No superoxide dismutase activity of cellular prion protein in vivo

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.     

Tunable, monochromatic X-rays: an enabling technology for molecular/cellular imaging and therapy

Pulsed, tunable, monochromatic X-rays hold great potential as a cellular and molecular probe. These beams can be tuned to the binding energy of orbital electrons in atoms, making them extremely useful in diagnostic k-edge imaging and Auger cascade radiotherapy. Their wide tunability makes them ideal for the performance of various techniques as disparate as protein crystallography and three-dimensional, compressionless, monochromatic mammography. Since only the frequency best suited to the task at hand is used, radiation exposure to patients or animals is exceedingly low when compared to standard X-ray techniques.     

Adjacent and spontaneous neurotization after distal digital replantation in children

In an exclusively pediatric population, this retrospective study examined the functional and aesthetic results after distal replantation without nerve suture. The aim was to demonstrate, in the child, the presence of spontaneous nervous regeneration resulting in a fingertip pulp with discriminatory sensation. Eight amputations in eight children with a mean age of 9 years and 2 months on the day of the accident were reviewed. The cases were managed by a single surgeon over a period of 8 years and were collected from two different hand centers. The patients were then examined by a different surgeon, and the data were collected. Sensibility was evaluated using the Weber, Semmes-Weinstein, and wrinkle tests. The results were excellent, with mean values of 4.6 mm for the Weber test, 3.3 for the Semmes-Weinstein test, and a positive wrinkle test in all subjects. All patients thus recovered discriminatory sensation with minimal aesthetic sequelae. The usual factors adversely affecting the results of the replantation (ischemic time, level and mechanism of the amputation, and quality of the venous return) were examined, but no statistical analysis was performed because of the small sample size. This study demonstrates the presence of the clinical phenomenon of adjacent neurotization in the absence of nerve repair. It thus confirms that children are excellent candidates for replantation of the distal extremities, even when nerve suture is not performed.     

Effect of linker sequence on the stability of circularly permuted variants of ribonuclease T1

Circularly permuted variants of ribonuclease T1 were constructed with a library of residues covalently linking the original amino and carboxyl terminal ends of the wild-type protein. The library of linking peptides consisted of three amino acids containing any combination of proline, aspartate, asparagine, serine, threonine, tyrosine, alanine, and histidine. Forty two unique linker sequences were isolated and 10 of these mutants were further characterized with regard to catalytic activity and overall thermodynamic stability. The 10 mutants with the different linking sequences (HPD, TPH, DTD, TPD, PYH, PAT, PHP, DSS, SPP, and TPS), in addition to GGG and GPG, were 4.0-6.2 kcal/mol less stable than the wild-type ribonuclease T1. However, these circular permuted variants were only 0.4-2.6 kcal/mol less stable than the direct parent protein that is missing the disulfide bond connecting residues 2 and 10. The most stable linking peptide was HPD.     

Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.     

Diffusional properties of methanogenic granular sludge: 1H NMR characterization

The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22 degrees C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H(2)O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H(2)O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55 degrees C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10 degrees C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90 degrees C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl(2) did not affect the H(2)O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.     

Membrane fluidity adjustments in ethanol-stressed Oenococcus oeni cells

The effect of ethanol on the cytoplasmic membrane of Oenococcus oeni cells and the role of membrane changes in the acquired tolerance to ethanol were investigated. Membrane tolerance to ethanol was defined as the resistance to ethanol-induced leakage of preloaded carboxyfluorescein (cF) from cells. To probe the fluidity of the cytoplasmic membrane, intact cells were labeled with doxyl-stearic acids and analyzed by electron spin resonance spectroscopy. Although the effect of ethanol was noticeable across the width of the membrane, we focused on fluidity changes at the lipid-water interface. Fluidity increased with increasing concentrations of ethanol. Cells responded to growth in the presence of 8% (vol/vol) ethanol by decreasing fluidity. Upon exposure to a range of ethanol concentrations, these adapted cells had reduced fluidity and cF leakage compared with cells grown in the absence of ethanol. Analysis of the membrane composition revealed an increase in the degree of fatty acid unsaturation and a decrease in the total amount of lipids in the cells grown in the presence of 8% (vol/vol) ethanol. Preexposure for 2 h to 12% (vol/vol) ethanol also reduced membrane fluidity and cF leakage. This short-term adaptation was not prevented in the presence of chloramphenicol, suggesting that de novo protein synthesis was not involved. We found a strong correlation between fluidity and cF leakage for all treatments and alcohol concentrations tested. We propose that the protective effect of growth in the presence of ethanol is, to a large extent, based on modification of the physicochemical state of the membrane, i.e., cells adjust their membrane permeability by decreasing fluidity at the lipid-water interface.     

