 microL min(-1) were evaluated in these capture beds.     

Conformational changes monitored by fluorescence study on reconstituted hemoglobins

Intrinsic steady state fluorescence measurements were performed on a series of reconstituted metal ion and hybrid hemoglobins (Hbs). At 296 nm excitation, the spectrum exhibits a broad and asymmetric feature in the case of copper and nickel reconstituted hemoglobins. Deconvolution of the fluorescence bands clearly reveals the existence of two definite peaks. A similar trend was also observed for hybrid hemoglobins (CuNi, NiCu, CuFe-CO, and NiFe-CO). A guassian fit of the fluorescence bands in these proteins again yields two prominent peaks, which are assigned as due to two different tryptophan (Trp) environments. A relative ratio of the amplitudes of these peaks indicates the percentage of T-character in these molecules. This is in support to our previous findings by other spectroscopic studies on the same molecules. These studies therefore, suggest the presence of two different environments of a tryptophan thereby revealing structural heterogeneity among the subunits.     

A Light-Responsive Metal–Organic Framework Hybrid Membrane with High On/Off Photoswitchable Proton Conductivity

Mimicking biological proton pumps to achieve stimuli-responsive protonic solids has long been of great interest for their diverse applications in fuel cells, chemical sensors, and bio-electronic devices. Now, dynamic light-responsive metal–organic framework hybrid membranes can be obtained by in situ encapsulation of photoactive molecules (sulfonated spiropyran, SSP), as the molecular valve, into the cavities of the host ZIF-8. The configuration of SSP can be changed and switched reversibly in response to light, generating different mobile acidic protons and thus high on/off photoswitchable proton conductivity in the hybrid membranes and device. This device exhibits a high proton conductivity, fast response time, and extremely large on/off ratio upon visible-light irradiation. This approach might provide a platform for creating emerging smart protonic solids with potential applications in the remote-controllable chemical sensors or proton-conducting field-effect transistors.     

Electron microscopic observation and rotational diffusion measurement of bacteriorhodopsin in lipid vesicles

The morphology of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine and egg-phosphatidylcholine vesicles was observed by freeze-fracture electron microscopy. The rotational diffusion of bacteriorhodopsin at different concentrations of melittin was measured by observing flash-induced transient dichroism in dimyristoylphosphatidylcholine vesicles. In the presence of melittin, bacteriorhodopsin molecules in dimyristoylphosphatidylcholine vesicles were aggregated into large particles or patches, and the ability of rotational diffusion of bacteriorhodopsin in vesicles was decreased. This suggests that melittin produces its effect via direct electrostatic interaction with bacteriorhodopsin. Low temperature-induced aggregation of bacteriorhodopsin was also observed in dimyristoylphosphatidylcholine vesicles. Low temperature may cause phase separation. Bacteriorhodopsin was also successfully reconstituted into egg-phosphatidylcholine vesicles, but low temperature-induced aggregation of bacteriorhod     

Fluorescence emission and absorption spectra of single Anabaena sp. strain PCC7120 cells

The fluorescence emission and absorption spectra of single Anabaena sp. strain PCC7120 cells including vegetative cells and heterocysts have been studied in intact filaments in vivo with confocal microscopy and grating spectrography. The diameters of the excitation and detection areas in the cells are less than 1.0 microm. Heterogeneities within the same cell and among different cells are observed. The evident spectral heterogeneities in heterocysts, not reported previously, are attributed to the different stages of the evolution of phycobilisomes in heterocysts. The photosystem II in heterocysts were found to be not metabolized completely.     

单个蓝藻细胞的光谱

利用共焦显微和光栅光谱技术，得到了蓝藻Anabaena strain PCC 7120完整藻丝体中的单细胞吸收和荧光发射光谱.发现了前人未观察到的不同异形胞之间有显著光谱差异的现象，该现象可能与异形胞不同发育阶段的藻胆体和光系统的演化相关联.     

Comprehensive analysis system using liquid chromatography-mass spectrometry for the biosynthetic study of peptides produced by cyanobacteria

Microcystins are hepatotoxic heptapeptides and general tumor promoters produced by several species of the genera Microcystis, Anabaena, Oscillatoria and Nostoc. They are non-ribosomally synthesized via a mixed polyketide synthase/non-ribosomal peptide synthetase system called microcystin synthetase. We have carried out the detection, isolation and structural determination of non-toxic peptides produced together with microcystins by toxic cyanobacteria, which are classified into several groups on the basis of their structures and some of these non-toxic peptides are also non-ribosomally synthesized as well as microcystins. In the present study, we tried to correlate the secondary metabolic peptides produced by the hepatotoxic cyanobacteria with the corresponding peptide synthetase genes. An analytical method using LC-electroscopy ionization MS and photodiode array detection was developed for the exhaustive screening of cyanobacterial peptides in Japanese strains and it was successfully applied to the peptide fractions extracted from these strains. The established method was advantageous over conventional ones using the usual HPLC and matrix-assisted laser desorption ionization time-of-flight MS, because more structural information could be obtained and it is easier to distinguish microcystins from other peptides using this method. Small amounts of other peptides could also be detected by this method. The established method will contribute to the investigation of the relationship between genes encoding the peptide synthetase and secondary metabolic peptides.     

