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Sample records for conditional lethal mutants

  1. A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice.

    PubMed

    Hidalgo, Alejandro A; Villagra, Nicolás A; Jerez, Sebastián A; Fuentes, Juan A; Mora, Guido C

    2016-02-01

    Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 10(6) bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 10(6) bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 10(4) oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

  2. Construction of a conditional lethal Salmonella mutant via genetic recombination using the ara system and asd gene.

    PubMed

    Kim, Sam Woong; Kang, Ho Young; Hur, Jin; Gal, Sang Wan; Bang, Woo Young; Cho, Kwang-Keun; Kim, Chul Wook; Bahk, Jeong Dong; Lee, John Hwa

    2011-11-01

    In order to construct a conditional lethal Salmonella mutant, an arabinose-regulated recombinant genetic system was used. The Salmonella aspartate semialdehyde dehydrogenase (asd) gene was localized under the control of araC P(araBAD) in a plasmid to create the araC P(araBAD)::asd cassette. The cassette was cloned into a plasmid carrying a p15A replication origin to create the recombinant plasmid pMMP55. The growth of Salmonella MMP10 harboring pMMP55 was dependent on the presence of arabinose. In the presence of arabinose, the Asd deficiency due to chromosomal deletion of asd in the Salmonella host was complemented by the asd gene transcribed and translated under the P(araBAD) promoter and araBAD Shine-Dalgarno (SD) sequence in pMMP55. Growth inhibition of the strain was demonstrated by arabinose depletion in M9 minimal medium, indicating that the strain were unable to grow in an arabinose-limited environment. In addition, the analysis of a 50% lethal dose (LD50) using mice revealed that the strain MMP10 exhibited attenuation by approximately 100-fold relative to that of the unmodified strain. In conclusion, these data suggest that the araC P(araBAD)::asd system developed in this study can be used to construct conditional lethal Salmonella mutants for application as safe, live-attenuated Salmonella vaccines.

  3. Conditional lethality of cell shape mutations of Salmonella typhimurium: rodA and mre mutants are lethal on solid but not in liquid medium.

    PubMed

    Costa, C S; Antón, D N

    1999-03-01

    Round-cell (rodA, mre, divD) derivatives of a conditional alaS mutant of Salmonella typhimurium were studied under conditions allowing expression of tolerance to lethal cell shape mutations (41 degrees C), and under nontolerant conditions (30 degrees C). The rodA22::Tn10d(Kan) derivative grew normally (OD650 nm) in LB-broth at 30 degrees C; however, doubling of total cell count took much longer (130 min) than at 41 degrees C (57 min). Although the cells were able to divide in LB-broth at 30 degrees C, viable count on LB-agar at 30 degrees C was 10(3)-fold lower than on LB-agar at 41 degrees C. Phase-contrast microscopy of rodA cells incubated under different conditions showed that their size increased on LB-soft agar at 30 degrees C, but they failed to divide and finally lysed. In contrast, division occurred in LB-broth at 30 degrees C and also in LB-broth and LB-soft agar at 41 degrees C. The mre-17::Tn10d(Kan) derivative acted like the rodA strain whereas the divD135::Tn10d(Kan) mutant behaved normally both at 30 degrees C and 41 degrees C. It is concluded that rodA and mre mutations delay cell division, but are lethal only on solid medium. Mutations conferring tolerance to "lethal" rodA and mre mutations improve division performance both in liquid and solid media.

  4. Conditional Lethal Mutants of Adenovirus 2-Simian Virus 40 Hybrids I. Host Range Mutants of Ad2+ND1

    PubMed Central

    Grodzicker, Terri; Anderson, Carl; Sharp, Phillip A.; Sambrook, Joe

    1974-01-01

    Human adenovirus type 2 (Ad2) grows poorly in monkey cells, although this defect can be overcome by co-infection with simian virus 40 (SV40). The nondefective Ad2-SV40 hybrid virus, Ad2+ND1, replicates efficiently in both human and African green monkey kidney cells, presumably due to the insertion of SV40 sequences into the Ad2 DNA. Several mutants of Ad2+ND1 have been isolated that grow and plaque poorly in monkey cells, although they retain the ability to replicate and plaque efficiently in HeLa cells. One of these mutants (H39) has been examined in detail. Studies comparing the DNA of the mutant with Ad2+ND1 either by the cleavage patterns produced by Escherichia coli R·RI restriction endonuclease digestion or by heteroduplexing reveal no differences. The pattern of protein synthesis of Ad2+ND1 and H39 in monkey cells is quite different with the mutant resembling Ad2, which is defective in the synthesis of late proteins. However, in human cells, the proteins synthesized by H39 and the parent Ad2+ND1 are very similar. The production of SV40 U antigen in H39-infected cells is different from that in Ad2+ND1-infected cells. Finally, the growth of H39 in monkey cells can be complemented by SV40. Images PMID:4364898

  5. [Synthetic lethal genes to mutant p53].

    PubMed

    Tongyang, Liu; Haiqiang, Guo; Meiyan, Zhu; Yingze, Huang; Shuting, Jia; Ying, Luo; Jihong, Zhang

    2015-04-01

    Targeted therapy has become a powerful approach for cancer treatment. Better understanding of oncogenes as well as synthetic lethal interactions with oncogenes will lead to new strategies for tumor-specific treatment. It is well known that mutant p53 plays an important role in tumorigenesis and tumor development. Thus, understanding the synthetic lethal relationship between p53 mutations and interacting genes in tumor is critical for the personalized treatments of p53 mutant tumors. Synthetic lethal genes to mutant p53 can be divided into cell cycle regulators and non-cell cycle regulators. This paper review show these two types of target genes contribute to synthetic lethal interactions with p53 mutations and potential applications of these interactions in anticancer therapy.

  6. Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases*

    PubMed Central

    Boitz, Jan M.; Ullman, Buddy

    2010-01-01

    Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice. PMID:20363738

  7. Lethal infection by Bordetella pertussis mutants in the infant mouse model.

    PubMed Central

    Weiss, A A; Goodwin, M S

    1989-01-01

    Different aspects of lethal infection of infant mice with Bordetella pertussis were examined. Mutants deficient in vir-regulated genes were tested for the ability to cause a lethal infection in the infant mouse model. Adenylate cyclase toxin-hemolysin and pertussis toxin were required to cause a lethal infection at low doses. Mixed infection caused by challenging the mice with an equal number of pertussis toxin and adenylate cyclase toxin-hemolysin mutants at a dose at which neither alone was lethal was also unable to cause a lethal infection. Production of the filamentous hemagglutinin and the dermonecrotic toxin was not required to cause a lethal infection. Nine other mutants in vir-regulated genes whose phenotypes have yet to be determined were also tested. Only two of these mutants were impaired in the ability to cause a lethal infection. Expression of fimbriae does not appear to affect the dose required to cause a lethal infection; however, fimbrial expression was correlated with the later stages of a nonlethal, persistent infection. Growth of the bacteria in MgSO4, a condition which reversibly suppresses expression of the genes required for virulence, did not alter the ability of the bacteria to cause a lethal infection. Auxotrophic mutants deficient in leucine biosynthesis were as virulent as the parental strain; however, mutants deficient in methionine biosynthesis were less virulent. A B. parapertussis strain was much less effective in promoting a lethal infection than any of the wild-type B. pertussis strains examined. A persistent infection in the lungs was observed for weeks after challenge for mice given a sublethal dose of B. pertussis, and transmission from infected infants to the mother was never observed. PMID:2572561

  8. Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants

    PubMed Central

    Liu, Yuanli; Zhou, Jinan; Qu, Yilin; Yang, Xinguang; Shi, Guojing; Wang, Xiuhong; Hong, Yuzhi; Drlica, Karl; Zhao, Xilin

    2016-01-01

    Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability. PMID:27045517

  9. Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

    PubMed Central

    Ohya, Y.; Botstein, D.

    1994-01-01

    Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

  10. Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus.

    PubMed

    Zhang, Y; Keck, J G; Moss, B

    1992-11-01

    There are three temporal classes of vaccinia virus genes: early, intermediate, and late. The object of this study was to determine the effects on virus replication of regulating the expression of G8R, an intermediate gene that encodes a late transcription factor. We inserted the lac operator adjacent to the RNA start site of the G8R gene in a recombinant vaccinia virus that constitutively expresses the Escherichia coli lac repressor to make expression of the G8R gene dependent on the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). In case repression would not be complete, we also weakened the promoter of the G8R gene by making a single-nucleotide substitution designed to reduce its basal level of transcription. The mutant virus replicated well in the presence of the inducer, although synthesis of the G8R-encoded 30,000-M(r) protein was only 10% of that of the wild-type virus. In the absence of IPTG, (i) synthesis of the G8R protein was inhibited by more than 99% relative to that of the wild-type virus, (ii) synthesis of early and intermediate mRNAs appeared to be unaffected, (iii) intermediate proteins accumulated to higher than normal levels, (iv) synthesis of late mRNA and protein was reduced by about 90%, (v) viral DNA was replicated but incompletely resolved concatemeric molecules accumulated, (vi) not even the earliest stages of virion assembly were detectable by transmission electron microscopy, and (vii) virus yield under one-step growth conditions and plaque formation were 10(-3) and 10(-4) times the wild-type values, respectively. The defect in late gene expression could be overcome by transfection of a G8R gene that was not under lac operator control, as well as by addition of IPTG, further demonstrating the specificity of the repression. The correlation between decreased expression of the G8R intermediate gene and inhibition of late mRNA synthesis is consistent with the notion that the G8R product serves as an essential late transcription factor

  11. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

    PubMed Central

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  12. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

    PubMed

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  13. Conditional lethality strains for the biological control of Anastrepha species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pro-apoptotic cell death genes are promising candidates for biologically-based autocidal control of pest insects as demonstrated by tetracycline (tet)-suppressible systems for conditional embryonic lethality in Drosophila melanogaster (Dm) and the medfly, Ceratitis capitata (Cc). However, for medfly...

  14. Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

    PubMed Central

    Elefant, Felice; Palter, Karen B.

    1999-01-01

    The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins’ dominant negative effects in vivo. PMID:10397752

  15. Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

    PubMed

    Miller, Rowan E; Brough, Rachel; Bajrami, Ilirjana; Williamson, Chris T; McDade, Simon; Campbell, James; Kigozi, Asha; Rafiq, Rumana; Pemberton, Helen; Natrajan, Rachel; Joel, Josephine; Astley, Holly; Mahoney, Claire; Moore, Jonathan D; Torrance, Chris; Gordan, John D; Webber, James T; Levin, Rebecca S; Shokat, Kevan M; Bandyopadhyay, Sourav; Lord, Christopher J; Ashworth, Alan

    2016-07-01

    New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

  16. Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene.

    PubMed

    Stacey, A; Bateman, J; Choi, T; Mascara, T; Cole, W; Jaenisch, R

    1988-03-10

    Substitutions of single glycine residues of alpha 1(I) collagen have previously been associated with the inherited disease osteogenesis imperfecta type II. Transgenic mice bearing a mutant alpha 1(I) collagen gene into which specific glycine substitutions have been engineered show a dominant lethal phenotype characteristic of the human disease, and demonstrate that as little as 10% mutant gene expression can disrupt normal collagen function.

  17. Developmental studies on an apparent cell-lethal mutant gene-ut-in the Mexican axolotl, Ambystoma mexicanum.

    PubMed

    Humphrey, R R; Malacinski, G M; Chung, H M

    1978-04-01

    The discovery of a new mutant gene in stocks of the Mexican axolotl derived from breeding stock of the Hubrecht Laboratory, the Netherlands, is described. The gene appears to be a simple recessive and displays complete penetrance. ut/ut larvae develop normally through hatching, but begin to lag in growth and display characteristics defects in gill and limb formation shortly thereafter. The results of parabiosis of normal and mutant embryos, as well as embryological transplants of mutant limb and branchial rudiments, support the conclusion that the gene ut is expressed as an 'autonomous-cell lethal'. Despite gross morphological defects in ut/ut larvae, comparisons between normal and mutant animals of the protein spectra of various tissues and organs revealed no substantial differences. A subtle change in the metabolism of ut/ut larvae apparently, therefore, leads to developmental arrest.

  18. A Coccidioides posadasii CPS1 Deletion Mutant Is Avirulent and Protects Mice from Lethal Infection.

    PubMed

    Narra, Hema P; Shubitz, Lisa F; Mandel, M Alejandra; Trinh, Hien T; Griffin, Kurt; Buntzman, Adam S; Frelinger, Jeffrey A; Galgiani, John N; Orbach, Marc J

    2016-10-01

    The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals. PMID:27481239

  19. Conditional Lethal Amber Mutations in Essential Escherichia coli Genes†

    PubMed Central

    Herring, Christopher D.; Blattner, Frederick R.

    2004-01-01

    The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as “tagalong” mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development. PMID:15090508

  20. Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

    PubMed Central

    White, W. H.; Johnson, D. I.

    1997-01-01

    Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance. PMID:9286667

  1. The Structural Variation Is Associated with the Embryonic Lethality of a Novel Red Egg Mutant Fuyin-lre of Silkworm, Bombyx mori

    PubMed Central

    Li, Qiongyan; Zhao, Qiaoling; Yang, Weike; Zhu, Shuifen; Wu, Fang; He, Rongfan; Dong, Zhanpeng; Huang, Ping

    2015-01-01

    Bombyx mori presents several types of egg color mutations, all of which have been extensively discussed in sericulture. While the red egg mutation has been previously observed, lethal red-egg mutants have not been reported. In the present work, the red egg mutant Fuyin-lre (Fuyin-lethal red egg) was discovered from the Fuyin germplasm resource of B. mori. This mutant features red-colored eggs and embryonic lethality. Genetic analysis showed that Fuyin-lre follows recessive inheritance, with the red egg gene re governing the egg color, and the embryonic lethality of Fuyin-lre may be caused by mutations of other genes closely linked to re. Digital gene expression (DGE) was employed to compare the transcription profiles of Fuyin and Fuyin-lre eggs after 24 and 48 h of incubation. A total of 48 differentially expressed genes followed the same expression patterns in both groups at both time points (FDR < 0.01 and log 2 Ratio ≥ 1). Further analyses indicated that 8 out of the 48 genes (including re) were closely linked to re. These 8 genes were highly expressed in wild-type Fuyin and the red egg mutant re but showed nearly absent expression in Fuyin-lre. Sequencing of the re gene confirmed that the re gene itself does not induce embryonic lethality, and structure analysis showed that the structural variation of the region where the 8 genes were located may be associated with the embryonic lethality of Fuyin-lre. The present work provides a good foundation for future studies on the mechanism of embryonic lethality and embryonic development in Fuyin-lre. PMID:26030868

  2. The Structural Variation Is Associated with the Embryonic Lethality of a Novel Red Egg Mutant Fuyin-lre of Silkworm, Bombyx mori.

    PubMed

    Chen, Anli; Liao, Pengfei; Li, Qiongyan; Zhao, Qiaoling; Yang, Weike; Zhu, Shuifen; Wu, Fang; He, Rongfan; Dong, Zhanpeng; Huang, Ping

    2015-01-01

    Bombyx mori presents several types of egg color mutations, all of which have been extensively discussed in sericulture. While the red egg mutation has been previously observed, lethal red-egg mutants have not been reported. In the present work, the red egg mutant Fuyin-lre (Fuyin-lethal red egg) was discovered from the Fuyin germplasm resource of B. mori. This mutant features red-colored eggs and embryonic lethality. Genetic analysis showed that Fuyin-lre follows recessive inheritance, with the red egg gene re governing the egg color, and the embryonic lethality of Fuyin-lre may be caused by mutations of other genes closely linked to re. Digital gene expression (DGE) was employed to compare the transcription profiles of Fuyin and Fuyin-lre eggs after 24 and 48 h of incubation. A total of 48 differentially expressed genes followed the same expression patterns in both groups at both time points (FDR < 0.01 and log 2 Ratio ≥ 1). Further analyses indicated that 8 out of the 48 genes (including re) were closely linked to re. These 8 genes were highly expressed in wild-type Fuyin and the red egg mutant re but showed nearly absent expression in Fuyin-lre. Sequencing of the re gene confirmed that the re gene itself does not induce embryonic lethality, and structure analysis showed that the structural variation of the region where the 8 genes were located may be associated with the embryonic lethality of Fuyin-lre. The present work provides a good foundation for future studies on the mechanism of embryonic lethality and embryonic development in Fuyin-lre.

  3. Lethality in PARP-1/Ku80 double mutant mice reveals physiologicalsynergy during early embryogenesis

    SciTech Connect

    Henrie, Melinda S.; Kurimasa, Akihiro; Burma, Sandeep; Menissier-de Murcia, Josiane; de Murcia, Gilbert; Li, Gloria C.; Chen,David J.

    2002-09-24

    Ku is an abundant heterodimeric nuclear protein, consisting of 70-kDa and 86-kDa tightly associated subunits that comprise the DNA binding component of DNA-dependent protein kinase. Poly(ADP)ribose polymerase-1 (PARP-1) is a 113-kDa protein that catalyzes the synthesis of poly(ADP-ribose) on target proteins. Both Ku and PARP-1 recognize and bind to DNA ends. Ku functions in the non-homologous end joining (NHEJ) repair pathway whereas PARP-1 functions in the single strand break repair and base excision repair (BER) pathways. Recent studies have revealed that PARP-1 and Ku80 interact in vitro. To determine whether the association of PARP-1 and Ku80 has any physiological significance or synergistic function in vivo, mice lacking both PARP-1 and Ku80 were generated. The resulting offspring died during embryonic development displaying abnormalities around the gastrulation stage. In addition, PARP-1-/-Ku80-/- cultured blastocysts had an increased level of apoptosis. These data suggest that the functions of both Ku80 and PARP-1 are essential for normal embryogenesis and that a loss of genomic integrity leading to cell death through apoptosis is likely the cause of the embryonic lethality observed in these mice.

  4. Lethal Accumulation of Guanylic Nucleotides in Saccharomyces cerevisiae HPT1-Deregulated Mutants

    PubMed Central

    Breton, Annick; Pinson, Benoît; Coulpier, Fanny; Giraud, Marie-France; Dautant, Alain; Daignan-Fornier, Bertrand

    2008-01-01

    Guanylic nucleotide biosynthesis is a conserved and highly regulated process. Drugs reducing GMP synthesis affect the immunological response and mutations enabling guanylic-derivative recycling lead to severe mental retardation. While the effects of decreased GMP synthesis have been well documented, the consequences of GMP overproduction in eukaryotes are poorly understood. In this work, we selected and characterized several mutations making yeast hypoxanthine–guanine phosphoribosyltransferase insensitive to feedback inhibition by GMP. In these mutants, accumulation of guanylic nucleotides can be triggered by addition of extracellular guanine. We show that such an accumulation is highly toxic for yeast cells and results in arrest of proliferation and massive cell death. This growth defect could be partially suppressed by overexpression of Rfx1p, a transcriptional repressor of the DNA damage response pathway. Importantly, neither guanylic nucleotide toxicity nor its suppression by Rfx1p was associated with an alteration of forward mutation frequency. PMID:18245832

  5. Strategy for enhanced transgenic strain development for embryonic conditional lethality in Anastrepha suspensa.

    PubMed

    Schetelig, Marc F; Handler, Alfred M

    2012-06-12

    Here the first reproductive sterility system for the tephritid fruit fly pest, Anastrepha suspensa, is presented, based on lethality primarily limited to embryos heterozygous for a conditional lethal transgene combination. This tetracycline (Tet)-suppressible system uses a driver construct having the promoter from the newly isolated embryo-specific A. suspensa serendipity α gene linked to the Tet-transactivator. This was used to drive expression of a phosphomutated variant of the pro-apoptotic cell death gene, hid, from A. ludens, that was isolated, based on its identity to A. suspensa hid. The Alhid(Ala2) variant was shown to have the highest cell death activity in an in vitro A. suspensa cell death assay compared to the orthologous genes Ashid, Dmhid, and the variant Dmhid(Ala5). These cell death assays also allowed a determination of the most-efficient driver-effector cassette combinations for use in A. suspensa transformants, resulting in two hybrid strains exhibiting 100% lethality. One strain was 96% lethal in embryos in the absence of tetracycline, with none surviving past the first larval instar, which is critical for pests that are most damaging in late-larval stages. We demonstrate that the isolation and in vitro validation of species-specific promoters and lethal effector genes can greatly improve the efficiency of creating high-performance conditional lethality strains that may be extended to other insect pest species. PMID:22647610

  6. Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

    SciTech Connect

    Grider, A. Jr.

    1986-01-01

    The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the /sup 109/Cd-saturation/hemolysate method, and by the /sup 65/Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the /sup 65/Zn-MT binding assay (3-fold) and by the /sup 109/Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of /sup 65/Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age.

  7. Pathogenesis of Lethal Cardiac Arrhythmias in Mecp2 Mutant Mice: Implication for Therapy in Rett Syndrome

    PubMed Central

    McCauley, Mark D.; Wang, Tiannan; Mike, Elise; Herrera, Jose; Beavers, David L.; Huang, Teng-Wei; Ward, Christopher S.; Skinner, Steven; Percy, Alan K.; Glaze, Daniel G.; Wehrens, Xander H. T.; Neul, Jeffrey L.

    2013-01-01

    Rett Syndrome is a neurodevelopmental disorder typically caused by mutations in Methyl-CpG-Binding Protein 2 (MECP2) in which 26% of deaths are sudden and of unknown cause. To explore the hypothesis that these deaths may be due to cardiac dysfunction, we characterized the electrocardiograms (ECGs) in 379 people with Rett syndrome and found that 18.5% show prolongation of the corrected QT interval (QTc), indicating a repolarization abnormality that can predispose to the development of an unstable fatal cardiac rhythm. Male mice lacking MeCP2 function, Mecp2Null/Y, also have prolonged QTc and show increased susceptibility to induced ventricular tachycardia. Female heterozygous null mice, Mecp2Null/+, show an age-dependent prolongation of QTc associated with ventricular tachycardia and cardiac-related death. Genetic deletion of MeCP2 function in only the nervous system was sufficient to cause long QTc and ventricular tachycardia, implicating neuronally-mediated changes to cardiac electrical conduction as a potential cause of ventricular tachycardia in Rett syndrome. The standard therapy for prolonged QTc in Rett syndrome, β-adrenergic receptor blockers, did not prevent ventricular tachycardia in Mecp2Null/Y mice. To determine whether an alternative therapy would be more appropriate, we characterized cardiomyocytes from Mecp2Null/Y mice and found increased persistent sodium current, which was normalized when cells were treated with the sodium channel-blocking anti-seizure drug phenytoin. Treatment with phenytoin reduced both QTc and sustained ventricular tachycardia in Mecp2Null/Y mice. These results demonstrate that cardiac abnormalities in Rett syndrome are secondary to abnormal nervous system control, which leads to increased persistent sodium current. Our findings suggest that treatment in people with Rett syndrome would be more effective if it targeted the increased persistent sodium current in order to prevent lethal cardiac arrhythmias. PMID:22174313

  8. Isolation of New Gravitropic Mutants under Hypergravity Conditions

    PubMed Central

    Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

    2016-01-01

    Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791

  9. Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

    PubMed

    Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

    2016-09-12

    Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. PMID:27498563

  10. A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

    PubMed Central

    van der Geize, R.; de Jong, W.; Hessels, G. I.; Grommen, A. W. F.; Jacobs, A. A. C.; Dijkhuizen, L.

    2008-01-01

    A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive. PMID:18984616

  11. When a Fly Has to Fly to Reproduce: Selection against Conditional Recessive Lethals in "Drosophila"

    ERIC Educational Resources Information Center

    Plunkett, Andrea D.; Yampolsky, Lev Y.

    2010-01-01

    We propose an experimental model suitable for demonstrating allele frequency change in Drosophila melanogaster populations caused by selection against an easily scorable conditional lethal, namely recessive flightless alleles such as apterous and vestigial. Homozygotes for these alleles are excluded from reproduction because the food source used…

  12. sigE facilitates the adaptation of Bordetella bronchiseptica to stress conditions and lethal infection in immunocompromised mice

    PubMed Central

    2012-01-01

    Background The cell envelope of a bacterial pathogen can be damaged by harsh conditions in the environment outside a host and by immune factors during infection. Cell envelope stress responses preserve the integrity of this essential compartment and are often required for virulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have been described in these species. Among the putative Bordetella transcription factors are a number of genes belonging to the extracytoplasmic function (ECF) group of alternative sigma factors, some of which are known to mediate cell envelope stress responses in other bacteria. Here we investigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetella bronchiseptica. Results We demonstrate that sigE encodes a functional sigma factor that mediates a cell envelope stress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive to high temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain β-lactam antibiotics. Using a series of immunocompromised mice deficient in different components of the innate and adaptive immune responses, we show that SigE plays an important role in evading the innate immune response during lethal infections of mice lacking B cells and T cells. SigE is not required, however, for colonization of the respiratory tract of immunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed by peripheral blood polymorphonuclear leukocytes (PMNs) than RB50, and exhibits decreased cytotoxicity toward macrophages. These altered interactions with phagocytes could contribute to the defects observed during lethal infection. Conclusions Much of the work on transcriptional regulation during infection in B. bronchiseptica has focused on the BvgAS two-component system. This study reveals that the SigE regulon also mediates a

  13. Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

    PubMed Central

    Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

    2014-01-01

    The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

  14. Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems.

    PubMed

    Handler, Alfred M

    2016-04-01

    The genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably the Sterile Insect Technique (SIT), for both plant and animal insect pests. Tetracycline-suppressible (Tet-off) conditional lethal systems may function together so that transgenic strains will be viable and fertile on a tetracycline-containing diet, but female-lethal and male sterile in tetracycline-free conditions. This would allow their most efficacious use in a unified system for sterile male-only production for SIT. A critical consideration for the field release of such transgenic insect strains, however, is a determination of the frequency and genetic basis of lethality revertant survival. This will provide knowledge essential to evaluating the genetic stability of the lethality system, its environmental safety, and provide the basis for modifications ensuring optimal efficacy. For Tet-off lethal survival determinations, development of large-scale screening protocols should also allow the testing of these modifications, and test the ability of other conditional lethal systems to fully suppress propagation of rare Tet-off survivors. If a dominant temperature sensitive (DTS) pupal lethality system proves efficient for secondary lethality in Drosophila, it may provide the safeguard needed to support the release of sexing/sterility strains, and potentially, the release of unisex lethality strains as a form of genetic male sterility. Should the DTS Prosβ2(1) mutation prove effective for redundant lethality, its high level of structural and functional conservation should allow host-specific cognates to be created for a wide range of insect species.

  15. Study of radiosensitive Drosophila lines. XI. Induction of recessive sex-linked lethal mutations in females of the mutant line rad(2)201/sup G1/

    SciTech Connect

    Varentsova, E.R.

    1986-05-01

    The authors have studied the frequency of occurrence of recessive sex-linked lethal mutations (RSLLM) after treatment of the females with ..gamma..-rays as a function of the dose (from 5 to 20 Gy) and oogenesis stage. They have shown that within the dose range used the oocytes of the 14th and 7th development stage are more sensitive in females of the mutant line than in those of the control. They detected significant differences in the frequency of occurrence of RSLLM between the 14th and 7th stages of development of oocytes for both Drosophila lines investigated.

  16. Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum.

    PubMed

    Lemanski, L F; Fitzharris, T P

    1989-05-01

    Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.

  17. We want what’s best for our baby: Prenatal Parenting of Babies with Lethal Conditions

    PubMed Central

    Côté-Arsenault, Denise; Krowchuk, Heidi; Hall, Wendasha Jenkins; Denney-Koelsch, Erin

    2015-01-01

    This article reports on qualitative research into the experience of couples who chose to continue their pregnancies after receiving a lethal fetal diagnosis, and to embrace the parenting of their baby in the shortened time they have. This analysis of interview data is part of a larger research project describing parents’ experiences of continuing pregnancy with a known lethal fetal diagnosis (LFD). PMID:26594107

  18. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences

    SciTech Connect

    Cai, Yuping; Wolk, C.P. )

    1990-06-01

    Use of the sacB gene provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, they mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.

  19. Conditional embryonic lethality to improve the sterile insect technique in Ceratitis capitata (Diptera: Tephritidae)

    PubMed Central

    Schetelig, Marc F; Caceres, Carlos; Zacharopoulou, Antigone; Franz, Gerald; Wimmer, Ernst A

    2009-01-01

    Background The sterile insect technique (SIT) is an environment-friendly method used in area-wide pest management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann; Diptera: Tephritidae). Ionizing radiation used to generate reproductive sterility in the mass-reared populations before release leads to reduction of competitiveness. Results Here, we present a first alternative reproductive sterility system for medfly based on transgenic embryonic lethality. This system is dependent on newly isolated medfly promoter/enhancer elements of cellularization-specifically-expressed genes. These elements act differently in expression strength and their ability to drive lethal effector gene activation. Moreover, position effects strongly influence the efficiency of the system. Out of 60 combinations of driver and effector construct integrations, several lines resulted in larval and pupal lethality with one line showing complete embryonic lethality. This line was highly competitive to wildtype medfly in laboratory and field cage tests. Conclusion The high competitiveness of the transgenic lines and the achieved 100% embryonic lethality causing reproductive sterility without the need of irradiation can improve the efficacy of operational medfly SIT programs. PMID:19173707

  20. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes. PMID:9007229

  1. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice

    PubMed Central

    Puig, Berta; Altmeppen, Hermann C.; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y.; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrPC) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrPSc leading to prion diseases. We show that a deletion in the C-terminal domain of PrPC (PrPΔ214–229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrPC. Transgenic (Tg(PrPΔ214–229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214–229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  2. Producing Conditional Mutants for Studying Plant Microtubule Function

    SciTech Connect

    Richard Cyr

    2009-09-29

    The cytoskeleton, and in particular its microtubule component, participates in several processes that directly affect growth and development in higher plants. Normal cytoskeletal function requires the precise and orderly arrangement of microtubules into several cell cycle and developmentally specific arrays. One of these, the cortical array, is notable for its role in directing the deposition of cellulose (the most prominent polymer in the biosphere). An understanding of how these arrays form, and the molecular interactions that contribute to their function, is incomplete. To gain a better understanding of how microtubules work, we have been working to characterize mutants in critical cytoskeletal genes. This characterization is being carried out at the subcellular level using vital microtubule gene constructs. In the last year of funding colleagues have discovered that gamma-tubulin complexes form along the lengths of cortical microtubules where they act to spawn new microtubules at a characteristic 40 deg angle. This finding complements nicely the finding from our lab (which was funded by the DOE) showing that microtubule encounters are angle dependent; high angles encounters results in catastrophic collisions while low angle encounters result in favorable zippering. The finding of a 40 deg spawn of new microtubules from extant microtubule, together with aforementioned rules of encounters, insures favorable co-alignment in the array. I was invited to write a New and Views essay on this topic and a PDF is attached (News and Views policy does not permit funding acknowledgments and so I was not allowed to acknowledge support from the DOE).

  3. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

    PubMed Central

    Nonet, M; Scafe, C; Sexton, J; Young, R

    1987-01-01

    We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis. Images PMID:3299050

  4. Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.

    PubMed

    Rojas-Rejón, Oscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Martínez-Jiménez, Alfredo; Cristiani-Urbina, Eliseo; de la Torre Martínez, Mayra; Ponce-Noyola, Teresa

    2011-01-01

    Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

  5. Lethal body residues for pentachlorophenol in zebra mussels (Dreissena polymorpha) under varying conditions of temperature and pH.

    PubMed

    Fisher, S W; Hwang, H; Atanasoff, M; Landrum, P F

    1999-07-01

    Pentachlorophenol (PCP) toxicity was measured in the zebra mussel under varying conditions of pH (6.5, 7.5, or 8.5) and temperature (10, 17, or 25 degrees C). Toxicity decreased significantly with increasing pH at all temperatures. At a given pH level, toxicity increased significantly with increasing temperature. PCP was most toxic at pH 6.5, 25 degrees C and least toxic at pH 8.5, 10 degrees C. Toxicokinetic parameters were determined at trace PCP concentrations under each combination of pH and temperature. Increasing temperature generally increased the PCP uptake clearance (ku) although elimination rate constants (kd) were unaffected. The effect of pH on toxicokinetic parameters was inconsistent but ku tended to decrease as pH and ionization of PCP increased. Lethal body residues (LR50s), estimated from kinetic parameters determined at trace PCP concentrations and the LC50 values, varied by a factor of 122 as a function of environmental conditions while LC50s varied by a factor of 381. LR50s were also estimated from the measured PCP tissue concentrations and varied by a factor of 8 across conditions. Calculated LR50s were always higher than measured LR50s, determined under identical conditions, by at least a factor of five. However, when LR50 values were recalculated using ku values measured at the LC25 concentration, the resulting adjusted LR50s varied only by a factor of 2.5 across the range of conditions studied and were more consistent with measured LR50 values. Thus, variance in the PCP concentration required to produce toxicity is reduced when LR50s are used in place of LC50s. Further, the method by which lethal residues (LR50 values) are determined can significantly affect the results and their interpretation.

  6. Characterization of the Two Maize Embryo-Lethal Defective Kernel Mutants Rgh*-1210 and Fl*-1253b: Effects on Embryo and Gametophyte Development

    PubMed Central

    Clark, J. K.; Sheridan, W. F.

    1988-01-01

    We have examined the effects on embryonic and gametophytic development of two nonallelic defective-kernel mutants of maize. Earlier studies indicated that both mutants are abnormal in embryonic morphogenesis as well as in the formation of their endosperm. Mutant rgh*-1210 embryos depart from the normal embryogenic pathway at the proembryo and transition stage, by developing meristematic lobes and losing bilateral symmetry. They continue growth as irregular cell masses that enlarge and become necrotic. Somatic embryos arising in rgh*-1210 callus cultures display the rgh*-1210 mutant phenotype. Mutant fl*-1253B embryos are variably blocked from the coleoptilar stage through stage 2. Following formation of the shoot apex in the mutant embryos the leaf primordia and tissues surrounding the embryonic axis continue growth and cell division, while the scutellum ceases development and becomes hypertrophied. Mutant fl*-1253B embryos are unable to germinate, either in mutant kernels or as immature embryos in culture, and the mutant scutellar tissue does not produce regenerable callus. Expression of the fl*-1253B locus during male gametophytic development is revealed by a marked reduction in pollen transmission as a result of mutant expression during the interval between meiosis and the initiation of pollen tube growth. In both mutants, there is considerable proliferation of the aleurone cells of the endosperm. Mutant expression of rgh*-1210 in the female gametophyte is revealed by the abnormal antipodal cells of the embryo sac. These results show that these two gene loci play unique and crucial roles in normal morphogenesis of the embryo. In addition, it is evident that both mutants are pleiotropic in affecting the development of the endosperm and gametophyte as well as the embryo. These pleiotropisms suggest some commonality in the gene regulation of development in these three tissues. PMID:17246478

  7. Studies on radiosensitive lines of Drosophila. IX. Analysis of fertility and frequency of dominant lethal mutations in the gamma-irradiated females of the mutant line rad(2)201/sup G1/

    SciTech Connect

    Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.M.

    1986-03-01

    Fertility and frequency of dominant lethal mutations (DLM) induced by gamma rays in females at the age of 0-5 h and 5-7 days were studied in the radiosensitive mutant rad(2)201/sup G1/ of Drosophila. It has been found that the oocytes of mutant lines are more radiosensitive as compared to those of the wild type flies when compared on the basis of DLM frequency obtained through the entire maturation period. The early oocytes of stages 2-7, i.e., at the stages corresponding to the recombination-defective properties of mutation rad(2)201/sup g1/ are the most sensitive. It has also been demonstrated that the gamma-ray doses exceeding 10 Gy cause a strong sterilizing effect in the mutant females as a result of destruction and resorption of the egg chamber, irradiated at the stages of previtellogenic growth of oocytes. In the radiosensitive mutant females, the sensitivity of the oocytes for DLM induction does not correlate with the sensitivity of the ovarian follicles toward the resorbing effect of gamma rays. The possible involvement of the mutant locus in the genetic processes in different specialized cells of the sexual pathway in Drosophila is discussed.

  8. A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

    PubMed

    Mbikay, Majambu; Croissandeau, Gilles; Sirois, Francine; Anini, Younes; Mayne, Janice; Seidah, Nabil G; Chrétien, Michel

    2007-06-15

    Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele. PMID:17490633

  9. Swimming Behaviour and Otolith Characteristics of wildtype and mutant Zebrafish (AIE) under diminished Gravity Conditions

    NASA Astrophysics Data System (ADS)

    Weigele, J.; Anken, R.; Hilbig, R.

    During microgravity humans often suffer from sensorimotor disorders e g motion sickness a kinetosis Using fish as vertebrate model systems we could previously provide ample evidence that the individually different susceptibility to such disorders is based on an individually differently pronounced asymmetric mineralisation calcification of inner ear stones otoliths In the course of a preliminary study we subjected mutant zebrafish Danio rerio due to malformation of the inner ear - see below - this mutant was termed Asymmetric Inner Ear AIE to diminished gravity conditions during parabolic aircraft flight PF As compared to wildtype WT animals the mutants showed a pronounced kinetotic behaviour The gross-morphology of the inner ears of AIE and WT animals strikingly differed In WT specimens the saccular otoliths were located at the periphery of the inner ear whereas the utricular stones were positioned mediad as it is usually the case in teleosts in most AIE animals dissected however the respective otoliths were positioned in an opposite arrangement Moreover the mutants sported transparent otoliths whereas the otoliths of WT specimens had an opaque appearance This finding clearly indicates that mutant otoliths differed from wildtype ones in their lattice structure i e the calcium carbonate polymorph and thus the compostion of the proteinacious matrix which is a template for calcium carbonate deposition In the course of the present study the PF experiment is scheduled to be carried out in March 2006 we intend to statistically verify

  10. Physiological and genetic analysis of Arabidopsis thaliana anthocyanin biosynthesis mutants under chronic adverse environmental conditions.

    PubMed

    Misyura, Maksym; Colasanti, Joseph; Rothstein, Steven J

    2013-01-01

    Anthocyanin production is a characteristic response of flowering plants to unfavourable environmental conditions. The potential roles of flavonoids and anthocyanins in plant growth were investigated by growing Arabidopsis thaliana anthocyanin production mutants (transparent testa) under limiting nitrogen and high light conditions. Inability to produce kaempferol or subsequent intermediate compounds by some transparent testa lines was correlated with less biomass accumulation in mature plants compared with wild-type control plants under all growth conditions tested. However, under both limiting nitrogen and high light chronic stress conditions, mutant lines defective in later steps of the anthocyanin production pathway produced the same or more biomass than wild-type plants. No difference in senescence between transparent testa and wild-type plants was found using chlorophyll catabolism and SAG12 expression measurements, and no mutants were impaired in the ability to remobilize nutrients from the vegetative to reproductive tissues. Moreover, the absence of anthocyanin and/or upstream flavonoids does not affect the ability of plants to respond to limiting nitrogen by reducing photosynthetic capacity. These results support a role for kaempferol and quercetin accumulation in normal plant growth and development. Further, the absence of anthocyanins has no effect on plant growth under the chronic stress conditions tested.

  11. Physiological and genetic analysis of Arabidopsis thaliana anthocyanin biosynthesis mutants under chronic adverse environmental conditions

    PubMed Central

    Rothstein, Steven J.

    2013-01-01

    Anthocyanin production is a characteristic response of flowering plants to unfavourable environmental conditions. The potential roles of flavonoids and anthocyanins in plant growth were investigated by growing Arabidopsis thaliana anthocyanin production mutants (transparent testa) under limiting nitrogen and high light conditions. Inability to produce kaempferol or subsequent intermediate compounds by some transparent testa lines was correlated with less biomass accumulation in mature plants compared with wild-type control plants under all growth conditions tested. However, under both limiting nitrogen and high light chronic stress conditions, mutant lines defective in later steps of the anthocyanin production pathway produced the same or more biomass than wild-type plants. No difference in senescence between transparent testa and wild-type plants was found using chlorophyll catabolism and SAG12 expression measurements, and no mutants were impaired in the ability to remobilize nutrients from the vegetative to reproductive tissues. Moreover, the absence of anthocyanin and/or upstream flavonoids does not affect the ability of plants to respond to limiting nitrogen by reducing photosynthetic capacity. These results support a role for kaempferol and quercetin accumulation in normal plant growth and development. Further, the absence of anthocyanins has no effect on plant growth under the chronic stress conditions tested. PMID:23162120

  12. Thiamine deficiency in self-induced refeeding syndrome, an undetected and potentially lethal condition.

    PubMed

    Hershkowitz, Einat; Reshef, Alon; Munich, Olga; Yosefi, Bracha; Markel, Arie

    2014-01-01

    Rapid restoration of nutrients and electrolytes after prolonged starvation could result in a life threatening condition characterized by sensory and neurological dysfunction and severe metabolic imbalance that has been designated as refeeding syndrome. Its diagnosis is frequently missed resulting in severe complications and even death. We describe a 25-years-old female patient with mental disorders and severe malnutrition who developed severe clinical manifestations and biochemical abnormalities characteristic of the refeeding syndrome, after restarting oral feeding on her own. Schizophrenia was later diagnosed. Increased awareness of this condition and its complications is necessary to prevent its detrimental complications.

