Cell division transforms mutagenic lesions into deletion-recombinagenic lesions in yeast cells.

Cell proliferation has been recognized as an important factor in human and experimental carcinogenesis. Point mutations as well as larger chromosomal rearrangements are involved in the initiation of cancer. In this paper we compared the relative potencies of radiation and chemical carcinogens for inducing point mutations vs. deletions in cell cycle arrested with dividing cells of Saccharomyces cerevisiae. Point mutation substrates and deletion (DEL) recombination substrates were constructed with the genes CDC28 and TUB2 that are required for cell cycle progression through G1 and G2, respectively. The carcinogens ionizing radiation, UV, MMS, EMS and 4-NQO induced point mutations in G1 and in G2 arrested as well as in dividing cells. UV, MMS, EMS and 4-NQO caused very weak if any increases in DEL recombination in G1 or G2 arrested cells, but large increases in dividing cells. When cells treated with carcinogen either in G1 or G2 were allowed to progress through the cell cycle, a time-dependent increase in DEL recombination was seen. Ionizing radiation and the site-specific endonuclease I-SceI, which both directly create double-strand breaks, induced DEL recombination in G1 as well as in G2 arrested cells. In conclusion, UV-, MMS-, EMS- and 4-NQO-induced DNA damage was converted during DNA replication to a lesion capable of inducing DEL recombination which is probably a DNA strand break. Thus, cell proliferation is not necessary to turn DNA alkylation or UV damage into a mutagenic lesion but to convert the damage into a lesion that induces DNA deletions. These results are discussed with respect to mechanisms of carcinogenesis.     

De novo-designed peptide transforms Golgi-specific lipids into Golgi-like nanotubules.

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex protein-lipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic alpha-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.     

ER stress transforms random olfactory receptor choice into axon targeting precision

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Drosophila dMyc is required for ovary cell growth and endoreplication

Although the Myc oncogene has long been known to play a role in many human cancers, the mechanisms that mediate its effects in both normal cells and cancer cells are not fully understood. We have initiated a genetic analysis of the Drosophila homolog of the Myc oncoprotein (dMyc), which is encoded by the dm locus. We carried out mosaic analysis to elucidate the functions of dMyc in the germline and somatic cells of the ovary during oogenesis, a process that involves cell proliferation, differentiation and growth. Germline and somatic follicle cells mutant for dm exhibit a profound decrease in their ability to grow and to carry out endoreplication, a modified cell cycle in which DNA replication occurs in the absence of cell division. In contrast to its dramatic effects on growth and endoreplication, dMyc is dispensable for the mitotic division cycles of both germline and somatic components of the ovary. Surprisingly, despite their impaired ability to endoreplicate, dm mutant follicle cells appeared to carry out chorion gene amplification normally. Furthermore, in germline cysts in which the dm mutant cells comprised only a subset of the 16-cell cluster, we observed strictly cell-autonomous growth defects. However, in cases in which the entire germline cyst or the whole follicular epithelium was mutant for dm, the growth of the entire follicle, including the wild-type cells, was delayed. This observation indicates the existence of a signaling mechanism that acts to coordinate the growth rates of the germline and somatic components of the follicle. In summary, dMyc plays an essential role in promoting the rapid growth that must occur in both the germline and the surrounding follicle cells for oogenesis to proceed.     

Peroxynitrite transforms nerve growth factor into an apoptotic factor for motor neurons

Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.     

The Drosophila F box protein archipelago regulates dMyc protein levels in vivo

BACKGROUND: The Myc oncoprotein is an important regulator of cellular growth in metazoan organisms. Its levels and activity are tightly controlled in vivo by a variety of mechanisms. In normal cells, Myc protein is rapidly degraded, but the mechanism of its degradation is not well understood. RESULTS: Here we present genetic and biochemical evidence that Archipelago (Ago), the F box component of an SCF-ubiquitin ligase and the Drosophila ortholog of a human tumor suppressor, negatively regulates the levels and activity of Drosophila Myc (dMyc) protein in vivo. Mutations in archipelago (ago) result in strongly elevated dMyc protein levels and increased tissue growth. Genetic interactions indicate that ago antagonizes dMyc function during development. Archipelago binds dMyc and regulates its stability, and the ability of Ago to bind dMyc in vitro correlates with its ability to inhibit dMyc accumulation in vivo. CONCLUSIONS: Our data indicate that archipelago is an important inhibitor of dMyc in developing tissues. Because archipelago can also regulate Cyclin E levels and Notch activity, these results indicate how a single F box protein can be responsible for the degradation of key components of multiple pathways that control growth and cell cycle progression.     

Activated H-ras transforms rat intestinal epithelial cells with expression of alpha-TGF

We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat alpha-TGF mRNA.     

