Gregory J.S. Fowler

THE PHOTOKILLING OF BLADDER CARCINOMA CELLS in vitro BY PHENOTHIAZINE DYES

Abstract— The potential photodynamic therapy photosensitizers Methylene Blue, Azure C, Methylene Violet, Thionine, Methylene Green, Haematoporphyrin, Nile Blue A, chloroaluminium phthalocyanine and bis‐aluminium phthalocyanine were examined for their photoeffects and dark toxicity against a human superficial bladder carcinoma cell‐line. By examination of [3H]thymidine uptake into dye‐treated cells after irradiation with a copper‐vapour pumped dye laser, it was found that Methylene Blue was the most phototoxic and dark toxic of all the dyes tested, suggesting that the dye might be of some use as a topically applied photodrug for use in photodynamic therapy of superficial or early‐recurring carcinomas.

HETEROLOGOUS EXPRESSION OF GENES ENCODING BACTERIAL LIGHT-HARVESTING COMPLEXES IN RHODOBACTER SPHAEROIDES

One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants; DBC/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional “foreign” light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.

Non-fouling microfluidic chip produced by radio frequency tetraglyme plasma deposition

This Technical Note presents the direct surface modification of a glass/PTFE hybrid microfluidic chip, via radio frequency glow discharge plasma polymerisation of tetraethlylene glycol dimethylether (tetraglyme), to produce hydrophilic, non-fouling, PEO-like surfaces. We use several techniques including X-ray photoelectron spectroscopy (XPS), direct enzyme-linked immunosorbent assays (ELISA) and immunofluorescent imaging to investigate the channel coatings. Our results indicate the successful deposition of a PEO-like coating onto microchannel surfaces that has both solution and shelf stability (>3 months) and is capable of preventing fibrinogen adsorption to the microchannel surfaces.

TILRR, a novel IL-1RI co-receptor, potentiates MyD88 recruitment to control Ras-dependent amplification of NF-κB.

Host defense against infection is induced by Toll-like and interleukin (IL)-1 receptors, and controlled by the transcription factor NF-κB. Our earlier studies have shown that IL-1 activation impacts cytoskeletal structure and that IL-1 receptor (IL-1RI) function is substrate-dependent. Here we identify a novel regulatory component, TILRR, which amplifies activation of IL-1RI and coordinates IL-1-induced control with mechanotransduction. We show that TILRR is a highly conserved and widely expressed enhancer of IL-1-regulated inflammatory responses and, further, that it is a membrane-bound glycosylated protein with sequence homology to members of the FRAS-1 family. We demonstrate that TILRR is recruited to the IL-1 receptor complex and magnifies signal amplification by increasing receptor expression and ligand binding. In addition, we show that the consequent potentiation of NF-κB is controlled through IL-1RI-associated signaling components in coordination with activation of the Ras GTPase. Using mutagenesis, we demonstrate that TILRR function is dependent on association with its signaling partner and, further, that formation of the TILRR-containing IL-1RI complex imparts enhanced association of the MyD88 adapter during ligand-induced activation of NF-κB. We conclude that TILRR is an IL-1RI co-receptor, which associates with the signaling receptor complex to enhance recruitment of MyD88 and control Ras-dependent amplification of NF-κB and inflammatory responses.

Excited-state dynamics of mutated antenna complexes of purple bacteria studied by hole-burning

Absorption and fluorescence excitation spectra of various LH2 antenna complexes of two purple bacteria at low temperature (1.2 and 4.2 K) have been measured, and energy transfer rates within these complexes have been determined by spectra hole-burning. The systems studied were membranes of a wild-type strain of Rhodobacter sphaeroides , membrane samples from four LH2-only strains containing specifically mutated LH2 complexes of the same bacterium, and the isolated B800–820 complex of Rhodopseudomonas acidophila (strain 7050). The mutants exhibit blue-shifted B850 absorption bands with their spectral positions depending on the specific amino acid residues replaced in the α-polypeptide sequence. Energy transfer rates from B800 to B850 (or to their respectively blue-shifted bands) have been obtained by hole-burning experiments in the B800 band. The mutants of Rb. sphaeroides and the LH2 complex of Rps. acidophila yielded transfer times similar to those of the B800–850 complex of Rb. sphaeroides . These values, which for the various complexes vary between 1.7 and 2.5 ps in the wavelength region from 798 to 805 nm, do not decrease monotonically with the spectral distance between the bands. Various models based on Forsters energy transfer mechanism are discussed, of which only one is consistent with the results. In this model the energy is assumed to be transferred not directly from the Q y 0-0 band of B800 to that of the (blue-shifted) B850, but indirectly through the excitation of a vibrational mode.

Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. II. A low-temperature spectroscopy study of structural aspects of the pigment-protein conformation

Abstract Low-temperature absorbance, fluorescence, linear and circular dichroism spectra were measured for two site-specific mutants (βHis21 → and βArg29 → Glu) of the peripheral (B800–850) light-harvesting complex of Rb. sphaeroides in order to obtain information on the possible changes in the binding site of the bacteriochlorophyll a pigments. From the absorbance and fluorescence measurements we conclude that when βHis21, the putative ligand of the BChl 800 pigment, is replaced by serine the pigment is not incorporated in the complex. In addition, this modification induces a 4 nm red-shift of the B850 band. Linear and circular dichroism measurements indicate that the specific orientation of the BChl 850 pigments is retained despite the absence of a pigment in the B800 binding-site. A second mutant, in which the conserved arginine at position 29 on the same polypeptide is replaced by glutamate, was also studied. This replacement causes a significant blue shift as well as a marked broadening of of the B800 band, which indicates that this binding site is more heterogeneous in this mutant. The overall orientation however is not drastically changed and energy transfer from the B800 to B850 still takes place efficiently. We conclude that neither residue is exclusively involved in the binding pocket for BChl 800, but both βHis21 and βArg29 are important in determining the BChl 800 binding.

Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. I: Mutagenesis, effects on binding, function and electrochromic behaviour of its carotenoids

Abstract The light-harvesting LH2 complex of Rhodobacter sphaeroides contains two amino acid residues, βHis21 and βArg29, which are conserved in all LH2 β-polypeptides of purple nonsulfur bacteria sequenced so far. These residues have been changed into serine and glutamic acid, respectively. Both mutations lead to severe changes in the spectroscopic characteristics of the antenna complex. Changing βArg29 into Glu results in a blue shift and a broadening of the B800 bacteriochlorophyll absorbance, suggesting a role of this residue in creating the binding pocket for B800 (see also Visschers et al. (1994) Biochim. Biophys Acta 1183, 483–490). Similar blue shifts, of approx. 6 nm, are also observed in the carotenoid absorbance peaks. This is accompanied by a large change in the electrochromic behaviour of the carotenoids, which suggests a major role of βArg29 in creating a local field near the responsive carotenoid. The second mutation, βHis21 to Ser, results in an inability to create a B800 domain. This mutation also causes changes in the carotenoid absorbance and electrochromic behaviour, suggesting a direct or indirect (via the bacteriochlorophyll B800 molecule) effect on the local dipole field of the sensitive carotenoid. Neither of the mutated complexes has lost the ability to bind carotenoids; in both complexes energy transfer from the carotenoids to B850 appears unaltered, indicating that all carotenoids can transfer energy directly to this bacteriochlorophyll, despite the loss of B800.

Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

Unlike the α and β polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the α and β polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 β polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 β polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position −10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position −10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position −10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 β polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 β polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position −10 was changed to Arg, as found in LH2 at this position, and in the other His at position −18 was changed to Val. The results from these mutants allow us to conclude that the residue at the −10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at −18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.

SITE-DIRECTED MUTAGENESIS OF THE LH2 LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides: CHANGING βLys23 TO Gin RESULTS IN A SHIFT IN THE 850 nm ABSORPTION PEAK

The present study describes the construction of a Rhodobacter sphaeroides light‐harvesting (LH2) mutant in which the charged residue βSLys23 is changed by site‐directed mutagenesis to a Gin residue, and the characterization of the resulting mutant complex by a range of spectroscopic techniques. In the 77 K absorption spectrum of the mutant, the peak equivalent to the 850 nm peak in the wild‐type membrane is blue‐shifted by approximately 18 nm to 837 nm; except for this blue‐shift, the 77 K. fluorescence excitation and emission spectra and the circular dichroism spectrum of the mutant are very similar to the equivalent spectra from the wild‐type membranes, suggesting that the mutation βLys23 → Gin probably does not cause any major changes in the conformation or aggregation state of these membranes. Possible causes of the 18 nm blue‐shift in the absorption spectrum are discussed.

Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode

Exoelectrogens have the ability to generate electricity in mediator-less microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigate the anode-specific responses of Arcobacter butzleri ED-1, the first identified exoelectrogenic Epsilonproteobacterium. iTRAQ and 2D-LC MS/MS driven proteomics were used to compare protein abundances in A. butzleri ED-1 when generating an electronegative potential (-225 mV) in an anaerobic half-cell - either growing as an electrogenic biofilm or suspended in the liquid medium - versus a microaerobic culture. This is the first quantitative proteomic study concentrating on growth of an exoelectrogen during current generation. From 720 proteins identified and quantified (soluble and insoluble sub-proteomes), statistical analysis reveals 75 differentially-expressed proteins. This dataset was enriched in proteins regulating energy and intermediary metabolism, electron and protein transport. Flagellin up-regulation was concomitant with electron transport in the anodic cells, while decreased abundance of a methyl-accepting chemotaxis protein suggested that flagella were involved in communication with the anode surface and electrogenesis, rather than motility. Two novel cytochromes potentially related to electron transport were up-regulated in anaerobic cultures. We demonstrate that employing an insoluble extracellular electron acceptor for anaerobic growth regulates multiple proteins involved in cell surface properties, electron transport and the methylcitrate cycle.