Chalcogenoxanthylium photosensitizers for the photodynamic purging of blood-borne viral and bacterial pathogens

Thio- and selenoxanthylium dyes were prepared by the addition of 2-lithiothiophene, 4-N,N-dimethylaminophenylmagnesium bromide, and 1-naphthylmagnesium bromide to the appropriate 2,7-bis-N,N-dimethylaminochalcogenoxanthen-9-one, followed by dehydration and ion exchange to the chloride salts. The corresponding chalcogenoxanthylium dyes were evaluated as photosensitizers for the inactivation of intracellular and extracellular virus in red blood cell suspensions and for the inactivation of selected strains of gram (+) and gram (-) bacteria in red blood cell suspensions. Selected combinations of photosensitizer and light gave >6 log10 inactivation of intracellular and extracellular virus, and >4 log10 inactivation of extracellular bacteria with varying levels of hemolyis, following a 42-day storage of red blood cell suspensions. Photocleavage experiments with plasmid DNA and the chalcogenoxanthylium dyes suggested the genomic material contained in the virus and in the bacteria as one possible target for the photodynamic action of some of these dyes.     

Involvement of acetosyringone in plant-pathogen recognition

In this study, acetosyringone was identified as one of the major extracellular phenolics in tobacco suspension cells and was shown to have bioactive properties that influence early events in plant-bacterial pathogenesis. In our model system, tobacco cell suspensions treated with bacterial isolate Pseudomonas syringae WT (HR+) undergo a resistant interaction characterized by a burst in oxygen uptake several hours after inoculation. When the extracellular concentration of acetosyringone in tobacco cell suspensions was supplemented with exogenous acetosyringone, the burst in oxygen uptake occurred as much as 1.5h earlier. The exogenous acetosyringone had no effect on tobacco suspensions undergoing susceptible interactions with Pseudomonas tabaci or a non-resistant interaction with a near-isogenic mutant derivative of isolate P. syringae WT (HR+). Resistant interactions with isolate P. syringae WT (HR+) also produce an oxidative burst which oxidizes the extracellular acetosyringone. This study demonstrates that acetosyringone, and likely other extracellular phenolics, may have bioactive characteristics that can influence plant-bacterial pathogenesis.     

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

Strong binding of myosin heads stretches and twists the actin helix

Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by 0.22% when relaxed fibers were put into low-tension rigor (<10 kN/m(2)) demonstrating that strongly bound myosin heads elongate the actin filaments even in the absence of external tension. The pitch of the 5.9-nm actin layer line increased by approximately 0.62% and that of the 5.1-nm layer line decreased by approximately 0.26%, suggesting that the elongation is accompanied by a decrease in its helical angle (approximately 166 degrees) by approximately 0.8 degrees. This effect explains the difference between actin compliance revealed from mechanical experiments with single fibers and from x-ray diffraction on whole muscles. Our measurement of actin compliance obtained by applying tension to fibers in rigor is consistent with the results of mechanical measurements.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

Recognition and selective binding of DNA by ionenes of different charge density

The ability of aliphatic ionenes to recognize and bind DNA or poly(methacrylic acid) (PMA) in the equimolar mixture of these polyanions was studied by fluorescence quenching technique. Within a particular system, the selectivity of competitive interactions was shown to be determined by a component with the lowest degree of polymerization (DP). Ionene polycations with lowest DP values did not exhibit pronounced selectivity in binding DNA or PMA with higher values of DP. Increase in ionene DP resulted in a steady increase in selectivity of interaction and ultimately in almost exclusive binding of one of the two polyanions. The ability of the ionene to recognize and bind DNA in the mixture of polyanions was shown not to correlate with the affinity of the ionene to DNA in their binary mixture. Although ionenes with a higher charge density exhibited preferential binding to PMA, the ionenes with the lowest charge density selectively bound DNA.     

Endothelin 1 induces beta 1Pix translocation and Cdc42 activation via protein kinase A-dependent pathway

p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.     

Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca(2+), phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.