  13. Thiamine Deficiency in Self-Induced Refeeding Syndrome, an Undetected and Potentially Lethal Condition

    PubMed Central

    Hershkowitz, Einat; Reshef, Alon; Munich, Olga; Yosefi, Bracha; Markel, Arie

    2014-01-01

    Rapid restoration of nutrients and electrolytes after prolonged starvation could result in a life threatening condition characterized by sensory and neurological dysfunction and severe metabolic imbalance that has been designated as refeeding syndrome. Its diagnosis is frequently missed resulting in severe complications and even death. We describe a 25-years-old female patient with mental disorders and severe malnutrition who developed severe clinical manifestations and biochemical abnormalities characteristic of the refeeding syndrome, after restarting oral feeding on her own. Schizophrenia was later diagnosed. Increased awareness of this condition and its complications is necessary to prevent its detrimental complications. PMID:25614745

  14. The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis

    PubMed Central

    Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun

    2013-01-01

    Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine. PMID:23220998

  15. Growth stimulation in inflorescences of an Arabidopsis tubulin mutant under microgravity conditions in space.

    PubMed

    Hoson, T; Soga, K; Wakabayashi, K; Hashimoto, T; Karahara, I; Yano, S; Tanigaki, F; Shimazu, T; Kasahara, H; Masuda, D; Kamisaka, S

    2014-01-01

    Cortical microtubules are involved in plant resistance to hypergravity, but their roles in resistance to 1 g gravity are still uncertain. To clarify this point, we cultivated an Arabidopsis α-tubulin 6 mutant (tua6) in the Cell Biology Experiment Facility on the Kibo Module of the International Space Station, and analyzed growth and cell wall mechanical properties of inflorescences. Growth of inflorescence stems was stimulated under microgravity conditions, as compared with ground and on-orbit 1 g conditions. The stems were 10-45% longer and their growth rate 15-55% higher under microgravity conditions than those under both 1 g conditions. The degree of growth stimulation tended to be higher in the tua6 mutant than the wild-type Columbia. Under microgravity conditions, the cell wall extensibility in elongating regions of inflorescences was significantly higher than the controls, suggesting that growth stimulation was caused by cell wall modifications. No clear differences were detected in any growth or cell wall property between ground and on-orbit 1 g controls. These results support the hypothesis that cortical microtubules generally play an important role in plant resistance to the gravitational force.

  16. A physiological, biochemical and proteomic characterization of Saccharomyces cerevisiae trk1,2 transport mutants grown under limiting potassium conditions.

    PubMed

    Gelis, Samuel; González-Fernández, Raquel; Herrera, Rito; Jorrín, Jesús; Ramos, José

    2015-06-01

    Saccharomyces cerevisiae mutants lacking both isoforms of the main plasma membrane potassium transporter display impaired potassium transport and defective growth at limiting concentrations of the cation. Moreover, they are hyperpolarized and have a lower intracellular pH than wild-type. In order to unravel global physiological processes altered in trk1,2 mutants, we have established conditions at which both wild-type and mutants can grow at different rates. Using a combination of physiological, biochemical and proteomic approaches, we show that during growth at suboptimal potassium concentrations, double trk1,2 mutants accumulate less potassium and reach lower yields. In contrast, the mutants maintain increased viability in the stationary phase and retain more potassium. Moreover, the mutants show increased expression of stress-related proteins such as catalase T, thioredoxin peroxidase or hexokinase 2, suggesting that they are better adapted to the additional stress factors associated with entry into stationary growth phase. PMID:25777080

  17. Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice.

    PubMed

    Petit, Fabrice G; Deng, Chuxia; Jamin, Soazik P

    2016-01-01

    Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.

  18. Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice

    PubMed Central

    Petit, Fabrice G.; Deng, Chuxia; Jamin, Soazik P.

    2016-01-01

    Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism. Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct. PMID:27194944

  19. Development of synthetic lethality anticancer therapeutics.

    PubMed

    Fang, Bingliang

    2014-10-01

    The concept of synthetic lethality (the creation of a lethal phenotype from the combined effects of mutations in two or more genes) has recently been exploited in various efforts to develop new genotype-selective anticancer therapeutics. These efforts include screening for novel anticancer agents, identifying novel therapeutic targets, characterizing mechanisms of resistance to targeted therapy, and improving efficacies through the rational design of combination therapy. This review discusses recent developments in synthetic lethality anticancer therapeutics, including poly ADP-ribose polymerase inhibitors for BRCA1- and BRCA2-mutant cancers, checkpoint inhibitors for p53 mutant cancers, and small molecule agents targeting RAS gene mutant cancers. Because cancers are caused by mutations in multiple genes and abnormalities in multiple signaling pathways, synthetic lethality for a specific tumor suppressor gene or oncogene is likely cell context-dependent. Delineation of the mechanisms underlying synthetic lethality and identification of treatment response biomarkers will be critical for the success of synthetic lethality anticancer therapy.

  20. Mutant DISC1 affects methamphetamine-induced sensitization and conditioned place preference: a comorbidity model

    PubMed Central

    Pogorelov, Vladimir; Nomura, Jun; Kim, Jongho; Kannan, Geetha; Yang, Chunxia; Taniguchi, Yu; Abazyan, Bagrat; Valentine, Heather; Krasnova, Irina N.; Kamiya, Atsushi; Cadet, Jean Lud; Wong, Dean F.; Pletnikov, Mikhail V.

    2011-01-01

    Genetic factors involved in neuroplasticity have been implicated in major psychiatric illnesses such as schizophrenia, depression, and substance abuse. Given its extended interactome, variants in the Disrupted-In-Schizophrenia-1 (DISC1) gene could contribute to drug addiction and psychiatric diseases. Thus, we evaluated how dominant-negative mutant DISC1 influenced the neurobehavioral and molecular effects of methamphetamine (METH). Control and mutant DISC1 mice were studied before or after treatment with non-toxic escalating dose (ED) of METH. In naïve mice, we assessed METH-induced conditioned place preference (CPP), dopamine (DA) D2 receptor density and the basal and METH-induced activity of DISC1 partners, AKT and GSK-3β in the ventral striatum. In ED treated mice, 4 weeks after METH treatment, we evaluated fear conditioning, depression-like responses in forced swim test, and the basal and METH-induced activity of AKT and GSK-3β in the ventral striatum. We found impairment in METH-induced CPP, decreased DA D2 receptor density and altered METH-induced phosphorylation of AKT and GSK-3β in naïve DISC1 female mice. The ED regimen was not neurotoxic as evidenced by unaltered brain regional monoamine tissue content. Mutant DISC1 significantly delayed METH ED-produced sensitization and affected drug-induced phosphorylation of AKT and GSK-3β in female mice. Our results suggest that perturbations in DISC1 functions in the ventral striatum may impact the molecular mechanisms of reward and sensitization, contributing to comorbidity between drug abuse and major mental diseases. PMID:21315744

  1. Mutant DISC1 affects methamphetamine-induced sensitization and conditioned place preference: a comorbidity model.

    PubMed

    Pogorelov, Vladimir M; Nomura, Jun; Kim, Jongho; Kannan, Geetha; Ayhan, Yavuz; Yang, Chunxia; Taniguchi, Yu; Abazyan, Bagrat; Valentine, Heather; Krasnova, Irina N; Kamiya, Atsushi; Cadet, Jean Lud; Wong, Dean F; Pletnikov, Mikhail V

    2012-03-01

    Genetic factors involved in neuroplasticity have been implicated in major psychiatric illnesses such as schizophrenia, depression, and substance abuse. Given its extended interactome, variants in the Disrupted-In-Schizophrenia-1 (DISC1) gene could contribute to drug addiction and psychiatric diseases. Thus, we evaluated how dominant-negative mutant DISC1 influenced the neurobehavioral and molecular effects of methamphetamine (METH). Control and mutant DISC1 mice were studied before or after treatment with non-toxic escalating dose (ED) of METH. In naïve mice, we assessed METH-induced conditioned place preference (CPP), dopamine (DA) D2 receptor density and the basal and METH-induced activity of DISC1 partners, AKT and GSK-3β in the ventral striatum. In ED-treated mice, 4 weeks after METH treatment, we evaluated fear conditioning, depression-like responses in forced swim test, and the basal and METH-induced activity of AKT and GSK-3β in the ventral striatum. We found impairment in METH-induced CPP, decreased DA D2 receptor density and altered METH-induced phosphorylation of AKT and GSK-3β in naïve DISC1 female mice. The ED regimen was not neurotoxic as evidenced by unaltered brain regional monoamine tissue content. Mutant DISC1 significantly delayed METH ED-produced sensitization and affected drug-induced phosphorylation of AKT and GSK-3β in female mice. Our results suggest that perturbations in DISC1 functions in the ventral striatum may impact the molecular mechanisms of reward and sensitization, contributing to comorbidity between drug abuse and major mental diseases. PMID:21315744

  2. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase

    PubMed Central

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M.; Vega, José M.; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J.; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress. PMID:26442073

  3. Structure, expression, and chromosome location of the gene for the beta subunit of brain-specific Ca2+/calmodulin-dependent protein kinase II identified by transgene integration in an embryonic lethal mouse mutant.

    PubMed Central

    Karls, U; Müller, U; Gilbert, D J; Copeland, N G; Jenkins, N A; Harbers, K

    1992-01-01

    The transgenic mouse strain CAT40 carries in its germ line one copy of a DNA construct consisting of the chloramphenicol acetyltransferase gene and the immunoglobulin heavy-chain enhancer. We show that transgene integration has resulted in a recessive lethal mutation that leads to death of homozygous CAT40 embryos shortly after implantation. The transgene has integrated adjacent to the 3' end of the gene coding for the beta subunit of the brain-specific Ca2+/calmodulin-dependent protein kinase II (Camk-2). The complete cDNA sequence of the Camk-2 gene and most of its exon/intron structure was determined. The deduced amino acid sequence is highly homologous to the previously described rat protein. The chromosomal location of the Camk-2 locus was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 11. This region lacks previously identified recessive embryonic lethal mutations. During embryonic development, Camk-2-specific transcripts are first seen in the head section of 12.5-day-old embryos, and in adult mice the gene is expressed almost exclusively in the brain. Although transcription of the Camk-2 gene in heterozygous CAT40 mice is affected by transgene integration, it is unlikely that this gene is responsible for the mutant phenotype, since it is not expressed in blastocysts and the first transcripts during normal development are detected after the death of homozygous CAT40 embryos. Transgene integration is accompanied by a large deletion of cellular DNA; death is therefore most likely caused by the loss of a gene or genes that are important for early postimplantation development. Images PMID:1321343

  4. Normalization of transposon-mutant library sequencing datasets to improve identification of conditionally essential genes.

    PubMed

    DeJesus, Michael A; Ioerger, Thomas R

    2016-06-01

    Sequencing of transposon-mutant libraries using next-generation sequencing (TnSeq) has become a popular method for determining which genes and non-coding regions are essential for growth under various conditions in bacteria. For methods that rely on quantitative comparison of counts of reads at transposon insertion sites, proper normalization of TnSeq datasets is vitally important. Real TnSeq datasets are often noisy and exhibit a significant skew that can be dominated by high counts at a small number of sites (often for non-biological reasons). If two datasets that are not appropriately normalized are compared, it might cause the artifactual appearance of Differentially Essential (DE) genes in a statistical test, constituting type I errors (false positives). In this paper, we propose a novel method for normalization of TnSeq datasets that corrects for the skew of read-count distributions by fitting them to a Beta-Geometric distribution. We show that this read-count correction procedure reduces the number of false positives when comparing replicate datasets grown under the same conditions (for which no genuine differences in essentiality are expected). We compare these results to results obtained with other normalization procedures, and show that it results in greater reduction in the number of false positives. In addition we investigate the effects of normalization on the detection of DE genes.

  5. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.

    PubMed

    Takeshige, K; Baba, M; Tsuboi, S; Noda, T; Ohsumi, Y

    1992-10-01

    confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

  6. Isolation and characterization of Methanobacterium thermoautotrophicum DeltaH mutants unable to grow under hydrogen-deprived conditions.

    PubMed

    Pennings, J L; Keltjens, J T; Vogels, G D

    1998-05-01

    By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen. PMID:9573152

  7. Conditionally lethal mutations in chinese hamster cells. Characterization of a cell line with a possible defect in the Krebs cycle.

    PubMed

    DeFrancesco, L; Werntz, D; Scheffler, I E

    1975-04-01

    A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of 14CO2 from 14C-labeled compounds was measured using pyruvate, acetate, beta-hydroxybutyrate, palmitate and glutamate, and in all cases it was neglibible in the mutant. In contrast to this, significant amounts of 14CO2 were produced from 14C-aspartate and 14C-succinate which suggest that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between alpha-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.

  8. The Activity of Nodules of the Supernodulating Mutant Mtsunn Is not Limited by Photosynthesis under Optimal Growth Conditions

    PubMed Central

    Cabeza, Ricardo A.; Lingner, Annika; Liese, Rebecca; Sulieman, Saad; Senbayram, Mehmet; Tränkner, Merle; Dittert, Klaus; Schulze, Joachim

    2014-01-01

    Legumes match the nodule number to the N demand of the plant. When a mutation in the regulatory mechanism deprives the plant of that ability, an excessive number of nodules are formed. These mutants show low productivity in the fields, mainly due to the high carbon burden caused through the necessity to supply numerous nodules. The objective of this study was to clarify whether through optimal conditions for growth and CO2 assimilation a higher nodule activity of a supernodulating mutant of Medicago truncatula (M. truncatula) can be induced. Several experimental approaches reveal that under the conditions of our experiments, the nitrogen fixation of the supernodulating mutant, designated as sunn (super numeric nodules), was not limited by photosynthesis. Higher specific nitrogen fixation activity could not be induced through short- or long-term increases in CO2 assimilation around shoots. Furthermore, a whole plant P depletion induced a decline in nitrogen fixation, however this decline did not occur significantly earlier in sunn plants, nor was it more intense compared to the wild-type. However, a distinctly different pattern of nitrogen fixation during the day/night cycles of the experiment indicates that the control of N2 fixing activity of the large number of nodules is an additional problem for the productivity of supernodulating mutants. PMID:24727372

  9. The activity of nodules of the supernodulating mutant Mtsunn is not limited by photosynthesis under optimal growth conditions.

    PubMed

    Cabeza, Ricardo A; Lingner, Annika; Liese, Rebecca; Sulieman, Saad; Senbayram, Mehmet; Tränkner, Merle; Dittert, Klaus; Schulze, Joachim

    2014-04-10

    Legumes match the nodule number to the N demand of the plant. When a mutation in the regulatory mechanism deprives the plant of that ability, an excessive number of nodules are formed. These mutants show low productivity in the fields, mainly due to the high carbon burden caused through the necessity to supply numerous nodules. The objective of this study was to clarify whether through optimal conditions for growth and CO2 assimilation a higher nodule activity of a supernodulating mutant of Medicago truncatula (M. truncatula) can be induced. Several experimental approaches reveal that under the conditions of our experiments, the nitrogen fixation of the supernodulating mutant, designated as sunn (super numeric nodules), was not limited by photosynthesis. Higher specific nitrogen fixation activity could not be induced through short- or long-term increases in CO2 assimilation around shoots. Furthermore, a whole plant P depletion induced a decline in nitrogen fixation, however this decline did not occur significantly earlier in sunn plants, nor was it more intense compared to the wild-type. However, a distinctly different pattern of nitrogen fixation during the day/night cycles of the experiment indicates that the control of N2 fixing activity of the large number of nodules is an additional problem for the productivity of supernodulating mutants.

  10. The tomato res mutant which accumulates JA in roots in non-stressed conditions restores cell structure alterations under salinity.

    PubMed

    Garcia-Abellan, José O; Fernandez-Garcia, Nieves; Lopez-Berenguer, Carmen; Egea, Isabel; Flores, Francisco B; Angosto, Trinidad; Capel, Juan; Lozano, Rafael; Pineda, Benito; Moreno, Vicente; Olmos, Enrique; Bolarin, Maria C

    2015-11-01

    Jasmonic acid (JA) regulates a wide spectrum of plant biological processes, from plant development to stress defense responses. The role of JA in plant response to salt stress is scarcely known, and even less known is the specific response in root, the main plant organ responsible for ionic uptake and transport to the shoot. Here we report the characterization of the first tomato (Solanum lycopersicum) mutant, named res (restored cell structure by salinity), that accumulates JA in roots prior to exposure to stress. The res tomato mutant presented remarkable growth inhibition and displayed important morphological alterations and cellular disorganization in roots and leaves under control conditions, while these alterations disappeared when the res mutant plants were grown under salt stress. Reciprocal grafting between res and wild type (WT) (tomato cv. Moneymaker) indicated that the main organ responsible for the development of alterations was the root. The JA-signaling pathway is activated in res roots prior to stress, with transcripts levels being even higher in control condition than in salinity. Future studies on this mutant will provide significant advances in the knowledge of JA role in root in salt-stress tolerance response, as well as in the energy trade-off between plant growth and response to stress.

  11. Fluctuation Analysis: The Probability Distribution of the Number of Mutants under Different Conditions

    PubMed Central

    Stewart, F. M.; Gordon, D. M.; Levin, B. R.

    1990-01-01

    In the 47 years since fluctuation analysis was introduced by Luria and Delbruck, it has been widely used to calculate mutation rates. Up to now, in spite of the importance of such calculations, the probability distribution of the number of mutants that will appear in a fluctuation experiment has been known only under the restrictive, and possibly unrealistic, assumptions: (1) that the mutation rate is exactly proportional to the growth rate and (2) that all mutants grow at a rate that is a constant multiple of the growth rate of the original cells. In this paper, we approach the distribution of the number of mutants from a new point of view that will enable researchers to calculate the distribution to be expected using assumptions that they believe to be closer to biological reality. The new idea is to classify mutations according to the number of observable mutants that derive from the mutation when the culture is selectively plated. This approach also simplifies the calculations in situations where two, or many, kinds of mutation may occur in a single culture. PMID:2307353

  12. A spontaneous eggplant (Solanum melongena L.) color mutant conditions anthocyanin-free fruit pigmentation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induced or spontaneously occuring color mutants in plants provide valuable tools for elucidating the genetic and developmental regulation of genes that influence pigmentation. We identified a single plant of the eggplant (Solanum melongena) cultivar Black Beauty bearing green fruit. Black Beauty no...

  13. Chinese hamster V79 cells harbor potentially lethal damage which is neither fixed nor repaired for long times after attaining maximal survival under growth conditions

    SciTech Connect

    Reddy, N.M.S.; Nori, D.; Mayer, P.J.; Lange, C.S.

    1995-03-01

    The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth medium but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation. 21 refs., 4 figs., 1 tab.

  14. The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions.

    PubMed

    Arce, Débora Pamela; Godoy, Andrea Verónica; Tsuda, Kenichi; Yamazaki, Ken-Ichi; Valle, Estela Marta; Iglesias, María José; Di Mauro, María Florencia; Casalongué, Claudia Anahí

    2010-02-15

    Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant (abc-) for the Arabidopsis thaliana MBF1 genes AtMBF1a, AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc- mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc- was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc- phenotype was partially or completely rescued by AtMBF1c cDNA over-expression (abc- +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc- mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

  15. A Conditional Mutant Having Paralyzed Cilia and a Block in Cytokinesis Is Rescued by Cytoplasmic Exchange in Tetrahymena Thermophila

    PubMed Central

    Pennock, D. G.; Thatcher, T.; Bowen, J.; Bruns, P. J.; Gorovsky, M. A.

    1988-01-01

    Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding α-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38°), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis. PMID:3224807

  16. Perinatal lethal osteogenesis imperfecta.

    PubMed Central

    Cole, W G; Dalgleish, R

    1995-01-01

    Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes that encode the alpha 1(I) and alpha 2(I) chains of type I collagen, respectively. Point mutations resulting in the substitution of Gly residues in Gly-X-Y amino acid triplets of the triple helical domain of the alpha 1(I) or alpha 2(I) chains are the most frequent mutations. They interrupt the repetitive Gly-X-Y structure that is mandatory for the formation of a stable triple helix. Most babies have their own private de novo mutation. However, the recurrence rate is about 7% owing to germline mosaicism in one parent. The mutations act in a dominant negative manner as the mutant pro alpha chains are incorporated into type I procollagen molecules that also contain normal pro alpha chains. The abnormal molecules are poorly secreted, more susceptible to degradation, and impair the formation of the extracellular matrix. The collagen fibres are abnormally organised and mineralisation is impaired. The severity of the clinical phenotype appears to be related to the type of mutation, its location in the alpha chain, the surrounding amino acid sequences, and the level of expression of the mutant allele. Images PMID:7643358

  17. Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice

    PubMed Central

    Yao, Ikuko; Takao, Keizo; Miyakawa, Tsuyoshi; Ito, Seiji; Setou, Mitsutoshi

    2011-01-01

    SCRAPPER, an F-box protein coded by FBXL20, is a subunit of SCF type E3 ubiquitin ligase. SCRAPPER localizes synapses and directly binds to Rab3-interacting molecule 1 (RIM1), an essential factor for synaptic vesicle release, thus it regulates neural transmission via RIM1 degradation. A defect in SCRAPPER leads to neurotransmission abnormalities, which could subsequently result in neurodegenerative phenotypes. Because it is likely that the alteration of neural transmission in Scrapper mutant mice affect their systemic condition, we have analyzed the behavioral phenotypes of mice with decreased or increased the amount of SCRAPPER. We carried out a series of behavioral test batteries for Scrapper mutant mice. Scrapper transgenic mice overexpressing SCRAPPER in the hippocampus did not show any significant difference in every test argued in this manuscript by comparison with wild-type mice. On the other hand, heterozygotes of Scrapper knockout [SCR (+/−)] mice showed significant difference in the contextual but not cued fear conditioning test. In addition, SCR (+/−) mice altered in some tests reflecting anxiety, which implies the loss of functions of SCRAPPER in the hippocampus. The behavioral phenotypes of Scrapper mutant mice suggest that molecular degradation conferred by SCRAPPER play important roles in hippocampal-dependent fear memory formation. PMID:21390313

  18. Enhancing the stability and ecological safety of mass-reared transgenic strains for field release by redundant conditional lethality systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advances in the genetic manipulation of agriculturally important insects now allows the development of genetic sexing and male sterility systems for more highly efficient biologically-based population control programs, most notably SIT, in fruit pests throughout the world. Potentially, these condit...

  19. Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

    PubMed

    Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

    2016-01-01

    RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal

  20. Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

    PubMed Central

    Kirkegaard, K; Nelsen, B

    1990-01-01

    Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

  1. Optimisation of fermentation conditions for gluconic acid production by a mutant of Aspergillus niger.

    PubMed

    Singh, O V; Sharma, A; Singh, R P

    2001-11-01

    Aspergillus niger ORS-4, isolated from the sugarcane industry waste materials was found to produce notable level of gluconic acid. From this strain, a mutant Aspergillus niger ORS-4.410 having remarkable increase in gluconic acid production was isolated and compared for fermentation properties. Among the various substrates used, glucose resulted into maximum production of gluconic acid (78.04 g/L). 12% concentration led to maximum production. Effect of spore age and inoculum level on fermentation indicated an inoculum level of 2% of the 4-7 days old spores were best suited for gluconic acid production. Maximum gluconate production could be achieved after 10-12 days of the fermentation at 30 degrees C and at a pH of 5.5. Kinetic analysis of production indicated that growth of the mutant was favoured during initial stages of the fermentation (4-8 days) and production increased during the subsequent 8-12 days of the fermentation. CaCO3 and varying concentrations of different nutrients affected the production of gluconic acid. Analysis of variance for the factors evaluated the significant difference in the production levels.

  2. Conditions supporting repair of potentially lethal damage cause a significant reduction of ultraviolet light-induced division delay in synchronized and plateau-phase Ehrlich ascites tumor cells

    SciTech Connect

    Iliakis, G.; Nusse, M.

    1982-09-01

    Repair of potentially lethal damage (PLD) induced by uv light in synchronized and in plateau-phase cultures of Ehrlich ascites tumor cells was studied by measuring cell survival. In particlar the influence of conditions supporting repair of PLD on growth kinetics was investigated. In synchronized G/sub 1/, S, or G/sub 2/ + M cells as well as in plateau-phase cells, uv light induced, almost exclusively, delay in the next S phase. A significant decrease of this delay was observed when the cells were incubated for 24 hr in balanced salt solution. Repair of PLD after uv irradiation was found to occur in plateau-phase cells and in cells in different phases of the cell cycle provided that after irradiation these were kept under conditions inhibiting cell multiplication (incubation in balanced salt solution or in conditioned medium). The repair time constant t/sub 50/ was significantly higher than those found for X irradiation (5-10 hr compared to 2 hr), and repair was not significantly inhibited by either 20 ..mu..g/ml cycloheximide or 2 mM caffeine in 24 hr.

  3. Synthetic lethal interaction between the pel1 and op1 mutations in Saccharomyces cerevisiae.

    PubMed

    Dzugasová, V; Subík, J

    2005-01-01

    The Saccharomyces cerevisiae mutant strain containing the op1 mutation affecting the function of a mitochondrial ATP/ADP translocator has been crossed to the pel1 and crd1 mutants deficient in the biosynthesis of mitochondrial phosphatidylglycerol (PG) and cardiolipin (CL). Using tetrad analysis of diploids issued from corresponding crosses a synthetic lethal interaction has been observed between the op1 and pel1 mutations resulting in the lack of growth of a corresponding double mutant on minimal medium containing glucose. The op1 pel1 double mutant also displayed a decreased susceptibility to fluconazole and a compromised growth even in complex medium containing glucose. The viability of mutant cells was strongly reduced, corresponding to <30 % and 10 % of colony-forming units observed after growth in complex and minimal medium, respectively. A lower viability of the double mutant in minimal medium was accompanied by an increased formation of mitochondrial petite mutants (as determined by mtDNA rescue into diploid cells). The results indicate that in the simultaneous absence of mitochondrial anionic phospholipids (PG plus CL) and ATP/ADP exchange across the inner mitochondrial membrane the yeast mitochondrial functions are severely limited, leading to a strongly compromised cell multiplication. Since under similar conditions the op1 crd1 double mutant was able to grow on minimal medium this deleterious effect of anionic phospholipid deficiency could be at least partially substituted by PG accumulated in the cardiolipin deficient delta crd1 mutant cells.

  4. SHORT-ROOT Deficiency Alleviates the Cell Death Phenotype of the Arabidopsis catalase2 Mutant under Photorespiration-Promoting Conditions.

    PubMed

    Waszczak, Cezary; Kerchev, Pavel I; Mühlenbock, Per; Hoeberichts, Frank A; Van Der Kelen, Katrien; Mhamdi, Amna; Willems, Patrick; Denecker, Jordi; Kumpf, Robert P; Noctor, Graham; Messens, Joris; Van Breusegem, Frank

    2016-08-01

    Hydrogen peroxide (H2O2) can act as a signaling molecule that influences various aspects of plant growth and development, including stress signaling and cell death. To analyze molecular mechanisms that regulate the response to increased H2O2 levels in plant cells, we focused on the photorespiration-dependent peroxisomal H2O2 production in Arabidopsis thaliana mutants lacking CATALASE2 (CAT2) activity (cat2-2). By screening for second-site mutations that attenuate the PSII maximum efficiency (Fv'/Fm') decrease and lesion formation linked to the cat2-2 phenotype, we discovered that a mutation in SHORT-ROOT (SHR) rescued the cell death phenotype of cat2-2 plants under photorespiration-promoting conditions. SHR deficiency attenuated H2O2-dependent gene expression, oxidation of the glutathione pool, and ascorbate depletion in a cat2-2 genetic background upon exposure to photorespiratory stress. Decreased glycolate oxidase and catalase activities together with accumulation of glycolate further implied that SHR deficiency impacts the cellular redox homeostasis by limiting peroxisomal H2O2 production. The photorespiratory phenotype of cat2-2 mutants did not depend on the SHR functional interactor SCARECROW and the sugar signaling component ABSCISIC ACID INSENSITIVE4, despite the requirement for exogenous sucrose for cell death attenuation in cat2-2 shr-6 double mutants. Our findings reveal a link between SHR and photorespiratory H2O2 production that has implications for the integration of developmental and stress responses. PMID:27432873

  5. Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system.

    PubMed

    Abdulaal, Wesam H; Walker, Catherine R; Costello, Ryan; Redondo-Castro, Elena; Mufazalov, Ilgiz A; Papaemmanouil, Athina; Rothwell, Nancy J; Allan, Stuart M; Waisman, Ari; Pinteaux, Emmanuel; Müller, Werner

    2016-04-01

    IL-1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL-1 actions as part of the inflammatory response in vivo, we generated a conditional IL-1 receptor 1 (IL-1R1) mouse mutant using the Cre/LoxP system (IL-1R1(fl/fl) ). In the mutant generated, exon 5, which encodes part of the extracellular-binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL-1R1 gene transcripts after Cre-mediated recombination. Using keratin 14-Cre driver mice, new IL-1R1 deficient (-/-) mice were subsequently generated, in which all signaling IL-1 receptor isoforms are deleted ubiquitously. Furthermore, using vav-iCre driver mice, we deleted IL-1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL-17 and IL-22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL-1R1(fl/fl) mice with different Cre-expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.

  6. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

  7. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants. PMID:26081484

  8. [The "lethal white foal" syndrome].

    PubMed

    Blendinger, C; Müller, G; Bostedt, H

    1994-06-01

    The lethal white foal syndrome (congenital intestinal aganglionosis) was diagnosed by history, clinical signs and pathological findings in a female foal, born in March 1992, that was an offspring of two overo-spotted paint horses. The syndrome is a congenital innervation defect of the gastrointestinal tract. A literature review of this condition, relatively unknown in Germany, is given.

  9. Assessing the essentiality of the decaprenyl-phospho-d-arabinofuranose pathway in Mycobacterium tuberculosis using conditional mutants.

    PubMed

    Kolly, Gaëlle S; Boldrin, Francesca; Sala, Claudia; Dhar, Neeraj; Hartkoorn, Ruben C; Ventura, Marcello; Serafini, Agnese; McKinney, John D; Manganelli, Riccardo; Cole, Stewart T

    2014-04-01

    In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

  10. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein

    PubMed Central

    Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

    The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles. PMID:26925228

  11. Increased dopaminergic innervation in the brain of conditional mutant mice overexpressing Otx2: effects on locomotor behavior and seizure susceptibility.

    PubMed

    Tripathi, P P; Di Giovannantonio, L G; Sanguinetti, E; Acampora, D; Allegra, M; Caleo, M; Wurst, W; Simeone, A; Bozzi, Y

    2014-03-01

    The homeobox-containing transcription factor Otx2 controls the identity, fate and proliferation of mesencephalic dopaminergic (mesDA) neurons. Transgenic mice, in which Otx2 was conditionally overexpressed by a Cre recombinase expressed under the transcriptional control of the Engrailed1 gene (En1(Cre/+); tOtx2(ov/+)), show an increased number of mesDA neurons during development. In adult mice, Otx2 is expressed in a subset of neurons in the ventral tegmental area (VTA) and its overexpression renders mesDA more resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-HCl (MPTP) neurotoxin. Here we further investigated the neurological consequences of the increased number of mesDA neurons in En1(Cre/+); tOtx2(ov/+) adult mice. Immunohistochemistry for the active, glycosylated form of the dopamine transporter (glyco-Dat) showed that En1(Cre/+); tOtx2(ov/+) adult mice display an increased density of mesocortical DAergic fibers, as compared to control animals. Increased glyco-Dat staining was accompanied by a marked hypolocomotion in En1(Cre/+); tOtx2(ov/+) mice, as detected in the open field test. Since conditional knockout mice lacking Otx2 in mesDA precursors (En1(Cre/+); Otx2(floxv/flox) mice) show a marked resistance to kainic acid (KA)-induced seizures, we investigated the behavioral response to KA in En1(Cre/+); tOtx2(ov/+) and control mice. No difference was observed between mutant and control mice, but En1(Cre/+); tOtx2(ov/+) mice showed a markedly different c-fos mRNA induction profile in the cerebral cortex and hippocampus after KA seizures, as compared to controls. Accordingly, an increased density of parvalbumin (PV)-positive inhibitory interneurons was detected in the deep layers of the frontal cortex of naïve En1(Cre/+); tOtx2(ov/+) mice, as compared to controls. These data indicate that Otx2 overexpression results in increased DAergic innervation and PV cell density in the fronto-parietal cortex, with important consequences on spontaneous locomotor

  12. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes

    PubMed Central

    Effendi, Yunus; Ferro, Noel; Labusch, Corinna; Geisler, Markus; Scherer, Günther F. E.

    2015-01-01

    The function of the extracytoplasmic AUXIN-BINDING-PROTEIN1 (ABP1) is largely enigmatic. We complemented a homozygous T-DNA insertion null mutant of ABP1 in Arabidopsis thaliana Wassilewskia with three mutated and one wild-type (wt) ABP1 cDNA, all tagged C-terminally with a strepII–FLAG tag upstream the KDEL signal. Based on in silico modelling, the abp1 mutants were predicted to have altered geometries of the auxin binding pocket and calculated auxin binding energies lower than the wt. Phenotypes linked to auxin transport were compromised in these three complemented abp1 mutants. Red light effects, such as elongation of hypocotyls in constant red (R) and far-red (FR) light, in white light supplemented by FR light simulating shade, and inhibition of gravitropism by R or FR, were all compromised in the complemented lines. Using auxin- or light-induced expression of marker genes, we showed that auxin-induced expression was delayed already after 10min, and light-induced expression within 60min, even though TIR1/AFB or phyB are thought to act as receptors relevant for gene expression regulation. The expression of marker genes in seedlings responding to both auxin and shade showed that for both stimuli regulation of marker gene expression was altered after 10–20min in the wild type and phyB mutant. The rapidity of expression responses provides a framework for the mechanics of functional interaction of ABP1 and phyB to trigger interwoven signalling pathways. PMID:25392478

  13. Protection from genital herpes disease, seroconversion and latent infection in a non-lethal murine genital infection model by immunization with an HSV-2 replication-defective mutant virus.

    PubMed

    Diaz, Fernando M; Knipe, David M

    2016-01-15

    Viral vaccines have traditionally protected against disease, but for viruses that establish latent infection, it is desirable for the vaccine to reduce infection to reduce latent infection and reactivation. While seroconversion has been used in clinical trials of herpes simplex virus (HSV) vaccines to measure protection from infection, this has not been modeled in animal infection systems. To measure the ability of a genital herpes vaccine candidate to protect against various aspects of infection, we established a non-lethal murine model of genital HSV-2 infection, an ELISA assay to measure antibodies specific for infected cell protein 8 (ICP8), and a very sensitive qPCR assay. Using these assays, we observed that immunization with HSV-2 dl5-29 virus reduced disease, viral shedding, seroconversion, and latent infection by the HSV-2 challenge virus. Therefore, it may be feasible to obtain protection against genital disease, seroconversion and latent infection by immunization, even if sterilizing immunity is not achieved.

  14. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation.

  15. Autoinducer 2 Production by Streptococcus gordonii DL1 and the Biofilm Phenotype of a luxS Mutant Are Influenced by Nutritional Conditions

    PubMed Central

    Blehert, David S.; Palmer, Robert J.; Xavier, Joao B.; Almeida, Jonas S.; Kolenbrander, Paul E.

    2003-01-01

    The luxS gene, present in many bacterial genera, encodes the autoinducer 2 (AI-2) synthase. AI-2 has been implicated in bacterial signaling, and this study investigated its role in biofilm formation by Streptococcus gordonii, an organism that colonizes human tooth enamel within the first few hours after professional cleaning. Northern blotting and primer extension analyses revealed that S. gordonii luxS is monocistronic. AI-2 production was dependent on nutritional conditions, and maximum AI-2 induction was detected when S. gordonii was grown in the presence of serum and carbonate. In planktonic cultures, AI-2 production rose sharply during the transition from exponential to stationary phase, and the AI-2 concentration peaked approximately 4 h into stationary phase. An S. gordonii luxS mutant that did not produce AI-2 was constructed by homologous recombination. Complementation of the mutant by insertion of an intact luxS gene into the chromosome in tandem with the disrupted gene restored AI-2 production to a level similar to that of the wild-type strain. In planktonic culture, no growth differences were observed between the mutant and wild-type strains when five different media were used. However, when grown for 4 h as biofilms in 25% human saliva under flow, the luxS mutant formed tall microcolonies that differed from those formed by the wild-type and complemented mutant strains. Biofilms of the luxS mutant exhibited finger-like projections of cells that extended into the flow cell lumen. Thus, the inability to produce AI-2 is associated with altered microcolony architecture within S. gordonii biofilms formed in saliva during a time frame consistent with initial colonization of freshly cleaned enamel surfaces. PMID:12897005

  16. PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions.

    PubMed

    Summerfield, Tina C; Shand, Jackie A; Bentley, Fiona K; Eaton-Rye, Julian J

    2005-01-18

    A PsbQ homologue has been found associated with photosystem II complexes in Synechocystis sp. PCC 6803 where it is involved in optimal photoautotrophic growth and water splitting under CaCl(2)-depleted conditions [Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175]. By inactivating psbQ in strains carrying photosystem II-specific mutations, we have identified stringent requirements for PsbQ in vivo. Whereas under nutrient-replete conditions the DeltaPsbQ mutant was similar to wild type, a strain lacking PsbQ and PsbV was not photoautotrophic, exhibiting decreased oxygen evolution and decreased photosystem II assembly compared to the DeltaPsbV mutant. Combining the removal of PsbU and PsbQ introduced an altered requirement for Ca(2+) and Cl(-), and photoautotrophic growth of the DeltaPsbQ strain was prevented in nutrient-limiting media depleted in Ca(2+), Cl(-), and iron. Unlike other photosystem II extrinsic proteins PsbQ did not participate in the acquisition of thermotolerance; however, photoautotrophic growth at elevated temperatures was impaired in this mutant. Growth of the DeltaPsbV:DeltaPsbQ mutant was restored at pH 10.0: in contrast, an additional deletion between Arg-384 and Val-392 in the CP47 protein of photosystem II prevented recovery at alkaline pH. When conditions prevented photoautotrophy in strains lacking PsbQ, photoheterotrophic growth was indistinguishable to wild type, indicating that photosystem II had been inactivated. These data substantiate a role for PsbQ in optimizing photosystem II activity in Synechocystis sp. PCC 6803 and establish an absolute requirement for the subunit under specific biochemical and physiological conditions. PMID:15641809

  17. Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage.

    PubMed

    Gras, Diana E; Silveira, Henrique C S; Peres, Nalu T A; Sanches, Pablo R; Martinez-Rossi, Nilce M; Rossi, Antonio

    2009-01-01

    The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregulated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa.

  18. Conditional Mutants of Rpc160, the Gene Encoding the Largest Subunit of RNA Polymerase C in Saccharomyces Cerevisiae

    PubMed Central

    Gudenus, R.; Mariotte, S.; Moenne, A.; Ruet, A.; Memet, S.; Buhler, J. M.; Sentenac, A.; Thuriaux, P.

    1988-01-01

    A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO(3) and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants by failure to complement a fully defective mutation rpc160::URA3. The faithful transcription of a yeast tRNA gene by mutant cell-free extracts is strongly reduced as compared to wild-type. In vivo, the rpc160 mutations specifically affect the synthesis of tRNA in a temperature sensitive way, with comparatively little effect on the synthesis of 5S rRNA and no effect on 5.8S rRNA. An unlinked mutation (pci1-3) suppresses the temperature sensitive phenotype of the rpc160-41 mutation. PMID:2841184

  19. Metabolic flux analysis of Escherichia coli creB and arcA mutants reveals shared control of carbon catabolism under microaerobic growth conditions.

    PubMed

    Nikel, Pablo I; Zhu, Jiangfeng; San, Ka-Yiu; Méndez, Beatriz S; Bennett, George N

    2009-09-01

    Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of (13)C-labeling experiments in chemostat cultures of a wild-type strain, DeltacreB and DeltaarcA single mutants, and a DeltacreB DeltaarcA double mutant. Continuous cultures were conducted at D = 0.1 h(-1) under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its DeltaarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the DeltaarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.

