Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy

Nitric oxide synthase (EC 1.14.13.39; NOS) converts L-arginine into NO and L-citrulline in a two-step reaction with Nomega-hydroxy-L-arginine (NOHLA) as an intermediate. The active site iron in NOS has thiolate axial heme-iron ligation as found in the related monooxygenase cytochrome P450. In NOS, tetrahydrobiopterin (BH4) is an essential cofactor for both steps, but its function is controversial. Previous optical studies of the reaction between reduced NOS with O2 at -30 degrees C suggested that BH4 may serve as an one-electron donor in the first cycle, implying formation of a trihydrobiopterin radical. We investigated the same reaction under identical conditions with electron paramagnetic resonance spectroscopy. With BH4-containing full-length neuronal NOS we obtained an organic free radical (g-value 2.0042) in the presence of Arg, and a similar radical was observed with the endothelial NOS oxygenase domain in the presence of Arg and BH4. Without substrate the radical yield was greatly (10x) diminished. Without BH4, or with NOHLA instead of Arg, no radical was observed. With 6-methyltetrahydropterin or 5-methyl-BH4 instead of BH4, radicals with somewhat different spectra were formed. On the basis of simulations we assign the signals to trihydropterin radical cations protonated at N5. This is the first study that demonstrates the formation of a protonated trihydrobiopterin radical with the constitutive isoforms of NOS, and the first time the radical was obtained without exogenous BH4. These results offer strong support for redox cycling of BH4 in the first reaction cycle of NOS catalysis (BH4 <--> BH3.H+).     

Cetaben-induced changes on the morphology and peroxisomal enzymes in MH1C1 rat hepatoma cells and HepG2 human hepatoblastoma cells

Human HepG2 and rat MH1C1 hepatoma cell lines were examined for their response to cetaben, an exceptional type of peroxisome proliferator. Shape change and proliferation of peroxisomes as well as induction of selected peroxisomal enzymes catalase, acyl-CoA oxidase, and peroxisomal bifunctional enzyme, were assessed in response to cetaben. In MH1C1 cells, peroxisomes were seen in clusters displaying typical features of microperoxisomes. Cetaben caused little but reversible proliferation and morphological heterogeneity with the occurrence of dumbbell-shaped and cup-shaped peroxisomal profiles. Peroxisomes in HepG2 cells showed marked variation in size and shape. Cetaben treatment of HepG2 cells caused disintegration of Golgi regions and augmented mitochondrial matrix. Interestingly, MH1C1 cells showed different subunit composition of acyl-CoA oxidase in immunoblot analysis: only subunit A at 72 kDa was detected but not the cleavage products. In situ hybridization underlined the marked morphological heterogeneity observed, and both cell lines revealed different stages of gene expression. Our results indicate that cetaben represents an extraordinary type of peroxisomal proliferator with pleiotropic effects on human and rat hepatoma cells, and, at least in the human hepatoma cell line HepG2, these effects are not restricted to peroxisome proliferation.     

Erythrogenic toxins A, B and C: Occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.     

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.     

Coccidioidomycosis in Northern California—An Outbreak among Archeology Students near Red Bluff

An outbreak of coccidioidomycosis occurred among 39 archeology students in the summer of 1972. The students excavated Indian ruins near Red Bluff in Tehama County, California, 20 miles north of the previously recognized northernmost limit of endemicity. At least 17 persons contracted an illness clinically compatible with a diagnosis of coccidioidomycosis. Coccidioidomycosis was documented by skin test conversion as well as by specific serologic reactions. Coccidioides immitis was also isolated from two soil samples taken at the excavation site. In light of its ecological requirements, it is doubtful that C. immitis will be recovered much farther north than Red Bluff. The occupational hazard of coccidioidomycosis to archeologists and others employed in known endemic areas remains a substantial threat to health.     

