TREPR spectra of micelle-confined spin correlated radical pairs: II. Spectral decomposition and asymmetric line shapes.

In the second paper, spectral decomposition is used to explain the origin of the asymmetry of the anti-phase structure (APS) and its temperature dependence in dynamic spin correlated radical pairs (SCRPs) created via the photoreduction of benzophenone (BP) in sodium dodecyl sulfate (SDS) micelles. It is shown that the main parameters defining the spectral shape of the TREPR spectra are the effectiveness of the electron spin exchange in contact pairs, and the ratio of the frequency of enforced encounters (Z) to the frequency of singlet-triplet mixing (q) in the separated radical pairs. The Z/q ratio is particularly important for the creation of the APS asymmetry. The existence of different q values in the same TREPR spectrum in this system affords the observation of SCRPs in both regimes: exchange broadening (large |q|/Z) and exchange narrowing (small |q|/Z). An important observation, supported by the successful simulation of the TREPR spectra, is that the S-component of the APS can be shifted in a direction opposite to that predicted by the earlier Closs-Forbes-Norris (CFN) model. This result is naturally explained in terms of a spectral exchange approach. Dispersion-like components in the spectra further amplify the asymmetry of the APS.

TREPR spectra of micelle-confined spin correlated radical pairs: I. Molecular motion and simulations.

Radical pairs created by the photoreduction of benzophenone (BP) in sodium dodecyl sulfate (SDS) micelles exhibit strong asymmetry in the line shapes of their time-resolved electron paramagnetic resonance (TREPR) signals. The asymmetry is strongly dependent on the temperature from 16 °C to 66 °C. Simulations of the anti-phase structure (APS) line shape of these spin correlated radical pairs (SCRPs), based on a numerical solution of the Stochastic Liouville Equation with the spin exchange interaction depending exponentially on the distance between radicals, are presented and discussed. The proposed model takes into account the diffusive motion of the radicals along with the motion of the transverse magnetization and accounts satisfactorily and self-consistently for the asymmetry of the observed TREPR signals.

Kinetic analysis of nitroxide radical formation under oxygenated photolysis: toward quantitative singlet oxygen topology.

Reaction kinetics for two sterically hindered secondary amines with singlet oxygen have been studied in detail. A water soluble porphyrin sensitizer, 5,10,15,20-tetrakis-(4-sulfunatophenyl)-21,23H-porphyrin (TPPS), was irradiated in oxygenated aqueous solutions containing either 2,2,6,6-tetramethylpiperidin-4-one (TMPD) or 4-[N,N,N-trimethyl-ammonium]-2,2,6,6-tetramethylpiperidinyl chloride (N-TMPCl). The resulting sensitization reaction produced singlet oxygen in high yield, ultimately leading to the formation of the corresponding nitroxide free radicals (R2NO) which were detected using steady-state electron paramagnetic resonance (EPR) spectroscopy. Careful actinometry and EPR calibration curves, coupled with a detailed kinetic analysis, led to a simple and compact expression relating the nitroxide quantum yield ΦR2NO (from the doubly-integrated EPR signal intensity) to the initial amine concentration [R2NH]i. With all other parameters held constant, a plot of ΦR2NOvs. [R2NH]i gave a straight line with a slope proportional to the rate constant for nitroxide formation, kR2NO. This establishment of a rigorous quantitative relationship between the EPR signal and the rate constant provides a mechanism for quantifying singlet oxygen production as a function of its topology in heterogeneous media. Implications for in vivo assessment of singlet oxygen topology are briefly discussed.

A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum.

BACKGROUND: The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin) gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light. RESULTS: A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein). The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions. CONCLUSIONS: The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1.