  20. Growth-dependent DNA breakage and cell death in a gyrase mutant of Salmonella.

    PubMed Central

    Garí, E; Bossi, L; Figueroa-Bossi, N

    2001-01-01

    A class of gyrase mutants of Salmonella enterica mimics the properties of bacteria exposed to quinolones. These mutants suffer spontaneous DNA breakage during normal growth and depend on recombinational repair for viability. Unlike quinolone-treated bacteria, however, they do not show accumulation of cleavable gyrase-DNA complexes. In recA or recB mutant backgrounds, the temperature-sensitive (ts) allele gyrA208 causes rapid cell death at 43 degrees. Here, we isolated "suppressor-of-death" mutations, that is, secondary changes that allow a gyrA208 recB double mutant to survive a prolonged exposure to 43 degrees and subsequently to form colonies at 28 degrees. In most isolates, the secondary change was itself a ts mutation. Three ts alleles were mapped in genes coding for amino acyl tRNA synthetases (alaS, glnS, and lysS). Allele alaS216 completely abolished DNA breakage in a gyrA208 recA double mutant. Likewise, treating this mutant with chloramphenicol prevented death and DNA damage at 43 degrees. Additional suppressors of gyrA208 lethality include rpoB mutations and, surprisingly, icd mutations inactivating isocitrate dehydrogenase. We postulate that the primary effect of the gyrase alteration is to hamper replication fork movement. Inhibiting DNA replication under conditions of continuing macromolecular synthesis ("unbalanced growth") activates a mechanism that causes DNA breakage and cell death, reminiscent of "thymineless" lethality. PMID:11779784

  1. Theory of lethal mutagenesis for viruses.

    PubMed

    Bull, J J; Sanjuán, R; Wilke, C O

    2007-03-01

    Mutation is the basis of adaptation. Yet, most mutations are detrimental, and elevating mutation rates will impair a population's fitness in the short term. The latter realization has led to the concept of lethal mutagenesis for curing viral infections, and work with drugs such as ribavirin has supported this perspective. As yet, there is no formal theory of lethal mutagenesis, although reference is commonly made to Eigen's error catastrophe theory. Here, we propose a theory of lethal mutagenesis. With an obvious parallel to the epidemiological threshold for eradication of a disease, a sufficient condition for lethal mutagenesis is that each viral genotype produces, on average, less than one progeny virus that goes on to infect a new cell. The extinction threshold involves an evolutionary component based on the mutation rate, but it also includes an ecological component, so the threshold cannot be calculated from the mutation rate alone. The genetic evolution of a large population undergoing mutagenesis is independent of whether the population is declining or stable, so there is no runaway accumulation of mutations or genetic signature for lethal mutagenesis that distinguishes it from a level of mutagenesis under which the population is maintained. To detect lethal mutagenesis, accurate measurements of the genome-wide mutation rate and the number of progeny per infected cell that go on to infect new cells are needed. We discuss three methods for estimating the former. Estimating the latter is more challenging, but broad limits to this estimate may be feasible.

  2. Heat Shock Transcriptional Responses in an MC-Producing Cyanobacterium (Planktothrix agardhii) and Its MC-Deficient Mutant under High Light Conditions

    PubMed Central

    Tran, Thi Du Chi; Bernard, Cecile; Ammar, Myriam; Chaouch, Soraya; Comte, Katia

    2013-01-01

    Microcystins (MCs) are the most commonly-reported hepatotoxins produced by various cyanobacterial taxa in fresh waters to constitute a potential threat to human and animal health. The biological role of MCs in the producer organisms is not known, and it would be very useful to understand the driving force behind the toxin production. Recent studies have suggested that MCs may have a protective function in cells facing environmental stress. Following this starting premise, we speculate that under adverse conditions the expression of stress-related genes coding for Heat Shock Proteins (Hsp) might be different in an MC-producing strain and its MC-deficient mutant. We therefore used RT-qPCR to compare the expression of 13 hsp genes of an MC-producing strain of Planktothrix agardhii (CYA126/8) and its MC-deficient ΔmcyD mutant over different periods of exposure to high light stress (HL). Three reference genes (RGs) were selected from six candidates to normalize the RT-qPCR data. Of these three RGs (rsh, rpoD, and gltA), gltA is used here for the first time as an RG in prokaryotes. Under HL stress, five genes were found to be strongly up-regulated in both strains (htpG, dnaK, hspA, groES, and groEL). Unexpectedly, we found that the MC-producing wild type strain accumulated higher levels of htpG and dnaK transcripts in response to HL stress than the MC-deficient mutant. In addition, a significant increase in the mcyE transcript was detected in the mutant, suggesting that MCs are required under HL conditions. We discuss several possible roles of MCs in the response to HL stress through their possible involvement in the protective mechanisms of the cells. PMID:24023831

  3. Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions

    PubMed Central

    van Kleeff, P. J. M.; Jaspert, N.; Li, K. W.; Rauch, S.; Oecking, C.; de Boer, A. H.

    2014-01-01

    Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation. PMID:25189593

  4. Electroshock weapons can be lethal!

    NASA Astrophysics Data System (ADS)

    Lundquist, Marjorie

    2008-03-01

    Electroshock weapons (EWs)-stun guns, tasers, riot shields-are electroconductive devices designed to safely incapacitate healthy men neuromuscularly, so they are called nonlethal or less-lethal. EW firms seeking large nonmilitary markets targeted law enforcement and corrections personnel, who began using EWs in prisons/jails and on public patrol in 1980 in the USA. This shifted the EW-shocked population from healthy soldiers to a heterogeneous mix of both sexes, ages 6-92, in a wide variety of health conditions! An EW operates by disrupting normal physiological processes, producing transient effects in healthy people. But if a person's health is sufficiently compromised, the margin of safety can be lost, resulting in death or permanent health problems. 325 people have died after EW shock since 1980. Did the EW cause these deaths? Evidence indicates that EWs do play a causal role in most such deaths. EWs can be lethal for people in diabetic shock^1 (hypoglycemia), which may be why Robert Dziekanski-a Polish immigrant to Canada-died so quickly after he was tasered at Vancouver Airport: not having eaten for over 10 hours, he likely was severely hypoglycemic. The EW death rate in North America is 30 times higher than need be, because EW users have not been properly trained to use EWs on a heterogeneous population safely! ^1J. Clinical Engineering 30(3):111(2005).

  5. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8'-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition.

    PubMed

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-03-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8'-hydroxyase gene which was highly expressed during seed development (TaABA8'OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8'OH1 on the D genome (TaABA8'OH1-D) was identified in Japanese cultivars including 'Tamaizumi'. However, a single mutation in TaABA8'OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8'OH1 on the A genome (TaABA8'OH1-A), TM1833, was identified from gamma-ray irradiation lines of 'Tamaizumi'. TM1833 (a double mutant in TaABA8'OH1-A and TaABA8'OH1-D) showed lower TaABA8'OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in 'Tamaizumi' (a single mutant in TaABA8'OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8'OH1 may be effective in germination inhibition in field-grown wheat.

  6. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8′-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition

    PubMed Central

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-01-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8′-hydroxyase gene which was highly expressed during seed development (TaABA8′OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8′OH1 on the D genome (TaABA8′OH1-D) was identified in Japanese cultivars including ‘Tamaizumi’. However, a single mutation in TaABA8′OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8′OH1 on the A genome (TaABA8′OH1-A), TM1833, was identified from gamma-ray irradiation lines of ‘Tamaizumi’. TM1833 (a double mutant in TaABA8′OH1-A and TaABA8′OH1-D) showed lower TaABA8′OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in ‘Tamaizumi’ (a single mutant in TaABA8′OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8′OH1 may be effective in germination inhibition in field-grown wheat. PMID:23641187

  7. Deficits in Adult Neurogenesis, Contextual Fear Conditioning, and Spatial Learning in a Gfap Mutant Mouse Model of Alexander Disease

    PubMed Central

    Paylor, Richard; Messing, Albee

    2013-01-01

    Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease. PMID:24259590

  8. Strategy for enhanced transgenic strain development for embryonic conditionnal lethality in Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here the first reproductive sterility system for the tephritid pest, Anastrepha suspensa, is presented, based on lethality primarily in embryos heterozygous for a lethal conditional transgene combination. The tetracycline-suppressible system uses the cellularization-specific A. suspensa serendipity...

  9. Evidence that the premature death mutation (p) in the Mexican axolotl (Ambystoma mexicanum) is not an autonomous cell lethal.

    PubMed

    Mes-Hartree, M; Armstrong, J B

    1980-12-01

    Cell-lethal developmental mutations, which are presumed to affect the viability of all cells in a mutant embryo, have been distinguished from other development lethals on the basis of the results of parabiosis and transplant experiments. Premature death (p), previously classified as a cell lethal, does not survive parabiosis. However, transplants involving mutant eye, flank epidermis and primordial limb tissue all survived on a normal recipient. The mutant, therefore, cannot be considered a true cell lethal, though it suffers from serious and widespread abnormalities that cannot be corrected by parabiosis. In addition, transplants of mutant branchial mound tissue did not develop into normal gills on a normal recipient. These transplants were the only ones involving mutant endoderm, and their failure supports our hypothesis that the mutation leads to a specific endoderm defect.

  10. Conditional loss of ErbB3 delays mammary gland hyperplasia induced by mutant PIK3CA without affecting mammary tumor latency, gene expression, or signaling.

    PubMed

    Young, Christian D; Pfefferle, Adam D; Owens, Philip; Kuba, María G; Rexer, Brent N; Balko, Justin M; Sánchez, Violeta; Cheng, Hailing; Perou, Charles M; Zhao, Jean J; Cook, Rebecca S; Arteaga, Carlos L

    2013-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K), have been shown to transform mammary epithelial cells (MEC). Studies suggest this transforming activity requires binding of mutant p110α via p85 to phosphorylated YXXM motifs in activated receptor tyrosine kinases (RTK) or adaptors. Using transgenic mice, we examined if ErbB3, a potent activator of PI3K, is required for mutant PIK3CA-mediated transformation of MECs. Conditional loss of ErbB3 in mammary epithelium resulted in a delay of PIK3CA(H1047R)-dependent mammary gland hyperplasia, but tumor latency, gene expression, and PI3K signaling were unaffected. In ErbB3-deficient tumors, mutant PI3K remained associated with several tyrosyl phosphoproteins, potentially explaining the dispensability of ErbB3 for tumorigenicity and PI3K activity. Similarly, inhibition of ErbB RTKs with lapatinib did not affect PI3K signaling in PIK3CA(H1047R)-expressing tumors. However, the p110α-specific inhibitor BYL719 in combination with lapatinib impaired mammary tumor growth and PI3K signaling more potently than BYL719 alone. Furthermore, coinhibition of p110α and ErbB3 potently suppressed proliferation and PI3K signaling in human breast cancer cells harboring PIK3CA(H1047R). These data suggest that PIK3CA(H1047R)-driven tumor growth and PI3K signaling can occur independently of ErbB RTKs. However, simultaneous blockade of p110α and ErbB RTKs results in superior inhibition of PI3K and mammary tumor growth, suggesting a rational therapeutic combination against breast cancers harboring PIK3CA activating mutations.

  11. Stem cell expansion during carcinogenesis in stem cell-depleted conditional telomeric repeat factor 2 null mutant mice.

    PubMed

    Bojovic, B; Ho, H-Y; Wu, J; Crowe, D L

    2013-10-24

    To examine the role of telomeric repeat-binding factor 2 (TRF2) in epithelial tumorigenesis, we characterized conditional loss of TRF2 expression in the basal layer of mouse epidermis. These mice exhibit some characteristics of dyskeratosis congenita, a human stem cell depletion syndrome caused by telomere dysfunction. The epidermis in conditional TRF2 null mice exhibited DNA damage response and apoptosis, which correlated with stem cell depletion. The stem cell population in conditional TRF2 null epidermis exhibited shorter telomeres than those in control mice. Squamous cell carcinomas induced in conditional TRF2 null mice developed with increased latency and slower growth due to reduced numbers of proliferating cells as the result of increased apoptosis. TRF2 null epidermal stem cells were found in both primary and metastatic tumors. Despite the low-grade phenotype of the conditional TRF2 null primary tumors, the number of metastatic lesions was similar to control cancers. Basal cells from TRF2 null tumors demonstrated extreme telomere shortening and dramatically increased numbers of telomeric signals by fluorescence in situ hybridization due to increased genomic instability and aneuploidy in these cancers. DNA damage response signals were detected at telomeres in TRF2 null tumor cells from these mice. The increased genomic instability in these tumors correlated with eightfold expansion of the transformed stem cell population compared with that in control cancers. We concluded that genomic instability resulting from loss of TRF2 expression provides biological advantages to the cancer stem cell population.

  12. Biogenesis of thylakoid membranes is controlled by light intensity in the conditional chlorophyll b-deficient CD3 mutant of wheat

    PubMed Central

    1988-01-01

    Biogenesis of thylakoid membranes in the conditional chlorophyll b- deficient CD3 mutant of wheat is dramatically altered by relatively small differences in the light intensity under which seedlings are grown. When the CD3 mutant is grown at 400 microE/m2 S (high light, about one-fifth full sunlight) plants are deficient in chlorophyll b (chlorophyll a/b ratio greater than 6.0) and lack or contain greatly reduced amounts of the chlorophyll a/b-binding complexes CPII/CPII (mobile or peripheral LHCII), CP29, CP24 and LHCI, as shown by mildly denaturing 'green gel' electrophoresis, by fully denaturing SDS-PAGE, and by Western blot analysis. High light CD3 chloroplasts display an unusual morphology characterized by large, sheet-like stromal thylakoids formed into parallel unstacked arrays and a limited number of small grana stacks displaced toward the edges of the arrays. Changes in the supramolecular organization of CD3 thylakoids, seen with freeze- fracture electron microscopy, include a reduction in the size of EFs particles, which correspond to photosystem II centers with variable amounts of attached LHCII, and a redistribution of EF particles from the stacked to the unstacked regions. When CD3 seedlings are grown at 150 microE/m2 S (low light) there is a substantial reversal of all of these effects. Thus, chlorophyll b and the chlorophyll a/b-binding proteins accumulate to near wild-type levels (chlorophyll a/b ratio = 3.5-4.5) and thylakoid morphology is more nearly wild type in appearance. Growth of the CD3 mutant in the presence of chloramphenicol stimulates the accumulation of chlorophyll b and its binding proteins (Duysen, M. E., T. P. Freeman, N. D. Williams, and L. L. Huckle. 1985. Plant Physiol. 78:531-536). We show that this partial rescue of the CD3 high light phenotype is accompanied by large changes in thylakoid structure. The CD3 mutant, which defines a new class of chlorophyll b- deficient phenotype, is discussed in the more general context of

  13. Elemental concentrations in the seed of mutants and natural variants of Arabidopsis thaliana grown under varying soil conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The concentrations of mineral nutrients in seeds are critical to both the life cycle of plants as well as human nutrition. These concentrations are strongly influenced by soil conditions, as shown here by quantifying the concentration of 14 elements in seeds from Arabidopsis thaliana plants grown un...

  14. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions

    PubMed Central

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant “Jingling Huangfeng” turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration. PMID:26788511

  15. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

    PubMed

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration.

  16. Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

    PubMed

    Li, Shu-Shun; Li, Qian-Zhong; Rong, Li-Ping; Tang, Ling; Zhang, Bo

    2015-01-01

    Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration. PMID:26788511

  17. Cortical Efferents Lacking Mutant huntingtin Improve Striatal Neuronal Activity and Behavior in a Conditional Mouse Model of Huntington's Disease

    PubMed Central

    Estrada-Sánchez, Ana María; Burroughs, Courtney L.; Cavaliere, Stephen; Barton, Scott J.; Chen, Shirley; Yang, X. William

    2015-01-01

    Abnormal electrophysiological activity in the striatum, which receives dense innervation from the cerebral cortex, is believed to set the stage for the behavioral phenotype observed in Huntington's disease (HD), a neurodegenerative condition caused by mutation of the huntingtin (mhtt) protein. However, cortical involvement is far from clear. To determine whether abnormal striatal processing can be explained by mhtt alone (cell-autonomous model) or by mhtt in the corticostriatal projection cell–cell interaction model, we used BACHD/Emx1–Cre (BE) mice, a conditional HD model in which full-length mhtt is genetically reduced in cortical output neurons, including those that project to the striatum. Animals were assessed beginning at 20 weeks of age for at least the next 40 weeks, a range over which presymptomatic BACHD mice become symptomatic. Both open-field and nest-building behavior deteriorated progressively in BACHD mice relative to both BE and wild-type (WT) mice. Neuronal activity patterns in the dorsal striatum, which receives input from the primary motor cortex (M1), followed a similar age progression because BACHD activity changed more rapidly than either BE or WT mice. However, in the M1, BE neuronal activity differed significantly from both WT and BACHD. Although abnormal cortical activity in BE mice likely reflects input from mhtt-expressing afferents, including cortical interneurons, improvements in BE striatal activity and behavior suggest a critical role for mhtt in cortical output neurons in shaping the onset and progression of striatal dysfunction. PMID:25762686

  18. A conditional mouse mutant in the tumor suppressor SdhD gene unveils a link between p21(WAF1/Cip1) induction and mitochondrial dysfunction.

    PubMed

    Millán-Uclés, Africa; Díaz-Castro, Blanca; García-Flores, Paula; Báez, Alicia; Pérez-Simón, José Antonio; López-Barneo, José; Piruat, José I

    2014-01-01

    Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh) genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive". Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the "pseudo-hypoxic" response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21(WAF1/Cip1), a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21(WAF1/Cip1) will open new avenues for the study of the mechanisms that cause tumors in Sdh

  19. Specific ultrasonographic features of perinatal lethal hypophosphatasia.

    PubMed

    Zankl, Andreas; Mornet, Etienne; Wong, Shell

    2008-05-01

    Prenatal diagnosis of perinatal lethal hypophosphatasia (PL-HPH) by ultrasonography is difficult as PL-HPH must be differentiated from other skeletal dysplasias with short long bones and poor mineralization of the skeleton, such as osteogenesis imperfecta type II and achondrogenesis/hypochondrogenesis. Here we present a case of molecularly confirmed PL-HPH and illustrate specific ultrasonographic findings that help to distinguish PL-HPH from similar conditions. PMID:18386808

  20. Genetic relationships among three chlorophyll-deficient mutants in peanut, Arachis hypogaea L.

    PubMed

    Tai, P Y; Hammons, R O; Matlock, R S

    1977-01-01

    The genetic relationships of three chlorophyll-deficient mutant peanuts, lutescens (lu), aureus (au), and virescent (v) were studied under field and greenhouse conditions. The F1 plants from crosses between these mutants produced phenotypically normal green. In F2, aureus X virescent segregated 675 normal green : 225 virescent : 45 aureus : 15 virescent aureus : 64 seedling lethal, and lutescens X virescent segregated 45 normal green : 15 virescent : 3 lutescens : 1 seedling lethal. (Lutescens peanuts were seedling lethal in the field.) As previously reported, the F2 of aureus X lutescens gave 225 normal green : 15 aureus :15 lutescens : 1 seedling lethal. The three chlorophyll-deficient factors (au, lu, and v) show independent inheritance. The recessive combinations from the parental types between aureus and virescent and between aureus and lutescens would produce plants with a combination of their respective parental characteristics, but the recessive combination between lutescens and virescent was nearly albino. The v-au and lu-au seedlings have a longer life span than the v-lu seedling has. The genotypes for the three mutants are tentatively identified as lutescens VV Au 1 Au 1 Au 2 Au 2 lu 1 lu 1 lu 2 lu 2 L 1 L 1 L 2 L 2, aureus VV au1au1 au2au2 Lu1Lu1 Lu2Lu2 L1L1 l2l2, and virescent vv Au1Au1 Au2Au2 lu1lu1 Lu2Lu2 l1l1 L2L2.

  1. Lethal outcome in xanthogranulomatous endometritis.

    PubMed

    Noack, Frank; Briese, Juliane; Stellmacher, Florian; Hornung, Daniela; Horny, Hans-Peter

    2006-05-01

    Xanthogranulomatous inflammation is rare, mainly involving the kidneys, while primary xanthogranulomatous endometritis (XE) is a very unusual finding, histologically characterized by partial or complete replacement of the mucosa by granulation tissue with an abundance of foamy histiocytes, siderophages and multinucleated giant cells. We present the case of a 69-year-old woman with a short history of abdominal pain and a palpable mass in the pouch of Douglas. Dilatation of the cervix drained a pyometra. Histological examination of the curettage rendered the diagnosis of XE. Microbiological studies revealed enterococcus spp. and Peptostreptococcus magnus. Despite antibiotic treatment the patient died of heart failure due to systemic inflammation. Autopsy confirmed the diagnosis of XE with transmural extension into the peritoneal cavity. Such a lethal course of XE is extraordinary. Proposed causes of XE include obstruction, infection and hemorrhage. Demonstration of enterococcus spp. and P. magnus supports the probable significance of bacteria in the development of XE. Because this condition may mimic malignant disease macroscopically and histologically, knowledge of XE is of major importance for both pathologists and gynecologists. PMID:16725016

  2. Chemically induced conditional rescue of the reduced epidermal fluorescence8 mutant of Arabidopsis reveals rapid restoration of growth and selective turnover of secondary metabolite pools.

    PubMed

    Kim, Jeong Im; Ciesielski, Peter N; Donohoe, Bryon S; Chapple, Clint; Li, Xu

    2014-02-01

    The phenylpropanoid pathway is responsible for the biosynthesis of diverse and important secondary metabolites including lignin and flavonoids. The reduced epidermal fluorescence8 (ref8) mutant of Arabidopsis (Arabidopsis thaliana), which is defective in a lignin biosynthetic enzyme p-coumaroyl shikimate 3'-hydroxylase (C3'H), exhibits severe dwarfism and sterility. To better understand the impact of perturbation of phenylpropanoid metabolism on plant growth, we generated a chemically inducible C3'H expression construct and transformed it into the ref8 mutant. Application of dexamethasone to these plants greatly alleviates the dwarfism and sterility and substantially reverses the biochemical phenotypes of ref8 plants, including the reduction of lignin content and hyperaccumulation of flavonoids and p-coumarate esters. Induction of C3'H expression at different developmental stages has distinct impacts on plant growth. Although early induction effectively restored the elongation of primary inflorescence stem, application to 7-week-old plants enabled them to produce new rosette inflorescence stems. Examination of hypocotyls of these plants revealed normal vasculature in the newly formed secondary xylem, presumably restoring water transport in the mutant. The ref8 mutant accumulates higher levels of salicylic acid than the wild type, but depletion of this compound in ref8 did not relieve the mutant's growth defects, suggesting that the hyperaccumulation of salicylic acid is unlikely to be responsible for dwarfism in this mutant.

  3. Potential lethal and non-lethal effects of predators on dispersal of spider mites.

    PubMed

    Otsuki, Hatsune; Yano, Shuichi

    2014-11-01

    Predators can affect prey dispersal lethally by direct consumption or non-lethally by making prey hesitate to disperse. These lethal and non-lethal effects are detectable only in systems where prey can disperse between multiple patches. However, most studies have drawn their conclusions concerning the ability of predatory mites to suppress spider mites based on observations of their interactions on a single patch or on heavily infested host plants where spider mites could hardly disperse toward intact patches. In these systems, specialist predatory mites that penetrate protective webs produced by spider mites quickly suppress the spider mites, whereas generalist predators that cannot penetrate the webs were ineffective. By using a connected patch system, we revealed that a generalist ant, Pristomyrmex punctatus Mayr (Hymenoptera: Formicidae), effectively prevented dispersal of spider mites, Tetranychus kanzawai Kishida (Acari: Tetranychidae), by directly consuming dispersing individuals. We also revealed that a generalist predatory mite, Euseius sojaensis Ehara (Acari: Phytoseiidae), prevented between-patch dispersal of T. kanzawai by making them hesitate to disperse. In contrast, a specialist phytoseiid predatory mite, Neoseiulus womersleyi Schicha, allowed spider mites to escape an initial patch, increasing the number of colonized patches within the system. Our results suggest that ants and generalist predatory mites can effectively suppress Tetranychus species under some conditions, and should receive more attention as agents for conservation biological control in agroecosystems.

  4. Structural basis for a lethal mutation in U6 RNA.

    PubMed

    Sashital, Dipali G; Allmann, Anne M; Van Doren, Steven R; Butcher, Samuel E

    2003-02-18

    U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3' intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67 x G80), and disrupts a protonated C67 x A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (Delta Delta G(fold) = -3.6 +/- 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4. PMID:12578359

  5. Lethal otogenic Candida meningitis.

    PubMed

    Koch, S; Rudel, B; Tietz, H-J

    2004-10-01

    History revealed a chronic obstructive pulmonary condition which had been treated with prednisolone for a long time. There was a raised temperature with further signs of an acute inflammatory underlying disease and internal hydrocephalus. After performing trepanation, the symptoms of raised intercerebral pressure ceased. Candida albicans could be detected microbiologically in the cerebrospinal fluid. There was no pneumonia at the time of admission. Despite instituting immediate intensive care with administration of antibiotics and antimycotics, the patient died 11 days after inpatient admission. Autopsy revealed a C. albicans mycosis originating from the right middle ear with extensive suppurative meningitis, which was the immediate cause of death. Confluent bronchopneumonia had developed in both lower lung lobes at the time of death, but did not show any signs of mycosis and had contributed indirectly to the death of the patient.

  6. Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme

    PubMed Central

    Thøgersen, Mariane S.; Delpin, Marina W.; Melchiorsen, Jette; Kilstrup, Mogens; Månsson, Maria; Bunk, Boyke; Spröer, Cathrin; Overmann, Jörg; Nielsen, Kristian F.; Gram, Lone

    2016-01-01

    It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7–10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea. PMID:27695447

  7. Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme

    PubMed Central

    Thøgersen, Mariane S.; Delpin, Marina W.; Melchiorsen, Jette; Kilstrup, Mogens; Månsson, Maria; Bunk, Boyke; Spröer, Cathrin; Overmann, Jörg; Nielsen, Kristian F.; Gram, Lone

    2016-01-01

    It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7–10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

  8. Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

    PubMed Central

    Budziszewski, G J; Lewis, S P; Glover, L W; Reineke, J; Jones, G; Ziemnik, L S; Lonowski, J; Nyfeler, B; Aux, G; Zhou, Q; McElver, J; Patton, D A; Martienssen, R; Grossniklaus, U; Ma, H; Law, M; Levin, J Z

    2001-01-01

    We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype. PMID:11779813

  9. Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells

    PubMed Central

    Craven, Ryan

    2015-01-01

    ABSTRACT Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. One of the principle virulence factors is TcsL, a large protein toxin which glucosylates host GTPases to cause cytopathic and cytotoxic effects. TcsL has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. This study describes the use of conditionally immortalized murine pulmonary microvascular endothelial cells as a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities were introduced into a recombinant version of TcsL, and the activities of these mutants were compared to those of wild-type toxin. We observed that all mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL. IMPORTANCE Clostridium sordellii is a bacterium that can infect humans and cause serious disease and death. The principle virulence factor associated with clinical symptoms is a large protein toxin known as lethal toxin. The mechanism of lethal-toxin intoxication is assumed to be similar to that of the homologous toxins from C. difficile, but very few studies have been done in the context of endothelial cells, a relevant target in C. sordellii infections. This study was designed to test the role of the lethal-toxin enzymatic activities and membrane localization in endothelial cell toxicity and host substrate modification. PMID:27303685

  10. Characterization of cottonseed nutrients composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines for fuzzless seed trait under well-watered and water stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton mutant near isogenic lines (NILs) for fuzzless seed trait has been used to investigate cell biology, genetic, and molecular processes of fiber initiation, development, fiber yield and quality. However, there is no information available on the effect of fuzzless seed trait on cottonseed nutrie...

  11. Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

    PubMed Central

    Babitzke, P; Granger, L; Olszewski, J; Kushner, S R

    1993-01-01

    RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed. Images PMID:8416898

  12. Starch mutants of Chlamydomonas

    SciTech Connect

    Berry-Lowe, S.L.; Schmidt, G.W. )

    1990-05-01

    Wild type Chlamydomonas accumulates starch and triglycerides when grown under nitrogen limiting conditions. Toward elucidation of the mechanisms for control of starch biosynthesis, we isolated mutants impaired int he accumulation of storage carbohydrates. Chlamydomonas reinhardtii (strain ya-12) was mutagenized by UV irradiation and colonies were screened by iodine staining after growth in darkness. Mutants, denoted ais for altered in iodine staining, have been characterized by electron microscopy and assays for starch synthease, ADPG-pyrophosphorylase, phosphoglucose isomerase (PGI), phosphoglucomutase and fructose 1,6-bisphosphatase, and amylase activities. Transcript analysis of wild type and mutant RNAs with PGI, ADPG-pyrophosphorylase, and waxy probes have also been carried out. No deficiencies of any of these components have been detected. Furthermore, long-term cultures of ya-12 and ais-1d in nitrogen-limited chemostats have been studied; starch also does not accumulate in ais-1d under these conditions. Thus, the lesion affects an essential factor of unknown identity that is required for starch synthesis.

  13. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.

    PubMed Central

    Robinson, J S; Klionsky, D J; Banta, L M; Emr, S D

    1988-01-01

    Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature

  14. Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ΔATpase pgr5 and ΔrbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii.

    PubMed

    Johnson, Xenie; Steinbeck, Janina; Dent, Rachel M; Takahashi, Hiroko; Richaud, Pierre; Ozawa, Shin-Ichiro; Houille-Vernes, Laura; Petroutsos, Dimitris; Rappaport, Fabrice; Grossman, Arthur R; Niyogi, Krishna K; Hippler, Michael; Alric, Jean

    2014-05-01

    The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation. PMID:24623849

  15. Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

    PubMed Central

    Lun, Shichun; Miranda, David; Kubler, Andre; Guo, Haidan; Maiga, Mariama C.; Winglee, Kathryn; Pelly, Shaaretha

    2014-01-01

    ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. PMID:25227469

  16. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth.

    PubMed Central

    Costigan, C; Gehrung, S; Snyder, M

    1992-01-01

    The Saccharomyces cerevisiae SPA2 protein localizes at sites involved in polarized cell growth in budding cells and mating cells. spa2 mutants have defects in projection formation during mating but are healthy during vegetative growth. A synthetic lethal screen was devised to identify mutants that require the SPA2 gene for vegetative growth. One mutant, called slk-1 (for synthetic lethal kinase), has been characterized extensively. The SLK1 gene has been cloned, and sequence analysis predicts that the SLK1 protein is 1,478 amino acid residues in length. Approximately 300 amino acids at the carboxy terminus exhibit sequence similarity with the catalytic domains of protein kinases. Disruption mutations have been constructed in the SLK1 gene. slk1 null mutants cannot grow at 37 degrees C, but many cells can grow at 30, 24, and 17 degrees C. Dead slk1 mutant cells usually have aberrant cell morphologies, and many cells are very small, approximately one-half the diameter of wild-type cells. Surviving slk1 cells also exhibit morphogenic defects; these cells are impaired in their ability to form projections upon exposure to mating pheromones. During vegetative growth, a higher fraction of slk1 cells are unbudded compared with wild-type cells, and under nutrient limiting conditions, slk1 cells exhibit defects in cell cycle arrest. The different slk1 mutant defects are partially rescued by an extra copy of the SSD1/SRK1 gene. SSD1/SRK1 has been independently isolated as a suppressor of mutations in genes involved in growth control, sit4, pde2, bcy1, and ins1 (A. Sutton, D. Immanuel, and K.T. Arnat, Mol. Cell. Biol. 11:2133-2148, 1991; R.B. Wilson, A.A. Brenner, T.B. White, M.J. Engler, J.P. Gaughran, and K. Tatchell, Mol. Cell. Biol. 11:3369-3373, 1991). These data suggest that SLK1 plays a role in both cell morphogenesis and the control of cell growth. We speculate that SLK1 may be a regulatory link for these two cellular processes. Images PMID:1545797

  17. Phosphate Homeostasis in Conditions of Phosphate Proficiency and Limitation in the Wild Type and the phoP Mutant of Streptomyces lividans.

    PubMed

    Smirnov, Aleksey; Esnault, Catherine; Prigent, Magali; Holland, Ian Barry; Virolle, Marie-Joelle

    2015-01-01

    Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

  18. Human ARF4 expression rescues sec7 mutant yeast cells.

    PubMed Central

    Deitz, S B; Wu, C; Silve, S; Howell, K E; Melançon, P; Kahn, R A; Franzusoff, A

    1996-01-01

    Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. PMID:8668142

  19. Lethal mutagenesis and evolutionary epidemiology.

    PubMed

    Martin, Guillaume; Gandon, Sylvain

    2010-06-27

    The lethal mutagenesis hypothesis states that within-host populations of pathogens can be driven to extinction when the load of deleterious mutations is artificially increased with a mutagen, and becomes too high for the population to be maintained. Although chemical mutagens have been shown to lead to important reductions in viral titres for a wide variety of RNA viruses, the theoretical underpinnings of this process are still not clearly established. A few recent models sought to describe lethal mutagenesis but they often relied on restrictive assumptions. We extend this earlier work in two novel directions. First, we derive the dynamics of the genetic load in a multivariate Gaussian fitness landscape akin to classical quantitative genetics models. This fitness landscape yields a continuous distribution of mutation effects on fitness, ranging from deleterious to beneficial (i.e. compensatory) mutations. We also include an additional class of lethal mutations. Second, we couple this evolutionary model with an epidemiological model accounting for the within-host dynamics of the pathogen. We derive the epidemiological and evolutionary equilibrium of the system. At this equilibrium, the density of the pathogen is expected to decrease linearly with the genomic mutation rate U. We also provide a simple expression for the critical mutation rate leading to extinction. Stochastic simulations show that these predictions are accurate for a broad range of parameter values. As they depend on a small set of measurable epidemiological and evolutionary parameters, we used available information on several viruses to make quantitative and testable predictions on critical mutation rates. In the light of this model, we discuss the feasibility of lethal mutagenesis as an efficient therapeutic strategy.

  20. Targeting Oncogenic Mutant p53 for Cancer Therapy

    PubMed Central

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

    Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels. PMID:26732534

  1. The acquisition of Clostridium tyrobutyricum mutants with improved bioproduction under acidic conditions after two rounds of heavy-ion beam irradiation

    PubMed Central

    Zhou, Xiang; Yang, Zhen; Jiang, Ting-Ting; Wang, Shu-Yang; Liang, Jian-Ping; Lu, Xi-Hong; Wang, Liang

    2016-01-01

    End-product inhibition is a key factor limiting the production of organic acid during fermentation. Two rounds of heavy-ion beam irradiation may be an inexpensive, indispensable and reliable approach to increase the production of butyric acid during industrial fermentation processes. However, studies of the application of heavy ion radiation for butyric acid fermentation engineering are lacking. In this study, a second 12C6+ heavy-ion irradiation-response curve is used to describe the effect of exposure to a given dose of heavy ions on mutant strains of Clostridium tyrobutyricum. Versatile statistical elements are introduced to characterize the mechanism and factors contributing to improved butyric acid production and enhanced acid tolerance in adapted mutant strains harvested from the fermentations. We characterized the physiological properties of the strains over a large pH value gradient, which revealed that the mutant strains obtained after a second round of radiation exposure were most resistant to harsh external pH values and were better able to tolerate external pH values between 4.5 and 5.0. A customized second round of heavy-ion beam irradiation may be invaluable in process engineering. PMID:27426447

  2. The acquisition of Clostridium tyrobutyricum mutants with improved bioproduction under acidic conditions after two rounds of heavy-ion beam irradiation.

    PubMed

    Zhou, Xiang; Yang, Zhen; Jiang, Ting-Ting; Wang, Shu-Yang; Liang, Jian-Ping; Lu, Xi-Hong; Wang, Liang

    2016-01-01

    End-product inhibition is a key factor limiting the production of organic acid during fermentation. Two rounds of heavy-ion beam irradiation may be an inexpensive, indispensable and reliable approach to increase the production of butyric acid during industrial fermentation processes. However, studies of the application of heavy ion radiation for butyric acid fermentation engineering are lacking. In this study, a second (12)C(6+) heavy-ion irradiation-response curve is used to describe the effect of exposure to a given dose of heavy ions on mutant strains of Clostridium tyrobutyricum. Versatile statistical elements are introduced to characterize the mechanism and factors contributing to improved butyric acid production and enhanced acid tolerance in adapted mutant strains harvested from the fermentations. We characterized the physiological properties of the strains over a large pH value gradient, which revealed that the mutant strains obtained after a second round of radiation exposure were most resistant to harsh external pH values and were better able to tolerate external pH values between 4.5 and 5.0. A customized second round of heavy-ion beam irradiation may be invaluable in process engineering. PMID:27426447

  3. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  4. The "Lethal Chamber": Further Evidence of the Euthanasia Option.

    ERIC Educational Resources Information Center

    Elks, Martin A.

    1993-01-01

    Historical discussions of the euthanasia or "lethal chamber" option in relation to people with mental retardation are presented. The paper concludes that eugenic beliefs in the primacy of heredity over environment and the positive role of natural selection may have condoned the poor conditions characteristic of large, segregated institutions and…

  5. Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance

    PubMed Central

    Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

    2014-01-01

    Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes. PMID:25177317

  6. Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

    PubMed

    Baldin, Clara; Valiante, Vito; Krüger, Thomas; Schafferer, Lukas; Haas, Hubertus; Kniemeyer, Olaf; Brakhage, Axel A

    2015-07-01

    The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores.

  7. Arenavirus extinction through lethal mutagenesis.

    PubMed

    de la Torre, Juan Carlos

    2005-02-01

    Viral hemorrhagic fevers represent serious human public health problems causing devastating and often lethal disease. Several hemorrhagic fevers are caused by arenaviruses including Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In recent years, increased air travel between Africa and other areas has led to the importation of LFV into the US, Europe, Japan, and Canada. This has raised awareness about arenaviruses as potential emerging viruses. Moreover, because of its severe morbidity and high mortality, and transmissibility from human to human, weaponized forms of LFV poses a real threat as agent of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these infections. Therefore, the importance of developing novel effective antiviral drugs to combat HF arenaviruses, for which the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) provides us with an excellent model system. Recent findings have shown that LCMV multiplication both in cultured cells and in vivo is highly susceptible to the mutagenic agent 5-fluorouracil (FU). FU-mediated extinction of LCMV was associated with only modest increases in virus mutation frequencies, but did not significantly affect virus replication and transcription, or virus particle formation. These findings indicate that, as with other riboviruses, lethal mutagenesis is effective also against LCMV raising the possibility of using this novel antiviral strategy to combat pathogenic arenaviruses. PMID:15649566

  8. [Hormone-dependent insertional Arabidopsis thaliana mutants with decreased viability and fertility].

    PubMed

    Tomilova, N B; Tomilov, A A; Ogarkova, O A; Soldatova, O P; Tarasov, V A

    2001-09-01

    We present data on the phenotype identification and genetic analysis of offspring in three lines of dominant morphological mutants of Arabidopsis thaliana having drastically reduced fertility (a sterile calluslike mutant, a flower mutant, and a dwarf mutant) and in five lines of recessive morphological mutants (four mutants with lethal seedlings and one pigmentation mutant). The mutants were selected from a collection of transgenic plants that had genomes carrying a T-DNA insertion of plasmid vectors pLD3 and pPCVRN4; the collection was created earlier via agrobacterial transformation of germinating seeds. The results presented here were obtained using compensation of hormonal imbalance in the insertional morphological mutants of A. thaliana by exogenous hormones. PMID:11642128

  9. DNA polymerase I is crucial for the repair of potentially lethal damage caused by the indirect effects of X irradiation in Escherichia coli

    SciTech Connect

    Billen, D.

    1985-07-01

    The radiosensitivity of an Escherichia coli mutant deficient in DNA polymerase I was measured in the presence of OH radical scavengers. The extreme X-ray sensitivity of the mutant could be abolished by OH radical scavengers if a sufficiently high level of radioprotector was present. There was a direct correlation between the OH radical scavenging activity of the chemicals tested (NO/sub 2//sup -/, n-butanol, glycerol, t-amyl alcohol, and t-butanol) and their protective ability. The author interprets the data as showing that the indirect actions of X rays (primarily OH radicals) result in major damage to the bacterial DNA which in large part consists of potentially lethal lesions. This potentially lethal damage is repaired through an enzymatic pathway requiring DNA polymerase I. I. In the mutant lacking DNA polymerase I, these potentially lethal lesions are expressed as cell lethality.

  10. The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

    PubMed Central

    Uchiyama, Junzo; Koshimizu, Eriko; Qi, Jie; Nanjappa, Purushothama; Imamura, Shintaro; Islam, Asiful; Neuberg, Donna; Amsterdam, Adam; Roberts, Thomas M.

    2008-01-01

    There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and

  11. Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

    PubMed

    Mori, Hirotada; Baba, Tomoya; Yokoyama, Katsushi; Takeuchi, Rikiya; Nomura, Wataru; Makishi, Kazuichi; Otsuka, Yuta; Dose, Hitomi; Wanner, Barry L

    2015-01-01

    Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein.