Transdermal enhancement through rat skin of luciferase plasmid DNA loaded in elastic nanovesicles

Transdermal absorption of luciferase plasmid (pLuc) was enhanced by loading in elastic cationic liposomes and niosomes and the application of iontophoresis or the stratum corneum (SC) stripping method. Cationic liposomes (DPPC/Chol/DDAB at a 1:1:1 molar ratio) and niosomes (Tween61/Chol/DDAB at a 1:1:0.5 molar ratio) were prepared by the freeze-dried empty liposomes method. The elastic vesicles were prepared by hydrating the lipid or surfactant film by 25% of ethanol instead of distilled water. Gel electrophoresis of all nanovesicles showed the 100% pLuc entrapment efficiency. All nanovesicles loaded with pLuc showed larger vesicular sizes than the nonloaded vesicles of about 1.4 times for liposomes and 1.7 times for niosomes. The nanovesicles loaded with pLuc demonstrated less positive zeta potential than the nonloaded vesicles. The pLuc loaded in elastic vesicles kept at 4 ± 2 and 27 ± 2°C for 8 weeks gave the remaining pLuc of about 70 and 60% for liposomes and 85 and 73% for niosomes, respectively. For nonelastic vesicles kept at 4 ± 2°C, 56 and 61% of the remaining pLuc were observed for liposomes and niosomes, respectively, while at 27 ± 2°C, all pLuc were degraded. The deformability indices of the elastic liposomes and niosomes loaded with the pLuc were 16.64 ± 2.92 and 20.72 ± 0.82, whereas the nonelastic vesicles gave 9.35 ± 0.09 and 10.08 ± 0.12, respectively. Transdermal absorption through rat skin pretreated with SC stripping or treated with iontophoresis of pLuc loaded in nanovesicles by vertical Franz diffusion cells was investigated at 37°C. The cells were stopped and the skin and the receiving solution were withdrawn at 1, 3, and 6 hours and the pLuc contents in the stripped SC, whole skin (viable epidermis and dermis; VED), and the receiving solution were assayed by the modified gel electrophoresis and gel documentation. Without the SC stripping technique or iontophoresis, the pLuc loaded and nonloaded in nonelastic cationic liposomes or niosomes were not found in SC, VED, and receiving solution. The fluxes in the whole skin of pLuc loaded in nonelastic liposomes and niosomes with SC stripping and iontophoresis at 6 hours gave 2.73 ± 0.46 and 3.83 ± 0.73, and 7.01 ± 1.22 and 9.60 ± 1.31 g/cm²/h, respectively, while pLuc loaded in elastic liposomes and niosomes without the SC stripping and iontophoresis at 6 hours showed 2.79 ± 0.09 and 2.84 ± 0.04 g/cm²/h, respectively. The pLuc loaded in elastic niosomes or in nonelastic niosomes with iontophoresis was found in the receiving solution with a higher amount than that loaded in elastic liposomes or nonelastic liposomes with iontophoresis. The fluxes in the receiving solution of pLuc loaded in nonelastic liposomes and niosomes with iontophoresis at 6 hours were 6.71 ± 0.31 and 8.82 ± 0.28 g/cm²/h, respectively. For elastic liposomes and niosomes, the fluxes of the loaded pLuc in the receiving solution were the same, at about 1.9 g/cm²/h. Although pLuc loaded in nonelastic niosomes with iontophoresis gave the highest delivery of the plasmid in VED and receiving solution, a more promising applicable approach for gene delivery has been suggested to be the elastic niosomal systems, since no equipment is required.     

Controlling the adhesion of the diatom Navicula perminuta using poly(N-isopropylacrylamide-co–N-(1-phenylethyl) acrylamide) films

Poly(N-isopropylacrylamide)-co–N-(1-phenylethyl) acrylamide [P(NIPAAm-co-PEAAm)] thermo-responsive thin films with a lower critical solution temperature (LCST) adjusted to fit marine applications were used to investigate the effect of changes in the wetting properties of a surface on the adhesion of the diatom Navicula perminuta, an organism which forms slime films on surfaces immersed in an aquatic environment. Although the strength of attachment of cells was affected by whether the film was collapsed or expanded, no significant decrease in adhesion strength occurred upon temperature decrease. The effects were attributed to possible strong interactions between the hydrophobic segments of the responsive film when collapsed with components in the adhesive complex.