The genome of the archaeon Halobacterium salinarum contains two copies of the pst (phosphate-specific transport) operon, the genes of which are related to well-studied bacterial homologues. Both operons (pst1 and pst2) were shown to be polycistronic and, when under P(i)-limited conditions, transcription initiated 1 bp upstream of the translational starts. Under P(i) saturation, the pst1 operon utilized an additional transcription start site 59 bp upstream of the first one. The leaderless pst1 transcript was found to be more efficiently translated than the leadered transcript. Promoter strengths differed significantly between the two operons and when P(i) levels changed. The basal pst1 promoter activity in P(i)-saturated conditions was minimal while the pst2 promoter was active. In contrast, phosphate limitation induced the pst1 operon threefold more than the pst2 operon. We identified basic and phosphate-dependent cis-acting elements in both promoters. Phosphate-uptake assays conducted with several Pst1 and Pst2 mutant strains revealed differences in the substrate affinities between the two transporters and also suggested that the P(i)-binding proteins PstS1 and PstS2 can interact with either of the two permease subunits of the transporters. The tactic behaviour of wild type and pst-deletion strains showed that the Pst1 transporter plays an important role for phosphate-directed chemotaxis.

Electrostatic control of spin exchange between mobile spin-correlated radical pairs created in micellar solutions.

A series of photoinduced H-atom abstraction reactions between anthraquinone-2,6,-disulfonate, disodium salt (AQDS) and differently charged micellar substrates is presented. After a 248 nm excimer laser flash, the first excited triplet state of AQDS is rapidly formed and then quenched by abstraction of a hydrogen atom from the alkyl chain of the micelle surfactant, leading to a spin-correlated radical pair (SCRP). The SCRP is detected 500 ns after the laser flash using time-resolved (direct detection) electron paramagnetic resonance (TREPR) spectroscopy at X-band (9.5 GHz). By changing the charge on the surfactant headgroup from negative (sodium dodecyl sulfate, SDS) to positive (dodecyltrimethylammonium chloride, DTAC), TREPR spectra with different degrees of antiphase structure (APS) in their line shape were observed. The first derivative-like APS line shape is the signature of an SCRP experiencing an electron spin exchange interaction between the radical centers, which was clearly observable in DTAC micelles and absent in SDS micellar solutions. Solutions with surfactant concentrations well below the critical micelle concentration (cmc) or solutions where micellar formation had been disrupted (1:1 v/v CH(3)CN/H(2)O) also showed no APS line shapes in their TREPR spectra. These results support the conclusion that electrostatic forces between the sensitizer (AQDS) charge and the substrate (surfactant) headgroup charge are responsible for the observed effects. The results represent a new example of electrostatic control of a spin exchange interaction in mobile radical pairs.

The missing link between molecular triplets and spin-polarized free radicals: room temperature triplet states of nanocrystalline radical pairs.

Photochemical reactions of organic molecules in the solid state have an excellent potential in green chemistry technologies as they often proceed in high yields to give a single product without generating volatile organic solvent waste. While recent synthetic applications highlight the need for a better understanding of reaction mechanisms and kinetics, spectroscopic observations of excited states and short-lived intermediates in single crystals and powdered samples have been extremely challenging due to the high optical density and scattering power of single crystals and powdered samples. In this communication, we report the first direct observation of a radical pair triplet state by time-resolved electron paramagnetic resonance (TREPR) with nanocrystals of 4,4'-dimethoxy-dicumyl ketone (1OMe) suspended in water. Steady state irradiation of 1OMe had previously shown that reactions in dry powders and nanocrystalline suspensions proceed with high efficiency and chemoselectivity to generate 4,4'-dimethoxy-dicumene 2OMe by decarbonylation and radical coupling. The nanocrystalline suspensions were excited with an 25 ns laser pulse at 308 nm using a flow system within the cavity of a time-resolved EPR spectrometer. The resulting TREPR spectra showed strong spin polarization with enhanced absorption A and emission E signals in an AAAEEE pattern characteristic of a randomly oriented triplet with zero-field splitting parameters D = 243 G and E = 11 G as well as an isotropic exchange integral J = -45,000 G. The assignment of this spectrum to a radical pair triplet state was supported by measurements carried out in fluid solution and in frozen glasses, which allowed for the characterization of the free radical and the triplet excited molecular state of the starting ketone 1OMe.

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1.

BACKGROUND: Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. RESULTS: It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. CONCLUSION: The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

The effect of ultrasound treatment on the interaction of brine with pork meat proteins.