  12. Identification of essential genes and synthetic lethal gene combinations in Escherichia coli K-12.

    PubMed

    Mori, Hirotada; Baba, Tomoya; Yokoyama, Katsushi; Takeuchi, Rikiya; Nomura, Wataru; Makishi, Kazuichi; Otsuka, Yuta; Dose, Hitomi; Wanner, Barry L

    2015-01-01

    Here we describe the systematic identification of single genes and gene pairs, whose knockout causes lethality in Escherichia coli K-12. During construction of precise single-gene knockout library of E. coli K-12, we identified 328 essential gene candidates for growth in complex (LB) medium. Upon establishment of the Keio single-gene deletion library, we undertook the development of the ASKA single-gene deletion library carrying a different antibiotic resistance. In addition, we developed tools for identification of synthetic lethal gene combinations by systematic construction of double-gene knockout mutants. We introduce these methods herein. PMID:25636612

  13. Ethical language and decision-making for prenatally diagnosed lethal malformations.

    PubMed

    Wilkinson, Dominic; de Crespigny, Lachlan; Xafis, Vicki

    2014-10-01

    In clinical practice, and in the medical literature, severe congenital malformations such as trisomy 18, anencephaly, and renal agenesis are frequently referred to as 'lethal' or as 'incompatible with life'. However, there is no agreement about a definition of lethal malformations, nor which conditions should be included in this category. Review of outcomes for malformations commonly designated 'lethal' reveals that prolonged survival is possible, even if rare. This article analyses the concept of lethal malformations and compares it to the problematic concept of 'futility'. We recommend avoiding the term 'lethal' and suggest that counseling should focus on salient prognostic features instead. For conditions with a high chance of early death or profound impairment in survivors despite treatment, perinatal and neonatal palliative care would be ethical. However, active obstetric and neonatal management, if desired, may also sometimes be appropriate. PMID:25200733

  14. RAS Synthetic Lethal Screens Revisited: Still Seeking the Elusive Prize?

    PubMed

    Downward, Julian

    2015-04-15

    The RAS genes are critical oncogenic drivers activated by point mutation in some 20% of human malignancies. However, no pharmacologic approaches to targeting RAS proteins directly have yet succeeded, leading to suggestions that these proteins may be "undruggable." This has led to two alternative indirect approaches to targeting RAS function in cancer. One has been to target RAS signaling pathways downstream at tractable enzymes such as kinases, particularly in combination. The other, which is the focus of this review, has been to seek targets that are essential in cells bearing an activated RAS oncogene, but not those without. This synthetic lethal approach, while rooted in ideas from invertebrate genetics, has been inspired most strongly by the successful use of PARP inhibitors, such as olaparib, in the clinic to treat BRCA defective cancers. Several large-scale screens have been carried out using RNA interference-mediated expression silencing to find genes that are uniquely essential to RAS-mutant but not wild-type cells. These screens have been notable for the low degree of overlap between their results, with the possible exception of proteasome components, and have yet to lead to successful new clinical approaches to the treatment of RAS-mutant cancers. Possible reasons for these disappointing results are discussed here, along with a reevaluation of the approaches taken. On the basis of experience to date, RAS synthetic lethality has so far fallen some way short of its original promise and remains unproven as an approach to finding effective new ways of tackling RAS-mutant cancers. Clin Cancer Res; 21(8); 1802-9. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers." PMID:25878361

  15. RAS Synthetic Lethal Screens Revisited: Still Seeking the Elusive Prize?

    PubMed Central

    Downward, Julian

    2015-01-01

    The RAS genes are critical oncogenic drivers activated by point mutation in some 20% of human malignancies. However, no pharmacological approaches to targeting RAS proteins directly have yet succeeded, leading to suggestions that these proteins may be “undruggable.” This has led to two alternative indirect approaches to targeting RAS function in cancer. One has been to target RAS signaling pathways downstream at tractable enzymes such as kinases, particularly in combination. The other, which is the focus of this review, has been to seek targets that are essential in cells bearing an activated RAS oncogene, but not those without. This synthetic lethal approach, while rooted in ideas from invertebrate genetics, has been inspired most strongly by the successful use of PARP inhibitors, such as olaparib, in the clinic to treat BRCA defective cancers. Several large-scale screens have been carried out using RNA interference-mediated expression silencing to find genes that are uniquely essential to RAS mutant but not wild type cells. These screens have been notable for the low degree of overlap between their results, with the possible exception of proteasome components, and have yet to lead to successful new clinical approaches to the treatment of RAS mutant cancers. Possible reasons for these disappointing results are discussed here, along with a re-evaluation of the approaches taken. Based on experience to date, RAS synthetic lethality has so far fallen some way short of its original promise and remains unproven as an approach to finding effective new ways of tackling RAS mutant cancers. PMID:25878361

  16. Misonidazole and potentially lethal damage

    SciTech Connect

    Korbelik, M.; Palcic, B.; Skov, K.; Skarsgard, L.

    1982-03-01

    The existence of potentially lethal damage (PLD) is demonstrated in exponentially growing CHO cells exposed to misonidazole in hypoxia. The method of hypertonic post-treatment of cells was used in these studies. Misonidazole-induced PLD differs in many characteristics from radiation-induced PLD.The repair kinetics of misonidazole-induced PLD are much slower than for the repair of radiation-induced PLD (hours vs. minutes). No significant repair of misonidazole-induced PLD took place at 25/sup 0/C. Other differences are discussed. Hypertonic post-treatment of irradiated cells which had been pre-incubated with misonidazole to non-toxic levels, gave survival data consistent with the interpretation that no radiation PLD can be induced in such cells.

  17. Acute and sub-lethal response to mercury in Arctic and boreal calanoid copepods.

    PubMed

    Overjordet, Ida Beathe; Altin, Dag; Berg, Torunn; Jenssen, Bjørn Munro; Gabrielsen, Geir Wing; Hansen, Bjørn Henrik

    2014-10-01

    Acute lethal toxicity, expressed as LC50 values, is a widely used parameter in risk assessment of chemicals, and has been proposed as a tool to assess differences in species sensitivities to chemicals between climatic regions. Arctic Calanus glacialis and boreal Calanus finmarchicus were exposed to mercury (Hg(2+)) under natural environmental conditions including sea temperatures of 2° and 10°C, respectively. Acute lethal toxicity (96 h LC50) and sub-lethal molecular response (GST expression; in this article gene expression is used as a synonym of gene transcription, although it is acknowledged that gene expression is also regulated, e.g., at translation and protein stability level) were studied. The acute lethal toxicity was monitored for 96 h using seven different Hg concentrations. The sub-lethal experiment was set up on the basis of nominal LC50 values for each species using concentrations equivalent to 50, 5 and 0.5% of their 96 h LC50 value. No significant differences were found in acute lethal toxicity between the two species. The sub-lethal molecular response revealed large differences both in response time and the fold induction of GST, where the Arctic species responded both faster and with higher mRNA levels of GST after 48 h exposure. Under the natural exposure conditions applied in the present study, the Arctic species C. glacialis may potentially be more susceptible to mercury exposure on the sub-lethal level.

  18. Construction of Listeria monocytogenes mutants with in-frame deletions in the Phosphotransferase Transport System (PTS) and analysis of their growth under stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a food-borne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. ...

  19. Construction of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette (ABC) transporters and analysis of their growth under stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate since it can survive under multiple stress conditions such as low pH and low temperature. Understanding its survival under stress conditions is important to control this pathogen in food. ABC transporters have been shown...

  20. Alcohol Consumption and Nearly Lethal Suicide Attempts.

    ERIC Educational Resources Information Center

    Powell, Kenneth E.; Kresnow, Marcie-jo; Mercy, James A.; Potter, Lloyd B.; Swann, Alan C.; Frankowski, Ralph F.; Lee, Roberta K.; Bayer, Timothy L.

    2002-01-01

    Presents a case-control study of the association between nearly lethal suicide attempts and facets of alcohol consumption; namely, drinking frequency, drinking quantity, binge drinking, alcoholism, drinking within 3 hours of suicide attempt, and age began drinking. In bivariate analyses, all measures were associated with nearly lethal suicide…

  1. A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum.

    PubMed

    Berghammer, A; Bucher, G; Maderspacher, F; Klingler, M

    1999-06-01

    Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species.

  2. Lethal entanglement in baleen whales.

    PubMed

    Cassoff, Rachel M; Moore, Kathleen M; McLellan, William A; Barco, Susan G; Rotsteins, David S; Moore, Michael J

    2011-10-01

    Understanding the scenarios whereby fishing gear entanglement of large whales induces mortality is important for the development of mitigation strategies. Here we present a series of 21 cases involving 4 species of baleen whales in the NW Atlantic, describing the available sighting history, necropsy observations, and subsequent data analyses that enabled the compilation of the manners in which entanglement can be lethal. The single acute cause of entanglement mortality identified was drowning from entanglement involving multiple body parts, with the animal's inability to surface. More protracted causes of death included impaired foraging during entanglement, resulting in starvation after many months; systemic infection arising from open, unresolved entanglement wounds; and hemorrhage or debilitation due to severe gear-related damage to tissues. Serious gear-induced injury can include laceration of large vessels, occlusion of the nares, embedding of line in growing bone, and massive periosteal proliferation of new bone in an attempt to wall off constricting, encircling lines. These data show that baleen whale entanglement is not only a major issue for the conservation of some baleen whale populations, but is also a major concern for the welfare of each affected individual.

  3. Lethal photosensitization of Helicobacter species

    NASA Astrophysics Data System (ADS)

    Millson, Charles E.; Wilson, Michael; MacRobert, Alexander J.; Thurrell, Wendy; Mlkvy, Peter; Davies, Claire; Bown, Stephen G.

    1995-01-01

    Helicobacter pylori (H. pylori) is associated with a large number of gastroduodenal disorders. Clearance of the bacteria has been shown to benefit patients with duodenal ulcers, gastric ulcers, and certain rare types of gastric tumors. Broad-spectrum antibiotics are the mainstay of current treatment strategies but side-effects, poor compliance, and drug resistance limit their usefulness. We sensitized H. pylori with toluidine blue, haematoporphyrin derivative, aluminum disulphonated phthalocyanine, methylene blue or protoporphyrin IX prior to exposure to low-power laser light from either a gallium aluminum arsenide laser or a helium neon gas laser. All 5 sensitizers caused reductions of greater than 1000-fold in the number of viable bacteria. Light alone had no effect and only HpD caused a significant decrease in bacterial numbers without laser light. Next, we sensitized H. mustelae on explanted ferret gastric mucosa (ex vivo) with the same sensitizers and exposed them to light from a copper vapor pumped dye laser tuned appropriately. MB caused significant reductions in bacterial counts. Successful lethal photosensitization of Helicobacter pylori both in vitro and ex vivo raises the possibility of a local method for eradicating the bacteria, especially as the bacteria are only found in those parts of the upper gastrointestinal tract that are accessible to the endoscope.

  4. Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N'-nitro-nitrosoguanidine

    SciTech Connect

    Avalos, J.; Casadesus, J.; Cerda-Olmedo, E.

    1985-01-01

    N-methyl-N'-nitrosoguanidine and to a lesser extent UV radiation are very mutagenic for Gibberella microconidia. The recommended nitrosoguanidine doses lead to much higher frequencies of mutants than are found in other microorganisms. The frequency of mutants among the survivors increases linearly with the nitrosoguanidine dose (molar concentration x time); the absolute number of viable mutants in a given population reaches a maximum for a dose of ca. 0.7 M x s. The microconidia are uninucleate. The onset of germination brings about increased lethality of nitrosoguanidine, but it does not modify the action of UV radiation. Mycelia are more resistant than spores to both agents. Visible illumination effectively prevents lethality when given immediately after UV irradiation. Auxotrophs and color mutants are very easily obtained. Pink adenine auxotrophs and several classes of color mutants are affected in the biosynthesis of the carotenoid pigment, neurosporaxanthin.

  5. Influence of potentially lethal temperature and food on the behavior of juvenile chum salmon, Oncorhynchus keta, under simulated marine conditions. Canadian data report of fisheries and aquatic sciences Number 1040

    SciTech Connect

    Korstrom, J.S.

    1998-12-31

    This is one of a series of reports that describe results of field and laboratory studies on the effect of heated sea water on juvenile chum salmon. The studies were initiated in response to potential increases in the thermal discharge from BC Hydro`s Burrard Generating Station into the marine waters of Port Moody Arm, Burrard Inlet. The report presents results of the first of two 1997 studies, in which the behaviour of chum salmon in response to heated sea water was investigated in the laboratory using a water column simulator that mimicked conditions the fish may encounter in Port Moody Arm. The behaviour of the salmon was examined under controlled conditions during a changing thermal regime and under thermally stratified conditions. The response of the fish to food, their swimming, and school positions were quantified in relation to experimental conditions.

  6. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir

    PubMed Central

    de Ávila, Ana I.; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens. PMID:27755573

  7. Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure.

    PubMed

    Jacobs, M F; Andersen, J B; Borchert, T V; Kontinen, V P

    1995-07-01

    High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42 degrees C, but not at 30 degrees C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the secA/div locus.

  8. Dominant lethal phenotype of a mutation in the -35 recognition region of Escherichia coli sigma 70.

    PubMed Central

    Keener, J; Nomura, M

    1993-01-01

    A dominant lethal mutation in the Escherichia coli rpoD gene, which encodes sigma 70, the promoter recognition subunit of RNA polymerase, was isolated after random mutagenesis. The lethal gene was maintained under control of the lac repressor on a low copy plasmid. An amount of lethal sigma 70 that was nearly equimolar with the chromosomally encoded sigma 70 was sufficient to cause cessation of growth. RNA synthesis per unit cell mass was unaffected, but protein synthesis was inhibited by the mutant sigma 70. The amino acid change (Glu-585 to Gln) was in a region of sigma 70 thought to bind the -35 hexamer of the promoter, and the mutant sigma 70 caused increased expression from promoters with nonconsensus bases in the third position of the -35 hexamer. A null mutation of the fis gene could partially suppress the mutant phenotype. These properties are consistent with those expected of a sigma 70 insensitive to growth rate control of rRNA and tRNA promoters. Images Fig. 2 PMID:7680477

  9. Lens Extrusion from Laminin Alpha 1 Mutant Zebrafish

    PubMed Central

    Semina, Elena V.; Duncan, Melinda K.

    2014-01-01

    We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1a69/a69. Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in the lama1a69/a69 mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye. PMID:24526906

  10. Gravity-controlled asymmetrical transport of auxin regulates a gravitropic response in the early growth stage of etiolated pea (Pisum sativum) epicotyls: studies using simulated microgravity conditions on a three-dimensional clinostat and using an agravitropic mutant, ageotropum.

    PubMed

    Hoshino, Tomoki; Miyamoto, Kensuke; Ueda, Junichi

    2007-09-01

    Increased expression of the auxin-inducible gene PsIAA4/5 was observed in the elongated side of epicotyls in early growth stages of etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown in a horizontal or an inclined position under 1 g conditions. Under simulated microgravity conditions on a 3D clinostat, accumulation of PsIAA4/5 mRNA was found throughout epicotyls showing automorphosis. Polar auxin transport in the proximal side of epicotyls changed when the seedlings were grown in a horizontal or an inclined position under 1 g conditions, but that under clinorotation did not, regardless of the direction of seed setting. Accumulation of PsPIN1 and PsPIN2 mRNAs in epicotyls was affected by gravistimulation, but not by clinorotation. Under 1 g conditions, auxin-transport inhibitors made epicotyls of seedlings grown in a horizontal or inclined position grow toward the proximal direction to cotyledons. These inhibitors led to epicotyl bending toward the cotyledons in seedlings grown in an inclined position under clinorotation. Polar auxin transport, as well as growth direction, of epicotyls of the agravitropic mutant ageotropum did not respond to various gravistimulation. These results suggest that alteration of polar auxin transport in the proximal side of epicotyls regulates the graviresponse of pea epicotyls. PMID:17712525

  11. Cardiac-Specific Activation of IKK2 Leads to Defects in Heart Development and Embryonic Lethality.

    PubMed

    Kraut, Bärbel; Maier, Harald J; Kókai, Enikö; Fiedler, Katja; Boettger, Thomas; Illing, Annett; Kostin, Sawa; Walther, Paul; Braun, Thomas; Wirth, Thomas

    2015-01-01

    The transcription factor NF-κB has been associated with a range of pathological conditions of the heart, mainly based on its function as a master regulator of inflammation and pro-survival factor. Here, we addressed the question what effects activation of NF-κB can have during murine heart development. We expressed a constitutively active (CA) mutant of IKK2, the kinase activating canonical NF-κB signaling, specifically in cardiomyocytes under the control of the α-myosin heavy chain promoter. Expression of IKK2-CA resulted in embryonic lethality around E13. Embryos showed defects in compact zone formation and the contractile apparatus, and overall were characterized by widespread inflammation with infiltration of myeloid cells. Gene expression analysis suggested an interferon type I signature, with increased expression of interferon regulatory factors. While apoptosis of cardiomyocytes was only increased at later stages, their proliferation was decreased early on, providing an explanation for the disturbed compact zone formation. Mechanistically, this could be explained by activation of the JAK/STAT axis and increased expression of the cell cycle inhibitor p21. A rescue experiment with an IκBα superrepressor demonstrated that the phenotype was dependent on NF-κB. We conclude that activation of NF-κB is detrimental during normal heart development due to excessive activation of pro-inflammatory pathways.

  12. On lethal injections and the death penalty.

    PubMed

    1982-10-01

    A brief account is given of the recent adoption by several states of capital punishment by lethal injection and the objections that this move has aroused. Critics argue that, because of difficulties in administering the drugs and in determining the proper dosage, this method may actually be less humane than other means of execution. Articles in medical journals have forcefully expressed the profession's opposition to lethal injections and to physician involvement in their administration.

  13. Lethal factor, but not edema factor, is required to cause fatal anthrax in cynomolgus macaques after pulmonary spore challenge.

    PubMed

    Hutt, Julie A; Lovchik, Julie A; Drysdale, Melissa; Sherwood, Robert L; Brasel, Trevor; Lipscomb, Mary F; Lyons, C Rick

    2014-12-01

    Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti-protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores.

  14. A search for radiosensitive mouse mutants by use of the micronucleus technique.

    PubMed

    van Buul, P P; Tuinenburg-Bolraap, A; Searle, A G; Natarajan, A T

    1987-01-01

    In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel (Slcon), scabby (scb), viable dominant spotting (Wv), quaking (qk), fidget (fi) and postaxial hemimelia (px), heterozygous lurcher (Lc), hemizygous gyro (Gy), the compounds Slcon/grizzle-belly (SlgbH) and Wv/rump-white (Rw) and MS/Ae mice, were regarded as radiosensitive, with Slcon/Slcon the highest in rank order. Homozygous wabbler-lethal (wl) and wasted (wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.

  15. Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

    PubMed Central

    Lai, Chiou-Yan; Cronan, John E.

    2004-01-01

    FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature. PMID:14996818

  16. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology. PMID:26472760

  17. Gene essentiality and synthetic lethality in haploid human cells.

    PubMed

    Blomen, Vincent A; Májek, Peter; Jae, Lucas T; Bigenzahn, Johannes W; Nieuwenhuis, Joppe; Staring, Jacqueline; Sacco, Roberto; van Diemen, Ferdy R; Olk, Nadine; Stukalov, Alexey; Marceau, Caleb; Janssen, Hans; Carette, Jan E; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio; Brummelkamp, Thijn R

    2015-11-27

    Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.

  18. A new lethal chondrodysplasia with spondylocostal dysostosis, multiple internal anomalies and Dandy-Walker cyst.

    PubMed

    Moerman, P; Vandenberghe, K; Fryns, J P; Haspeslagh, M; Lauweryns, J M

    1985-02-01

    We describe here a female infant, exhibiting lethal short-limbed dwarfism. The condition superficially resembled achondrogenesis. However, unlike achondrogenesis there was an associated severe spondylocostal dysostosis and major non-skeletal anomalies, particularly a cerebellar Dandy-Walker cyst, cardiovascular and urogenital malformations. The chondroosseous morphology was nonspecific. The case is believed to be unique. It is therefore suggested that this constellation of anomalies constitutes a "new" lethal syndrome, different from the delineated chondrodysplasias. PMID:3884191

  19. Effects of fuzzless cottonseed phenotype on cottonseed nutrient composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines under well-watered and water stress conditions.

    PubMed

    Bellaloui, Nacer; Turley, Rickie B

    2013-01-01

    There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using five sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (N) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (F) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds.Generally and with few exceptions, seed protein was higher in fuzzy lines than in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality

  20. Effects of fuzzless cottonseed phenotype on cottonseed nutrient composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines under well-watered and water stress conditions1

    PubMed Central

    Bellaloui, Nacer; Turley, Rickie B.

    2013-01-01

    There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using five sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (N) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (F) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds.Generally and with few exceptions, seed protein was higher in fuzzy lines than in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality

  1. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  2. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  3. Crystallographic studies of the Anthrax lethal toxin. Annual report

    SciTech Connect

    Frederick, C.A.

    1996-07-01

    The lethal form of Anthrax results from the inhalation of anthrax spores. Death is primarily due to the effects of the lethal toxin (Protective Antigen (PA) + Lethal Factor) from the causative agent, Bacillus anthracis. All the Anthrax vaccines currently in use or under development contain or produce PA, the major antigenic component of anthrax toxin, and there is a clear need for an improved vaccine for human use. In the previous report we described the first atomic resolution structure of PA, revealing that the molecule is composed largely of beta-sheets organized into four domains. This information can be used in the design. of recombinant PA vaccines. In this report we describe additional features of the full-length PA molecule derived from further crystallographic refinement and careful examination of the structure. We compare two crystal forms of PA grown at different pH values and discuss the functional implications. A complete definition of the function of each domain must await the crystal structure of the PA63 heptamer. We have grown crystals of the heptamer under both detergent and detergent-free conditions, and made substantial progress towards the crystal structure. The mechanism of anthrax intoxication in the light of our results is reviewed.

  4. New form of platyspondylic lethal chondrodysplasia.

    PubMed

    Akaba, K; Nishimura, G; Hashimoto, M; Wakabayashi, T; Kanasugi, H; Hayasaka, K

    1996-12-30

    We report on a sporadic case of hitherto unknown lethal skeletal dysplasia. The cardinal clinical manifestations consisted of frontal bossing, cloudy corneae, low nasal ridge, and micrognathia, hypoplastic thorax, and rhizomelic micromelia. Laryngoscopy and neck CT disclosed laryngeal stenosis, and brain CT demonstrated hypoplasia of the corpus callosum. Skeletal survey demonstrated hypoplasia of facial bones and short skull base, extremely severe platyspondyly, hypoplastic ilia, and delayed epiphyseal ossification and rhizomelic shortness of tubular bones. The long bones appeared overtubulated with exaggerated metaphyseal flaring. The humeri were particularly short and bowed. Bowing of the radii and ulnae with subluxation of radial heads presented as a Madelung-like deformity. Unlike the long bones, the short tubular bones were not short and normally modeled. The skeletal changes were superficially similar to those in a group of lethal platyspondylic chondrodysplasias, but were inconsistent with any known subtypes of this group or other lethal skeletal dysplasias. PMID:8989469

  5. Inadequate anaesthesia in lethal injection for execution.

    PubMed

    Koniaris, Leonidas G; Zimmers, Teresa A; Lubarsky, David A; Sheldon, Jonathan P

    Anaesthesia during lethal injection is essential to minimise suffering and to maintain public acceptance of the practice. Lethal injection is usually done by sequential administration of thiopental, pancuronium, and potassium chloride. Protocol information from Texas and Virginia showed that executioners had no anaesthesia training, drugs were administered remotely with no monitoring for anaesthesia, data were not recorded and no peer-review was done. Toxicology reports from Arizona, Georgia, North Carolina, and South Carolina showed that post-mortem concentrations of thiopental in the blood were lower than that required for surgery in 43 of 49 executed inmates (88%); 21 (43%) inmates had concentrations consistent with awareness. Methods of lethal injection anaesthesia are flawed and some inmates might experience awareness and suffering during execution.

  6. Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae.

    PubMed

    Toyoshima, Yoshiyuki; Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo; Hamada, Ryoko; Iwashita, Kazuhiro; Satoh, Katsuya; Narumi, Issay

    2012-12-01

    Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/μm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation.

  7. Live deaths online: internet suicide and lethality.

    PubMed

    Klein, Carolina A

    2012-01-01

    The Internet provides an infinite platform for the portrayal of lethal events. Beyond mere display, however, it dispenses information, allows for participation and sharing of content, and constitutes a virtual interactive forum. The Internet may ultimately shape society's approach to perceiving and dealing with death. Thus, psychiatrists may wish to be aware of these matters so that they may be considered in assessments and clinical care. In this article, the author attempts to identify key online locations where lethality is portrayed and how it may affect the individual patient and practitioner and the population at large.

  8. Therapeutically targeting RNA viruses via lethal mutagenesis.

    PubMed

    Graci, Jason D; Cameron, Craig E

    2008-11-01

    RNA viruses exhibit increased mutation frequencies relative to other organisms. Recent work has attempted to exploit this unique feature by increasing the viral mutation frequency beyond an extinction threshold, an antiviral strategy known as lethal mutagenesis. A number of novel nucleoside analogs have been designed around this premise. Herein, we review the quasispecies nature of RNA viruses and survey the antiviral, biological and biochemical characteristics of mutagenic nucleoside analogs, including clinically-used ribavirin. Biological implications of modulating viral replication fidelity are discussed in the context of translating lethal mutagenesis into a clinically-useful antiviral strategy.

  9. Lethality of sortase depletion in Actinomyces oris caused by excessive membrane accumulation of a surface glycoprotein.

    PubMed

    Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

    2014-12-01

    Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harbouring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalysed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens.

  10. Lethality of Sortase Depletion in Actinomyces oris Caused by Excessive Membrane Accumulation of a Surface Glycoprotein

    PubMed Central

    Wu, Chenggang; Huang, I-Hsiu; Chang, Chungyu; Reardon-Robinson, Melissa Elizabeth; Das, Asis; Ton-That, Hung

    2014-01-01

    Sortase, a cysteine-transpeptidase conserved in Gram-positive bacteria, anchors on the cell wall many surface proteins that facilitate bacterial pathogenesis and fitness. Genetic disruption of the housekeeping sortase in several Gram-positive pathogens reported thus far attenuates virulence, but not bacterial growth. Paradoxically, we discovered that depletion of the housekeeping sortase SrtA was lethal for Actinomyces oris; yet, all of its predicted cell wall-anchored protein substrates (AcaA-N) were individually dispensable for cell viability. Using Tn5-transposon mutagenesis to identify factors that upend lethality of srtA deletion, we uncovered a set of genetic suppressors harboring transposon insertions within genes of a locus encoding AcaC and a LytR-CpsA-Psr (LCP)-like protein. AcaC was shown to be highly glycosylated and dependent on LCP for its glycosylation. Upon SrtA depletion, the glycosylated form of AcaC, hereby renamed GspA, was accumulated in the membrane. Overexpression of GspA in a mutant lacking gspA and srtA was lethal; conversely, cells overexpressing a GspA mutant missing a membrane-localization domain were viable. The results reveal a unique glycosylation pathway in A. oris that is coupled to cell wall anchoring catalyzed by sortase SrtA. Significantly, this novel phenomenon of glyco-stress provides convenient cell-based assays for developing a new class of inhibitors against Gram-positive pathogens. PMID:25230351

  11. Development of ELISA based detection system for lethal toxin of Clostridium sordellii

    PubMed Central

    Arya, Preetika; Ponmariappan, S.; Singh, Lokendra; Kumar, Om

    2013-01-01

    Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund's complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples. PMID:23852299

  12. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development

    PubMed Central

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  13. Use of a Conditional Ubr5 Mutant Allele to Investigate the Role of an N-End Rule Ubiquitin-Protein Ligase in Hedgehog Signalling and Embryonic Limb Development.

    PubMed

    Kinsella, Elaine; Dora, Natalie; Mellis, David; Lettice, Laura; Deveney, Paul; Hill, Robert; Ditzel, Mark

    2016-01-01

    Hedgehog (Hh) signalling is a potent regulator of cell fate and function. While much is known about the events within a Hh-stimulated cell, far less is known about the regulation of Hh-ligand production. Drosophila Hyperplastic Discs (Hyd), a ubiquitin-protein ligase, represents one of the few non-transcription factors that independently regulates both hh mRNA expression and pathway activity. Using a murine embryonic stem cell system, we revealed that shRNAi of the mammalian homologue of hyd, Ubr5, effectively prevented retinoic-acid-induced Sonic hedgehog (Shh) expression. We next investigated the UBR5:Hh signalling relationship in vivo by generating and validating a mouse bearing a conditional Ubr5 loss-of-function allele. Conditionally deleting Ubr5 in the early embryonic limb-bud mesenchyme resulted in a transient decrease in Indian hedgehog ligand expression and decreased Hh pathway activity, around E13.5. Although Ubr5-deficient limbs and digits were, on average, shorter than control limbs, the effects were not statistically significant. Hence, while loss of UBR5 perturbed Hedgehog signalling in the developing limb, there were no obvious morphological defects. In summary, we report the first conditional Ubr5 mutant mouse and provide evidence for a role for UBR5 in influencing Hh signalling, but are uncertain to whether the effects on Hedgehog signaling were direct (cell autonomous) or indirect (non-cell-autonomous). Elaboration of the cellular/molecular mechanism(s) involved may help our understanding on diseases and developmental disorders associated with aberrant Hh signalling. PMID:27299863

  14. Appearance of recessive lethal mutations in derivatives of an unstable X{sup Z} chromosome of Drosophila melanogaster

    SciTech Connect

    Yurchenko, N.N.; Koryakov, D.E.; Zakharov, I.K.

    1995-09-01

    An X{sup Z} chromosome isolated from a natural population of Drosophila melanogaster is characterized by spontaneous mutability of the genes yellow, white, and singed and the appearance of chromosomal rearrangements. In mutant lines derived from the line carrying the X{sup Z} chromosome that had one, two, or three unstable vision mutations (markers), the rate of appearance of sex-linked lethal mutations was analyzed. This rate was shown to increase with an increase in the number of markers in a line. This phenomenon, termed {open_quotes}marker induction,{close_quotes} might explain the phenotypic homogeneity of natural Drosophila populations. Spontaneous lethal mutations were mapped, and their nonrandom distribution along the X{sup Z} chromosome was shown. Along with common {open_quotes}hot spot{close_quotes} sites of lethal mutations, the derivatives of the X{sup Z} chromosomes had their own specific sites for lethal mutations. In some cases, the appearance of lethal mutations was accompanied by the formation of inversions in the X{sup Z} chromosome. The lethal destabilization of the X{sup Z} derivatives, caused by selection for accumulation of visible mutations, is associated with an increase in the number of hot spots for nuclear mutations. Presumably, these hot spots are hot sites for the transposition of mobile genetic elements. 18 refs., 4 figs., 3 tabs.

  15. Macrophage cytotoxicity in lethal and non-lethal murine malaria and the effect of vaccination.

    PubMed Central

    Taverne, J; Treagust, J D; Playfair, J H

    1986-01-01

    We investigated the development of cell-mediated immunity in lethal and non-lethal malarial infections by assaying the cytotoxic activity of spleen cells for L929 tumour cells at different times after infection of mice with the lethal P. berghei, a lethal variant of Plasmodium yoelii and the non-lethal P. yoelii and P. chabaudi. In all cases the cytotoxicity increased to a peak during the first week and then diminished but the time of the peak varied with the infection; its activity was lowest with P. berghei. A second peak occurred in the non-lethal infections at the time of recovery. A protective vaccine accelerated and enhanced the early peak of cytotoxicity. The activity was mediated by adherent phagocytic cells, probably through the release of tumour necrosis factor (TNF) by macrophages since it was inhibited by antiserum against recombinant mouse TNF and did not destroy TNF-resistant L929 cells. Its induction was not dependent on T cells since it occurred in T cell-deficient mice infected with non-lethal P. yoelii. However, the accelerated increase associated with vaccination could be adoptively transferred by spleen lymphocytes from vaccinated mice. PMID:3542317

  16. Neonatal lethality of neural crest cell-specific Rest knockout mice is associated with gastrointestinal distension caused by aberrations of myenteric plexus.

    PubMed

    Aoki, Hitomi; Hara, Akira; Oomori, Yoshiyuki; Shimizu, Yasutake; Yamada, Yasuhiro; Kunisada, Takahiro

    2014-10-01

    RE1-silencing transcription factor (REST), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes. Rest null mice have embryonic lethality which prevents further investigations of the functions of the Rest gene in vivo. We studied neonatal but not embryonic lethality that was characterized by gastrointestinal tract dilation in the neural crest cell (NCC)-specific Rest conditional knockout (CKO) mice. While no histological abnormalities except the thinning of the digestive tract as a consequence of the gas accumulation were found in the digestive tract of the mutant mice, they do not have proper gastric retention after oral dye administration and the reduction of acetylcholinesterase (AChE) activity in NCC-derived myenteric plexus in the stomach was detected. High CO2 concentration in the dilated digestive tract of the Rest CKO mice indicates a failure of gut function by underdeveloped cholinergic transmission in the enteric nervous system. The observed gastrointestinal distension phenotype provides a model for understanding the genetic and molecular basis of NCC defects in humans. PMID:25135772

  17. Suicide intent and accurate expectations of lethality: predictors of medical lethality of suicide attempts.

    PubMed

    Brown, Gregory K; Henriques, Gregg R; Sosdjan, Daniella; Beck, Aaron T

    2004-12-01

    The degree of intent to commit suicide and the severity of self-injury were examined in individuals (N = 180) who had recently attempted suicide. Although a minimal association was found between the degree of suicide intent and the degree of lethality of the attempt, the accuracy of expectations about the likelihood of dying was found to moderate the relationship between suicide intent and lethality. Specifically, higher levels of suicide intent were associated with more lethal attempts but only for those individuals who had more accurate expectations about the likelihood of dying from their attempts.

  18. Deadly Lessons: Understanding Lethal School Violence.

    ERIC Educational Resources Information Center

    Moore, Mark H., Ed.; Petrie, Carol V., Ed.; Braga, Anthony A., Ed.; McLaughlin, Brenda L., Ed.

    This collection of papers is the outcome of the National Academies' effort to glean information from six different case studies of student-perpetrated school shootings. Part 1, "Case Studies of Lethal School Violence," includes: "The Copycat Factor: Mental Illness, Guns, and the Shooting Incident at Heritage High School, Rockdale County, Georgia"…

  19. The evolution of lethal intergroup violence

    PubMed Central

    Kelly, Raymond C.

    2005-01-01

    Recent findings and analyses in evolutionary biology, archaeology, and ethnology provide a favorable conjuncture for examining the evolution of lethal intergroup violence among hominids during the 2.9-million-year Paleolithic time span. Here, I seek to identify and investigate the main turning points in this evolutionary trajectory and to delineate the periodization that follows from this inquiry. PMID:16129826

  20. Cooperative Interaction Within RNA Virus Mutant Spectra.

    PubMed

    Shirogane, Yuta; Watanabe, Shumpei; Yanagi, Yusuke

    2016-01-01

    RNA viruses usually consist of mutant spectra because of high error rates of viral RNA polymerases. Growth competition occurs among different viral variants, and the fittest clones predominate under given conditions. Individual variants, however, may not be entirely independent of each other, and internal interactions within mutant spectra can occur. Examples of cooperative and interfering interactions that exert enhancing and suppressing effects on replication of the wild-type virus, respectively, have been described, but their underlying mechanisms have not been well defined. It was recently found that the cooperation between wild-type and variant measles virus genomes produces a new phenotype through the heterooligomer formation of a viral protein. This observation provides a molecular mechanism underlying cooperative interactions within mutant spectra. Careful attention to individual sequences, in addition to consensus sequences, may disclose further examples of internal interactions within mutant spectra. PMID:26162566

  1. Cholesterol Metabolism and Prostate Cancer Lethality.

    PubMed

    Stopsack, Konrad H; Gerke, Travis A; Sinnott, Jennifer A; Penney, Kathryn L; Tyekucheva, Svitlana; Sesso, Howard D; Andersson, Swen-Olof; Andrén, Ove; Cerhan, James R; Giovannucci, Edward L; Mucci, Lorelei A; Rider, Jennifer R

    2016-08-15

    Cholesterol metabolism has been implicated in prostate cancer pathogenesis. Here, we assessed the association of intratumoral mRNA expression of cholesterol synthesis enzymes, transporters, and regulators in tumor specimen at diagnosis and lethal prostate cancer, defined as mortality or metastases from prostate cancer in contrast to nonlethal disease without evidence of metastases after at least 8 years of follow-up. We analyzed the prospective prostate cancer cohorts within the Health Professionals Follow-up Study (n = 249) and the Physicians' Health Study (n = 153) as well as expectantly managed patients in the Swedish Watchful Waiting Study (n = 338). The expression of squalene monooxygenase (SQLE) was associated with lethal cancer in all three cohorts. Men with high SQLE expression (>1 standard deviation above the mean) were 8.3 times (95% confidence interval, 3.5 to 19.7) more likely to have lethal cancer despite therapy compared with men with the mean level of SQLE expression. Absolute SQLE expression was associated with lethal cancer independently from Gleason grade and stage, as was a SQLE expression ratio in tumor versus surrounding benign prostate tissue. Higher SQLE expression was tightly associated with increased histologic markers of angiogenesis. Collectively, this study establishes the prognostic value of intratumoral cholesterol synthesis as measured via SQLE, its second rate-limiting enzyme. SQLE expression at cancer diagnosis is prognostic for lethal prostate cancer both after curative-intent prostatectomy and in a watchful waiting setting, possibly by facilitating micrometastatic disease. Cancer Res; 76(16); 4785-90. ©2016 AACR.

  2. Differential expression of the lethal gene Luteus-Pa in cacao of the Parinari series.

    PubMed

    Rehem, B C; Almeida, A-A F; Figueiredo, G S F; Gesteira, A S; Santos, S C; Corrêa, R X; Yamada, M M; Valle, R R

    2016-01-01

    The recessive lethal character Luteus-Pa is found in cacao (Theobroma cacao) genotypes of the Parinari series (Pa) and is characterized by expression of leaf chlorosis and seedling death. Several genotypes of the Pa series are bearers of the gene responsible for the expression of the Luteus-Pa character, which can be used as a tool for determining relationships between genotypes of this group. To evaluate this phenomenon, we analyzed the differential expression of genes between mutant seedlings and wild-type hybrid Pa 30 x 169 seedlings, with the aim of elucidating the possible lethal mechanisms of the homozygous recessive character Luteus-Pa. Plant material was harvested from leaves of wild and mutant seedlings at different periods to construct a subtractive library and perform quantitative analysis using real-time PCR. The 649 sequences obtained from the subtractive library had an average length of 500 bp, forming 409 contigs. The probable proteins encoded were grouped into 10 functional categories. Data from ESTs identified genes associated with Rubisco, peroxidases, and other proteins and enzymes related to carbon assimilation, respiration, and photosystem 2. Mutant seedlings were characterized by synthesizing defective PsbO and PsbA proteins, which were overexpressed from 15 to 20 days after seedling emergence. PMID:26910005

  3. Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    PubMed

    Svensson, Malin; Fast, Jonas; Mossberg, Ann-Kristin; Düringer, Caroline; Gustafsson, Lotta; Hallgren, Oskar; Brooks, Charles L; Berliner, Lawrence; Linse, Sara; Svanborg, Catharina

    2003-12-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.

  4. Carbon monoxide and lethal arrhythmias

    SciTech Connect

    Farber, J.P.; Schwartz, P.J.; Vanoli, E.; Stramba-Badiale, M.; De Ferrari, G.M. )

    1990-12-01

    The effect of acute exposure to carbon monoxide on ventricular arrhythmias was studied in a previously described chronically maintained animal model of sudden cardiac death. In 60 percent of dogs with a healed anterior myocardial infarction, the combination of mild exercise and acute myocardial ischemia induces ventricular fibrillation. The events in this model are highly reproducible, thus allowing study by internal control analysis. Dogs that develop ventricular fibrillation during the test of exercise and acute myocardial ischemia are considered at high risk for sudden death and are defined as 'susceptible'; dogs that survive the test without a fatal arrhythmia are considered at low risk for sudden death and are defined as 'resistant.' In the current study, the effects of carboxyhemoglobin levels ranging from 5 to 15 percent were tested in resistant and susceptible dogs. A trend toward higher heart rates was observed at all levels of carboxyhemoglobin, although significant differences were observed only with 15 percent carboxyhemoglobin. This trend was observed at rest and during exercise in both resistant and susceptible dogs. In resistant animals, in which acute myocardial ischemia is typically associated with bradycardia even under the control condition, this reflex response occurred earlier and was augmented after exposure to carbon monoxide. This effect may depend on the increased hypoxic challenge caused by carbon monoxide, and thus on an augmentation of the neural reflex activation or a sensitization of the sinus node to acetylcholine induced by hypoxia. In both resistant and susceptible dogs, carbon monoxide exposure induced a worsening of ventricular arrhythmias in a minority of cases. This worsening was not reproducible in subsequent trials. These data indicate that acute exposure to carbon monoxide is seldom arrhythmogenic in dogs that have survived myocardial infarction. (Abstract Truncated)

  5. Developmental arrest of Caenorhabditis elegans BRAP-2 mutant exposed to oxidative stress is dependent on BRC-1.

    PubMed

    Koon, Janet C; Kubiseski, Terrance J

    2010-04-30

    Oxidative damage by reactive oxygen species is believed to be a contributor to the development of cancer and the physiological deterioration associated with aging. In this report, we describe the effect of reactive oxygen species exposure to a developing Caenorhabditis elegans organism containing a deletion in the homolog of BRCA1-associated protein 2 (BRAP-2). A mutant containing a deletion of brap-2 was highly sensitive to oxidizing conditions and demonstrated early larval arrest and lethality at low concentrations of the oxidative stress-inducing drug paraquat compared with the wild-type. This developmental arrest occurred early in the L1 stage and was dependent specifically on the function of the C. elegans ortholog of BRCA-1 tumor suppressor brc-1. We also show that developmental arrest in brap-2 mutants when exposed to oxidative stress was due to enhanced expression levels of the cell cycle inhibitor cki-1, and this increase in the expression levels of cki-1 requires brc-1 in brap-2 mutant animals. Our findings demonstrate that BRAP-2 is necessary for preventing an inappropriate response to elevated levels of reactive oxygen species by countering premature activation of BRC-1 and CKI-1.

