Multimeric arrays of the yeast retrotransposon Ty.

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.     

Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.     

Concordance between Parental Origin of Chromosome 13q Loss and 6p Duplication in Sporadic Retinoblastoma

Two hypotheses are capable of explaining nonrandom loss of one parent's alleles at tumor suppressor loci in sporadic cases of several pediatric cancers, including retinoblastoma—namely, preferential germ-line mutation or chromosome imprinting. We have examined 74 cases of sporadic retinoblastoma for tumors in which at least two genetic events—loss of heterozygosity for chromosome 13q markers and formation of an isochromosome 6p—have occurred. Sixteen cases were found to contain both events. In 13 of 16 such tumors, the chromosomes 13q that were lost and chromosomes 6p that were duplicated are derived from the same parent. These data may be explained within the framework of the genome imprinting model but are not predicted by preferential germ-line mutation.     

Disruption of a Silencer Domain by a Retrotransposon

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.     

A phase III comparison of methyl-CCNU + vincristine with or without BCG + allogeneic tumor cells in metastatic melanoma

Summary Sixty-two patients with metastatic malignant melanoma were randomized to treatment with either (a) methyl-CCNU (200 mg/m², PO every 8 weeks) plus vincristine (2 mg IV every 4 weeks), or (b) the same chemotherapy plus intradermal (ID) injections of irradiated (15,000 rads) allogeneic (fresh-frozen) melanoma cells (1–2×10⁸) admixed with BCG (Glaxo, 2–4.5×10⁶ organisms) every 2 weeks. Treatment cycles were repeated every 8 weeks until tumor progression. Seven (2 CR, 5 PR) objective remissions were noted among 31 patients (22.5%) treated with chemotherapy alone, whereas six (3 CR, 3 PR) objective remissions were noted among 31 patients (19%) treated with chemoimmunotherapy (P>0.05). The medians for remission duration (6 months) and survival (6.5 months) in the chemotherapy group did not differ significantly from the medians for remission duration (8 months) and survival (8 months) in the chemoimmunotherapy group. The patients manifested no unexpected toxicity. Hematologic toxicity was experienced by patients on both regimens; however, those receiving chemoimmunotherapy rebounded more quickly.     

Antagonism of membrane compression effects by high pressure gas mixtures in a phospholipid bilayer system

Binary mixtures of helium with nitrogen, xenon or nitrous oxide were applied to suspensions of phosphatidylcholine-cholesterol vesicles to determine those mixtures of lipid soluble gases which would exactly antagonize the membrane rigidifying effect of 100 ATA compression. A previous study has shown that the initial application of 100 ATA compression by gas produces a significant reduction in the fluidity of the phospholipid bilayer. However, as the high pressure gas dissolves into the lipid region it creates disorder and increases fluidity. Fluidity of the bilayer at equilibrium represents the sum of the compression-ordering and dissolved-gas disordering effects and is dependent on the gas/lipid partition coefficient of the particular gas. The beneficial effect of a narcotic gas added to Trimix mixtures to ameliorate HPNS in deep divers may be due to a balance of compression-ordering and solubility-disordering effects achieved within the nerve membrane. It is therefore valuable to determine those gas mixtures which achieve balance of these two effects and result in zero net change in phospholipid bilayer fluidity at an established pressure of 100 ATA. Binary mixtures of helium with 88% nitrogen, 3.8% xenon or 2.8% nitrous oxide resulted in zero net change in bilayer fluidity with our model system at 100 ATA. A graph of the percent of narcotic gas needed to produce zero net effect as a function of pressure, however, was nonlinear. This would suggest the ratio of gases in Trimix must be varied as a function of pressure. While the phosphatidylcholine-cholesterol bilayer is a good model for certain components of the nerve membrane, it does not allow for study of protein-lipid or gas-protein interactions. The data presented thus aid in our understanding of HPNS but are yet incomplete for precise use in predicting diving mixtures.     

Protective Role of Genetic Variants in HSP90 Genes-Complex in COPD Secondary to Biomass-Burning Smoke Exposure and Non-Severe COPD Forms in Tobacco Smoking Subjects

Background: Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory disease characterized by airflow obstruction, commonly present in smokers and subjects exposed to noxious particles product of biomass-burning smoke (BBS). Several association studies have identified single-nucleotide polymorphisms (SNP) in coding genes related to the heat shock proteins family-genes that codify the heat shock proteins (Hsp). Hsp accomplishes critical roles in regulating immune response, antigen-processing, eliminating protein aggregates and co-activating receptors. The presence of SNPs in these genes can lead to alterations in immune responses. We aimed to evaluate the association of SNPs in the HSP90 gene complex and COPD. Methods: We enrolled 1549 participants, divided into two comparison groups; 919 tobacco-smoking subjects (cases COPD-TS n = 294 and, controls SWOC n = 625) and 630 chronic exposed to BBS (cases COPD-BBS n = 186 and controls BBES n = 444). We genotyped 2 SNPs: the rs13296 in HSP90AB1 and rs2070908 in HSP90B1. Results: Through the dominant model (GC + CC), the rs2070908 is associated with decreased risk (p < 0.01, OR = 0.6) to suffer COPD among chronic exposed BBS subjects. We found an association between rs13296 GG genotype and lower risk (p = 0.01, OR = 0.22) to suffer severe COPD-TS forms in the severity analysis. Conclusions: single-nucleotide variants in the HSP90AB1 and HSP90B1 genes are associated with decreased COPD risk in subjects exposed to BBS and the most severe forms of COPD in tobacco-smoking subjects.     

Redox Control of Signal Transduction,Gene Expression and Cellular Senescence

Reactive oxygen species (ROS) act as subcellular messengers in such complex cellular processes as mitogenic signal transduction, gene expression, regulation of cell proliferation, replicative senescence, and apoptosis. They serve to maintain cellular homeostasis and their production is under strict control. However, the mechanisms whereby ROS act are still obscure. Here we review recent advances in our understanding of signaling mechanisms and recent data about the involvement of ROS in: (i) the regulation of the mitogenic transduction elements, particularly protein kinases and phosphatases; (ii) the regulation of gene expression; and (iii) the induction of replicative senescence and the role, if any, in aging and age-related disorders.     

Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.