Characterization of two tetrachloroethene-reducing,acetate-oxidizing anaerobic bacteria and their description as Desulfuromonas michiganensis sp. nov

Two tetrachlorethene (PCE)-dechlorinating populations, designated strains BB1 and BRS1, were isolated from pristine river sediment and chloroethene-contaminated aquifer material, respectively. PCE-to-cis-1,2-dichloroethene-dechlorinating activity could be transferred in defined basal salts medium with acetate as the electron donor and PCE as the electron acceptor. Taxonomic analysis based on 16S rRNA gene sequencing placed both isolates within the Desulfuromonas cluster in the delta subdivision of the Proteobacteria. PCE was dechlorinated at rates of at least 139 nmol min(-1) mg of protein(-1) at pH values between 7.0 and 7.5 and temperatures between 25 and 30 degrees C. Dechlorination also occurred at 10 degrees C. The electron donors that supported dechlorination included acetate, lactate, pyruvate, succinate, malate, and fumarate but not hydrogen, formate, ethanol, propionate, or sulfide. Growth occurred with malate or fumarate alone, whereas oxidation of the other electron donors depended strictly on the presence of fumarate, malate, ferric iron, sulfur, PCE, or TCE as an electron acceptor. Nitrate, sulfate, sulfite, thiosulfate, and other chlorinated compounds were not used as electron acceptors. Sulfite had a strong inhibitory effect on growth and dechlorination. Alternate electron acceptors (e.g., fumarate or ferric iron) did not inhibit PCE dechlorination and were consumed concomitantly. The putative fumarate, PCE, and ferric iron reductases were induced by their respective substrates and were not constitutively present. Sulfide was required for growth. Both strains tolerated high concentrations of PCE, and dechlorination occurred in the presence of free-phase PCE (dense non-aqueous-phase liquids). Repeated growth with acetate and fumarate as substrates yielded a BB1 variant that had lost the ability to dechlorinate PCE. Due to the 16S rRNA gene sequence differences with the closest relatives and the unique phenotypic characteristics, we propose that the new isolates are members of a new species, Desulfuromonas michiganensis, within the Desulfuromonas cluster of the Geobacteraceae.     

Effects of mesozooplankton removal and ammonium addition on planktonic trophic structure during a bloom of the Texas 'brown tide': a mesocosm study

A bloom of the alga Aureoumbra lagunensis, known as the Texas‘brown tide’, persisted in the Laguna Madre of Texasfor most of the 1990s. The dominant mesozooplankter in LagunaMadre, Acartia tonsa, does not feed on A. lagunensis, and duringblooms there are few other suitably sized phytoplankton cellsavailable to feed on. We hypothesized that these copepods increasedtheir feeding on microzooplankton, thereby reducing grazingpressure by microzooplankton on A. lagunensis and contributingto the persistence of this bloom. A mesocosm experiment wascarried out to test this hypothesis during the summer of 1999.Twelve fiberglass corral-type mesocosms were deployed in thefield for 16 days, each enclosing 1.2 m³ of Laguna Madre waterand 1.1 m² of natural benthos. Mesozooplankton were removedfrom six mesocosms with a 153 µm mesh dip net every 4days; the other six mesocosms were treated in the same way,except that the contents of the net were returned to the mesocosm.For each zooplankton treatment, half of the mesocosms were dosedwith 40 µm NH₄ at 4 day intervals, and half received noadditions. Phytoplankton populations in these mesocosms at thestart of the experiment were dominated by A. lagunensis andthe cyanobacterium Synechococcus spp. Growth rates of A. lagunensiswere higher in mesocosms without ammonium additions, providingno evidence for nitrogen limitation. Acartia tonsa populationswere reduced by 50% in the zooplankton removal mesocosms, andciliate populations were significantly higher. The increasein ciliate population had no significant impact on A. lagunensispopulation dynamics, however, providing no evidence to supportthe hypothesis that a trophic cascade reducing microzooplanktonpopulations contributed to the persistence of the brown-tidebloom. In contrast, populations of Synechococcus sp. showedevidence of both ‘top–down’ and ‘bottom–up’control; they grew faster in nutrient addition mesocosms andhad lower populations in mesocosms with increased densitiesof ciliate grazers.