  6. Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron-sulfur clusters.

    PubMed

    Tanaka, Naoyuki; Kanazawa, Miaki; Tonosaki, Keitaro; Yokoyama, Nao; Kuzuyama, Tomohisa; Takahashi, Yasuhiro

    2016-03-01

    Biological assembly of iron-sulfur (Fe-S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA-hscBA-fdx-iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe-S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe-S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe-S cluster-containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron-transfer Fe-S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe-S scaffold protein responsible for the de novo Fe-S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe-S cluster biosynthesis.

  7. Lesion mimic mutants

    PubMed Central

    Moeder, Wolfgang

    2008-01-01

    Over the last decade a substantial number of lesion mimic mutants (LMM) have been isolated and a growing number of the genes have been cloned. It is now becoming clear that these mutants are valuable tools to dissect various aspects of programmed cell death (PCD) and pathogen resistance pathways in plants. Together with other forward genetics approaches LMMs shed light on the PCD machinery in plant cells and revealed important roles for sphingolipids, Ca2+ and chloroplast-derived porphyrin-metabolites during cell death development. PMID:19513227

  8. Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

    PubMed Central

    Himmelfarb, H J; Simpson, E M; Friesen, J D

    1987-01-01

    Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain. PMID:3299061

  9. Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant.

    PubMed Central

    Machin, N A; Lee, J M; Barnes, G

    1995-01-01

    To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal beta-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSN1, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSN1 gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSN1 overexpression, showing that JSN1 is an allele-specific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSN1 during anaphase. Images PMID:8534919

  10. Disruption of the Rice Plastid Ribosomal Protein S20 Leads to Chloroplast Developmental Defects and Seedling Lethality

    PubMed Central

    Gong, Xiaodi; Jiang, Quan; Xu, Jianlong; Zhang, Jianhui; Teng, Sheng; Lin, Dongzhi; Dong, Yanjun

    2013-01-01

    Plastid ribosomal proteins (PRPs) are essential for ribosome biogenesis, plastid protein biosynthesis, chloroplast differentiation, and early chloroplast development. This study identifies the first rice PRP mutant, asl1 (albino seedling lethality1), which exhibits an albino lethal phenotype at the seedling stage. This albino phenotype was associated with altered chlorophyll (Chl) content and chloroplast development. Map-based cloning revealed that ASL1 encodes PRP S20 (PRPS20), which localizes to the chloroplast. ASL1 showed tissue-specific expression, as it was highly expressed in plumule and young seedlings but expressed at much lower levels in other tissues. In addition, ASL1 expression was regulated by light. The transcript levels of nuclear genes for Chl biosynthesis and chloroplast development were strongly affected in asl1 mutants; transcripts of some plastid genes for photosynthesis were undetectable. Our findings indicate that nuclear-encoded PRPS20 plays an important role in chloroplast development in rice. PMID:23979931

  11. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  12. Prevalence of lethal osteochondrodysplasias in Denmark.

    PubMed

    Andersen, P E

    1989-04-01

    The point prevalence at birth of lethal osteochondrodysplasias in a subregion of Denmark was estimated by a study of all children born January 1970 through December 1983. Two cases of thanatophoric dysplasia, one case of thanatophoric dysplasia with cloverleaf skull, two cases of micromelic bone dysplasia with cloverleaf skull, two cases of achondrogenesis type III, and three cases of achondrogenesis type IV were found. Two cases were unclassifiable due to lack of radiographs. In total, the point prevalence at birth was 15.4 per 100,000. Thus lethal osteochondrodysplasias seem to be more common than is generally assumed. The clinical and radiographic findings in micromelic bone dysplasia with cloverleaf skull are discussed in relation to thanatophoric dysplasia and achondrogenesis type IV. PMID:2789000

  13. Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles

    PubMed Central

    Andressoo, Jaan-Olle; Jans, Judith; de Wit, Jan; Coin, Frederic; Hoogstraten, Deborah; van de Ven, Marieke; Toussaint, Wendy; Huijmans, Jan; Thio, H. Bing; van Leeuwen, Wibeke J; de Boer, Jan; Egly, Jean-Marc; Hoeijmakers, Jan H. J; van der Horst, Gijsbertus T. J; Mitchell, James R

    2006-01-01

    Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of “null” alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals. PMID:17020410

  14. Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases

    PubMed Central

    Klein, Gracjana; Lindner, Buko; Brabetz, Werner; Brade, Helmut; Raina, Satish

    2009-01-01

    To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Δ(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 °C, besides showing accumulation of free lipid IVA precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Δ(waaC lpxL lpxM lpxP) strains showed primarily Kdo2-lipid IVA, indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 °C. These lipid IVA derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. ΔwaaA and Δ(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and σE signal transduction pathways. Δ(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Δ(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Δ(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II. PMID:19346244

  15. Lethal exposure: An integrated approach to pathogen transmission via environmental reservoirs

    PubMed Central

    Turner, Wendy C.; Kausrud, Kyrre L.; Beyer, Wolfgang; Easterday, W. Ryan; Barandongo, Zoë R.; Blaschke, Elisabeth; Cloete, Claudine C.; Lazak, Judith; Van Ert, Matthew N.; Ganz, Holly H.; Turnbull, Peter C. B.; Stenseth, Nils Chr.; Getz, Wayne M.

    2016-01-01

    To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1–2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways. PMID:27265371

  16. Lethal exposure: An integrated approach to pathogen transmission via environmental reservoirs.

    PubMed

    Turner, Wendy C; Kausrud, Kyrre L; Beyer, Wolfgang; Easterday, W Ryan; Barandongo, Zoë R; Blaschke, Elisabeth; Cloete, Claudine C; Lazak, Judith; Van Ert, Matthew N; Ganz, Holly H; Turnbull, Peter C B; Stenseth, Nils Chr; Getz, Wayne M

    2016-01-01

    To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1-2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways. PMID:27265371

  17. Brine shrimp lethality assay of Bacopa monnieri.

    PubMed

    D'Souza, Prashanth; Deepak, Mundkinajeddu; Rani, Padmaja; Kadamboor, Sandhya; Mathew, Anjana; Chandrashekar, Arun P; Agarwal, Amit

    2002-03-01

    Successive petroleum ether, chloroform, ethanol and water extracts, a saponin rich fraction (SRF) and bacoside A isolated from Bacopa monnieri were tested for brine shrimp lethality. Successive ethanol extracts and SRF showed potent activity. Bacoside A showed the maximum activity with a LC(50) of 38.3 microg/mL. The results confirmed the previous reports of an anticancer effect of Bacopa monnieri and suggest bacoside A as the active constituent. PMID:11933129

  18. Brine shrimp lethality assay of Bacopa monnieri.

    PubMed

    D'Souza, Prashanth; Deepak, Mundkinajeddu; Rani, Padmaja; Kadamboor, Sandhya; Mathew, Anjana; Chandrashekar, Arun P; Agarwal, Amit

    2002-03-01

    Successive petroleum ether, chloroform, ethanol and water extracts, a saponin rich fraction (SRF) and bacoside A isolated from Bacopa monnieri were tested for brine shrimp lethality. Successive ethanol extracts and SRF showed potent activity. Bacoside A showed the maximum activity with a LC(50) of 38.3 microg/mL. The results confirmed the previous reports of an anticancer effect of Bacopa monnieri and suggest bacoside A as the active constituent.

  19. Mice Expressing Mutant Trpv4 Recapitulate the Human TRPV4 Disorders††

    PubMed Central

    Chen, Yuqing; Lee, Brendan; Cohn, Daniel H.

    2014-01-01

    Activating mutations in TRPV4 are known to cause a spectrum of skeletal dysplasias ranging from autosomal dominant brachyolmia to lethal metatropic dysplasia. To develop an animal model of these disorders, we created transgenic mice expressing either wild-type or mutant TRPV4. Mice transgenic for wild-type Trpv4 showed no morphological changes at embryonic day 16.5, but did have a delay in bone mineralization. Overexpression of a mutant TRPV4 caused a lethal skeletal dysplasia that phenocopied many abnormalities associated with metatropic dysplasia in humans, including dumbbell-shaped long bones, a small ribcage, abnormalities in the autopod, and abnormal ossification in the vertebrae. The difference in phenotype between embryos transgenic for wild-type or mutant Trpv4 demonstrates that an increased amount of wild-type protein can be tolerated and that an activating mutation of this protein is required to produce a skeletal dysplasia phenotype. PMID:24644033

  20. Lethal interpersonal violence in the Middle Pleistocene.

    PubMed

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin. PMID:26018668

  1. Lethal Interpersonal Violence in the Middle Pleistocene

    PubMed Central

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M.; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin. PMID:26018668

  2. Lethal interpersonal violence in the Middle Pleistocene.

    PubMed

    Sala, Nohemi; Arsuaga, Juan Luis; Pantoja-Pérez, Ana; Pablos, Adrián; Martínez, Ignacio; Quam, Rolf M; Gómez-Olivencia, Asier; Bermúdez de Castro, José María; Carbonell, Eudald

    2015-01-01

    Evidence of interpersonal violence has been documented previously in Pleistocene members of the genus Homo, but only very rarely has this been posited as the possible manner of death. Here we report the earliest evidence of lethal interpersonal violence in the hominin fossil record. Cranium 17 recovered from the Sima de los Huesos Middle Pleistocene site shows two clear perimortem depression fractures on the frontal bone, interpreted as being produced by two episodes of localized blunt force trauma. The type of injuries, their location, the strong similarity of the fractures in shape and size, and the different orientations and implied trajectories of the two fractures suggest they were produced with the same object in face-to-face interpersonal conflict. Given that either of the two traumatic events was likely lethal, the presence of multiple blows implies an intention to kill. This finding shows that the lethal interpersonal violence is an ancient human behavior and has important implications for the accumulation of bodies at the site, supporting an anthropic origin.

  3. Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2

    PubMed Central

    Gaussand, Gwénaël M. D. J.-M.; Jia, Qi; van der Graaff, Eric; Lamers, Gerda E. M.; Fransz, Paul F.; Hooykaas, Paul J. J.; de Pater, Sylvia

    2011-01-01

    From a pool of transgenic Arabidopsis (Arabidopsis thaliana) plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D) on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type. DNA fragmentation in the nuclei of cells in the necrotic spots and a significant increase of caspase-3 and caspase-6 like activities in sns-D leaf extracts indicated that the sns-D mutation caused programmed cell death (PCD). The integration of the activator T-DNA caused an increase of the expression level of At1g13020, which encodes the eukaryotic translation initiation factor eIF4B2. The expression level of eIF4B2 was positively correlated with the severity of sns-D mutant phenotype. Overexpression of the eIF4B2 cDNA mimicked phenotypic traits of the sns-D mutant indicating that the sns-D mutant phenotype is indeed caused by activation tagging of eIF4B2. Thus, incorrect regulation of translation initiation may result in PCD. PMID:22639576

  4. The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin-regulated genes.

    PubMed

    Effendi, Yunus; Rietz, Steffen; Fischer, Urs; Scherer, Günther F E

    2011-01-01

    AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

  5. Lethal and sub-lethal effects on the Asian common toad Duttaphrynus melanostictus from exposure to hexavalent chromium.

    PubMed

    Fernando, Vindhya A K; Weerasena, Jagathpriya; Lakraj, G Pemantha; Perera, Inoka C; Dangalle, Chandima D; Handunnetti, Shiroma; Premawansa, Sunil; Wijesinghe, Mayuri R

    2016-08-01

    Chromium discharged in industrial effluents frequently occurs as an environmental pollutant, but the lethal and sub-lethal effects the heavy metal might cause in animals exposed to it have been insufficiently investigated. Selecting the amphibian Duttaphrynus melanostictus, we carried out laboratory tests to investigate the effects of short and long term exposure to hexavalent chromium (Cr(VI)) in both tadpoles and adult toads. The concentrations used were 0.002, 0.02, 0.2, 1.0 and 2.0mg/L, the first three corresponding to field levels. In vitro exposures were also carried out using toad erythrocytes and Cr(VI) concentrations of 0.0015, 0.003, 0.015, 0.03, 0.15mg/L. Mortality, growth retardation, developmental delays and structural aberrations were noted in the metal-treated tadpoles, with increasing incidence corresponding to increase in Cr(VI) level and duration of exposure. Many of the sub-lethal effects were evident with long term exposure to environmentally relevant levels of the toxicant. Changes in selected blood parameters and erythrocyte morphometry were also detected in Cr(VI) exposed toads, indicating anaemic and leucopenic conditions. In the genotoxicity study, DNA damage indicated by comet assay and increased micronuclei frequency, occurred at the low Cr(VI) concentrations tested. The multiple deleterious effects of exposure to chromium signal the need for monitoring and controlling the discharge of chromium to the environment. The dose-dependency and genotoxic effects observed in this widely distributed Asian toad indicates its suitability for monitoring heavy metal pollution in aquatic systems. PMID:27262939

  6. Lethal and sub-lethal effects of UVB on juvenile Biomphalaria glabrata (Mollusca: Pulmonata).

    PubMed

    Ruelas, Debbie S; Karentz, Deneb; Sullivan, John T

    2006-11-01

    Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects. PMID:16996081

  7. Lethal and sub-lethal effects of UVB on juvenile Biomphalaria glabrata (Mollusca: Pulmonata).

    PubMed

    Ruelas, Debbie S; Karentz, Deneb; Sullivan, John T

    2006-11-01

    Although Schistosoma mansoni occurs mainly in the tropics, where intense levels of solar radiation are present, the impact of ultraviolet (UV) light on schistosome transmission is not known. The purpose of this study was to investigate potential effects of UVB (290-320nm) on juvenile Biomphalaria glabrata, the snail intermediate host of S. mansoni. Albino and wild-type snails were exposed to doses of UVB from UV-fluorescent lamps, and the following were measured: survival, photoreactivation (light-mediated DNA repair), effects on feeding behavior, and morphological tissue abnormalities. Irradiation with UVB is lethal to B. glabrata in a dose-dependent manner. Exposure to white light subsequent to UVB irradiation enhances survival, probably by photoreactivation. The shell offers some, but not complete, protection. Experiments in which UVB transmittance through the shell was blocked with black nail polish suggest that injury to both exposed (headfoot) and shell-enclosed (mantle and visceral mass) tissues contributes to mortality in lethally irradiated snails. Wild-type (pigmented) snails are less susceptible to lethal effects of UVB than albino snails, and they may be more capable of photoreactivation. UVB exposure inhibits snail feeding behavior, and causes tentacle forks and growths on the headfoot. Thus, UVB may influence the life cycle of S. mansoni by both lethal and sub-lethal damage to the snail intermediate host. However, the ability of snails to photoreactivate may mitigate these effects.

  8. Reduced LPS phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants

    PubMed Central

    Rotman, Ella; Bratcher, Preston; Kuzminov, Andrei

    2009-01-01

    Summary The seqA defect in E. coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA co-lethality). To understand the nature of this chromosomal fragmentation, we characterized ΔseqA mutants and isolated suppressors of the ΔseqA recA lethality. We demonstrate that our ΔseqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of ΔseqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the ΔseqA mutants, but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the ΔseqA mutants. We also found that co-lethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane. PMID:19432803

  9. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

    PubMed Central

    2014-01-01

    Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

  10. Rapid Antibiotic Resistance Evolution of GASP Mutants

    NASA Astrophysics Data System (ADS)

    Zhang, Qiucen; Kim, Hyunsung; Pourmand, Nader; Austin, Robert

    2012-02-01

    The GASP phenotype in bacteria is due to a mutation which enables the bacteria to grow under high stress conditions where other bacteria stop growing. We probe using our Death Galaxy microenvironment how rapidly the GASP mutant can evolve resistance to mutagenic antibiotics compared to wild-type bacteria, and explore the genomic landscape changes due to the evolution of resistance.

  11. Engineered female-specific lethality for control of pest Lepidoptera.

    PubMed

    Jin, Li; Walker, Adam S; Fu, Guoliang; Harvey-Samuel, Timothy; Dafa'alla, Tarig; Miles, Andrea; Marubbi, Thea; Granville, Deborah; Humphrey-Jones, Nerys; O'Connell, Sinead; Morrison, Neil I; Alphey, Luke

    2013-03-15

    The sterile insect technique (SIT) is a pest control strategy involving the mass release of radiation-sterilized insects, which reduce the target population through nonviable matings. In Lepidoptera, SIT could be more broadly applicable if the deleterious effects of sterilization by irradiation could be avoided. Moreover, male-only release can improve the efficacy of SIT. Adequate methods of male-only production in Lepidoptera are currently lacking, in contrast to some Diptera. We describe a synthetic genetic system that allows male-only moth production for SIT and also replaces radiation sterilization with inherited female-specific lethality. We sequenced and characterized the doublesex (dsx) gene from the pink bollworm (Pectinophora gossypiella). Sex-alternate splicing from dsx was used to develop a conditional lethal genetic sexing system in two pest moths: the diamondback moth (Plutella xylostella) and pink bollworm. This system shows promise for enhancing existing pink bollworm SIT, as well as broadening SIT-type control to diamondback moth and other Lepidoptera.

  12. "Terminal burrowing behaviour"--a phenomenon of lethal hypothermia.

    PubMed

    Rothschild, M A; Schneider, V

    1995-01-01

    Between 1978 and 1994, 69 cases of death due to lethal hypothermia were examined in our Institute. In addition to the common findings associated with hypothermia we especially wanted to examine the so-called paradox reaction which refers to the undressing of persons in a state of severe (lethal) hypothermia. This is obviously the result of a peripheral vasodilatation effecting a feeling of warmth. In our material this paradoxical undressing occurred in 25% of the cases and nearly all exhibited an additional phenomenon which has not yet been described in the literature. Nearly all bodies with partial or complete disrobement were found in a position which indicated a final mechanism of protection i.e. under a bed, behind a wardrobe, in a shelf etc.. This is obviously an autonomous process of the brain stem, which is triggered in the final state of hypothermia and produces a primitive and burrowing-like behaviour of protection, as seen in (hibernating) animals. This phenomenon, which we refer to as "terminal burrowing behaviour", occurred predominantly with slow decreases in temperature and moderately cold conditions. PMID:7632602

  13. Architectural phenotypes in the transparent testa mutants of Arabidopsis thaliana

    PubMed Central

    Buer, Charles S.; Djordjevic, Michael A.

    2009-01-01

    Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants are compromised in the enzymatic steps or transcriptional regulators affecting flavonoid synthesis. tt mutant seedlings were grown on hard-slanted agar (a stress condition), under varying light conditions, and in soil to examine the resulting growth patterns. These tt mutants revealed a wide variety of architectural phenotypes in root and aerial tissues. Mutants with increased inflorescences, siliques, and lateral root density or reduced stature are traits that could affect plant yield or performance under certain environmental conditions. The regulatory genes affected in architectural traits may provide useful molecular targets for examination in other plants. PMID:19129166

  14. Cartilage matrix deficiency (cmd): a new autosomal recessive lethal mutation in the mouse.

    PubMed

    Rittenhouse, E; Dunn, L C; Cookingham, J; Calo, C; Spiegelman, M; Dooher, G B; Bennett, D

    1978-02-01

    A new autosomal recessive lethal mutation in the mouse designated cartilage matrix deficiency (cmd) is described. Homozygotes are dwarfed, and have abnormally short trunk, limbs, tail and snout, as well as a protruding tongue and cleft palate. The abdomen is distended because the foreshortened rib cage and spinal column forces the liver ventrad from its normal location. Histological and electron microscopic study reveals a deficiency of cartilage matrix in tracheal cartilage and in all cartilagenous bones examined. The syndrome closely resembles the rare lethal condition achondrogenesis, found in human infants, which is also believed to be due to an autosomal recessive gene. PMID:632744

  15. Execution by lethal injection, euthanasia, organ-donation and the proper goals of medicine.

    PubMed

    Varelius, Jukka

    2007-03-01

    In a recent issue of this journal, David Silver and Gerald Dworkin discuss the physicians' role in execution by lethal injection. Dworkin concludes that discussion by stating that, at that point, he is unable to think of an acceptable set of moral principles to support the view that it is illegitimate for physicians to participate in execution by lethal injection that would not rule out certain other plausible moral judgements, namely that euthanasia is under certain conditions legitimate and that organ-donation surgery is sometimes permissible. This article draws attention to some problems in the views of Silver and Dworkin and suggests moral principles which support the three moral views just mentioned.

  16. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    PubMed Central

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  17. Growth and development of maize that contains mutant tubulin genes

    SciTech Connect

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  18. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    SciTech Connect

    Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G; Chesler, Elissa J; Johnson, Dabney K; Goldowitz, Daniel

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  19. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, Oliver E.; Pan, David

    1994-01-01

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating.

  20. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, O.E.; Pan, D.

    1994-07-19

    A maize plant has in its genome a non-mutable form of a mutant allele designated vitX-8132. The allele is located at a locus designated as glt which conditions kernels having an altered starch characteristic. Maize plants including such a mutant allele produce a starch that does not increase in viscosity on cooling, after heating. 2 figs.

  1. [Lethal intoxication with arsenic using prepared butter].

    PubMed

    Weller, Jens-Peter; Larsch, Klaus-Peter; Teske, Jörg; Tröger, Hans Dieter

    2008-01-01

    The present case report deals with a lethal intoxication with arsenic mixed into butter. It describes the course of events over about two days on the basis of the statements by the persons involved, the clinical findings after the belated hospitalisation of the victim, the results of the first pathological autopsy, the forensic autopsy performed after exhumation and the results of the chemical-toxicological investigations. The results are discussed in relation to the later confession of the female perpetrator and her statements regarding a previous unsuccessful murder attempt by poisoning. It also presents the judgement pronounced by the court and the reasons given for it.

  2. Lethal predators: psychopathic, sadistic, and sane.

    PubMed

    Ochberg, Frank M; Brantley, Alan C; Hare, R D; Houk, Peter D; Ianni, Robert; James, Earl; O'Toole, Mary Ellen; Saathoff, Gregory

    2003-01-01

    The violent criminals defined in this article are a small, exceptionally dangerous group of offenders designated by the authors as "lethal predators." They have a history of sexual predation, have killed at least once, and are mentally abnormal but legally sane. They are highly likely to keep killing as long as they are free. Laws permitting civil commitment of dangerous and mentally abnormal sexual predators after they have completed criminal prison sentences have been upheld by the U.S. Supreme Court. Such laws can provide a legal means of keeping these highly dangerous killers confined so they cannot kill again.

  3. Lethal mobilization of DDT by cowbirds

    USGS Publications Warehouse

    Van Velzen, A.C.; Stiles, W.B.; Stickel, L.F.

    1972-01-01

    This study is an experimental demonstration of lethal mobilization of DDT by brown-headed cowbirds (Molothrus ater) and the effects of food deprivation on the distribution and loss of DDT, DDD, and DDE. The principal experimental group consisted of 20 birds fed a dietary dosage of 100 ppm of DDT for 13 days. After 2 days of full rations of untreated food, they were subjected to food restriction. Food was reduced to 43 percent of normal. Seven of the 20 birds died within 4 days. No birds died in the three control groups, treated as follows: ( 1 ) 20 birds fed 100 ppm DDT for 13 days and full rations of untreated food thereafter, (2) 20 birds fed only untreated food but subjected to food restriction, and (3) 20 birds fed full rations of untreated food throughout. In a pilot study, birds were fed 100, 200, or 300 ppm of DDT and subjected to two periods of food restriction, the first of these immediately after dosage ceased and the second 4 months later. DDT-dosed birds from all dosage levels died in each period of food restriction. Before the weight loss that accompanied food restriction, the brains of DDT-dosed birds had concentrations of DDT and DDD that were far below the lethal range. Concentrations increased rapidly to lethal levels. In these birds, DDT in carcasses decreased while DDD increased. DDT-dosed birds that died during food restriction lost 16 percent of their total body burden of DDT + DDD + DDE, 21 percent of their weight, and 81 percent of their fat. The DDT-dosed birds that were subjected to food restriction but survived lost a significantly greater proportion of their body burden of residues than similarly dosed birds not subjected to weight loss. Brain levels of DDT and DDD in birds that died during food restriction soon after dosage did not differ significantly from brain levels of birds that died in a period of food restriction 4 months after dosage. Concentrations of DDE were significantly higher in the latter group, although they were lower

  4. Direct selection of cloned DNA in Bacillus subtilis based on sucrose-induced lethality.

    PubMed Central

    Bramucci, M G; Nagarajan, V

    1996-01-01

    Expression of the Bacillus subtilis or Bacillus amyloliquefaciens sacB gene in the presence of sucrose is lethal for a variety of bacteria. Sucrose-induced lethality can be used to select for inactivation of sacB by insertion of heterologous DNA in sensitive bacteria. This procedure has not been applicable to B. subtilis heretofore because expression of wild-type sacB is not detrimental to B. subtilis. The W29 mutation in the B. amyloliquefaciens sacB gene interferes with processing of the levansucrase signal peptide. The W29 mutation does not affect growth of B. subtilis in media lacking sucrose. However, this mutation inhibited growth of B. subtilis in media containing sucrose. Inactivation of the fructose polymerase activity encoded by sacB indicated that levan production was essential for sucrose-induced lethality. As a result, it was possible to select for cloned DNA in B. subtilis by insertional inactivation of the mutant sacB gene located on a multicopy plasmid vector in medium containing sucrose. PMID:8899981

  5. Deciphering the Mechanisms of Developmental Disorders (DMDD): a new programme for phenotyping embryonic lethal mice.

    PubMed

    Mohun, Timothy; Adams, David J; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J; Hemberger, Myriam; Houart, Corinne; Hurles, Matt E; Robertson, Elizabeth; Smith, James C; Weaver, Tom; Weninger, Wolfgang

    2013-05-01

    International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The 'Deciphering the Mechanisms of Developmental Disorders' (DMDD) programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013)] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes. PMID:23519034

  6. Enhancement of Overgrowth by Gene Interactions in Lethal(2)giant Discs Imaginal Discs from Drosophila Melanogaster

    PubMed Central

    Buratovich, M. A.; Bryant, P. J.

    1997-01-01

    Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts as a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation. PMID:9335602

  7. Lethal infection thresholds of Paenibacillus larvae for honeybee drone and worker larvae (Apis mellifera).

    PubMed

    Behrens, Dieter; Forsgren, Eva; Fries, Ingemar; Moritz, Robin F A

    2010-10-01

    We compared the mortality of honeybee (Apis mellifera) drone and worker larvae from a single queen under controlled in vitro conditions following infection with Paenibacillus larvae, a bacterium causing the brood disease American Foulbrood (AFB). We also determined absolute P. larvae cell numbers and lethal titres in deceased individuals of both sexes up to 8 days post infection using quantitative real-time PCR (qPCR). Our results show that in drones the onset of infection induced mortality is delayed by 1 day, the cumulative mortality is reduced by 10% and P. larvae cell numbers are higher than in worker larvae. Since differences in bacterial cell titres between sexes can be explained by differences in body size, larval size appears to be a key parameter for a lethal threshold in AFB tolerance. Both means and variances for lethal thresholds are similar for drone and worker larvae suggesting that drone resistance phenotypes resemble those of related workers.

  8. Lethal infection thresholds of Paenibacillus larvae for honeybee drone and worker larvae (Apis mellifera).

    PubMed

    Behrens, Dieter; Forsgren, Eva; Fries, Ingemar; Moritz, Robin F A

    2010-10-01

    We compared the mortality of honeybee (Apis mellifera) drone and worker larvae from a single queen under controlled in vitro conditions following infection with Paenibacillus larvae, a bacterium causing the brood disease American Foulbrood (AFB). We also determined absolute P. larvae cell numbers and lethal titres in deceased individuals of both sexes up to 8 days post infection using quantitative real-time PCR (qPCR). Our results show that in drones the onset of infection induced mortality is delayed by 1 day, the cumulative mortality is reduced by 10% and P. larvae cell numbers are higher than in worker larvae. Since differences in bacterial cell titres between sexes can be explained by differences in body size, larval size appears to be a key parameter for a lethal threshold in AFB tolerance. Both means and variances for lethal thresholds are similar for drone and worker larvae suggesting that drone resistance phenotypes resemble those of related workers. PMID:20545737

  9. Combination of opium smoking and hypercholesterolemia augments susceptibility for lethal cardiac arrhythmia and atherogenesis in rabbit.

    PubMed

    Najafipour, Hamid; Joukar, Siyavash

    2012-09-01

    Opium consumption is increasing in some eastern societies, where it is grown. We investigated the effect of opium smoking on plasma atherogenic index and incidence of lethal cardiac arrhythmia, i.e. ventricular tachycardia (VT) and ventricular fibrillation (VF) in rabbits. Animals were divided into two-, normo- and hyper-cholesterolemic main groups fed with normal or high cholesterol diet prior and during short-term and long-term exposure to opium smoke. Then, isoproterenol (3mg/kg, i.p.) was injected to induce cardiac ischemia and animals were followed for 3h for counting of lethal arrhythmia incidence. Long-term opium smoking significantly increased the plasma atherogenic index. In ischemic hearts, opium smoking along with hypercholesterolemia significantly enhanced the incidence of fatal arrhythmia. This vulnerability was not mediated by changes in QT interval. These data suggest that opium smoking, especially in hypercholesterolemic conditions, can be a predisposing factor for atherogenesis and lethal arrhythmia.

  10. Isolation of Aneuploid-Generating Mutants of ASPERGILLUS NIDULANS, One of Which Is Defective in Interphase of the Cell Cycle

    PubMed Central

    Upshall, A.; Mortimore, I. D.

    1984-01-01

    A method is described for isolating mutants potentially defective in loci involved in mitotic chromosome segregation. Conditional lethal, heat-sensitive (42°) mutants were assayed at a subrestrictive temperature of 37° for an inflated production of colonies displaying phenotypes and behavior patterns of whole chromosome aneuploids. Of 14 mutants, three showed specificity for one disomic phenotype, whereas 11 generated colonies mosaic for different aneuploid phenotypes. This latter group is designated hfa ( high frequency of aneuploid). For ten of the 11 mutants temperature sensitivity and aneuploid production cosegregated, indicating a single mutation in each. These mutations were recessive and nonallelic. Analysis was concentrated on the hfaB3 mutation which is mapped to chromosome VI tightly linked to the methB and tsB loci. The disruptive influence of hfaB3 on mitosis at 37° was shown by (1) ploidy and whole chromosome-type segregation of markers in the breakdown sectors of phenotypically aneuploid colonies obtained from multiply marked homozygous hfaB3 disploids; (2) a high frequency of haploid and nondisjunctional diploid segregants among spontaneous yellow-spored parasexual recombinants taken from green-spored homozygous hfaB3 diploids. The mutation had no effect on meiotic chromosome segregation at 37°. The single interphase nucleus in germlings at 42°, coupled with changes in the mitotic index in temperature exchange experiments, showed hfaB3 to arrest the cell cycle in interphase at restrictive temperature. A conclusion drawn is that the hfaB gene product is required both for entry into mitosis and for normal chromosome segregation in dividing nuclei. PMID:6479583

  11. [Molecular analysis of space mutant line of kidney bean].

    PubMed

    Zhang, J; Li, J G; Wang, P S; Wang, X Q; Jiang, X C

    2000-12-01

    Objective. To identify the occurrence of gene mutant in mutant lines in the offspring of Kidney bean seeds under space flight condition. Method. Kidney bean seeds were carried onboard a recoverable satellite for 15 days in space and were planted on the ground after recovery. Five mutant lines showing variation in the form of leaf blade and their parents were analyzed with RAPD technique. Result. 50 random 10-mer primers were used in this study, among which 20 primers generated 180 polymorphic DNA bands, their size ranged from 200 bp to 2000 bp. 3 primers amplified obviously different bands in the DNA of mutant lines in comparison with that of the control. Conclusion. This is the first molecular analysis of the mutant lines of Kidney bean generated by space mutagenesis at DNA level. The result of RAPD analysis indicated that distinct variations were demonstrated in the DNA of mutant lines as compared with that of the original control.

  12. Lethal mutagenesis of foot-and-mouth disease virus involves shifts in sequence space.

    PubMed

    Perales, Celia; Henry, Michel; Domingo, Esteban; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2011-12-01

    Lethal mutagenesis or virus transition into error catastrophe is an antiviral strategy that aims at extinguishing a virus by increasing the viral mutation rates during replication. The molecular basis of lethal mutagenesis is largely unknown. Previous studies showed that a critical substitution in the foot-and-mouth disease virus (FMDV) polymerase was sufficient to allow the virus to escape extinction through modulation of the transition types induced by the purine nucleoside analogue ribavirin. This substitution was not detected in mutant spectra of FMDV populations that had not replicated in the presence of ribavirin, using standard molecular cloning and nucleotide sequencing. Here we selectively amplify and analyze low-melting-temperature cDNA duplexes copied from FMDV genome populations passaged in the absence or presence of ribovirin Hypermutated genomes with high frequencies of A and U were present in both ribavirin -treated and untreated populations, but the major effect of ribavirin mutagenesis was to accelerate the occurrence of AU-rich mutant clouds during the early replication rounds of the virus. The standard FMDV quasispecies passaged in the absence of ribavirin included the salient transition-modulating, ribavirin resistance mutation, whose frequency increased in populations treated with ribavirin. Thus, even nonmutagenized FMDV quasispecies include a deep, mutationally biased portion of sequence space, in support of the view that the virus replicates close to the error threshold for maintenance of genetic information.

  13. Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae.

    PubMed

    Stanley, Dragana; Fraser, Sarah; Chambers, Paul J; Rogers, Peter; Stanley, Grant A

    2010-02-01

    Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l(-1). At a glucose concentration of 100 g l(-1), SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD(+)/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.

  14. Bacillus anthracis Lethal Toxin Reduces Human Alveolar Epithelial Barrier Function

    PubMed Central

    Langer, Marybeth; Duggan, Elizabeth Stewart; Booth, John Leland; Patel, Vineet Indrajit; Zander, Ryan A.; Silasi-Mansat, Robert; Ramani, Vijay; Veres, Tibor Zoltan; Prenzler, Frauke; Sewald, Katherina; Williams, Daniel M.; Coggeshall, Kenneth Mark; Awasthi, Shanjana; Lupu, Florea; Burian, Dennis; Ballard, Jimmy Dale; Braun, Armin

    2012-01-01

    The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness. PMID:23027535

  15. Issues surrounding lethal injection as a means of capital punishment.

    PubMed

    Romanelli, Frank; Whisman, Tyler; Fink, Joseph L

    2008-12-01

    Lethal injection as a method of state-sanctioned capital punishment was initially proposed in the United States in 1977 and used for the first time in 1982. Most lethal injection protocols use a sequential drug combination of sodium thiopental, pancuronium bromide, and potassium chloride. Lethal injection was originally introduced as a more humane form of execution compared with existing mechanical methods such as electrocution, toxic gassing, hanging, or firing squad. Lethal injection has not, however, been without controversy. Several states are considering whether lethal injection meets constitutional scrutiny forbidding cruel and unusual punishment. Recently in the case of Ralph Baze and Thomas C. Bowling, Petitioners, v John D. Rees, Commissioner, Kentucky Department of Corrections et al, the United States Supreme Court upheld the constitutionality of the lethal injection protocol as carried out in the Commonwealth of Kentucky. Most of the debate has surrounded the dosing and procedures used in lethal injection and whether the drug combinations and measures for administering the drugs truly produce a timely, pain-free, and fail-safe death. Many have also raised issues regarding the "medicalization" of execution and the ethics of health care professionals' participation in any part of the lethal injection process. As a result of all these issues, the future of lethal injection as a means of execution in the United States is under significant scrutiny. Outcomes of ongoing legislative and judicial reviews might result in cessation of lethal injection in totality or in alterations involving specific drug combinations or administration procedures.

  16. Antenatal diagnosis of lethal skeletal dysplasias.

    PubMed

    Tretter, A E; Saunders, R C; Meyers, C M; Dungan, J S; Grumbach, K; Sun, C C; Campbell, A B; Wulfsberg, E A

    1998-02-17

    Lethal skeletal dysplasias (LSD) are a heterogeneous group of rare but important genetic disorders characterized by abnormal growth and development of bone and cartilage. We describe the diagnosis and outcome of 29 cases of lethal skeletal dysplasias evaluated between January 1989 and December 1996 at the University of Maryland Medical Center and the Ultrasound Institute of Baltimore. Two cases presented at delivery with no prenatal care while the remaining 27 cases were identified by antenatal sonography. Final diagnoses included thanatophoric dysplasia (14), osteogenesis imperfecta, type II (6), achondrogenesis (2), short rib syndromes (3), campomelic syndrome (2), atelosteogenesis (1), and no evidence of a skeletal dysplasia (1). Twenty out of 27 pregnancies were terminated with an average at detection of 21.6 weeks. The other 7 pregnancies that went on to deliver had an average age at detection of 29.2 weeks. Fetal abnormalities in the terminated pregnancies were identified at a significantly earlier gestational age (P = 0.0016) than the pregnancies that continued. While the identification of LSD by sonography was excellent (26/27), only 13/27 (48%) were given an accurate specific antenatal diagnosis. In 8/14 (57%) cases with an inaccurate or nonspecific diagnosis there was a significant or crucial change in the genetic counseling. Thus, while antenatal sonography is an excellent method for discovering LSD, clinical examination, radiographs, and autopsy are mandatory for making a specific diagnosis. PMID:9489797

  17. Suicide Intent and Accurate Expectations of Lethality: Predictors of Medical Lethality of Suicide Attempts

    ERIC Educational Resources Information Center

    Brown, Gregory K.; Henriques, Gregg R.; Sosdjan, Daniella; Beck, Aaron T.

    2004-01-01

    The degree of intent to commit suicide and the severity of self-injury were examined in individuals (N = 180) who had recently attempted suicide. Although a minimal association was found between the degree of suicide intent and the degree of lethality of the attempt, the accuracy of expectations about the likelihood of dying was found to moderate…

  18. Gonadosomatic mosaicism for lethal mutations in Drosophila lethal mutations disturbing larval development

    SciTech Connect

    Ivanov, A.I.; Sakharova, N.Yu.

    1988-11-01

    Phenogenetic analysis of autonomous lethal mutations obtained by the method of gonadosomatic mosaicism which manifested during larval stages, established that the nuclei of hypodermal cells, salivary glands suprapharyngeal ganglion, pharynx, esophagus, gizzard, and hindgut are the derivatives of the same nucleus (from the first two nuclei of cleavage) as the nuclei of the cells of the imaginal-somatic tissues.

  19. Synthetic Lethality of Drosophila in the Absence of the MUS81 Endonuclease and the DmBlm Helicase Is Associated With Elevated Apoptosis

    PubMed Central

    Trowbridge, Kirsten; McKim, Kim; Brill, Steven J.; Sekelsky, Jeff

    2007-01-01

    Mus81-Mms4 (Mus81-Eme1 in some species) is a heterodimeric DNA structure-specific endonuclease that has been implicated in meiotic recombination and processing of damaged replication forks in fungi. We generated and characterized mutations in Drosophila melanogaster mus81 and mms4. Unlike the case in fungi, we did not find any role for MUS81-MMS4 in meiotic crossing over. A possible role for this endonuclease in repairing double-strand breaks that arise during DNA replication is suggested by the finding that mus81 and mms4 mutants are hypersensitive to camptothecin; however, these mutants are not hypersensitive to other agents that generate lesions that slow or block DNA replication. In fungi, mus81, mms4, and eme1 mutations are synthetically lethal with mutations in genes encoding RecQ helicase homologs. Similarly, we found that mutations in Drosophila mus81 and mms4 are synthetically lethal with null mutations in mus309, which encodes the ortholog of the Bloom Syndrome helicase. Synthetic lethality is associated with high levels of apoptosis in proliferating tissues. Lethality and elevated apoptosis were partially suppressed by a mutation in spn-A, which encodes the ortholog of the strand invasion protein Rad51. These findings provide insights into the causes of synthetic lethality. PMID:17603121

  20. Recovery of microorganisms from potentially lethal radiation damage

    NASA Astrophysics Data System (ADS)

    Borsa, Joseph; Lucht, Lisa; Blank, Greg

    1995-02-01

    Dose response curves for inactivation of microorganisms are central in the design of any process intending to use irradiation for the improvement of the microbiological quality of any treated materials, be it food or medical supplies. Under some conditions a fraction of irradiated microorganisms is able to recover from a potentially lethal dose. This recovery phenomenon must be considered in determining the efficacy of irradiation in microbial inactivation. In this work the recovery phenomenon was examined in eleven species of microorganisms. Variables examined included dose, radiation type, post-irradiation holding temperature, and nutritient medium used to culture the organism. Kinetics of damage repair and fixation were also examined. Results indicate that, for certain species of microorganisms, recovery can significantly lower the killing efficacy of irradiation.

  1. Role of macrophage oxidative burst in the action of anthrax lethal toxin.

    PubMed Central

    Hanna, P. C.; Kruskal, B. A.; Ezekowitz, R. A.; Bloom, B. R.; Collier, R. J.