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2

Platelet rich plasma transforms exogenous prostaglandin endoperoxide H2 into thromboxane A2 immediately prior to the initiation of irreversible aggregation. Selective thromboxane synthetase inhibitors block thromboxane A2 formation and aggregation. Thromboxane A2 formation appears to be essential during arachidonate mediated aggregation. The results presented reconcile the previously accepted paradoxical behavior of thromboxane synthetase in platelet rich plasma toward the prostaglandin endoperoxide H2 substrate.     

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.     

Selective use of the primary literature transforms the classroom into a virtual laboratory

CREATE (consider, read, elucidate hypotheses, analyze and interpret the data, and think of the next experiment) is a new method for teaching science and the nature of science through primary literature. CREATE uses a unique combination of novel pedagogical tools to guide undergraduates through analysis of journal articles, highlighting the evolution of scientific ideas by focusing on a module of four articles from the same laboratory. Students become fluent in the universal language of data analysis as they decipher the figures, interpret the findings, and propose and defend further experiments to test their own hypotheses about the system under study. At the end of the course students gain insight into the individual experiences of article authors by reading authors' responses to an e-mail questionnaire generated by CREATE students. Assessment data indicate that CREATE students gain in ability to read and critically analyze scientific data, as well as in their understanding of, and interest in, research and researchers. The CREATE approach demystifies the process of reading a scientific article and at the same time humanizes scientists. The positive response of students to this method suggests that it could make a significant contribution to retaining undergraduates as science majors.     

Deprotonated carboxylic group of amino acids transforms adenine into its rare prototropic tautomers

By UV spectroscopic data for anhydrous DMSO solutions and ab initio HF/6-31G** calculations in vacuum it was shown for the first time that deprotonated amino acid carboxylic group is able to change tautomeric state of a nucleotide base, exactly to convert the N9H ground-state prototropic tautomer of adenine into the N7H and N1H rare ones.     

Regionalisation of the mouse visceral endoderm as the blastocyst transforms into the egg cylinder

Background  

Reciprocal interactions between two extra-embryonic tissues, the extra-embryonic ectoderm and the visceral endoderm, and the pluripotent epiblast, are required for the establishment of anterior-posterior polarity in the mouse. After implantation, two visceral endoderm cell types can be distinguished, in the embryonic and extra-embryonic regions of the egg cylinder. In the embryonic region, the specification of the anterior visceral endoderm (AVE) is central to the process of anterior-posterior patterning. Despite recent advances in our understanding of the molecular interactions underlying the differentiation of the visceral endoderm, little is known about how cells colonise the three regions of the tissue.     

Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity.     

Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1?Np), which was previously found to be an active form in the ?snf2 mutant. Overexpression of Dga1?Np in the ?snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1?Np in lipid accumulation. We found that the overexpression of Dga1?Np in the ?dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1?Np to stimulate lipid accumulation, the ?dga1-1 mutant, in which the 3′-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ?dga1-2 mutant, in which the 3′-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1?Np in the ?dga1-1 mutant had a lower lipid content than the original ?dga1 mutant, whereas overexpression in the ?dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3′ terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1?Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1?Np in the ?dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1?Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ?dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1?Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.     

Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis

The best-characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes. The endonuclease responsible for DNA degradation in caspase-dependent apoptosis is caspase-activated DNase. In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult. Among the known effectors of caspase-independent cell death, L-DNase II (LEI [leukocyte elastase inhibitor]-derived DNase II) has been previously characterized by our laboratory. We have thus shown that this endonuclease derives from the serpin superfamily member LEI by posttranslational modification (A. Torriglia, P. Perani, J. Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, and M. F. Counis, Mol. Cell. Biol. 18:3612-3619, 1998). In this work, we assessed the molecular mechanism involved in the change in the enzymatic activity of this molecule from an antiprotease to an endonuclease. We report that the cleavage of LEI by elastase at its reactive center loop abolishes its antiprotease activity and leads to a conformational modification that exposes an endonuclease active site and a nuclear localization signal. This represents a novel molecular mechanism for a complete functional conversion induced by changing the conformation of a serpin. We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II are needed to induce cell death.     

The BCR-ABL oncogene transforms Rat-1 cells and cooperates with v-myc.

The tyrosine kinase P210 is the gene product of the rearranged BCR-ABL locus on the Philadelphia chromosome (Ph1), which is found in leukemic cells of patients with chronic myelogenous leukemia. It has a weakly oncogenic effect in immature murine hematopoietic cells and does not transform NIH 3T3 cells. We have found that P210 has a strikingly different effect in Rat-1 cells, another line of established rodent fibroblasts. Stable expression of P210 in Rat-1 cells caused a distinct morphological change and conferred both tumorigenicity and capacity for anchorage-independent growth. The introduction of v-myc into Rat-1 cells expressing P210 led to complete morphological transformation and enhanced tumorigenicity. No such interaction took place in NIH 3T3 cells. Thus, Rat-1 cells can be used to detect cooperation between BCR-ABL and other oncogenes and may prove useful for the identification of secondary oncogenic events in chronic myelogenous leukemia.