Unraveling the biological roles of reactive oxygen species

Reactive oxygen species are not only harmful agents that cause oxidative damage in pathologies, they also have important roles as regulatory agents in a range of biological phenomena. The relatively recent development of this more nuanced view presents a challenge to the biomedical research community on how best to assess the significance of reactive oxygen species and oxidative damage in biological systems. Considerable progress is being made in addressing these issues, and here we survey some recent developments for those contemplating research in this area.     

Zinc Lactate and Sapindin Act Synergistically with Lactocin 160 Against Gardnerella vaginalis

Lactocin 160 is a vaginal probiotic-derived bacteriocin shown to selectively inhibit the growth of Gardenerella vaginalis and some other pathogens commonly associated with bacterial vaginosis. The natural origin of this peptide, its safety, and selective antimicrobial properties make it a promising candidate for successful treatment and prophylaxis of bacterial vaginosis (BV). This study evaluated interactions between lactocin 160 and four other natural antimicrobials in the ability to inhibit G. vaginalis. We report that zinc lactate and soapnut extract act synergistically with lactocin 160 against this pathogen and therefore have a potential to be successfully used as the components of the multiple-hurdle antimicrobial formulation for the treatment of BV.     

Reactive oxygen and nitrogen species: Friends or foes?

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS)and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as electric cables, as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive ROS induced ROS release.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 265–272.Original Russian Text Copyright ¢ 2005 by Zorov, Bannikova, Belousov, Vyssokikh, Zorova, Isaev, Krasnikov, Plotnikov.This revised version was published online in April 2005 with corrections to the post codes.     

Genetically encoded fluorescent indicator for intracellular hydrogen peroxide

We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Proton transfer via the external proton channel of bacteriorhodopsin

The process of proton transfer across the membrane via the external proton channel in bacteriorhodopsin is considered. A possible amino acid composition of the channel is suggested and the step-by-step mechanism of proton transfer is proposed which agrees with the experimental data. The rate of proton transfer between fixed centers at several chains of the channel was estimated for which the spectroscopic data are available.     

Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.     

Marker Analysis of Quantitative Traits in Maize by ISSR–PCR

The segregating maize population (GK26 × Mo17)F₂ has been used for identification of ISSR markers able to reveal a significant difference between alleles by a quantitative index. Confidence ranges have been determined for variation in 17 quantitative traits. Variations in the traits under study correlate with the inheritance of 16 marker loci have been found. The nature of these correlations and the possibility of chromosomal mapping of genetic markers are discussed.     

“Our standpoint different from common...” (Scientific heritage of Alexander Gurwitsch)

The work of prominent Russian biologist Alexander Gavrilovich Gurwitsch (1874–1954) on the theory of organism development are reviewed. Alexander Gurwitsch introduced the concept of embryonic (morphogenetic, biological, and cellular) field and proposed several revisions of it from 1912 to 1944. Although neither of them can be considered as a final theory of development, his the persistent search for the invariant law that allows the shape (spatial structure) to be proposed for each next developmental stage from the previous shape is of imperishable methodological interest. Alexander Gurwitsch anticipated many ideas of the future theory of self-organization. His theoretical constructions are explicit and experiment-oriented but absolutely not esoteric. They represent a highly important and original contribution to theoretical biology and are an essential step to further development of the ontogenetic theory.