Inhibition of oxidative hemolysis in erythrocytes by mitochondria-targeted antioxidants of SkQ series

In the present work we studied the effect of antioxidants of the SkQ1 family (10-(6′-plastoquinonyl)decyltriphenylphosphonium) on the oxidative hemolysis of erythrocytes induced by a lipophilic free radical initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and a water-soluble free radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). SkQ1 was found to protect erythrocytes from hemolysis, 2 μM being the optimal concentration. Both the oxidized and reduced SkQ1 forms exhibited protective properties. Both forms of SkQ1 also inhibited lipid peroxidation in erythrocytes induced by the lipophilic free radical initiator AMVN as detected by accumulation of malondialdehyde. However, in the case of induction of erythrocyte oxidation by AAPH, the accumulation of malondialdehyde was not inhibited by SkQ1. In the case of AAPH-induced hemolysis, the rhodamine-containing analog SkQR1 exerted a comparable protective effect at the concentration of 0.2 μM. At higher SkQ1 and SkQR1 concentrations, the protective effect was smaller, which was attributed to the ability of these compounds to facilitate hemolysis in the absence of oxidative stress. We found that plastoquinone in the oxidized form of SkQ1 could be reduced by erythrocytes, which apparently accounted for its protective action. Thus, the protective effect of SkQ in erythrocytes, which lack mitochondria, proceeded at concentrations that are two to three orders of magnitude higher than those that were active in isolated mitochondria.     

Flow and Diffusion in Channel-Guided Cell Migration

Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport.     

Oxidative Stress in Caenorhabditis elegans: Protective Effects of Spartin

Troyer syndrome is caused by a mutation in the SPG20 gene, which results in complete loss of expression of the protein spartin. We generated a genetic model of Troyer syndrome in worms to explore the locomotor consequences of a null mutation of the Caenorhabditis elegans SPG20 orthologue, F57B10.9, also known as spg-20. Spg-20 mutants showed decreased length, crawling speed, and thrashing frequency, and had a shorter lifespan than wild-type animals. These results suggest an age-dependent decline in motor function in mutant animals. The drug paraquat was used to induce oxidative stress for 4 days in the animals. We measured survival rate and examined locomotion by measuring crawling speed and thrashing frequency. After 4 days of paraquat exposure, 77% of wild-type animals survived, but only 38% of spg-20 mutant animals survived. Conversely, animals overexpressing spg-20 had a survival rate of 95%. We also tested lifespan after a 1 hour exposure to sodium azide. After a 24 hour recovery period, 87% of wild type animals survived, 57% of spg-20 mutant animals survived, and 82% of animals overexpressing spg-20 survived. In the behavioral assays, spg-20 mutant animals showed a significant decrease in both crawling speed and thrashing frequency compared with wild-type animals. Importantly, the locomotor phenotype for both crawling and thrashing was rescued in animals overexpressing spg-20. The animals overexpressing spg-20 had crawling speeds and thrashing frequencies similar to those of wild-type animals. These data suggest that the protein F57B10.9/SPG-20 might have a protective role against oxidative stress.     

Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting.     

Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris

Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

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It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

Analysis of synaptonemal complexes in a heterozygous human male carrier of a reciprocal translocation involving an acrocentric chromosome: heterosynapsis without previous homosynapsis

Summary Silver-stained synaptonemal complexes in surface-spread pachytene nuclei from an oligospermic man, heterozygous for a reciprocal translocation involving an acrocentric chromosome, were analyzed by electron microscopy. Contrary to the classically expected configuration, nonhomologous pairing was observed with asymetrical association of the lateral elements of the nonhomologous arms of the quadrivalents. A possible role of heterosynapsis in germ cell conservation is discussed.     

Control of tsetse and trypanosomiasis transmission in Uganda by applications of lambda-cyhalothrin

The pyrethroid insecticide lambda-cyhalothrin was evaluated in field trials against Glossina f.fuscipes and sleeping sickness transmission in Iyolwa sub-county, Tororo District, Uganda. The insecticide was applied selectively to the resting-sites of tsetse, by bush-spraying, using 10% wettable powder (10WP) formulation at an application rate of 11.6 g a.i./ha over an area of 28 km2, or by a 2% Electrodyn formulation (2ED) applied at 0.9 g a.i./ha over 30 km2. In a third trial area of 32 km2, 215 pyramidal traps treated with lambda-cyhalothrin 100 mg/m2 were set. The best impact was obtained with 10WP lambda-cyhalothrin which eliminated tsetse within 1-2 months, whereas G.f.fuscipes persisted at very low density in part of the area treated with 2ED lambda-cyhalothrin. In both treated areas the numbers of human sleeping sickness cases fell to no more than one per month, compared with four to twelve per month previously. The overall rate of cattle trypanosomiasis (T.brucei and T.vivax) was also reduced slightly. Insecticide-treated traps remained fully effective for at least 6 months under field conditions and catches were reduced 20-90-fold. These results in the control of tsetse and trypanosomiasis transmission lead us to recommend lambda-cyhalothrin for tsetse control operations.