Short and long term stability of the elemental composition of human body fluid reference materials and their use as master lots

Summary Studies have been made of both short and long term stability of trace elements in lyophilized human body fluid reference materials, as well as the stability of mercury in reconstituted urine solutions. No detectable concentration changes for mercury, lead and aluminium occurred during the 5-year period. There are large differences in the amounts of mercury loss among different reconstituted materials. Addition of traces of gold to the solutions minimized the mercury loss and increased the useable time of the reconstituted material from hours to 8 days. A certification process based on direct determinations by reference laboratories and by comparison against master lots of the same material and against similar certified reference materials is presented. Values for calcium, copper and mercury obtained from reference laboratories and using the data transfer principle have been assigned in new batches of urine and serum.     

A Light‐Responsive Metal–Organic Framework Hybrid Membrane with High On/Off Photoswitchable Proton Conductivity

Mimicking biological proton pumps to achieve stimuli‐responsive protonic solids has long been of great interest for their diverse applications in fuel cells, chemical sensors, and bio‐electronic devices. Now, dynamic light‐responsive metal–organic framework hybrid membranes can be obtained by in situ encapsulation of photoactive molecules (sulfonated spiropyran, SSP), as the molecular valve, into the cavities of the host ZIF‐8. The configuration of SSP can be changed and switched reversibly in response to light, generating different mobile acidic protons and thus high on/off photoswitchable proton conductivity in the hybrid membranes and device. This device exhibits a high proton conductivity, fast response time, and extremely large on/off ratio upon visible‐light irradiation. This approach might provide a platform for creating emerging smart protonic solids with potential applications in the remote‐controllable chemical sensors or proton‐conducting field‐effect transistors.     

Hybrid polymer/nanoparticle solar cells: preparation, principles and challenges

Hybrid polymer/nanoparticle solar cells have a light harvesting layer composed of semiconducting inorganic nanoparticles and a semiconducting conjugated polymer. They have potential to give high power conversion efficiencies (PCE). However, the PCE values reported for these solar cells are not currently as high as anticipated. This article reviews the main methods currently used for preparing hybrid polymer/nanoparticle solar cells from the colloid perspective. PCE data for the period of 2005-2011 are presented for hybrid polymer/nanoparticle solar cells and compared to those from polymer/fullerene cells. The key reasons for the relatively low PCE values for hybrid polymer/nanoparticle solar cells are uncontrolled aggregation and residual insulating ligands at the nanoparticle surface. Two hybrid polymer/nanoparticle systems studied at Manchester are considered in which the onset of aggregation and its affect on composite film morphology were studied from the colloidal perspective. It is concluded that step-change approaches are required to increase the PCEs of hybrid polymer/nanoparticle solar cells and move them toward the 10% value required for widespread commercialisation. A range of nanoparticles that have potential for application in possible longer term terawatt solar energy production are discussed.     

Synthesis,spectral and biological studies of organotin(IV) complexes of heteroscorpionate

A heteroscorpionate ligand, potassium hydrobis(benzoato)(salicylaldehyde)borate (KL), has been synthesized. This was converted into organotin complexes R₂SnL₂ and R₃SnL complexes by mixing and stirring with a methanolic solution/suspension of organotin chloride. The ligand and its complexes were characterized by elemental analyses and spectral studies (IR, ¹H NMR, ¹³C NMR, ESI mass spectra and Thermo gravimetric analysis (TGA)). Antibacterial and antifungal studies of these compounds were evaluated by the disc diffusion method at variable concentration against three species of bacteria (Staphylococcus aureus, Klebsiella pneumonia and Bacillius subtillis) and two species of fungi (Asperjillius fiavus and Candida albicans). It was found that triorganotin derivatives (R₃SnL) of the ligand were more effective as compared with diorganotin derivatives (R₂SnL₂). The organotin complexes of borates were tested for their algicidal activity on the cyanobacterial strains Aulosira fertilissma, Anabaena species, Anabaena variabilis and Nostoc muscorum and showed high to moderate toxicity towards the above species. The ligand and its complexes were also tested for its pH effect on soil in vitro for a duration of more than one month and it was found that they are able to kill pests without damaging the soil quality. Copyright © 2006 John Wiley & Sons, Ltd.