The objective of the study is to elucidate the effect of ultrasound treated salt solution on curing of pork meat. The interactions of salt (NaCl) solutions of 3 and 25% with the proteins of pork meat are studied. High intensity ultrasound operating at 20 kHz was used. The differential scanning calorimetry (DSC), NMR spin-spin relaxation time, unfrozen water and water diffusion coefficient measurements were carried out in meat cured with ultrasound treated and untreated salt solutions. The effect of ultrasonication was most evident from measured spin-spin relaxation times T21, the rate of chemical exchange of water protons k and the amount of unfrozen water Wunf in the meat. The measured diffusion coefficient of water Dw in meat cured with ultrasound treated and control salt solution did not show significant difference. The nuclear magnetic resonance (NMR) relaxation data, differential scanning calorimetry (DSC) and the diffusion coefficient data reliably show that the possible action of ultrasound to salt solution was manifested on the first 2 days of the experiment with a 3% salt solution.

CIDNP as a tool to unveil the reaction mechanism: interaction of mixed phosphonium-iodonium ylide with p-methoxyphenylacetylene.

In situ acquisition of the reaction between benzoyl phosphonium-iodonium ylide and p-methoxyphenylacetylene in an NMR spectrometer reveals the CIDNP effect in 31P and 1H NMR spectra of major products, substituted furan (emission) and phosphonium salt (enhanced absorption). The mechanism of products formation via radical pairs is discussed.

Photo-oxidation of diglycine in confined media Relaxation of longitudinal magnetization in spin correlated radical pairs.

Time-resolved electron paramagnetic resonance spectra (X-band) of correlated radical pairs created in AOT reverse micelles are presented and simulated using the microreactor model. They are discussed in terms of the two-site model with a particular emphasis on longitudinal relaxation mechanisms. The geminate radical pair is created by photo-oxidation of dyglicine by the excited triplet states of an anthraquinone salt. The strong chemically induced electron spin polarization observed is due to three mechanisms: TM, RPM, and SCRPM. Relative contributions from these mechanisms depend on the water pool volume and the time of observation. There are three types of longitudinal relaxation in radical pairs. The first is relaxation of the RPM induced longitudinal magnetization in spin correlated radical pairs. The second is the longitudinal relaxation in radical pairs which are not correlated (with a zero value of the double quantum coherence). In such pairs, the generation of longitudinal magnetization due to RPM is impossible, and the spin-selective recombination of the pairs is ineffective. Under all experimental conditions, the first type of relaxation is slower than the second type. For both, the physical mechanism leading to relaxation is modulation of the Heisenberg electron spin exchange interaction. This is an internal relaxation process. The third relaxation type occurs in radical pairs due to ordinary longitudinal relaxation in non-interacting radicals. Normally, relaxation of the third type is the slowest of the three. This explains time and micelle size dependence of the relative contribution of RPM into TREPR spectra. It seems reasonable to suggest that the creation of the initial spin state populations is partially adiabatic.

Production of No-Carrier Added Lutetium-177 by Irradiation of Enriched Ytterbium-176.

Two methods of Lu-177 production are reviewed: irradiation of isotopically enriched Lu- 176 (direct way) and irradiation of ytterbium enriched with Yb-176 (indirect way). Based on neutronphysical calculations Lu-177 yield and specific activity were estimated for both methods. Lu-177 specific activity strongly depends on neutron flux density in the direct way, that is 75,000 Ci/g for 10- days irradiation in a neutron flux of 2.10(15) cm(-2) s(-1), and only 13,000 Ci/g after 30 days irradiation at neutron flux 1.10(14) cm(-2) s(-1). Irradiation of Yb-176 provides Lu-177 specific activity close to theoretical value (110,000 Ci/g). Neutron flux density effect Lu-177 yield, that is 530 Ci/g for 2.1015 cm(-2) s(-1) neutron flux density after 30 days irradiation. A procedure of isolation and purification of Lu-177 from irradiated targets is described based on combination of galvanostatic extraction of ytterbium followed by cation-exchange chromatography from alfa-hydroxyisobutirate solutions on BioRad AG(®)50W-X8 resin.