    1994-01-01

    BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and

  2. Synthetic lethal approaches for assessing combinatorial efficacy of chemotherapeutic drugs.

    PubMed

    Jackson, Rebecca A; Chen, Ee Sin

    2016-06-01

    The recent advances in pharmacogenomics have made personalized medicine no longer a pipedream but a precise and powerful way to tailor individualized cancer treatment strategies. Cancer is a devastating disease, and contemporary chemotherapeutic strategies now integrate several agents in the treatment of some types of cancer, with the intent to block more than one target simultaneously. This constitutes the premise of synthetic lethality, an attractive therapeutic strategy already demonstrating clinical success in patients with breast and ovarian cancers. Synthetic lethal combinations offer the potential to also target the hitherto "undruggable" mutations that have challenged the cancer field for decades. However, synthetic lethality in clinical cancer therapy is very much still in its infancy, and selecting the most appropriate combinations-or synthetic lethal pairs-is not always an intuitive process. Here, we review some of the recent progress in identifying synthetic lethal combinations and their potential for therapy and highlight some of the tools through which synthetic lethal pairs are identified.

  3. Rest mutant zebrafish swim erratically and display atypical spatial preferences.

    PubMed

    Moravec, Cara E; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F; Weng, Wei; Sirotkin, Howard I

    2015-05-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest. PMID:25712696

  4. Functional analysis of an Orc6 mutant in Drosophila.

    PubMed

    Balasov, Maxim; Huijbregts, Richard P H; Chesnokov, Igor

    2009-06-30

    The origin recognition complex (ORC) is a 6-subunit complex required for the initiation of DNA replication in eukaryotic organisms. ORC is also involved in other cell functions. The smallest Drosophila ORC subunit, Orc6, is important for both DNA replication and cytokinesis. To study the role of Orc6 in vivo, the orc6 gene was deleted by imprecise excision of P element. Lethal alleles of orc6 are defective in DNA replication and also show abnormal chromosome condensation and segregation. The analysis of cells containing the orc6 deletion revealed that they arrest in both the G(1) and mitotic stages of the cell cycle. Orc6 deletion can be rescued to viability by a full-length Orc6 transgene. The expression of mutant transgenes of Orc6 with deleted or mutated C-terminal domain results in a release of mutant cells from G(1) arrest and restoration of DNA replication, indicating that the DNA replication function of Orc6 is associated with its N-terminal domain. However, these mutant cells accumulate at mitosis, suggesting that the C-terminal domain of Orc6 is important for the passage through the M phase. In a cross-species complementation experiment, the expression of human Orc6 in Drosophila Orc6 mutant cells rescued DNA replication, suggesting that this function of the protein is conserved among metazoans. PMID:19541634

  5. Rest Mutant zebrafish swim erratically and display atypical spatial preferences

    PubMed Central

    Moravec, Cara E.; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F.; Weng, Wei; Sirotkin, Howard I.

    2015-01-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest. PMID:25712696

  6. Rest mutant zebrafish swim erratically and display atypical spatial preferences.

    PubMed

    Moravec, Cara E; Li, Edward; Maaswinkel, Hans; Kritzer, Mary F; Weng, Wei; Sirotkin, Howard I

    2015-05-01

    The Rest/Nrsf transcriptional repressor modulates expression of a large set of neural specific genes. Many of these target genes have well characterized roles in nervous system processes including development, plasticity and synaptogenesis. However, the impact of Rest-mediated transcriptional regulation on behavior has been understudied due in part to the embryonic lethality of the mouse knockout. To investigate the requirement for Rest in behavior, we employed the zebrafish rest mutant to explore a range of behaviors in adults and larva. Adult rest mutants of both sexes showed abnormal behaviors in a novel environment including increased vertical swimming, erratic swimming patterns and a proclivity for the tank walls. Adult males also had diminished reproductive success. At 6 days post fertilization (dpf), rest mutant larva were hypoactive, but displayed normal evoked responses to light and sound stimuli. Overall, these results provide evidence that rest dysfunction produces atypical swimming patterns and preferences in adults, and reduced locomotor activity in larvae. This study provides the first behavioral analysis of rest mutants and reveals specific behaviors that are modulated by Rest.

  7. XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer | Office of Cancer Genomics

    Cancer.gov

    The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity.

  8. A synthetic lethal screen identifies a role for the cortical actin patch/endocytosis complex in the response to nutrient deprivation in Saccharomyces cerevisiae.

    PubMed Central

    Care, Alison; Vousden, Katherine A; Binley, Katie M; Radcliffe, Pippa; Trevethick, Janet; Mannazzu, Ilaria; Sudbery, Peter E

    2004-01-01

    Saccharomyces cerevisiae whi2Delta cells are unable to halt cell division in response to nutrient limitation and are sensitive to a wide variety of stresses. A synthetic lethal screen resulted in the isolation of siw mutants that had a phenotype similar to that of whi2Delta. Among these were mutations affecting SIW14, FEN2, SLT2, and THR4. Fluid-phase endocytosis is severely reduced or abolished in whi2Delta, siw14Delta, fen2Delta, and thr4Delta mutants. Furthermore, whi2Delta and siw14Delta mutants produce large actin clumps in stationary phase similar to those seen in prk1Delta ark1Delta mutants defective in protein kinases that regulate the actin cytoskeleton. Overexpression of SIW14 in a prk1Delta strain resulted in a loss of cortical actin patches and cables and was lethal. Overexpression of SIW14 also rescued the caffeine sensitivity of the slt2 mutant isolated in the screen, but this was not due to alteration of the phosphorylation state of Slt2. These observations suggest that endocytosis and the organization of the actin cytoskeleton are required for the proper response to nutrient limitation. This hypothesis is supported by the observation that rvs161Delta, sla1Delta, sla2Delta, vrp1Delta, ypt51Delta, ypt52Delta, and end3Delta mutations, which disrupt the organization of the actin cytoskeleton and/or reduce endocytosis, have a phenotype similar to that of whi2Delta mutants. PMID:15020461

  9. Altered cytoskeletal organization characterized lethal but not surviving Brtl+/- mice: insight on phenotypic variability in osteogenesis imperfecta.

    PubMed

    Bianchi, Laura; Gagliardi, Assunta; Maruelli, Silvia; Besio, Roberta; Landi, Claudia; Gioia, Roberta; Kozloff, Kenneth M; Khoury, Basma M; Coucke, Paul J; Symoens, Sofie; Marini, Joan C; Rossi, Antonio; Bini, Luca; Forlino, Antonella

    2015-11-01

    Osteogenesis imperfecta (OI) is a heritable bone disease with dominant and recessive transmission. It is characterized by a wide spectrum of clinical outcomes ranging from very mild to lethal in the perinatal period. The intra- and inter-familiar OI phenotypic variability in the presence of an identical molecular defect is still puzzling to the research field. We used the OI murine model Brtl(+/-) to investigate the molecular basis of OI phenotypic variability. Brtl(+/-) resembles classical dominant OI and shows either a moderately severe or a lethal outcome associated with the same Gly349Cys substitution in the α1 chain of type I collagen. A systems biology approach was used. We took advantage of proteomic pathway analysis to functionally link proteins differentially expressed in bone and skin of Brtl(+/-) mice with different outcomes to define possible phenotype modulators. The skin/bone and bone/skin hybrid networks highlighted three focal proteins: vimentin, stathmin and cofilin-1, belonging to or involved in cytoskeletal organization. Abnormal cytoskeleton was indeed demonstrated by immunohistochemistry to occur only in tissues from Brtl(+/-) lethal mice. The aberrant cytoskeleton affected osteoblast proliferation, collagen deposition, integrin and TGF-β signaling with impairment of bone structural properties. Finally, aberrant cytoskeletal assembly was detected in fibroblasts obtained from lethal, but not from non-lethal, OI patients carrying an identical glycine substitution. Our data demonstrated that compromised cytoskeletal assembly impaired both cell signaling and cellular trafficking in mutant lethal mice, altering bone properties. These results point to the cytoskeleton as a phenotypic modulator and potential novel target for OI treatment.

  10. Identification of Arabidopsis rat Mutants

    PubMed Central

    Zhu, Yanmin; Nam, Jaesung; Humara, Jaime M.; Mysore, Kirankumar S.; Lee, Lan-Ying; Cao, Hongbin; Valentine, Lisa; Li, Jingling; Kaiser, Anthony D.; Kopecky, Andrea L.; Hwang, Hau-Hsuan; Bhattacharjee, Saikat; Rao, Praveen K.; Tzfira, Tzvi; Rajagopal, Jyothi; Yi, HoChul; Veena; Yadav, Badam S.; Crane, Yan M.; Lin, Kui; Larcher, Yves; Gelvin, Matthew J.K.; Knue, Marnie; Ramos, Cynthia; Zhao, Xiaowen; Davis, Susan J.; Kim, Sang-Ic; Ranjith-Kumar, C.T.; Choi, Yoo-Jin; Hallan, Vipin K.; Chattopadhyay, Sudip; Sui, Xiangzhen; Ziemienowicz, Alicja; Matthysse, Ann G.; Citovsky, Vitaly; Hohn, Barbara; Gelvin, Stanton B.

    2003-01-01

    Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants. PMID:12805582

  11. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  12. The Wiggle Index: An Open Source Bioassay to Assess Sub-Lethal Insecticide Response in Drosophila melanogaster.

    PubMed

    Denecke, Shane; Nowell, Cameron J; Fournier-Level, Alexandre; Perry, Trent; Batterham, Phil

    2015-01-01

    Toxicological assays measuring mortality are routinely used to describe insecticide response, but sub-lethal exposures to insecticides can select for resistance and yield additional biological information describing the ways in which an insecticide impacts the insect. Here we present the Wiggle Index (WI), a high-throughput method to quantify insecticide response by measuring the reduction in motility during sub-lethal exposures in larvae of the vinegar fly Drosophila melanogaster. A susceptible wild type strain was exposed to the insecticides chlorantraniliprole, imidacloprid, spinosad, and ivermectin. Each insecticide reduced larval motility, but response times and profiles differed among insecticides. Two sets of target site mutants previously identified in mortality studies on the basis of imidacloprid or spinosad resistance phenotypes were tested. In each case the resistant mutant responded significantly less than the control. The WI was also able to detect a spinosad response in the absence of the primary spinosad target site. This response was not detected in mortality assays suggesting that spinosad, like many other insecticides, may have secondary targets affecting behaviour. The ability of the WI to detect changes in insecticide metabolism was confirmed by overexpressing the imidacloprid metabolizing Cyp6g1 gene in digestive tissues or the central nervous system. The data presented here validate the WI as an inexpensive, generic, sub-lethal assay that can complement information gained from mortality assays, extending our understanding of the genetic basis of insecticide response in D. melanogaster.

  13. The Wiggle Index: An Open Source Bioassay to Assess Sub-Lethal Insecticide Response in Drosophila melanogaster

    PubMed Central

    Denecke, Shane; Nowell, Cameron J.; Fournier-Level, Alexandre; Perry, Trent; Batterham, Phil

    2015-01-01

    Toxicological assays measuring mortality are routinely used to describe insecticide response, but sub-lethal exposures to insecticides can select for resistance and yield additional biological information describing the ways in which an insecticide impacts the insect. Here we present the Wiggle Index (WI), a high-throughput method to quantify insecticide response by measuring the reduction in motility during sub-lethal exposures in larvae of the vinegar fly Drosophila melanogaster. A susceptible wild type strain was exposed to the insecticides chlorantraniliprole, imidacloprid, spinosad, and ivermectin. Each insecticide reduced larval motility, but response times and profiles differed among insecticides. Two sets of target site mutants previously identified in mortality studies on the basis of imidacloprid or spinosad resistance phenotypes were tested. In each case the resistant mutant responded significantly less than the control. The WI was also able to detect a spinosad response in the absence of the primary spinosad target site. This response was not detected in mortality assays suggesting that spinosad, like many other insecticides, may have secondary targets affecting behaviour. The ability of the WI to detect changes in insecticide metabolism was confirmed by overexpressing the imidacloprid metabolizing Cyp6g1 gene in digestive tissues or the central nervous system. The data presented here validate the WI as an inexpensive, generic, sub-lethal assay that can complement information gained from mortality assays, extending our understanding of the genetic basis of insecticide response in D. melanogaster. PMID:26684454

  14. Lethal photosensitization of biofilm-grown bacteria

    NASA Astrophysics Data System (ADS)

    Wilson, Michael

    1997-12-01

    Antibacterial agents are increasingly being used for the prophylaxis and treatment of oral diseases. As these agents can be rendered ineffective by resistance development in the target organisms there is a need to develop alternative antimicrobial approaches. Light-activated antimicrobial agents release singlet oxygen and free radicals which can kill adjacent bacteria and a wide range of cariogenic and periodontopathogenic bacteria has been shown to be susceptible to such agents. In the oral cavity these organisms are present as biofilms (dental plaques) which are less susceptible to traditional antimicrobial agents than bacterial suspensions. The results of these studies have shown that biofilm-grown oral bacteria are also susceptible to lethal photosensitization although the light energy doses required are grater than those needed to kill the organisms when they are grown as aqueous suspensions.

  15. Alleged lethal sorcery in East Timor.

    PubMed

    Pollanen, Michael S

    2004-01-01

    A wide range of cultural and social perspectives exists on the concept of sudden and unexpected death. In countries, without a formal system of death investigation, sudden death is shrouded in mysticism often based on traditional belief systems. This cultural perspective on sudden death is often at variance with medical and forensic concepts and may include explanations such as sorcery, magic, and voodoo. In this case report, the postmortem findings in an alleged victim of lethal 'black magic', known as ema halo by the indigenous people of East Timor, is described. The alleged victim died suddenly in front of witnesses. At autopsy, marked dilation of a bicuspid aortic valve with annuloaortic ectasia and a sinus of Valsalva aneurysm was found after exhumation of the body. The findings mitigated the local belief in witchcraft and established a natural manner of death.

  16. Ants defend aphids against lethal disease.

    PubMed

    Nielsen, Charlotte; Agrawal, Anurag A; Hajek, Ann E

    2010-04-23

    Social insects defend their own colonies and some species also protect their mutualist partners. In mutualisms with aphids, ants typically feed on honeydew produced by aphids and, in turn guard and shelter aphid colonies from insect natural enemies. Here we report that Formica podzolica ants tending milkweed aphids, Aphis asclepiadis, protect aphid colonies from lethal fungal infections caused by an obligate aphid pathogen, Pandora neoaphidis. In field experiments, bodies of fungal-killed aphids were quickly removed from ant-tended aphid colonies. Ant workers were also able to detect infective conidia on the cuticle of living aphids and responded by either removing or grooming these aphids. Our results extend the long-standing view of ants as mutualists and protectors of aphids by demonstrating focused sanitizing and quarantining behaviour that may lead to reduced disease transmission in aphid colonies.

  17. Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline

    PubMed Central

    Linkevicius, Marius; Anderssen, Jytte Mark; Sandegren, Linus; Andersson, Dan I.

    2016-01-01

    Objectives The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Methods Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. Results The LPS mutants were 48–85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%–13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Conclusions Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system. PMID:26851608

  18. Collagen defects in lethal perinatal osteogenesis imperfecta.

    PubMed

    Bateman, J F; Chan, D; Mascara, T; Rogers, J G; Cole, W G

    1986-12-15

    Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.

  19. Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant

    PubMed Central

    Chen, Yuning; Ye, Dening; Held, Michael A.; Cannon, Maura C.; Ray, Tui; Saha, Prasenjit; Frye, Alexandra N.; Mort, Andrew J.; Kieliszewski, Marcia J.

    2015-01-01

    Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes. PMID:27135319

  20. An exome sequencing strategy to diagnose lethal autosomal recessive disorders.

    PubMed

    Ellard, Sian; Kivuva, Emma; Turnpenny, Peter; Stals, Karen; Johnson, Matthew; Xie, Weijia; Caswell, Richard; Lango Allen, Hana

    2015-03-01

    Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0-4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.

  1. An exome sequencing strategy to diagnose lethal autosomal recessive disorders

    PubMed Central

    Ellard, Sian; Kivuva, Emma; Turnpenny, Peter; Stals, Karen; Johnson, Matthew; Xie, Weijia; Caswell, Richard; Lango Allen, Hana

    2015-01-01

    Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0–4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies. PMID:24961629

  2. Characterization of New Punch Mutations: Identification of Two Additional Mutant Classes

    PubMed Central

    Reynolds, E. R.; O'Donnell, J. M.

    1988-01-01

    The Punch locus of Drosophila melanogaster which encodes the pteridine biosynthetic enzyme, GTP cyclohydrolase, is genetically complex. Lethal alleles of the locus resolve into an array of interallelic complementation groups, and at least one class of mutations is developmentally specific, affecting GTP cyclohydrolase activity only in the heads of adults. All previously isolated Punch alleles were identified on the basis of a mutant eye color phenotype. By screening mutagenized chromosomes over Punch region deficiencies, we have now isolated new alleles on the basis of lethal and visible phenotypes. Most of these alleles fall into previously identified genetic classes, but two new classes of mutations were also found. One class contains two alleles that behave as dominant lethal mutations in some genetic backgrounds. The other class represents a second developmentally specific set of alleles that affect the function of the Punch locus only during embryogenesis. PMID:3136053

  3. Syn-lethality: an integrative knowledge base of synthetic lethality towards discovery of selective anticancer therapies.

    PubMed

    Li, Xue-juan; Mishra, Shital K; Wu, Min; Zhang, Fan; Zheng, Jie

    2014-01-01

    Synthetic lethality (SL) is a novel strategy for anticancer therapies, whereby mutations of two genes will kill a cell but mutation of a single gene will not. Therefore, a cancer-specific mutation combined with a drug-induced mutation, if they have SL interactions, will selectively kill cancer cells. While numerous SL interactions have been identified in yeast, only a few have been known in human. There is a pressing need to systematically discover and understand SL interactions specific to human cancer. In this paper, we present Syn-Lethality, the first integrative knowledge base of SL that is dedicated to human cancer. It integrates experimentally discovered and verified human SL gene pairs into a network, associated with annotations of gene function, pathway, and molecular mechanisms. It also includes yeast SL genes from high-throughput screenings which are mapped to orthologous human genes. Such an integrative knowledge base, organized as a relational database with user interface for searching and network visualization, will greatly expedite the discovery of novel anticancer drug targets based on synthetic lethality interactions. The database can be downloaded as a stand-alone Java application.

  4. Chronic exposure of corals to fine sediments: lethal and sub-lethal impacts.

    PubMed

    Flores, Florita; Hoogenboom, Mia O; Smith, Luke D; Cooper, Timothy F; Abrego, David; Negri, Andrew P

    2012-01-01

    Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l(-1) TSS (25 mg cm(-2) day(-1)) for M. aequituberculata and 100 mg l(-1) TSS (83 mg cm(-2) day(-1)) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue. PMID:22662225

  5. Syn-Lethality: An Integrative Knowledge Base of Synthetic Lethality towards Discovery of Selective Anticancer Therapies

    PubMed Central

    Li, Xue-juan; Mishra, Shital K.; Wu, Min; Zhang, Fan

    2014-01-01

    Synthetic lethality (SL) is a novel strategy for anticancer therapies, whereby mutations of two genes will kill a cell but mutation of a single gene will not. Therefore, a cancer-specific mutation combined with a drug-induced mutation, if they have SL interactions, will selectively kill cancer cells. While numerous SL interactions have been identified in yeast, only a few have been known in human. There is a pressing need to systematically discover and understand SL interactions specific to human cancer. In this paper, we present Syn-Lethality, the first integrative knowledge base of SL that is dedicated to human cancer. It integrates experimentally discovered and verified human SL gene pairs into a network, associated with annotations of gene function, pathway, and molecular mechanisms. It also includes yeast SL genes from high-throughput screenings which are mapped to orthologous human genes. Such an integrative knowledge base, organized as a relational database with user interface for searching and network visualization, will greatly expedite the discovery of novel anticancer drug targets based on synthetic lethality interactions. The database can be downloaded as a stand-alone Java application. PMID:24864230

  6. Chronic Exposure of Corals to Fine Sediments: Lethal and Sub-Lethal Impacts

    PubMed Central

    Flores, Florita; Hoogenboom, Mia O.; Smith, Luke D.; Cooper, Timothy F.; Abrego, David; Negri, Andrew P.

    2012-01-01

    Understanding the sedimentation and turbidity thresholds for corals is critical in assessing the potential impacts of dredging projects in tropical marine systems. In this study, we exposed two species of coral sampled from offshore locations to six levels of total suspended solids (TSS) for 16 weeks in the laboratory, including a 4 week recovery period. Dose-response relationships were developed to quantify the lethal and sub-lethal thresholds of sedimentation and turbidity for the corals. The sediment treatments affected the horizontal foliaceous species (Montipora aequituberculata) more than the upright branching species (Acropora millepora). The lowest sediment treatments that caused full colony mortality were 30 mg l−1 TSS (25 mg cm−2 day−1) for M. aequituberculata and 100 mg l−1 TSS (83 mg cm−2 day−1) for A. millepora after 12 weeks. Coral mortality generally took longer than 4 weeks and was closely related to sediment accumulation on the surface of the corals. While measurements of damage to photosystem II in the symbionts and reductions in lipid content and growth indicated sub-lethal responses in surviving corals, the most reliable predictor of coral mortality in this experiment was long-term sediment accumulation on coral tissue. PMID:22662225

  7. A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

    PubMed Central

    Smith, M M; Yang, P; Santisteban, M S; Boone, P W; Goldstein, A T; Megee, P C

    1996-01-01

    The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation. PMID:8622646

  8. Characterization of Francisella tularensis Schu S4 defined mutants as live-attenuated vaccine candidates.

    PubMed

    Santiago, Araceli E; Mann, Barbara J; Qin, Aiping; Cunningham, Aimee L; Cole, Leah E; Grassel, Christen; Vogel, Stefanie N; Levine, Myron M; Barry, Eileen M

    2015-08-01

    Francisella tularensis (Ft), the etiological agent of tularemia and a Tier 1 select agent, has been previously weaponized and remains a high priority for vaccine development. Ft tularensis (type A) and Ft holarctica (type B) cause most human disease. We selected six attenuating genes from the live vaccine strain (LVS; type B), F. novicida and other intracellular bacteria: FTT0507, FTT0584, FTT0742, FTT1019c (guaA), FTT1043 (mip) and FTT1317c (guaB) and created unmarked deletion mutants of each in the highly human virulent Ft strain Schu S4 (Type A) background. FTT0507, FTT0584, FTT0742 and FTT1043 Schu S4 mutants were not attenuated for virulence in vitro or in vivo. In contrast, Schu S4 gua mutants were unable to replicate in murine macrophages and were attenuated in vivo, with an i.n. LD50 > 10(5) CFU in C57BL/6 mice. However, the gua mutants failed to protect mice against lethal challenge with WT Schu S4, despite demonstrating partial protection in rabbits in a previous study. These results contrast with the highly protective capacity of LVS gua mutants against a lethal LVS challenge in mice, and underscore differences between these strains and the animal models in which they are evaluated, and therefore have important implications for vaccine development.

  9. A genetic screen for zygotic embryonic lethal mutations affecting cuticular morphology in the wasp Nasonia vitripennis.

    PubMed Central

    Pultz, M A; Zimmerman, K K; Alto, N M; Kaeberlein, M; Lange, S K; Pitt, J N; Reeves, N L; Zehrung, D L

    2000-01-01

    We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera; Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. PMID:10866651

  10. Identification of a novel Drosophila melanogaster heat-shock gene, lethal(2)denticleless [l(2)dtl], coding for an 83-kDa protein.

    PubMed

    Kurzik-Dumke, U; Neubauer, M; Debes, A

    1996-06-01

    In this study, we describe the identification of a novel Drosophila melanogaster (Dm) gene, l(2)dtl, characterized by elevated expression under heat-shock (HS) conditions. It encodes a protein of 83 kDa with no homology to known members of the HSP90 family and other proteins. Gene l(2)dtl is located on the right arm of the second chromosome at locus 59F5, close to the tumor suppressor gene l(2)tid, a homolog of the dnaJ encoding a chaperone strongly conserved in evolution. In the following, we present the sequence of l(2)dtl, the putative protein it encodes, and its molecular localization in a closely interspaced gene cluster consisting of at least four nested genes spanning an approximately 10-kb genomic interval. Furthermore, we present the temporal expression of l(2)dtl in the wild type under normal and HS conditions, and describe the isolation and the phenotype of eight embryonic lethal l(2)dtl mutants.

  11. Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

    PubMed Central

    Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

    2015-01-01

    KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. PMID:26166481

  12. Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome.

    PubMed

    Alby, Caroline; Piquand, Kevin; Huber, Céline; Megarbané, André; Ichkou, Amale; Legendre, Marine; Pelluard, Fanny; Encha-Ravazi, Ferechté; Abi-Tayeh, Georges; Bessières, Bettina; El Chehadeh-Djebbar, Salima; Laurent, Nicole; Faivre, Laurence; Sztriha, László; Zombor, Melinda; Szabó, Hajnalka; Failler, Marion; Garfa-Traore, Meriem; Bole, Christine; Nitschké, Patrick; Nizon, Mathilde; Elkhartoufi, Nadia; Clerget-Darpoux, Françoise; Munnich, Arnold; Lyonnet, Stanislas; Vekemans, Michel; Saunier, Sophie; Cormier-Daire, Valérie; Attié-Bitach, Tania; Thomas, Sophie

    2015-08-01

    KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. PMID:26166481

  13. Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori.

    PubMed

    Fujii, Tsuguru; Yamamoto, Kimiko; Banno, Yutaka

    2016-06-01

    Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.

  14. Examining lethality risk for rodent studies of primary blast lung injury.

    PubMed

    Hubbard, William Brad; Hall, Christina; Siva Sai Suijith Sajja, Venkata; Lavik, Erink; VandeVord, Pamela

    2014-01-01

    While protective measures have been taken to mitigate injury to the thorax during a blast exposure, primary blast lung injury (PBLI) is still evident in mounted/in vehicle cases during military conflicts. Moreover, civilians, who are unprotected from blast exposure, can be severely harmed by terrorist attacks that use improvised explosive devices (IEDs). Since the lungs are the most susceptible organ due to their air-filled nature, PBLI is one of the most serious injuries seen in civilian blast cases. Determining lethality threshold for rodent studies is crucial to guide experimental designs centered on therapies for survival after PBLI or mechanistic understanding of the injury itself. Using an Advanced Blast Simulator, unprotected rats were exposed to a whole body blast to induce PBLI. The one-hour survival rate was assessed to determine operating conditions for a 50% lethality rate. Macroscopic and histological analysis of lung was conducted using hematoxylin and eosin staining. Results demonstrated lethality risk trends based on static blast overpressure (BOP) for rodent models, which may help standardized animal studies and contribute to scaling to the human level. The need for a standardized method of producing PBLI is pressing and establishing standard curves, such as a lethality risk curve for lung blasts, is crucial for this condensing of BOP methods. PMID:25405409

  15. Conditional Deletion of Indian Hedgehog in Limb Mesenchyme Results in Complete Loss of Growth Plate Formation but Allows Mature Osteoblast Differentiation.

    PubMed

    Amano, Katsuhiko; Densmore, Michael J; Lanske, Beate

    2015-12-01

    Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1-Cre;Ihh(fl/fl) mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show a significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show for the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb

  16. The Genetics of Catalase in Drosophila Melanogaster: Isolation and Characterization of Acatalasemic Mutants

    PubMed Central

    Mackay, W. J.; Bewley, G. C.

    1989-01-01

    Activated oxygen species have been demonstrated to be the important agents in oxygen toxicity by disrupting the structural and functional integrity of cells through lipid peroxidation events, DNA damage and protein inactivation. The biological consequences of free radical damage have long been hypothesized to be a causal agent in many aging-related diseases. Catalase (H(2)O(2):H(2)O(2) oxidoreductase; EC 1.15.1.1) is one of several enzymes involved in the scavenging of oxygen free radicals and free radical derivatives. The structural gene for catalase in Drosophila melanogaster has been localized to region 75D1-76A on chromosome 3L by dosage responses to segmental aneuploidy. This study reports the isolation of a stable deficiency, Df(3L)Cat(DH104)(75C1-2;75F1), that uncovers the catalase locus and the subsequent isolation of six acatalasemic mutants. All catalase mutants are viable under standard culture conditions and recessive lethal mutations within the 75C1-F1 interval have been shown not to affect catalase activity. Two catalase mutations are amorphic while four are hypomorphic alleles of the Cat(+) locus. The lack of intergenic complementation between the six catalase mutations strongly suggests that there is only one functional gene in Drosophila. One acatalesemic mutation was mapped to position 3-47.0 which resides within the catalase dosage sensitive region. While complete loss of catalase activity confers a severe viability effect, residual levels are sufficient to restore viability to wild type levels. These results suggest a threshold effect for viability and offer an explanation for the general lack of phenotypic effects associated with the known mammalian acatalasemics. PMID:2503418

  17. Lethal body burdens of polar narcotics: Chlorophenols

    SciTech Connect

    Wezel, A.P. van; Punte, S.S.; Opperhuizen, A.

    1995-09-01

    The goal of the present study was to measure in fathead minnow (Pimephales promelas) the lethal body burden (LBB) of three chlorophenols that are known as polar narcotic chemicals. The LBBs of the chlorophenols were compared to LBBs of nonpolar narcotic chemicals to consider if the two classes of narcotic chemicals differ on a body burden level. The LBB of the most acidic chlorophenol was measured at two different levels of pH exposure to determine the influence of the degree of ionization on the magnitude of the LBB. Both n-octanol/water partition coefficients and n-hexane/water partition coefficients of the chlorophenols were determined at different pH levels to consider the influence of ionization on the partition coefficient and to determine the importance of a polar group in the organic phase on the partitioning behavior. Partitioning to n-octanol and n-hexane was used as input in a model to simulate the equilibrium partitioning between hydrophobic and nonhydrophobic and target and nontarget compartments in the fish.

  18. Tumor clone dynamics in lethal prostate cancer.

    PubMed

    Carreira, Suzanne; Romanel, Alessandro; Goodall, Jane; Grist, Emily; Ferraldeschi, Roberta; Miranda, Susana; Prandi, Davide; Lorente, David; Frenel, Jean-Sebastien; Pezaro, Carmel; Omlin, Aurelius; Rodrigues, Daniel Nava; Flohr, Penelope; Tunariu, Nina; S de Bono, Johann; Demichelis, Francesca; Attard, Gerhardt

    2014-09-17

    It is unclear whether a single clone metastasizes and remains dominant over the course of lethal prostate cancer. We describe the clonal architectural heterogeneity at different stages of disease progression by sequencing serial plasma and tumor samples from 16 ERG-positive patients. By characterizing the clonality of commonly occurring deletions at 21q22, 8p21, and 10q23, we identified multiple independent clones in metastatic disease that are differentially represented in tissue and circulation. To exemplify the clinical utility of our studies, we then showed a temporal association between clinical progression and emergence of androgen receptor (AR) mutations activated by glucocorticoids in about 20% of patients progressing on abiraterone and prednisolone or dexamethasone. Resistant clones showed a complex dynamic with temporal and spatial heterogeneity, suggesting distinct mechanisms of resistance at different sites that emerged and regressed depending on treatment selection pressure. This introduces a management paradigm requiring sequential monitoring of advanced prostate cancer patients with plasma and tumor biopsies to ensure early discontinuation of agents when they become potential disease drivers.

  19. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    Goldberg, Allison B; Turk, Benjamin E

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LFinhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and highthroughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  20. Generation and identification of Arabidopsis EMS mutants.

    PubMed

    Qu, Li-Jia; Qin, Genji

    2014-01-01

    EMS mutant analysis is a routine experiment to identify new players in a specific biological process or signaling pathway using forward genetics. It begins with the generation of mutants by treating Arabidopsis seeds with EMS. A mutant with a phenotype of interest (mpi) is obtained by screening plants of the M2 generation under a specific condition. Once the phenotype of the mpi is confirmed in the next generation, map-based cloning is performed to locate the mpi mutation. During the map-based cloning, mpi plants (Arabidopsis Columbia-0 (Col-0) ecotype background) are first crossed with Arabidopsis Landsberg erecta (Ler) ecotype, and the presence or absence of the phenotype in the F1 hybrids indicates whether the mpi is recessive or dominant. F2 plants with phenotypes similar to the mpi, if the mpi is recessive, or those without the phenotype, if the mpi is dominant, are used as the mapping population. As few as 24 such plants are selected for rough mapping. After finding one marker (MA) linked to the mpi locus or mutant phenotype, more markers near MA are tested to identify recombinants. The recombinants indicate the interval in which the mpi is located. Additional recombinants and molecular markers are then required to narrow down the interval. This is an iterative process of narrowing down the mapping interval until no further recombinants or molecular markers are available. The genes in the mapping interval are then sequenced to look for the mutation. In the last step, the wild-type or mutated gene is cloned to generate binary constructs. Complementation or recapitulation provides the most convincing evidence in determining the mutation that causes the phenotype of the mpi. Here, we describe the procedures for generating mutants with EMS and analyzing EMS mutations by map-based cloning.

  1. Parent-of-Origin-Effect rough endosperm Mutants in Maize.

    PubMed

    Bai, Fang; Daliberti, Mary; Bagadion, Alyssa; Xu, Miaoyun; Li, Yubing; Baier, John; Tseung, Chi-Wah; Evans, Matthew M S; Settles, A Mark

    2016-09-01

    Parent-of-origin-effect loci have non-Mendelian inheritance in which phenotypes are determined by either the maternal or paternal allele alone. In angiosperms, parent-of-origin effects can be caused by loci required for gametophyte development or by imprinted genes needed for seed development. Few parent-of-origin-effect loci have been identified in maize (Zea mays) even though there are a large number of imprinted genes known from transcriptomics. We screened rough endosperm (rgh) mutants for parent-of-origin effects using reciprocal crosses with inbred parents. Six maternal rough endosperm (mre) and three paternal rough endosperm (pre) mutants were identified with three mre loci mapped. When inherited from the female parent, mre/+ seeds reduce grain fill with a rough, etched, or pitted endosperm surface. Pollen transmission of pre mutants results in rgh endosperm as well as embryo lethality. Eight of the mutants had significant distortion from the expected one-to-one ratio for parent-of-origin effects. Linked markers for mre1, mre2, and mre3 indicated that the mutant alleles have no bias in transmission. Histological analysis of mre1, mre2, mre3, and pre*-949 showed altered timing of starch grain accumulation and basal endosperm transfer cell layer (BETL) development. The mre1 locus delays BETL and starchy endosperm development, while mre2 and pre*-949 cause ectopic starchy endosperm differentiation. We conclude that many parent-of-origin effects in maize have incomplete penetrance of kernel phenotypes and that there is a large diversity of endosperm developmental roles for parent-of-origin-effect loci.

  2. Zebrafish Mutants calamity and catastrophe Define Critical Pathways of Gene–Nutrient Interactions in Developmental Copper Metabolism

    PubMed Central

    Madsen, Erik C.; Gitlin, Jonathan D.

    2008-01-01

    Nutrient availability is an important environmental variable during development that has significant effects on the metabolism, health, and viability of an organism. To understand these interactions for the nutrient copper, we used a chemical genetic screen for zebrafish mutants sensitive to developmental copper deficiency. In this screen, we isolated two mutants that define subtleties of copper metabolism. The first contains a viable hypomorphic allele of atp7a and results in a loss of pigmentation when exposed to mild nutritional copper deficiency. This mutant displays incompletely penetrant skeletal defects affected by developmental copper availability. The second carries an inactivating mutation in the vacuolar ATPase that causes punctate melanocytes and embryonic lethality. This mutant, catastrophe, is sensitive to copper deprivation revealing overlap between ion metabolic pathways. Together, the two mutants illustrate the utility of chemical genetic screens in zebrafish to elucidate the interaction of nutrient availability and genetic polymorphisms in cellular metabolism. PMID:19008952

  3. Selective targeting of KRAS-Mutant cells by miR-126 through repression of multiple genes essential for the survival of KRAS-Mutant cells

    PubMed Central

    Hara, Toshifumi; Jones, Matthew F.; Subramanian, Murugan; Li, Xiao Ling; Ou, Oliver; Zhu, Yuelin; Yang, Yuan; Wakefield, Lalage M.; Hussain, S. Perwez; Gaedcke, Jochen; Ried, Thomas; Luo, Ji; Caplen, Natasha J.; Lal, Ashish

    2014-01-01

    MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors. PMID:25245095

  4. Ethical language and decision-making for prenatally diagnosed lethal malformations

    PubMed Central

    Wilkinson, Dominic; de Crespigny, Lachlan; Xafis, Vicki

    2014-01-01

    Summary In clinical practice, and in the medical literature, severe congenital malformations such as trisomy 18, anencephaly, and renal agenesis are frequently referred to as ‘lethal’ or as ‘incompatible with life’. However, there is no agreement about a definition of lethal malformations, nor which conditions should be included in this category. Review of outcomes for malformations commonly designated ‘lethal’ reveals that prolonged survival is possible, even if rare. This article analyses the concept of lethal malformations and compares it to the problematic concept of ‘futility’. We recommend avoiding the term ‘lethal’ and suggest that counseling should focus on salient prognostic features instead. For conditions with a high chance of early death or profound impairment in survivors despite treatment, perinatal and neonatal palliative care would be ethical. However, active obstetric and neonatal management, if desired, may also sometimes be appropriate. PMID:25200733

  5. Bacillus anthracis cell wall produces injurious inflammation but paradoxically decreases the lethality of anthrax lethal toxin in a rat model

    PubMed Central

    Cui, Xizhong; Su, Junwu; Li, Yan; Shiloach, Joseph; Solomon, Steven; Kaufman, Jeanne B.; Mani, Haresh; Fitz, Yvonne; Weng, Jia; Altaweel, Laith; Besch, Virginia; Eichacker, Peter Q.

    2012-01-01

    Objectives The in vivo inflammatory effects of the Bacillus anthracis cell wall are unknown. We therefore investigated these effects in rats and, for comparison, those of known inflammatory stimulants, Staphylococcus aureus cell wall or lipopolysaccharide (LPS). Method and Results Sprague–Dawley rats (n = 103) were challenged with increasing B. anthracis cell wall doses (10, 20, 40, 80, or 160 mg/kg) or diluent (control) as a bolus or 24-h infusion. The three highest bolus doses were lethal (20–64% lethality rates) as were the two highest infused doses (13% with each). Comparisons among lethal or nonlethal doses on other measured parameters were not significantly different, and these were combined for analysis. Over the 24 h after challenge initiation with lethal bolus or infusion, compared to controls, ten inflammatory cytokines and NO levels were increased and circulating neutrophils and platelets decreased (P ≤ 0.05). Changes with lethal doses were greater than changes with nonlethal doses (P ≤ 0.01). Lethal bolus or infusion doses produced hypotension or hypoxemia, respectively (P ≤ 0.05). The effects with B. anthracis cell wall were similar to those of S. aureus cell wall or LPS. However, paradoxically administration of B. anthracis cell wall or LPS decreased the lethality of concurrently administered B. anthracis lethal toxin (P < 0.0001 and 0.04, respectively). Conclusion B. anthracis cell wall has the potential to produce inflammatory injury during anthrax infection clinically. However, understanding why cell wall or LPS paradoxically reduced lethality with lethal toxin may help understand this toxin’s pathogenic effects. PMID:19756496

  6. Increased Viral Dissemination in the Brain and Lethality in MCMV-Infected, Dicer-Deficient Neonates

    PubMed Central

    Ostermann, Eleonore; Macquin, Cécile; Krezel, Wojciech; Bahram, Seiamak; Georgel, Philippe

    2015-01-01

    Among Herpesviruses, Human Cytomegalovirus (HCMV or HHV-5) represents a major threat during congenital or neonatal infections, which may lead to encephalitis with serious neurological consequences. However, as opposed to other less prevalent pathogens, the mechanisms and genetic susceptibility factors for CMV encephalitis are poorly understood. This lack of information considerably reduces the prognostic and/or therapeutic possibilities. To easily monitor the effects of genetic defects on brain dissemination following CMV infection we used a recently developed in vivo mouse model based on the neonatal inoculation of a MCMV genetically engineered to express Luciferase. Here, we further validate this protocol for live imaging, and demonstrate increased lethality associated with viral infection and encephalitis in mutant mice lacking Dicer activity. Our data indicate that miRNAs are important players in the control of MCMV pathogenesis and suggest that miRNA-based endothelial functions and integrity are crucial for CMV encephalitis. PMID:25955106

  7. Increased Viral Dissemination in the Brain and Lethality in MCMV-Infected, Dicer-Deficient Neonates.

    PubMed

    Ostermann, Eleonore; Macquin, Cécile; Krezel, Wojciech; Bahram, Seiamak; Georgel, Philippe

    2015-05-01

    Among Herpesviruses, Human Cytomegalovirus (HCMV or HHV-5) represents a major threat during congenital or neonatal infections, which may lead to encephalitis with serious neurological consequences. However, as opposed to other less prevalent pathogens, the mechanisms and genetic susceptibility factors for CMV encephalitis are poorly understood. This lack of information considerably reduces the prognostic and/or therapeutic possibilities. To easily monitor the effects of genetic defects on brain dissemination following CMV infection we used a recently developed in vivo mouse model based on the neonatal inoculation of a MCMV genetically engineered to express Luciferase. Here, we further validate this protocol for live imaging, and demonstrate increased lethality associated with viral infection and encephalitis in mutant mice lacking Dicer activity. Our data indicate that miRNAs are important players in the control of MCMV pathogenesis and suggest that miRNA-based endothelial functions and integrity are crucial for CMV encephalitis. PMID:25955106

  8. Are high-lethality suicide attempters with bipolar disorder a distinct phenotype?

    PubMed

    Oquendo, Maria A; Carballo, Juan Jose; Rajouria, Namita; Currier, Dianne; Tin, Adrienne; Merville, Jessica; Galfalvy, Hanga C; Sher, Leo; Grunebaum, Michael F; Burke, Ainsley K; Mann, J John

    2009-01-01

    Because Bipolar Disorder (BD) individuals making highly lethal suicide attempts have greater injury burden and risk for suicide, early identification is critical. BD patients were classified as high- or low-lethality attempters. High-lethality attempts required inpatient medical treatment. Mixed effects logistic regression models and permutation analyses examined correlations between lethality, number, and order of attempts. High-lethality attempters reported greater suicidal intent and more previous attempts. Multiple attempters showed no pattern of incremental lethality increase with subsequent attempts, but individuals with early high-lethality attempts more often made high-lethality attempts later. A subset of high-lethality attempters make only high-lethality attempts. However, presence of previous low-lethality attempts does not indicate that risk for more lethal, possibly successful, attempts is reduced.

