Topoisomerase II activity in the replicating DNA of irradiated hamster cells.

The activity of DNA topoisomerase II in the replicating DNA of irradiated Chinese hamster ovary cells was estimated by determining protein-linked DNA double-strand breaks generated in the presence of the DNA intercalative drug 4'-(9-acridinylamino) methanesulfon-m-anisidide. In the presence of this drug, DNA double-strand breaks were produced at the same rate, and with the same overall frequency, in both the bulk and the newly synthesized DNA of control cells and cells irradiated with 10 Gy. The results indicate that DNA topoisomerase II is fully active in the replicating DNA of irradiated cells and is distributed at a frequency similar to that in parental DNA.     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

Sequence and genetic analysis of a dispensible 189 nucleotide snRNA from Saccharomyces cerevisiae.

The structure of a Saccharomyces cerevisiae gene that encodes a small nuclear RNA (snRNA) of 189 nucleotides is described. This gene, designated SNR189, is located 400 base pairs upstream of the CRY1 gene on yeast chromosome III. Gene replacement analysis revealed the SNR189 gene to be dispensable for growth under a variety of culture conditions. The snR189 sequence lacks homology with other sequenced yeast or metazoan snRNAs.     

A novel retinoid binding property of human annexin A6

Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.     

Arrestin interaction with rhodopsin

It is becoming increasingly apparent that G protein-coupled receptors (GPCRs) can exist and function as oligomers. This notion differs from the classical view of signaling wherein the receptor has been presumed to be monomeric. Despite this shift in views, the interpretation of data related to GPCR function is still largely carried out within the framework of a monomeric receptor. Rhodopsin is a prototypical GPCR that initiates phototransduction. Like other GPCRs, the activity of rhodopsin is regulated by phosphorylation and the binding of arrestin. In the current investigation, we have explored by modeling methods the interaction of rhodopsin and arrestin under the assumption that either one or two rhodopsin molecules bind each arrestin molecule. The dimeric receptor framework may provide a more accurate representation of the system and is therefore likely to lead to a better and more accurate understanding of GPCR signaling.     

A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.