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella.

We examine distribution of flagellins along multicomponent flagellar filaments (FF) and protofilaments (PF) of the haloalkaliphilic archaeon Natrialba magadii using immunogold electron microscopy. A high specific polyclonal antibody raised to one of the flagellin types bound homogeneously to the undissociated FF along the full length. At the same time both uniformly labelled and completely unlabelled PF, outwardly indistinguishable one from another, were observed.

Time-resolved high-frequency EPR studies on magnesium and zinc tetraphenylporphines in their lowest excited triplet states.

The lowest excited triplet (T(1)) states of magnesium and zinc tetraphenylporphines (MgTPP and ZnTPP) were studied by time-resolved (TR) high-frequency/high-field W-band electron paramagnetic resonance (hf-EPR) spectroscopy in rigid glasses at low temperatures. Inspections of the TR-hf-EPR spectra of the spin-polarized triplets revealed that the zero field splitting (ZFS) parameters, D and E, for MgTPP and ZnTPP triplets were nearly the same. At the same time, their g-tensors were found to be different. These results are interpreted quantitatively in terms of spin-orbit couplings (SOCs) and angular momenta among the excited states, giving a magnitude of SOC in the T(1) state of ZnTPP. For the first time, both the TR-hf-EPR spectra and corresponding time profiles were acquired on the ZnTPP's triplet at room temperature in liquid paraffin solution with the populations of the electron spin states being in Boltzmann equilibrium. Because of relatively fast paramagnetic relaxation in rotating triplet at room temperature, the spectra and time profiles were free from the effects of microwave saturation that allowed for the direct measurement of the absolute intersystem crossing ratios P(x):P(y):P(z) 0.085:0.085:0.83. All of these results have demonstrated advantages and new perspectives of the W-band EPR spectroscopy.

Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G(1) cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (2/6; 33.3%) and pancreatic (3/19; 15.7%) carcinoma cell lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32 samples; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53 after DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

A small protein from the bop-brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription.

Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin-regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site-directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.

Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest.

In a genome-wide screen for microRNAs regulated by the transcription factor encoded by the p53 tumor suppressor gene we found that after p53-activation the abundance of thirty-four miRNAs was significantly increased, whereas sixteen miRNAs were suppressed. The induction of miR-34a was most pronounced among all differential regulations. Also expression of the primary miR-34a transcript was induced after p53 activation and by DNA damage in a p53-dependent manner. p53 occupied an evolutionarily conserved binding site proximal to the first non-coding exon of miR-34a. Ectopic miR-34a induced apoptosis and a cell cycle arrest in the G1-phase, thereby suppressing tumor cell proliferation. Other p53-induced miRNAs identified here may also have tumor suppressive potential as they are known to suppress the anti-apoptotic factor Bcl2 (miR-15a/16) and the oncogenes RAS and HMGA2 (let-7a). Our results for the first time directly integrate the regulation of miRNA expression into the transcriptional network regulated by p53. siRNAs corresponding to p53-induced miRNAs may have potential as cancer therapeutic agents as RNA interference based therapies are currently emerging.

Photooxidation of diglycine in confined media. Application of the microreactor model for spin-correlated radical pairs in reverse micelles and water-in-oil microemulsions.

Time-resolved electron paramagnetic resonance spectra (X-band) of correlated radical pairs created in AOT reverse micelles and microemulsions are presented, simulated, and discussed using the microreactor model. The radicals are formed inside the water pool using photooxidation of diglycine by the excited triplet states of two different anthraquinone sulfonate salts. Water pool size and temperature effects on the spectra are reported, and the simulations allow for extraction of the diffusion coefficient in the interior, which monotonically increases with water pool size. The data directly correlate with the diffusional properties of correlated radical pairs in regular aqueous micelle solutions studied previously by similar methods. Competition between H-atom abstraction and electron transfer is observed with anthraquinone sulfonate, but electron transfer is the only reaction pathway observed when anthraquinone disulfonate triplet state is the sensitizing species.