  9. Bloomsbury report on mouse embryo phenotyping: recommendations from the IMPC workshop on embryonic lethal screening

    PubMed Central

    Adams, David; Baldock, Richard; Bhattacharya, Shoumo; Copp, Andrew J.; Dickinson, Mary; Greene, Nicholas D. E.; Henkelman, Mark; Justice, Monica; Mohun, Timothy; Murray, Stephen A.; Pauws, Erwin; Raess, Michael; Rossant, Janet; Weaver, Tom; West, David

    2013-01-01

    Identifying genes that are important for embryo development is a crucial first step towards understanding their many functions in driving the ordered growth, differentiation and organogenesis of embryos. It can also shed light on the origins of developmental disease and congenital abnormalities. Current international efforts to examine gene function in the mouse provide a unique opportunity to pinpoint genes that are involved in embryogenesis, owing to the emergence of embryonic lethal knockout mutants. Through internationally coordinated efforts, the International Knockout Mouse Consortium (IKMC) has generated a public resource of mouse knockout strains and, in April 2012, the International Mouse Phenotyping Consortium (IMPC), supported by the EU InfraCoMP programme, convened a workshop to discuss developing a phenotyping pipeline for the investigation of embryonic lethal knockout lines. This workshop brought together over 100 scientists, from 13 countries, who are working in the academic and commercial research sectors, including experts and opinion leaders in the fields of embryology, animal imaging, data capture, quality control and annotation, high-throughput mouse production, phenotyping, and reporter gene analysis. This article summarises the outcome of the workshop, including (1) the vital scientific importance of phenotyping embryonic lethal mouse strains for basic and translational research; (2) a common framework to harmonise international efforts within this context; (3) the types of phenotyping that are likely to be most appropriate for systematic use, with a focus on 3D embryo imaging; (4) the importance of centralising data in a standardised form to facilitate data mining; and (5) the development of online tools to allow open access to and dissemination of the phenotyping data. PMID:23519032

  10. Impact of non-constant concentration exposure on lethality of inhaled hydrogen cyanide.

    PubMed

    Sweeney, Lisa M; Sommerville, Douglas R; Channel, Stephen R

    2014-03-01

    The ten Berge model, also known as the toxic load model, is an empirical approach in hazard assessment modeling for estimating the relationship between the inhalation toxicity of a chemical and the exposure duration. The toxic load (TL) is normally expressed as a function of vapor concentration (C) and duration (t), with TL equaling C(n) × t being a typical form. Hypothetically, any combination of concentration and time that yields the same "toxic load" will give a constant biological response. These formulas have been developed and tested using controlled, constant concentration animal studies, but the validity of applying these assumptions to time-varying concentration profiles has not been tested. Experiments were designed to test the validity of the model under conditions of non-constant acute exposure. Male Sprague-Dawley rats inhaled constant or pulsed concentrations of hydrogen cyanide (HCN) generated in a nose-only exposure system for 5, 15, or 30 min. The observed lethality of HCN for the 11 different C versus t profiles was used to evaluate the ability of the model to adequately describe the lethality of HCN under the conditions of non-constant inhalation exposure. The model was found to be applicable under the tested conditions, with the exception of the median lethality of very brief, high concentration, discontinuous exposures.

  11. Toxin-induced resistance in Bacillus anthracis lethal toxin-treated macrophages

    PubMed Central

    Salles, Isabelle I.; Tucker, Amy E.; Voth, Daniel E.; Ballard, Jimmy D.

    2003-01-01

    In the current study, we show that macrophages adaptively resist anthrax lethal toxin (LT) through a toxin-activated process termed toxin-induced resistance (TIR). TIR was triggered by pretreatment of RAW 264.7 or J774A.1 macrophages with a low dose of LT for at least 6 h, which resulted in resistance to high doses of LT for 96 h. Activation of TIR required functional toxin, because LT subunits, mutants, and heat-inactivated toxin were unable to trigger resistance. TIR macrophages were not altered in toxin receptor levels or cell cycle profiles. Treatment of TIR macrophages with high doses of LT resulted in a sustained decline in full-length mitogen-activated protein kinase kinase 2, a known target of lethal factor, and a marked reduction in diphosphorylated extracellular response kinases 1,2 for 24 h. However, despite the sustained loss of full-length mitogen-activated protein kinase kinase 2, by 48 h, TIR macrophages regained diphosphorylated extracellular response kinases 1,2, suggesting an adaptation led to recovery of this signaling pathway. TIR macrophages were also able to maintain normal levels of ubiquitinylated proteins, whereas sensitive cells show a rapid reduction in ubiquitin-modified proteins before cell death, indicating a possible alteration in proteasome activity contributed to resistance. These results provide a paradigm for toxin-cell interactions and suggest macrophages are capable of adapting to and tolerating toxic doses of LT. PMID:14519843

  12. Structure and novel functional mechanism of Drosophila SNF in sex-lethal splicing.

    PubMed

    Hu, Jicheng; Cui, Gaofeng; Li, Congmin; Liu, Cong; Shang, Erchang; Lai, Luhua; Jin, Changwen; Wang, Jiwu; Xia, Bin

    2009-09-03

    Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B'', and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF(1621) binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination.

  13. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

    SciTech Connect

    Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

    2006-01-09

    Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

  14. A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

    NASA Technical Reports Server (NTRS)

    Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    2000-01-01

    A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

  15. Sirenomelia Phenotype in Bmp7;Shh Compound Mutants: A Novel Experimental Model for Studies of Caudal Body Malformations

    PubMed Central

    Garrido-Allepuz, Carlos; González-Lamuño, Domingo; Ros, Maria A.

    2012-01-01

    Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i) a primary defect in the generation of caudal mesoderm, ii) a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia. PMID:23028704

  16. Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK

    PubMed Central

    Wang, Jieqiong; Hu, Kewen; Guo, Jiawei; Cheng, Feixiong; Lv, Jing; Jiang, Wenhao; Lu, Weiqiang; Liu, Jinsong; Pang, Xiufeng; Liu, Mingyao

    2016-01-01

    No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1, leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21WAF1/CIP1, either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21WAF1/CIP1. Co-administration of BI-2536 and fasudil either in the LSL-KRASG12D mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers. PMID:27193833

  17. Genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of Escherichia coli.

    PubMed

    Moradpour, Zahra; Sepehrizadeh, Zargham; Rahbarizadeh, Fatemeh; Ghasemian, Abdollah; Yazdi, Mojtaba Tabatabaei; Shahverdi, Ahmad Reza

    2009-07-01

    Complications of chemotherapy, such as appearance of multidrug resistance, have persuaded researchers to consider phage therapy as a new method to combat bacterial infections. In vitro experiments were performed to assess the therapeutic value of genetically modified phages for controlling gastrointestinal Escherichia coli O157:H7 cells in Luria-Bertani (LB) media and contaminated cow milk. We constructed a modified nonreplicating M13-derived phage expressing a lethal catabolite gene activator protein (CAP) that is a Glu181Gln mutant of CAP. The modified phagemid was propagated in the lethal CAP-resistant strain XA3DII. Time-kill assay experiments showed a considerable reduction in the number of surviving bacteria in both LB media and contaminated cow milk. Our further study using other test strains demonstrated that the host range of lethal phage is limited to E. coli strains that produce pili. This study provides a possible strategy for the exploitation of genetically engineered nonlytic phages as bactericidal agents by minimizing the risk of release of progeny phages and endotoxins into the environment. The phage was engineered to remain lethal to its bacterial target, but incapable of replicating therein. Furthermore, the addition of an inducer to express the lethal protein is not required. PMID:19459966

  18. Demographic Toxicokinetic-Toxicodynamic Modeling of Lethal Effects.

    PubMed

    Gergs, André; Gabsi, Faten; Zenker, Armin; Preuss, Thomas G

    2016-06-01

    The aquatic effect assessment of chemicals is largely based on standardized measures of toxicity determined in short-term laboratory tests which are designed to reduce variability. For this purpose, uniform individuals of a species are kept under environmental and chemical exposure conditions which are as constant as possible. In nature, exposure often appears to be pulsed, effects might last longer than a few days, sensitivity might vary among different sized organisms and populations are usually size or age structured and are subject to demographic processes. To overcome this discrepancy, we tested toxicokinetic-toxicodynamic models of different complexities, including body size scaling approaches, for their ability to represent lethal effects observed for Daphnia magna exposed to triphenyltin. The consequences of the different toxicokinetic and toxicodynamic assumptions for population level responses to pulsed exposure are tested by means of an individual based model and are evaluated by confronting model predictions with population data for various pulsed exposure scenarios. We provide an example where increased model complexity reduces the uncertainty in model outputs. Furthermore, our results emphasize the importance of considering population demography in toxicokinetics and toxicodynamics for understanding and predicting potential chemical impacts at higher levels of biological organization. PMID:27158745

  19. Precursors of lethal violence: a death row sample.

    PubMed

    Freedman, D; Hemenway, D

    2000-06-01

    A qualitative methodology based on the standards of criminal defense investigation was used to analyze the social and family histories of 16 men sentenced to death in California. Using a multisource cross-validation methodology, we assessed patterns of impairment, injury and deficit at each of four ecological levels: family, individual, community and social institutions. Investigation documented consistent and pervasive patterns of serious impairment, injury and deficit across the cases and levels. The men share numerous risk factors and few resiliency factors associated with violence. We found family violence in all 16 cases, including severe physical and/or sexual abuse in 14 cases; individual impairments in 16, including 14 with post-traumatic stress disorder, 13 with severe depression and 12 with histories of traumatic brain injury; community isolation and violence in 12; and institutional failure in 15, including 13 cases of severe physical and/or sexual abuse while in foster care or under state youth authority jurisdiction. Appropriate interventions might have made a difference in reducing lethal violence and its precursor conditions.

  20. Transferrin Iron Starvation Therapy for Lethal Bacterial and Fungal Infections

    PubMed Central

    Lin, Lin; Pantapalangkoor, Paul; Tan, Brandon; Bruhn, Kevin W.; Ho, Tiffany; Nielsen, Travis; Skaar, Eric P.; Zhang, Yaofang; Bai, Ruipeng; Wang, Amy; Doherty, Terence M.; Spellberg, Brad

    2014-01-01

    New strategies to treat antibiotic-resistant infections are urgently needed. We serendipitously discovered that stem cell conditioned media possessed broad antimicrobial properties. Biochemical, functional, and genetic assays confirmed that the antimicrobial effect was mediated by supra-physiological concentrations of transferrin. Human transferrin inhibited growth of gram-positive (Staphylococcus aureus), gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens by sequestering iron and disrupting membrane potential. Serial passage in subtherapeutic transferrin concentrations resulted in no emergence of resistance. Infected mice treated with intravenous human transferrin had improved survival and reduced microbial burden. Finally, adjunctive transferrin reduced the emergence of rifampin-resistant mutants of S. aureus in infected mice treated with rifampin. Transferrin is a promising, novel antimicrobial agent that merits clinical investigation. These results provide proof of principle that bacterial infections can be treated in vivo by attacking host targets (ie, trace metal availability) rather than microbial targets. PMID:24446527

  1. Methods of producing protoporphyrin IX and bacterial mutants therefor

    DOEpatents

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  2. Suppression of viral infectivity through lethal defection

    PubMed Central

    Grande-Pérez, Ana; Lázaro, Ester; Lowenstein, Pedro; Domingo, Esteban; Manrubia, Susanna C.

    2005-01-01

    RNA viruses replicate with a very high error rate and give rise to heterogeneous, highly plastic populations able to adapt very rapidly to changing environments. Viral diseases are thus difficult to control because of the appearance of drug-resistant mutants, and it becomes essential to seek mechanisms able to force the extinction of the quasispecies before adaptation emerges. An alternative to the use of conventional drugs consists in increasing the replication error rate through the use of mutagens. Here, we report about persistent infections of lymphocytic choriomeningitis virus treated with fluorouracil, where a progressive debilitation of infectivity leading to eventual extinction occurs. The transition to extinction is accompanied by the production of large amounts of RNA, indicating that the replicative ability of the quasispecies is not strongly impaired by the mutagen. By means of experimental and theoretical approaches, we propose that a fraction of the RNA molecules synthesized can behave as a defective subpopulation able to drive the viable class extinct. Our results lead to the identification of two extinction pathways, one at high amounts of mutagen, where the quasispecies completely loses its ability to infect and replicate, and a second one, at lower amounts of mutagen, where replication continues while the infective class gets extinct because of the action of defectors. The results bear on a potential application of increased mutagenesis as an antiviral strategy in that low doses of a mutagenic agent may suffice to drive persistent virus to extinction. PMID:15767582

  3. Gill lesions in the major carp, Labeo rohita exposed to lethal and sublethal concentrations of tannery effluent.

    PubMed

    Dhanapakiam, P; Sampoorani, V; Kavitha, M; Ramasamy, V K; Chandrakala, A; Aruna, K C

    2004-07-01

    The major carp, Labeo rohita were exposed to (0.873%) lethal and sublethal (0.073%) concentrations of tannery effluent for 24h and 40 days respectively under static bioassay condition. The surface architecture of gill revealed severe damages such as : fusion and clumping in the middle and distal parts of the primary lamellae, swelling and deterioration of the cells. The interlamellar space was filled either with hyperplastic epithelial or mucous cells. Secondary lamellae lost their identity and appeared finger like in structure in the lethal concentration and necrosis was observed in the primary and secondary epithelium. Swelling of primary and secondary epithelial cells was evident in sublethal concentration.

  4. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    PubMed Central

    Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  5. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet.

    PubMed

    Vanderperre, Benoît; Herzig, Sébastien; Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-05-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  6. Isolation and characterization of nonspreading mutants of the gliding bacterium Cytophaga johnsonae.

    PubMed Central

    Chang, L E; Pate, J L; Betzig, R J

    1984-01-01

    Three approaches were taken to isolate a total of 153 nonspreading mutants derived from our laboratory strain of Cytophaga johnsonae, UW101, or from its auxotrophic derivative, UW10538. Characterization of 109 of these mutants led to their placement in five general categories: (i) motile, nonspreading (MNS) mutants whose cells are motile to various degrees but whose colonies fail to spread on agar gels under any conditions of incubation; (ii) conditional nonspreading (CNS) mutants with motile cells whose colonies require more moisture to spread on agar gels than do those of wild-type cells; (iii) filamentous conditional motility (FCM) mutants whose cells grow as nonmotile filaments or as motile cells with wild-type morphology, depending on conditions of incubation; (iv) short, tumbling, nonspreading (STN) mutants with short cells that tumble constantly; and (v) truly nonmotile (TNM) mutants whose cells never move and whose colonies never spread under any conditions tested. All TNM mutants exhibited a remarkable pleiotropy not seen in the other four classes of mutants: all were resistant to 39 phages to which wild-type cells are sensitive, and all were unable to digest chitin, which is digested by wild-type cells. The correlation between ability to move and phage sensitivity was strengthened further by showing that 150 additional TNM mutants derived from UW101 and 43 TNM mutants derived from 29 independent isolates of C. johnsonae were resistant to all phages to which their parents were sensitive. Furthermore, motile revertants of TNM mutants became phage sensitive, and temperature-sensitive mutants were motile and phage sensitive at 25 degrees C and nonmotile and phage resistant at 32 degrees C. Evidence supports the conclusion that any mutation rendering cells truly nonmotile invariably alters cell surface-associated properties such as phage sensitivity and chitin digestion merely as a consequence of changing a moving cell surface to a static surface. Images PMID

  7. Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

    PubMed

    Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

    2016-03-10

    Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. PMID:26608331

  8. A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (A{sup y}) mutation

    SciTech Connect

    Michaud, E.J.; Klebig, M.L.; Stubbs, L.J.; Russell, L.B.; Woychik, R.P.; Bultman, S.J. |; Vugt, M.J. van |

    1994-03-29

    Lethal yellow (A{sup y}) is a mutation at the mouse agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and an increased propensity to develop a variety of spontaneous and induced tumors. Additionally, homozygosity for A{sup y} results in preimplantation lethality, which terminates development by the blastocyst stage. The A{sup y} mutation is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transcriptional orientation as agouti and maps 280 kb proximal to the 3{prime} end of the agouti gene. The authors present a model for the structure of the A{sub y} allele that can explain the dominant pleiotropic effects associated with this mutation, as well as the recessive lethality, which is unrelated to the agouti gene.

  9. Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma.

    PubMed

    Kiessling, Michael K; Curioni-Fontecedro, Alessandra; Samaras, Panagiotis; Lang, Silvia; Scharl, Michael; Aguzzi, Adriano; Oldrige, Derek A; Maris, John M; Rogler, Gerhard

    2016-01-01

    High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies. PMID:26821351

  10. Targeting the mTOR Complex by Everolimus in NRAS Mutant Neuroblastoma

    PubMed Central

    Kiessling, Michael K.; Curioni-Fontecedro, Alessandra; Samaras, Panagiotis; Lang, Silvia; Scharl, Michael; Aguzzi, Adriano; Oldrige, Derek A.; Maris, John M.; Rogler, Gerhard

    2016-01-01

    High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies. PMID:26821351

  11. Genetics Home Reference: Amish lethal microcephaly

    MedlinePlus

    ... movement or the ability to track faces and sounds. Affected infants live only about six months. Related Information What does it mean if a disorder seems to run in my family? What is the prognosis of a genetic condition? ...

  12. Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

    PubMed

    Pierson, B K; Keith, L M; Leovy, J G

    1984-07-01

    Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.

  13. UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli.

    PubMed

    Lestini, Roxane; Michel, Bénédicte

    2008-09-01

    Rep and UvrD are two related Escherichia coli helicases, and inactivating both is lethal. Based on the observation that the synthetic lethality of rep and uvrD inactivation is suppressed in the absence of the recombination presynaptic proteins RecF, RecO, or RecR, it was proposed that UvrD is essential in the rep mutant to counteract a deleterious RecFOR-dependent RecA binding. We show here that the synthetic lethality of rep and uvrD mutations is also suppressed by recQ and recJ inactivation but not by rarA inactivation. Furthermore, it is independent of the action of UvrD in nucleotide excision repair and mismatch repair. These observations support the idea that UvrD counteracts a deleterious RecA binding to forks blocked in the rep mutant. An ATPase-deficient mutant of UvrD [uvrD(R284A)] is dominant negative in a rep mutant, but only in the presence of all RecQJFOR proteins, suggesting that the UvrD(R284A) mutant protein is deleterious when it counteracts one of these proteins. In contrast, the uvrD252 mutant (G30D), which exhibits a strongly decreased ATPase activity, is viable in a rep mutant, where it allows replication fork reversal. We conclude that the residual ATPase activity of UvrD252 prevents a negative effect on the viability of the rep mutant and allows UvrD to counteract the action of RecQ, RecJ, and RecFOR at forks blocked in the rep mutant. Models for the action of UvrD at blocked forks are proposed. PMID:18567657

  14. Cumulative Mutations Affecting Sterol Biosynthesis in the Yeast Saccharomyces cerevisiae Result in Synthetic Lethality That Is Suppressed by Alterations in Sphingolipid Profiles

    PubMed Central

    Valachovic, Martin; Bareither, Bart M.; Bhuiyan, M. Shah Alam; Eckstein, James; Barbuch, Robert; Balderes, Dina; Wilcox, Lisa; Sturley, Stephen L.; Dickson, Robert C.; Bard, Martin

    2006-01-01

    UPC2 and ECM22 belong to a Zn(2)–Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2Δ ecm22Δ in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2Δ ecm22Δ are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2Δ upc2Δ ecm22Δ triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1Δ, erg2Δ, erg6Δ, and erg28Δ in the upc2Δ ecm22Δ genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2Δ upc2Δ ecm22Δ. We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance. PMID:16702413

  15. Selective reconstitution of liver cholesterol biosynthesis promotes lung maturation but does not prevent neonatal lethality in Dhcr7 null mice

    PubMed Central

    Yu, Hongwei; Li, Man; Tint, G Stephen; Chen, Jianliang; Xu, Guorong; Patel, Shailendra B

    2007-01-01

    Background Targeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver. Results We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice. Conclusion The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity. PMID:17408495

  16. A novel balanced-lethal host-vector system based on glmS.

    PubMed

    Kim, Kwangsoo; Jeong, Jae Ho; Lim, Daejin; Hong, Yeongjin; Yun, Misun; Min, Jung-Joon; Kwak, Sahng-June; Choy, Hyon E

    2013-01-01

    During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.

  17. Empirical complexities in the genetic foundations of lethal mutagenesis.

    PubMed

    Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

    2013-10-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

  18. Chemotherapy agents and the induction of late lethal defects.

    PubMed

    Seymour, C B; Mothersill, C

    1991-01-01

    Radiation is now known to be capable of producing both lethal and non lethal lesions in a variety of mammalian cells which may not be expressed for several cell divisions after the initial insult. The mechanism by which such an effect occurs is unknown. Because of the possible implications for cancer treatment, if such an effect also occurred following chemotherapy exposure, cells were exposed to various cytotoxic chemotherapy agents with known and well characterised effects on DNA or other areas of cell function or structure. The results indicate that late lethal defects are not detectable after treatment with appropriate ranges of doses of cisplatinum, vincristine, BCNU or adriamycin, but that they are induced by Bleomycin and to a lesser extent by 5-Fluorouracil. Bleomycin is known to cause strand breaks and is regarded as a radiomimetic agent. 5-Fluorouracil may act by preventing efficient and faithful synthesis of DNA, allowing mutations to become integrated into the genome. The occurrence of lethal mutations with both these agents supports previous suggestions that error-prone repair of DNA base sequence abnormalities may be fundamental to the process of late lethal damage production in mammalian cells. The cloning efficiency of cells which survived exposure to Vincristine or BCNU over a wide dose range was found to be significantly increased; this may represent an adaptive response to the drugs.

  19. The Rorschach Suicide Constellation: assessing various degrees of lethality.

    PubMed

    Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

    2001-04-01

    In this article we examine the relation between the Rorschach Comprehensive System's Suicide Constellation (S-CON; Exner, 1993; Exner & Wiley, 1977) and lethality of suicide attempts during the course of patients' hospitalization at the Austen Riggs Center (Stockbridge, MA). Patient records were rated as nonsuicidal (n = 37), parasuicidal (n = 37), or near-lethal (n = 30) based on the presence and lethality of self-destructive acts. Diagnostic efficiency statistics utilizing a cutoff score of 7 or more positive indicators successfully predicted which patients would engage in near-lethal suicidal activity relative to parasuicidal patients (overall correct classification rate [OCC] = .79), nonsuicidal inpatients (OCC = .79), and college students (OCC = .89). Although these predictions were influenced by relatively high base rates in the hospital population (14.5%), base rate estimates were calculated for other hypothetical populations revealing different prediction estimates that should be considered when judging the relative efficacy of the S-CON. Logistic regression analysis revealed that an S-CON score of 7 or more was the sole predictor of near-lethal suicide attempts among 9 psychiatric and demographic variables.

  20. The Rorschach Suicide Constellation: assessing various degrees of lethality.

    PubMed

    Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

    2001-04-01

    In this article we examine the relation between the Rorschach Comprehensive System's Suicide Constellation (S-CON; Exner, 1993; Exner & Wiley, 1977) and lethality of suicide attempts during the course of patients' hospitalization at the Austen Riggs Center (Stockbridge, MA). Patient records were rated as nonsuicidal (n = 37), parasuicidal (n = 37), or near-lethal (n = 30) based on the presence and lethality of self-destructive acts. Diagnostic efficiency statistics utilizing a cutoff score of 7 or more positive indicators successfully predicted which patients would engage in near-lethal suicidal activity relative to parasuicidal patients (overall correct classification rate [OCC] = .79), nonsuicidal inpatients (OCC = .79), and college students (OCC = .89). Although these predictions were influenced by relatively high base rates in the hospital population (14.5%), base rate estimates were calculated for other hypothetical populations revealing different prediction estimates that should be considered when judging the relative efficacy of the S-CON. Logistic regression analysis revealed that an S-CON score of 7 or more was the sole predictor of near-lethal suicide attempts among 9 psychiatric and demographic variables. PMID:11393464

  1. Recapitulation of the ovum mutant (Om) phenotype and loss of Om locus polarity in cloned mouse embryos.

    PubMed

    Gao, Shaorong; Wu, Guangming; Han, Zhiming; de la Casa-Esperón, Elena; Sapienza, Carmen; Latham, Keith E

    2005-02-01

    The ovum mutant (Om) locus in mice affects early interactions between sperm and egg that in turn affect viability of embryos beyond the morula stage. Crosses of DDK females to males of many other inbred strains are 95% lethal around the morula stage, whereas reciprocal crosses are fully viable. Available data indicate that the early lethality is the result of an interaction between a factor in the ooplasm and the paternal genome. In this study, we examined whether this lethal interaction would likewise occur in cloned embryos produced by somatic cell nuclear transfer. We find that the Om effect is recapitulated but that the parental origin effect at the Om locus is no longer evident in cloned embryos.

  2. The population genetics of X-autosome synthetic lethals and steriles.

    PubMed

    Lachance, Joseph; Johnson, Norman A; True, John R

    2011-11-01

    Epistatic interactions are widespread, and many of these interactions involve combinations of alleles at different loci that are deleterious when present in the same individual. The average genetic environment of sex-linked genes differs from that of autosomal genes, suggesting that the population genetics of interacting X-linked and autosomal alleles may be complex. Using both analytical theory and computer simulations, we analyzed the evolutionary trajectories and mutation-selection balance conditions for X-autosome synthetic lethals and steriles. Allele frequencies follow a set of fundamental trajectories, and incompatible alleles are able to segregate at much higher frequencies than single-locus expectations. Equilibria exist, and they can involve fixation of either autosomal or X-linked alleles. The exact equilibrium depends on whether synthetic alleles are dominant or recessive and whether fitness effects are seen in males, females, or both sexes. When single-locus fitness effects and synthetic incompatibilities are both present, population dynamics depend on the dominance of alleles and historical contingency (i.e., whether X-linked or autosomal mutations occur first). Recessive synthetic lethality can result in high-frequency X-linked alleles, and dominant synthetic lethality can result in high-frequency autosomal alleles. Many X-autosome incompatibilities in natural populations may be cryptic, appearing to be single-locus effects because one locus is fixed. We also discuss the implications of these findings with respect to standing genetic variation and the origins of Haldane's rule.

  3. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase

    SciTech Connect

    Mat-Jan, F.; Alam, K.Y.; Clark, D.P. )

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.

  4. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants*

    PubMed Central

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J.; Milbrandt, Jeffrey

    2015-01-01

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD+ biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD+ synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. PMID:26018082

  5. Lethal combat and sex ratio evolution in a parasitoid wasp

    PubMed Central

    Innocent, Tabitha M.; Savage, Joanna; West, Stuart A.; Reece, Sarah E.

    2013-01-01

    Sex allocation theory provides excellent opportunities for testing how behavior and life histories are adjusted in response to environmental variation. One of the most successful areas from this respect is Hamilton’s local mate competition theory. As predicted by theory, a large number of animal species have been shown to adjust their offspring sex ratios (proportion male) conditionally, laying less female-biased sex ratios as the number of females that lay eggs on a patch increases. However, recent studies have shown that this predicted pattern is not followed by 2 parasitoid species in the genus Melittobia, which always produce extremely female-biased sex ratios. A possible explanation for this is that males fight fatally and that males produced by the first female to lay eggs on a patch have a competitive advantage over later emerging males. This scenario would negate the advantage of later females producing a less female-biased sex ratio. Here we examine fatal fighting and sex ratio evolution in another species, Melittobia acasta. We show that females of this species also fail to adjust their offspring sex ratio in response to the number of females laying eggs on a patch. We then show that although earlier emerging males do have an advantage in winning fights, this advantage 1) can be reduced by an interaction with body size, with larger males more likely to win fights and 2) only holds for a brief period around the time at which the younger males emerge from their pupae. This suggests that lethal male combat cannot fully explain the lack of sex ratio shift observed in Melittobia species. We discuss alternative explanations. PMID:24273326

  6. Lethal effects of short-wavelength visible light on insects

    NASA Astrophysics Data System (ADS)

    Hori, Masatoshi; Shibuya, Kazuki; Sato, Mitsunari; Saito, Yoshino

    2014-12-01

    We investigated the lethal effects of visible light on insects by using light-emitting diodes (LEDs). The toxic effects of ultraviolet (UV) light, particularly shortwave (i.e., UVB and UVC) light, on organisms are well known. However, the effects of irradiation with visible light remain unclear, although shorter wavelengths are known to be more lethal. Irradiation with visible light is not thought to cause mortality in complex animals including insects. Here, however, we found that irradiation with short-wavelength visible (blue) light killed eggs, larvae, pupae, and adults of Drosophila melanogaster. Blue light was also lethal to mosquitoes and flour beetles, but the effective wavelength at which mortality occurred differed among the insect species. Our findings suggest that highly toxic wavelengths of visible light are species-specific in insects, and that shorter wavelengths are not always more toxic. For some animals, such as insects, blue light is more harmful than UV light.

  7. Lethal effects of short-wavelength visible light on insects.

    PubMed

    Hori, Masatoshi; Shibuya, Kazuki; Sato, Mitsunari; Saito, Yoshino

    2014-12-09

    We investigated the lethal effects of visible light on insects by using light-emitting diodes (LEDs). The toxic effects of ultraviolet (UV) light, particularly shortwave (i.e., UVB and UVC) light, on organisms are well known. However, the effects of irradiation with visible light remain unclear, although shorter wavelengths are known to be more lethal. Irradiation with visible light is not thought to cause mortality in complex animals including insects. Here, however, we found that irradiation with short-wavelength visible (blue) light killed eggs, larvae, pupae, and adults of Drosophila melanogaster. Blue light was also lethal to mosquitoes and flour beetles, but the effective wavelength at which mortality occurred differed among the insect species. Our findings suggest that highly toxic wavelengths of visible light are species-specific in insects, and that shorter wavelengths are not always more toxic. For some animals, such as insects, blue light is more harmful than UV light.

  8. Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

    PubMed

    Klein, Gracjana; Lindner, Buko; Brabetz, Werner; Brade, Helmut; Raina, Satish

    2009-06-01

    To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Delta(waaC lpxL lpxM lpxP) strains showed primarily Kdo(2)-lipid IV(A), indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 degrees C. These lipid IV(A) derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. DeltawaaA and Delta(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and sigma(E) signal transduction pathways. Delta(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Delta(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Delta(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

  9. Cyclopeptide toxins of lethal amanitas: Compositions, distribution and phylogenetic implication.

    PubMed

    Tang, Shanshan; Zhou, Qian; He, Zhengmi; Luo, Tao; Zhang, Ping; Cai, Qing; Yang, Zhuliang; Chen, Jia; Chen, Zuohong

    2016-09-15

    Lethal amanitas (Amanita sect. Phalloideae) are responsible for 90% of all fatal mushroom poisonings. Since 2000, more than ten new lethal Amanita species have been discovered and some of them had caused severe mushroom poisonings in China. However, the contents and distribution of cyclopeptides in these lethal mushrooms remain poorly known. In this study, the diversity of major cyclopeptide toxins in seven Amanita species from Eastern Asia and three species from Europe and North America were systematically analyzed, and a new approach to inferring phylogenetic relationships using cyclopeptide profile was evaluated for the first time. The results showed that there were diversities of the cyclopeptides among lethal Amanita species, and cyclopeptides from Amanita rimosa and Amanita fuligineoides were reported for the first time. The amounts of amatoxins in East Asian Amanita species were significantly higher than those in European and North American species. The analysis of distribution of amatoxins and phallotoxins in various Amanita species demonstrated that the content of phallotoxins was higher than that of amatoxins in Amanita phalloides and Amanita virosa. In contrast, the content of phallotoxins was significantly lower than that of amatoxins in all East Asian lethal Amanita species tested. However, the distribution of amatoxins and phallotoxins in different tissues showed the same tendency. Eight cyclopeptides and three unknown compounds were identified using cyclopeptide standards and high-resolution MS. Based on the cyclopeptide profiles, phylogenetic relationships of lethal amanitas were inferred through a dendrogram generated by UPGMA method. The results showed high similarity to the phylogeny established previously based on the multi-locus DNA sequences. PMID:27476461

  10. [LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

    PubMed

    Semenova, G A; Chekunova, E M; Ladygin, V G

    2015-01-01

    The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed.

  11. [LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

    PubMed

    Semenova, G A; Chekunova, E M; Ladygin, V G

    2015-01-01

    The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed. PMID:26281212

  12. Lethal toxicity of cadmium to Cyprinus carpio and Tilapia aurea

    SciTech Connect

    Not Available

    1986-09-01

    There have been several studies of the lethal toxicity of cadmium to freshwater fishes, but further information is required on a number of points. For example, the shallow slope which is characteristic of the cadmium toxicity curve makes interspecific comparisons difficult. There also is a paucity of information on cadmium toxicity to non-Salmonid European species. As part of a study of the water quality requirements of cultured fish species in the Mediterranean, the authors report on the lethal toxicity of cadmium to two such species, the common carp Cyprinus carpio, and Tilapia aurea, for which little information has previously been reported.

  13. Approaches to Identifying Synthetic Lethal Interactions in Cancer

    PubMed Central

    Thompson, Jordan M.; Nguyen, Quy H.; Singh, Manpreet; Razorenova, Olga V.

    2015-01-01

    Targeting synthetic lethal interactions is a promising new therapeutic approach to exploit specific changes that occur within cancer cells. Multiple approaches to investigate these interactions have been developed and successfully implemented, including chemical, siRNA, shRNA, and CRISPR library screens. Genome-wide computational approaches, such as DAISY, also have been successful in predicting synthetic lethal interactions from both cancer cell lines and patient samples. Each approach has its advantages and disadvantages that need to be considered depending on the cancer type and its molecular alterations. This review discusses these approaches and examines case studies that highlight their use. PMID:26029013

  14. [Attenuation in the virulence of a mutant of Junin virus in suckling mice].

    PubMed

    Scolaro, L A; Mersich, S E; Damonte, E B

    1987-01-01

    The virulence in neonatal mice of a temperature-sensitive mutant of Junin virus, named C167, was studied. The thermosensitive properties of this mutant were tested by titration on Vero cells at 37 and 40 degrees C. The ratio of infectivity 40/37 was approximately 100-fold lower for C167 with respect to XJC13 (Table 1). The attenuation of C167 was determined by measurement of mean survival time and 50% lethal dose after intracerebral injection of 2 and 11 day old mice. For C167 the lethality index (expressed ad the ratio TCID50/LD50) was greater than 580, while for XJC13 the index was 4.4 (Table 2). The lack of virulence of C167 was correlated with a restricted ability to replicate in suckling mouse brains. By contrast, the mutant and the parental virus grew to a similar titre in Vero cells (Figure 2). Both viruses were indistinguishable in cross-neutralization tests using hyperimmune antisera.

  15. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines

    PubMed Central

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate. PMID:26519470

  16. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

    PubMed

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

  17. Design, overexpression, and purification of polymerization-blocked yeast αβ-tubulin mutants.

    PubMed

    Johnson, Vinu; Ayaz, Pelin; Huddleston, Patrick; Rice, Luke M

    2011-10-11

    Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αβ-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αβ-tubulin represents an obstacle to deeper insight into αβ-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αβ-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αβ-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αβ-tubulin mutants. These "blocked" forms of αβ-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αβ-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αβ-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αβ-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation. PMID:21888381

  18. Germination-defective mutant of Neurospora crassa that responds to siderophores

    NASA Technical Reports Server (NTRS)

    Charlang, G.; Williams, N. P.

    1977-01-01

    A conditionally germination-defective mutant of Neurospora crassa has been found to be partially curable by ferricrocin and other siderophores. The mutant conidia rapidly lose their membrane-bound siderophores when suspended in buffer or growth media. Germination is consequently delayed unless large numbers of conidia are present (positive population effect). This indicates that the mutant has a membrane defect involving the siderophore attachment site.

  19. Directed Synthesis of a Segmental Chromosomal Transposition: An Approach to the Study of Chromosomes Lethal to the Gametophyte Generation of Maize

    PubMed Central

    Birchler, J. A.; Levin, D. M.

    1991-01-01

    Because of the haploid nature of the gametophyte generation of plants, most mutations that are lethal or detrimental to the gametophytes cannot be recovered. Our laboratory is currently developing several techniques to overcome this situation. In this paper, a procedure is described to generate directed segmental chromosomal transpositions. The method involves recovery of recombinants between reciprocal translocation overlaps such that one region of the genome is inserted into a nonhomologous chromosome in a predetermined and directed manner. This duplicated segment then could serve to cover deficiencies or mutations, lethal to the gametophytes, in the region from whence it originated. The manipulation of segmental chromosomal transpositions for analyzing mutants lethal or detrimental to the gametophyte generation is discussed. The procedure to generate transpositions, the translocations between normal A and supernumerary B chromosomes that generate deficiencies in the male gametes, the r-X1 chromosome that generates deficiencies in the female gametes and other techniques available in maize form a system to analyze gametophyte lethal mutations. PMID:2016056

  20. Attenuated virulence of a Francisella mutant lacking the lipid A 4′-phosphatase

    PubMed Central

    Wang, Xiaoyuan; Ribeiro, Anthony A.; Guan, Ziqiang; Abraham, Soman N.; Raetz, Christian R. H.

    2007-01-01

    Francisella tularensis causes tularemia, a highly contagious disease of animals and humans, but the virulence features of F. tularensis are poorly defined. F. tularensis and the related mouse pathogen Francisella novicida synthesize unusual lipid A molecules lacking the 4′-monophosphate group typically found in the lipid A of Gram-negative bacteria. LpxF, a selective phosphatase located on the periplasmic surface of the inner membrane, removes the 4′-phosphate moiety in the late stages of F. novicida lipid A assembly. To evaluate the relevance of the 4′-phosphatase to pathogenesis, we constructed a deletion mutant of lpxF and compared its virulence with wild-type F. novicida. Intradermal injection of 106 wild-type but not 108 mutant F. novicida cells is lethal to mice. The rapid clearance of the lpxF mutant is associated with a stronger local cytokine response and a greater influx of neutrophils compared with wild-type. The F. novicida mutant was highly susceptible to the cationic antimicrobial peptide polymyxin. LpxF therefore represents a kind of virulence factor that confers a distinct lipid A phenotype, preventing Francisella from activating the host innate immune response and preventing the bactericidal actions of cationic peptides. Francisella lpxF mutants may be useful for immunization against tularemia. PMID:17360489

  1. Lethal neuroleptic malignant syndrome due to amisulpride.

    PubMed

    Musshoff, Frank; Doberentz, Elke; Madea, Burkhard

    2013-06-01

    A 42-year old-man was found lying in his bed having seizures. Later he became unconscious and hypotonic developing mydriasis as well as rigidity. The body core temperature (rectal temperature) was above 42 °C. Blood pH was decreased during treatment, and his general condition deteriorated. The patient developed gasping respiration, ventricular fibrillation, and died. During autopsy and histological investigation cerebral and pulmonary edema were noted together with general congestion of the internal organs. Further observations included contraction bands of myocytes, a contracted spleen, fibrosis of the liver, and gall stones. Toxicological analyses of peripheral blood revealed the following results: amisulpride 4.65 mg/l, biperiden 0.12 mg/l, imipramine 0.33 mg/l, and desipramine 0.68 mg/l. An amisulpride-induced neuroleptic malignant syndrome was therefore diagnosed as the patho-physiological mechanism leading to death.

  2. Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia.

    PubMed

    Shibata, H; Fukushi, M; Igarashi, A; Misumi, Y; Ikehara, Y; Ohashi, Y; Oda, K

    1998-05-01

    We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses ( approximately 80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218-->Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162-->Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162-->Thr) molecule caused by its aggregation is the molecular basis for the lethal

  3. Effects of sub-lethal and lethal doses of lambda-cyhalothrin on oviposition experience and host-searching behaviour of a parasitic wasp, Aphidius ervi.

    PubMed

    Desneux, Nicolas; Pham-Delègue, Minh-Hà; Kaiser, Laure

    2004-04-01

    In many parasitoid species, the recognition of chemical signals is essential to find specific hosts. This function is often impaired by exposure to insecticides that are usually neurotoxic. The behaviour of the Hymenopterous parasitoid Aphidius ervi (Haliday) (Hymenoptera: Aphidiinae) after surviving low doses of the pyrethroid lambda-cyhalothrin was examined in laboratory conditions. The host aphid was Myzus persicae (Sulzer) (Homoptera: Aphididae) on oilseed rape. Parasitoid females were exposed by contact with dry residues of the active ingredient at a lethal dose, LD20, and a sub-lethal dose, LD0.1. In a four-armed olfactometer, untreated and inexperienced females were attracted by the odour of M. persicae-infested plants and previous oviposition experience increased the duration of the attraction response. The response of inexperienced females decreased after an exposure to LD0.1 but not to LD20. No effect was observed when females had an oviposition experience prior to the olfactometer test. The oviposition activity was significantly decreased in the LD20-treated group but not in the LD0.1-treated one. All effects disappeared within 24h. Our work shows that orientation and oviposition behaviours may be impaired by low doses of lambda-cyhalothrin, depending on the dose, the parasitoid experience and the type of behaviour.

  4. Intracytoplasmic inclusion bodies in the chondrocytes of type I lethal achondrogenesis.

    PubMed

    Yang, S S; Heidelberger, K P; Bernstein, J

    1976-11-01

    Lethal achondrogenesis in the past has been frequently confused with achondroplasia. Clinical and radiologic advances in the last decade have led to clear differentiation of this condition from other types of bone dysplasia. It is further separated into two types on the basis of radiographic and pathologic findings. Re-evaluation of the histologic features has led to the recognition of heretofore unrecognized intracytoplasmic inclusion bodies in the chondrocytes of type 1 achondrogenesis. The finding of inclusions strengthens the differentiation of the two types and may have implications for the pathogenesis of the form of chondrodystrophy. PMID:186383

  5. Small Molecule Inhibitors of Anthrax Lethal Factor Toxin

    PubMed Central

    Williams, John D.; Khan, Atiyya R.; Cardinale, Steven C.; Butler, Michelle M.; Bowlin, Terry L.; Peet, Norton P.

    2014-01-01

    This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like compounds. PMID:24290062

  6. Dominant-lethal mutations and heritable translocations in mice

    SciTech Connect

    Generoso, W.M.

    1983-01-01

    Chromosome aberrations are a major component of radiation or chemically induced genetic damage in mammalian germ cells. The types of aberration produced are dependent upon the mutagen used and the germ-cell stage treated. For example, in male meiotic and postmeiotic germ cells certain alkylating chemicals induce both dominant-lethal mutations and heritable translocations while others induce primarily dominant-lethal mutations. Production of these two endpoints appears to be determined by the stability of alkylation products with the chromosomes. If the reaction products are intact in the male chromosomes at the time of sperm entry, they may be repaired in fertilized eggs. If repair is not effected and the alkylation products persist to the time of pronuclear chromosome replication, they lead to chromatid-type aberrations and eventually to dominant-lethality. The production of heritable translocations, on the other hand, requires a transformation of unstable alkylation products into suitable intermediate lesions. The process by which these lesions are converted into chromosome exchange within the male genome takes place after sperm enters the egg but prior to the time of pronuclear chromosome replication (i.e., chromosome-type). Thus, dominant-lethal mutations result from both chromatid- and chromosome-type aberrations while heritable translocations result primarily from the latter type. DNA target sites associated with the production of these two endpoints are discussed.

  7. Subcutaneous wounding postirradiation reduces radiation lethality in mice.

    PubMed

    Garrett, Joy; Orschell, Christie M; Mendonca, Marc S; Bigsby, Robert M; Dynlacht, Joseph R

    2014-06-01

    The detonation of an improvised nuclear device during a radiological terrorist attack could result in the exposure of thousands of civilians and first responders to lethal or potentially lethal doses of ionizing radiation (IR). There is a major effort in the United States to develop phamacological mitigators of radiation lethality that would be effective particularly if administered after irradiation. We show here that giving female C57BL/6 mice a subcutaneous surgical incision after whole body exposure to an LD50/30 X-ray dose protects against radiation lethality and increases survival from 50% to over 90% (P = 0.0001). The increase in survival, at least in part, appears to be due to enhanced recovery of hematopoiesis, notably red blood cells, neutrophils and platelets. While a definitive mechanism has yet to be elucidated, we propose that this approach may be used to identify potentially novel mechanisms and pathways that could aid in the development of novel pharmacological radiation countermeasures. PMID:24811864

  8. The Prevalence, Lethality and Intent of Suicide Attempts among Adolescents.

    ERIC Educational Resources Information Center

    Andrews, Judy A.; Lewinsohn, Peter M.

    Although suicide is the second leading cause of death among adolescents in the United States, little is known about the prevalence or characteristics of suicide attempts among adolescents. Data from 1,710 adolescents attending 9 high schools in 5 communities were examined to determine the prevalence of suicide attempts and the lethality and intent…

  9. An overview of the future of non-lethal weapons.

    PubMed

    Alexander, J B

    2001-01-01

    During the past decade, vast changes have occurred in the geopolitical landscape and the nature of the types of conflicts in which technologically developed countries have been involved. While the threat of conventional war remains, forces have been more frequently deployed in situations that require great restraint. Adversaries are often likely to be elusive and commingled with noncombatants. There has been some shift in public opinion away from tolerance of collateral casualties. Therefore there is a need to be able to apply force while limiting casualties. Non-lethal weapons provide part of the solution. Among the changes that will influence the future have been studies by the US and NATO concerning the use of non-lethal weapons, coincidental with increased funding for their development and testing. New concepts and policies have recently been formalized. Surprisingly, the most strident objections to the implementation of non-lethal weapons have come from organizations that are ostensibly designed to protect non-combatants. These arguments are specious and, while technically and academically challenging, actually serve to foster an environment that will result in the deaths of many more innocent civilians. They misconstrue technology with human intent. The reasons for use of force will not abate. Alternatives to bombs, missiles, tanks and artillery must therefore be found. Non-lethal weapons are not a panacea but do offer the best hope of minimizing casualties while allowing nations or alliances the means to use force in protection of national or regional interests.

  10. Histopathological effects of anthrax lethal factor on rat liver.

    PubMed

    Altunkaynak, Berrin Zuhal; Ozbek, Elvan

    2015-01-01

    Bacillus anthracis, the causative agent of anthrax, has become an increasingly important scientific topic due to its potential role in bioterrorism. The lethal toxin (LT) of B. anthracis consists of lethal factor (LF) and a protective antigen (PA). This study investigated whether only lethal factor was efficient as a hepatotoxin in the absence of the PA. To achieve this aim, LF (100 µg/kg body weight, dissolved in sterile distilled water) or distilled water vehicle were intraperitoneally injected once into adult rats. At 24 h post-injection, the hosts were euthanized and their livers removed and tissue samples examined under light and electron microscopes. As a result of LF application, hepatic injury - including cytoplasmic and nuclear damage in hepatocytes, sinusoidal dilatation, and hepatocellular lysis - became apparent. Further, light microscopic analyses of liver sections from the LF-injected rats revealed ballooning degeneration and cytoplasmic loss within hepatocytes, as well as peri-sinusoidal inflammation. Additionally, an increase in the numbers of Kupffer cells was evident. Common vascular injuries were also found in the liver samples; these injuries caused hypoxia and pathological changes. In addition, some cytoplasmic and nuclear changes were detected within the liver ultrastructure. The results of these studies allow one to suggest that LF could be an effective toxicant alone and that PA might act in situ to modify the effect of this agent (or the reverse situation wherein LF modifies effects of PA) such that lethality results.

  11. Moving ahead on harnessing synthetic lethality to fight cancer.

    PubMed

    Jerby-Arnon, Livnat; Ruppin, Eytan

    2015-01-01

    We have recently developed a data-mining pipeline that comprehensively identifies cancer unique susceptibilities, following the concept of Synthetic Lethality (SL). The approach enables, for the first time, to identify and harness genome-scale SL-networks to accurately predict gene essentiality, drug response, and clinical prognosis in cancer.

  12. An overview of the future of non-lethal weapons.

    PubMed

    Alexander, J B

    2001-01-01

    During the past decade, vast changes have occurred in the geopolitical landscape and the nature of the types of conflicts in which technologically developed countries have been involved. While the threat of conventional war remains, forces have been more frequently deployed in situations that require great restraint. Adversaries are often likely to be elusive and commingled with noncombatants. There has been some shift in public opinion away from tolerance of collateral casualties. Therefore there is a need to be able to apply force while limiting casualties. Non-lethal weapons provide part of the solution. Among the changes that will influence the future have been studies by the US and NATO concerning the use of non-lethal weapons, coincidental with increased funding for their development and testing. New concepts and policies have recently been formalized. Surprisingly, the most strident objections to the implementation of non-lethal weapons have come from organizations that are ostensibly designed to protect non-combatants. These arguments are specious and, while technically and academically challenging, actually serve to foster an environment that will result in the deaths of many more innocent civilians. They misconstrue technology with human intent. The reasons for use of force will not abate. Alternatives to bombs, missiles, tanks and artillery must therefore be found. Non-lethal weapons are not a panacea but do offer the best hope of minimizing casualties while allowing nations or alliances the means to use force in protection of national or regional interests. PMID:11578037

  13. 40 CFR 798.5450 - Rodent dominant lethal assay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... result, one of which is a statistically significant dose-related increase in the number of dominant... a statistically significant dose-related increase in the number of dominant lethals or a... of the uteri are examined to determine the numbers of implants and live and dead embryos....

  14. 40 CFR 798.5450 - Rodent dominant lethal assay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... result, one of which is a statistically significant dose-related increase in the number of dominant... a statistically significant dose-related increase in the number of dominant lethals or a... of the uteri are examined to determine the numbers of implants and live and dead embryos....

  15. The Lethal "Femme Fatale" in the Noir Tradition.

    ERIC Educational Resources Information Center

    Boozer, Jack

    2000-01-01

    Traces the lethal seductress through Hollywood's "noir" history from "Double Indemnity" (1944) to "The Last Seduction" (1996). Examines how this figure largely abjures traditional romance and passive domesticity, choosing instead to apply her sexuality to homicidal plots toward greed. Argues that her narrative positioning serves as a barometer of…

  16. Isolation and characterization of type III group B streptococcal mutants defective in biosynthesis of the type-specific antigen.

    PubMed Central

    Yeung, M K; Mattingly, S J

    1983-01-01

    Four classes of mutants of type III group B streptococcus were isolated by serial subculture of the wild-type strain in the presence of type III-specific rabbit antiserum. Class I mutants no longer synthesized sialic acid but still elaborated the core antigen. Class II mutants maintained the ability to synthesize sialic acid but could not attach it to the core antigen. Class III mutants did not produce the core antigen but still synthesized intracellular sialic acid. Class IV mutants synthesized the complete antigen; however, only approximately 4% of the antigen synthesized was found associated with the cell wall peptidoglycan (in the wild-type strain greater than 85% of the antigen synthesized is covalently attached to the cell wall peptidoglycan), whereas greater than 90% of the antigen was secreted into the growth medium. Production of other components (CAMP factor, group B antigen, beta-hemolysin, neuraminidase) by these mutants appeared similar to those of the wild-type strain. Mouse lethality studies of these strains indicated that all four classes have greater than 3 log10-higher 50% lethal dose values than that of the wild-type strain. To understand the basis for this variation, the invasive ability of the wild-type strain and the sialic acid-deficient mutant strain M-10 (class I) was examined. Mice received 10(5) CFU of each organism; they were then sacrificed at various times postinoculation, and viable group B streptococci from different organs were enumerated. Mice were able to clear M-10 more efficiently, with greater than 80% of M-10 cells being phagocytized by macrophages within 1 h, whereas the wild-type strain was able to evade phagocytic killing and disseminate to other tissues. These data, therefore, strongly indicate that the sialic acid moiety greatly enhances the virulence of the type III antigen. In addition, the level of cell-associated type-specific antigen appears to contribute significantly to the pathogenicity of the organism. PMID

  17. A multivariate model of stakeholder preference for lethal cat management.

    PubMed

    Wald, Dara M; Jacobson, Susan K

    2014-01-01

    Identifying stakeholder beliefs and attitudes is critical for resolving management conflicts. Debate over outdoor cat management is often described as a conflict between two groups, environmental advocates and animal welfare advocates, but little is known about the variables predicting differences among these critical stakeholder groups. We administered a mail survey to randomly selected stakeholders representing both of these groups (n=1,596) in Florida, where contention over the management of outdoor cats has been widespread. We used a structural equation model to evaluate stakeholder intention to support non-lethal management. The cognitive hierarchy model predicted that values influenced beliefs, which predicted general and specific attitudes, which in turn, influenced behavioral intentions. We posited that specific attitudes would mediate the effect of general attitudes, beliefs, and values on management support. Model fit statistics suggested that the final model fit the data well (CFI=0.94, RMSEA=0.062). The final model explained 74% of the variance in management support, and positive attitudes toward lethal management (humaneness) had the largest direct effect on management support. Specific attitudes toward lethal management and general attitudes toward outdoor cats mediated the relationship between positive (p<0.05) and negative cat-related impact beliefs (p<0.05) and support for management. These results supported the specificity hypothesis and the use of the cognitive hierarchy to assess stakeholder intention to support non-lethal cat management. Our findings suggest that stakeholders can simultaneously perceive both positive and negative beliefs about outdoor cats, which influence attitudes toward and support for non-lethal management.

  18. A Multivariate Model of Stakeholder Preference for Lethal Cat Management

    PubMed Central

    Wald, Dara M.; Jacobson, Susan K.

    2014-01-01

    Identifying stakeholder beliefs and attitudes is critical for resolving management conflicts. Debate over outdoor cat management is often described as a conflict between two groups, environmental advocates and animal welfare advocates, but little is known about the variables predicting differences among these critical stakeholder groups. We administered a mail survey to randomly selected stakeholders representing both of these groups (n = 1,596) in Florida, where contention over the management of outdoor cats has been widespread. We used a structural equation model to evaluate stakeholder intention to support non-lethal management. The cognitive hierarchy model predicted that values influenced beliefs, which predicted general and specific attitudes, which in turn, influenced behavioral intentions. We posited that specific attitudes would mediate the effect of general attitudes, beliefs, and values on management support. Model fit statistics suggested that the final model fit the data well (CFI = 0.94, RMSEA = 0.062). The final model explained 74% of the variance in management support, and positive attitudes toward lethal management (humaneness) had the largest direct effect on management support. Specific attitudes toward lethal management and general attitudes toward outdoor cats mediated the relationship between positive (p<0.05) and negative cat-related impact beliefs (p<0.05) and support for management. These results supported the specificity hypothesis and the use of the cognitive hierarchy to assess stakeholder intention to support non-lethal cat management. Our findings suggest that stakeholders can simultaneously perceive both positive and negative beliefs about outdoor cats, which influence attitudes toward and support for non-lethal management. PMID:24736744

  19. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  20. High-throughput simultaneous screen and counterscreen identifies homoharringtonine as synthetic lethal with von Hippel-Lindau loss in renal cell carcinoma

    PubMed Central

    Wolff, Nicholas C.; Pavía-Jiménez, Andrea; Tcheuyap, Vanina T.; Alexander, Shane; Vishwanath, Mridula; Christie, Alana; Xie, Xian-Jin; Williams, Noelle S.; Kapur, Payal; Posner, Bruce; McKay, Renée M.; Brugarolas, James

    2015-01-01

    Renal cell carcinoma (RCC) accounts for 85% of primary renal neoplasms, and is rarely curable when metastatic. Approximately 70% of RCCs are clear-cell type (ccRCC), and in >80% the von Hippel-Lindau (VHL) gene is mutated or silenced. We developed a novel, high-content, screening strategy for the identification of small molecules that are synthetic lethal with genes mutated in cancer. In this strategy, the screen and counterscreen are conducted simultaneously by differentially labeling mutant and reconstituted isogenic tumor cell line pairs with different fluorochromes and using a highly sensitive high-throughput imaging-based platform. This approach minimizes confounding factors from sequential screening, and more accurately replicates the in vivo cancer setting where cancer cells are adjacent to normal cells. A screen of ~12,800 small molecules identified homoharringtonine (HHT), an FDA-approved drug for treating chronic myeloid leukemia, as a VHL-synthetic lethal agent in ccRCC. HHT induced apoptosis in VHL-mutant, but not VHL-reconstituted, ccRCC cells, and inhibited tumor growth in 30% of VHL-mutant patient-derived ccRCC tumorgraft lines tested. Building on a novel screening strategy and utilizing a validated RCC tumorgraft model recapitulating the genetics and drug responsiveness of human RCC, these studies identify HHT as a potential therapeutic agent for a subset of VHL-deficient ccRCCs. PMID:26219258

  1. Determination of lethality rate constants and D-values for Bacillus atrophaeus (ATCC 9372) spores exposed to dry heat from 115 degrees C to 170 degrees C.

    PubMed

    Kempf, M J; Schubert, W W; Beaudet, R A

    2008-12-01

    Dry heat microbial reduction is the NASA-approved sterilization method to reduce the microbial bioburden on spaceflight hardware for missions with planetary protection requirements. The method involves heating the spaceflight hardware to temperatures between 104 degrees C and 125 degrees C for up to 50 hours, while controlling the humidity to very low values. Collection of lethality data at temperatures above 125 degrees C and with ambient (uncontrolled) humidity conditions would establish whether any microbial reduction credit can be offered to the flight project for processes that occur at temperatures greater than 125 degrees C. The goal of this research is to determine the survival rates of Bacillus atrophaeus (ATCC 9372) spores subjected to temperatures higher than 125 degrees C under both dry (controlled) and room ambient humidity (36-66% relative humidity) conditions. Spores were deposited inside thin, stainless steel thermal spore exposure vessels (TSEVs) and heated under ambient or controlled humidity conditions from 115 degrees C to 170 degrees C. After the exposures, the TSEVs were cooled rapidly, and the spores were recovered and plated. Survivor ratios, lethality rate constants, and D-values were calculated at each temperature. At 115 degrees C and 125 degrees C, the controlled humidity lethality rate constant was faster than the ambient humidity lethality rate constant. At 135 degrees C, the ambient and controlled humidity lethality rate constants were statistically identical. At 150 degrees C and 170 degrees C, the ambient humidity lethality rate constant was slightly faster than the controlled humidity lethality rate constant. These results provide evidence for possibly modifying the NASA dry heat microbial reduction specification. PMID:19191542

  2. Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants

    SciTech Connect

    Koch, A.K.; Fiechter, A.; Reiser, J. ); Kaeppeli, O. )

    1991-07-01

    The authors isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up {sup 14}C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C{sub 12} to C{sub 19}. However, growth on these alkanes and uptake of ({sup 14}C)hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up ({sup 14}C)hexadecane uptake. The addition of small amounts of rhamnolipids restored on alkanes and ({sup 14}C)hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P.aeruginosa.

  3. The repair of sub-lethal damage and the stimulated repair of potentially lethal damage in Saintpaulia.

    PubMed

    Leenhouts, H P; Sijsma, M J; Litwiniszyn, M; Chadwick, K H

    1981-10-01

    The repair of sublethal and potentially lethal damage in stationary resting epidermal cells of Saintpaulia has been investigated. Fractionation experiments reveal an efficient repair of sublethal damage with a half-life of 1.9 hours. No repair of potentially lethal damage was noted when cultivation of the leaves was delayed for 24 hours after irradiation. At delay times of 2, 3 and 4 days some repair of potentially lethal damage has been found. A small pre-dose given 24 hours before a challenging dose improved the cells' chance to regenerate and the improvement has been shown to be compatible with an improved repair of potentially lethal damage induced by X-rays and fast neutrons. It hs been shown that the stimulated repair process takes 12 to 24 hours to develop, is dependent on the size of the pre-dose, has single-hit dose kinetics, and an r.b.e. of 1 for neutrons. With delayed cultivation of 2 days the stimulated repair process leads to an alteration in the shape of the regeneration (survival)-dose relationship which increases the low dose r.b.e. for neutrons from 10 to 35. PMID:6975252

  4. (1)H NMR-based metabolomics of Daphnia magna responses after sub-lethal exposure to triclosan, carbamazepine and ibuprofen.

    PubMed

    Kovacevic, Vera; Simpson, André J; Simpson, Myrna J

    2016-09-01

    Pharmaceuticals and personal care products are a class of emerging contaminants that are present in wastewater effluents, surface water, and groundwater around the world. There is a need to determine rapid and reliable bioindicators of exposure and the toxic mode of action of these contaminants to aquatic organisms. (1)H nuclear magnetic resonance (NMR)-based metabolomics in combination with multivariate statistical analysis was used to determine the metabolic profile of Daphnia magna after exposure to a range of sub-lethal concentrations of triclosan (6.25-100μg/L), carbamazepine (1.75-14mg/L) and ibuprofen (1.75-14mg/L) for 48h. Sub-lethal triclosan exposure suggested a general oxidative stress condition and the branched-chain amino acids, glutamine, glutamate, and methionine emerged as potential bioindicators. The aromatic amino acids, serine, glycine and alanine are potential bioindicators for sub-lethal carbamazepine exposure that may have altered energy metabolism. The potential bioindicators for sub-lethal ibuprofen exposure are serine, methionine, lysine, arginine and leucine, which showed a concentration-dependent response. The differences in the metabolic changes were related to the dissimilar modes of toxicity of triclosan, carbamazepine and ibuprofen. (1)H NMR-based metabolomics gave an improved understanding of how these emerging contaminants impact the keystone species D. magna. PMID:26809854

  5. Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants

    SciTech Connect

    Winkelman, J.W.; Clark, D.P.

    1984-11-01

    A positive selection procedure was devised for bacterial mutants incapable of producing acid from sugars by fermentation. The method relied on the production of elemental bromine from a mixture of bromide and bromate under acidic conditions. When wild-type Escherichia coli cells were plated on media containing a fermentable sugar and an equimolar mixture of bromide and bromate, most of the cells were killed but a variety of mutants unable to produce acid from the sugar survived. Among these mutants were those defective in (i) sugar uptake, (ii) the glycolytic pathway, and (iii) the excretion. There were also novel mutants with some presumed regulatory defects affecting fermentation.

  6. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    PubMed Central

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  7. Candida albicans mutant construction and characterization of selected virulence determinants.

    PubMed

    Motaung, T E; Albertyn, J; Pohl, C H; Köhler, Gerwald

    2015-08-01

    Candida albicans is a diploid, polymorphic yeast, associated with humans, where it mostly causes no harm. However, under certain conditions it can cause infections ranging from superficial to life threatening. This ability to become pathogenic is often linked to the immune status of the host as well as the expression of certain virulence factors by the yeast. Due to the importance of C. albicans as a pathogen, determination of the molecular mechanisms that allow this yeast to cause disease is important. These studies rely on the ability of researchers to create deletion mutants of specific genes in order to study their function. This article provides a critical review of the important techniques used to create deletion mutants in C. albicans and highlights how these deletion mutants can be used to determine the role of genes in the expression of virulence factors in vitro.

  8. Characteristics of a glycerol utilization mutant of Neurospora crassa.

    PubMed Central

    Courtright, J B

    1975-01-01

    A mutant of Neurospora crassa able to grow on liquid minimal glycerol medium without evidence of conidiation and with high cell yields has been isolated and shown to be allelic to ff-1. The glycerol-specific induction of glycerokinase and glycerol-3-phosphate dehydrogenase was similar in both wild-type and mutant cells, although higher specific activities as well as higher glycerokinase cross-reacting material levels were found in fully induced mutant cells. After growth in minimal glycerol medium there is a significant reduction in wild-type cells of the activities of both pyruvate dehydrogenase and dihydrolipoyl transacetylase. This evidence indicates a relationship between the conditional acetate requirement by wild-type cells grown on glycerol medium and the levels of the pyruvate dehydrogenase complex. Images PMID:126227

  9. Properties of Mutants of Synechocystis sp. Strain PCC 6803 Lacking Inorganic Carbon Sequestration Systems

    SciTech Connect

    Xu, Min; Bernat, Gabor; Singh, Abhay K.; Mi, Hualing; Rogner, Matthias; Pakrasi, Himadri B.; Ogawa, Teruo

    2008-09-10

    A mutant ( 5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO2 or HCO3– and was unable to grow in air with or without glucose. The 4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO3– uptake and grew under these conditions but more slowly than the wild-type strain. The 5 mutant required 1.7% CO2 to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the 5 mutant more susceptible to high light than the 4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.

  10. Influence of sub-lethal stresses on the survival of lactic acid bacteria after spray-drying in orange juice.

    PubMed

    Barbosa, J; Borges, S; Teixeira, P

    2015-12-01

    The demand for new functional non-dairy based products makes the production of a probiotic orange juice powder an encouraging challenge. However, during drying process and storage, loss of viability of the dried probiotic cultures can occur, since the cells are exposed to various stresses. The influence of sub-lethal conditions of temperature, acidic pH and hydrogen peroxide on the viability of Pediococcus acidilactici HA-6111-2 and Lactobacillus plantarum 299v during spray drying in orange juice and subsequent storage under different conditions was investigated. At the end of storage, the survival of both microorganisms through simulated gastro-intestinal tract (GIT) conditions was also determined. The viability of cells previously exposed to each stress was not affected by the drying process. However, during 180 days of storage at room temperature, unlike P. acidilactici HA-6111-2, survival of L. plantarum 299v was enhanced by prior exposure to sub-lethal conditions. Previous exposure to sub-lethal stresses of each microorganism did not improve their viability after passage through simulated GIT. Nevertheless, as cellular inactivation during 180 days of storage was low, both microorganisms were present in numbers of ca. 10(7) cfu/mL at the end of GIT. This is an indication that both bacteria are good candidates for use in the development of an orange juice powder with functional characteristics.

  11. Complete blockage of the mevalonate pathway results in male gametophyte lethality.

    PubMed

    Suzuki, Masashi; Nakagawa, Shoko; Kamide, Yukiko; Kobayashi, Keiko; Ohyama, Kiyoshi; Hashinokuchi, Hiromi; Kiuchi, Reiko; Saito, Kazuki; Muranaka, Toshiya; Nagata, Noriko

    2009-01-01

    Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that approximately 50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.

  12. Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis.

    PubMed

    Kvitko, Brian H; Goodyear, Andrew; Propst, Katie L; Dow, Steven W; Schweizer, Herbert P

    2012-01-01

    Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba) and pyochelin (pch) gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B. pseudomallei may employ

  13. Development of a non-lethal method for evaluating transcriptomic endpoints in Arctic grayling (Thymallus arcticus).

    PubMed

    Veldhoen, Nik; Beckerton, Jean E; Mackenzie-Grieve, Jody; Stevenson, Mitchel R; Truelson, Robert L; Helbing, Caren C

    2014-07-01

    With increases in active mining and continued discharge associated with former mine operations, evaluating the health of watersheds in the Canadian Yukon Territory is warranted. Current environmental assessment approaches often employ guidelines established using sentinel species not relevant to Arctic monitoring programs. The present study focused on the successful development of a quantitative real-time polymerase chain reaction (qPCR) assay directed towards the indigenous Arctic grayling (Thymallus arcticus) and examines the feasibility of using non-lethal sampling from the caudal fin as a means for evaluation of mRNA abundance profiles reflective of environmental conditions. In a proof of concept study performed blind, qPCR results from animals in an area with elevated water concentrations of cadmium (Cd) and zinc (Zn) and higher body burdens of Cd, Zn, and lead (Pb) were compared to a reference location in the Yukon Territory. Lower condition factor and a higher abundance of hepatic and caudal fin gene transcripts encoding the metallothionein isoforms (mta/mtb), in addition to elevated heat shock protein 70 (hsp70) and catalase (cat) mRNAs in liver, were observed in fish from the test site. The strong positive correlation between metal body burden and caudal fin mta/mtb mRNA abundance demonstrates a high potential for use of the Arctic grayling assay in non-lethal environmental monitoring programs.

  14. TTSS2-deficient hha mutant of Salmonella Typhimurium exhibits significant systemic attenuation in immunocompromised hosts

    PubMed Central

    Vishwakarma, Vikalp; Pati, Niladri Bhusan; Ray, Shilpa; Das, Susmita; Suar, Mrutyunjay

    2014-01-01

    Non-typhoidal Salmonella (NTS) infections are emerging as leading problem worldwide and the variations in host immune status append to the concern of NTS. Salmonella enterica serovar Typhimurium is one of the causative agents of NTS infections and has been extensively studied. The inactivation of Salmonella pathogenicity island 2 (SPI2) encoded type-III secretion system 2 (TTSS2) has been reported rendering the strain incapable for systemic dissemination to host sites and has also been proposed as live-attenuated vaccine. However, infections from TTSS2-deficient Salmonella have also been reported. In this study, mutant strain MT15 was developed by inactivation of the hemolysin expression modulating protein (hha) in TTSS2-deficient S. Typhimurium background. The MT15 strain showed significant level of attenuation in immune-deprived murine colitis model when tested in iNos−/−, IL10−/−, and CD40L−/− mice groups in C57BL/6 background. Further, the mutation in hha does not implicate any defect in bacterial colonization to the host gut. The long-term infection of developed mutant strain conferred protective immune responses to suitably immunized streptomycin pre-treated C57BL/6 mice. The immunization enhanced the CD4+ and CD8+ cell types involved in bacterial clearance. The serum IgG and luminal secretory IgA (sIgA) was also found to be elevated after the due course of infection. Additionally, the immunized C57BL/6 mice were protected from the subsequent lethal infection of Salmonella Typhimurium. Collectively, these findings implicate the involvement of hemolysin expression modulating protein (Hha) in establishment of bacterial infection. In light of the observed attenuation of the developed mutant strain, this study proposes the possible significance of SPI2-deficient hha mutant as an alternative live-attenuated vaccine strain for use against lethal Salmonella infections. PMID:24401482

  15. Arabidopsis hot mutants define multiple functions required for acclimation to high temperatures.

    PubMed

    Hong, Suk-Whan; Lee, Ung; Vierling, Elizabeth

    2003-06-01

    Plants acquire thermotolerance to lethal high temperatures if first exposed to moderately high temperature or if temperature is increased gradually to an otherwise lethal temperature. We have taken a genetic approach to dissecting acquired thermotolerance by characterizing loss-of-function thermotolerance mutants in Arabidopsis. In previous work, we identified single recessive alleles of four loci required for thermotolerance of hypocotyl elongation, hot1-1, hot2-1, hot3-1, and hot4-1. Completed screening of M2 progeny from approximately 2500 M1 plants has now identified new alleles of three of these original loci, along with three new loci. The low mutant frequency suggests that a relatively small number of genes make a major contribution to this phenotype or that other thermotolerance genes encode essential or redundant functions. Further analysis of the original four loci was performed to define the nature of their thermotolerance defects. Although the HOT1 locus was shown previously to encode a major heat shock protein (Hsp), Hsp101, chromosomal map positions indicate that HOT2, 3, and 4 do not correspond to major Hsp or heat shock transcription factor genes. Measurement of thermotolerance at different growth stages reveals that the mutants have growth stage-specific heat sensitivity. Analysis of Hsp accumulation shows that hot2 and hot4 produce normal levels of Hsps, whereas hot3 shows reduced accumulation. Thermotolerance of luciferase activity and of ion leakage also varies in the mutants. These data provide the first direct genetic evidence, to our knowledge, that distinct functions, independent of Hsp synthesis, are required for thermotolerance, including protection of membrane integrity and recovery of protein activity/synthesis. PMID:12805605

  16. Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants.

    PubMed

    Sung, Dong-Yul; Lee, David; Harris, Hugh; Raab, Andrea; Feldmann, Jörg; Meharg, Andrew; Kumabe, Bryan; Komives, Elizabeth A; Schroeder, Julian I

    2007-03-01

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  17. Identification of An Arsenic Tolerant Double Mutant With a Thiol-Mediated Component And Increased Arsenic Tolerance in PhyA Mutants

    SciTech Connect

    Sung, D.Y.; Lee, D.; Harris, H.; Raab, A.; Feldmann, J.; Meharg, A.; Kumabe, B.; Komives, E.A.; Schroeder, J.I.; /SLAC, SSRL /Sydney U. /Aberdeen U. /UC, San Diego

    2007-04-06

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

  18. Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants.

    PubMed

    Sung, Dong-Yul; Lee, David; Harris, Hugh; Raab, Andrea; Feldmann, Jörg; Meharg, Andrew; Kumabe, Bryan; Komives, Elizabeth A; Schroeder, Julian I

    2007-03-01

    A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5. PMID:17335514

  19. CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii.

    PubMed

    Li, Fang; Scott, Maxwell J

    2016-01-22

    Drosophila suzukii (commonly called spotted wing Drosophila) is an invasive pest of soft-skinned fruit (e.g. blueberries, strawberries). A high quality reference genome sequence is available but functional genomic tools, such as used in Drosophila melanogaster, remain to be developed. In this study we have used the CRISPR/Cas9 system to introduce site-specific mutations in the D. suzukii white (w) and Sex lethal (Sxl) genes. Hemizygous males with w mutations develop white eyes and the mutant genes are transmissible to the next generation. Somatic mosaic females that carry mutations in the Sxl gene develop abnormal genitalia and reproductive tissue. The D. suzukii Sxl gene could be an excellent target for a Cas9-mediated gene drive to suppress populations of this highly destructive pest.

  20. CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii.

    PubMed

    Li, Fang; Scott, Maxwell J

    2016-01-22

    Drosophila suzukii (commonly called spotted wing Drosophila) is an invasive pest of soft-skinned fruit (e.g. blueberries, strawberries). A high quality reference genome sequence is available but functional genomic tools, such as used in Drosophila melanogaster, remain to be developed. In this study we have used the CRISPR/Cas9 system to introduce site-specific mutations in the D. suzukii white (w) and Sex lethal (Sxl) genes. Hemizygous males with w mutations develop white eyes and the mutant genes are transmissible to the next generation. Somatic mosaic females that carry mutations in the Sxl gene develop abnormal genitalia and reproductive tissue. The D. suzukii Sxl gene could be an excellent target for a Cas9-mediated gene drive to suppress populations of this highly destructive pest. PMID:26721433

  1. A framework for the assessment of non-lethal weapons.

    PubMed

    Rappert, Brian

    2004-01-01

    In many government, police and military circles, attention is being given to so-called 'non-lethal' weapons as means of reducing many of the negative effects directly or indirectly associated with the use of force. Despite the purported ability of the adoption of such weaponry to lessen grounds for contention and concern, past experience suggests the need for scepticism regarding the purported benefits. Rather than relying on poorly substantiated claims, comprehensive procedures are needed to ensure the appropriateness of force options. This article outlines some of the institutional structures required for 'carefully evaluating' and 'carefully controlling' non-lethal weapons, with a discussion of the perennial tensions associated with ensuring the relative 'acceptability' of the use of force. PMID:15015546

  2. Variability of platyspondylic lethal chondrodysplasia: another case report.

    PubMed

    Nishimura, G; Iwasawa, T; Fukuzawa, R; Hirabayashi, Y; Ito, T

    1998-07-01

    We report the radiological and histological findings of another case of platyspondylic lethal chondrodysplasia. The patient was a girl, who died of respiratory failure at 18 days of age. The radiological changes comprised moderate platyspondyly with ovoid-shaped vertebral bodies, broad and short ilia, rhizomelic shortening and mild bowing of the long bones (particularly of the humeri), relatively long short tubular bones, and retarded epiphyseal ossification and ragged metaphyses, which were most similar to those of a mild variant of this entity, the Luton type. However, the histological findings of cartilage, including hypercellularity of the reserve zone with round resting chondrocytes, relatively normal column formation of the proliferative and hypertrophic zones, and incorporation of hypertrophic cartilage with a columnar arrangement into metaphyseal bony trabeculae, resemble those of a severe variant of this entity, the Torrance type. Our observation provides an insight into the phenotypic variabilities of platyspondylic lethal chondrodysplasia. PMID:9689993

  3. Medical ethics, cultural values, and physician participation in lethal injection.

    PubMed

    Boehnlein, J K; Parker, R M; Arnold, R M; Bosk, C F; Sparr, L F

    1995-01-01

    Capital punishment by lethal injection has been discussed in the literature, but there has been no consideration of the sociocultural foundations of the ethical issues related to medical aspects of capital punishment. Lethal injection represents the inappropriate medicalization of a complex social issue whereby medical skills and procedures are used in ways that contradict established medical practice. Although physicians are socialized to their healing role during medical education and training, their behavior is influenced by social and cultural values that both precede and coexist with their professional life. Because of this dynamic interplay between professional and sociocultural values, physicians can neither exempt themselves from societal debate by merely invoking professional ethics, nor can they define their professional role exclusively in terms of societal values that potentially diminish personal and collective professional responsibility. It is essential that physicians have a broad historical perspective on the development of the profession's standards and values in order to deal effectively with present and future complex ethical issues.

  4. Lethal congenital contracture syndrome: further delineation and genetic aspects.

    PubMed Central

    Vuopala, K; Herva, R

    1994-01-01

    In a national morphology based study of lethal arthrogryposis between 1979 and 1992, 40 fetuses and infants with lethal congenital contracture syndrome (LCCS, McKusick 253310) were found in Finland. The incidence of LCCS in Finland was 1:19,000 births. There were 20 affected males and 20 affected females in 26 families. In 16 cases the pregnancy was terminated after the prenatal diagnosis of total akinesia and fetal hydrops on ultrasound. There were 19 stillborn infants and five were born showing signs of life, but died within one hour. The segregation analyses yielded 0.45 affected by the "singles" method and 0.34 by the "sib" method. The birthplaces of the grandparents were located in the sparsely populated north east of Finland. This finding supports the existence of an autosomal recessive LCCS gene in Finland, particularly in the north eastern part. Images PMID:7966188

  5. Paranodal permeability in `myelin mutants'

    PubMed Central

    Shroff, S.; Mierzwa, A.; Scherer, S.S.; Peles, E.; Arevalo, J.C.; Chao, M.V.; Rosenbluth, J.

    2011-01-01

    Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three `myelin mutant' mice, Caspr-null, cst-null and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3kDa, 10kDa), which penetrate most fibers, and to larger tracers (40kDa, 70kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of transverse bands in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of transverse bands. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of transverse bands but does depend on the length of the paranode and, in turn, on the length of `pathway 3', the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath. PMID:21618613

  6. Paranodal permeability in "myelin mutants".

    PubMed

    Shroff, Seema; Mierzwa, Amanda; Scherer, Steven S; Peles, Elior; Arevalo, Juan C; Chao, Moses V; Rosenbluth, Jack

    2011-10-01

    Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three "myelin mutant" mice, Caspr-null, cst-null, and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3 kDa and 10 kDa), which penetrate most fibers, and to larger tracers (40 kDa and 70 kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands (TBs) in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of TBs in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of TBs. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of TBs but does depend on the length of the paranode and, in turn, on the length of "pathway 3," the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath. PMID:21618613

  7. DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation.

    PubMed

    Burger, Amanda; Raymer, Jenny; Bockrath, R

    2002-10-01

    UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0-100min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining "fixation" of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

  8. A missense mutation in the endothelin-B receptor gene is associated with Lethal White Foal Syndrome: an equine version of Hirschsprung disease.

    PubMed

    Metallinos, D L; Bowling, A T; Rine, J

    1998-06-01

    Lethal White Foal Syndrome is a disease associated with horse breeds that register white coat spotting patterns. Breedings between particular spotted horses, generally described as frame overo, produce some foals that, in contrast to their parents, are all white or nearly all white and die shortly after birth of severe intestinal blockage. These foals have aganglionosis characterized by a lack of submucosal and myenteric ganglia from the distal small intestine to the large intestine, similar to human Hirschsprung Disease. Some sporadic and familial cases of Hirschsprung Disease are due to mutations in the endothelin B receptor gene (EDNRB). In this study, we investigate the role of EDNRB in Lethal White Foal Syndrome. A cDNA for the wild-type horse endothelin-B receptor gene was cloned and sequenced. In three unrelated lethal white foals, the EDNRB gene contained a 2-bp nucleotide change leading to a missense mutation (I118K) in the first transmembrane domain of the receptor, a highly conserved region of this protein among different species. Seven additional unrelated lethal white foal samples were found to be homozygous for this mutation. No other homozygotes were identified in 138 samples analyzed, suggesting that homozygosity was restricted to lethal white foals. All (40/40) horses with the frame overo pattern (a distinct coat color pattern that is a subset of overo horses) that were tested were heterozygous for this allele, defining a heterozygous coat color phenotype for this mutation. Horses with tobiano markings included some carriers, indicating that tobiano is epistatic to frame overo. In addition, horses were identified that were carriers but had no recognized overo coat pattern phenotype, demonstrating the variable penetrance of the mutation. The test for this mutant allele can be utilized in all breeds where heterozygous animals may be unknowingly bred to each other including the Paint Horse, Pinto horse, Quarter Horse, Miniature Horse, and Thoroughbred.

  9. A Sall4 Mutant Mouse Model Useful for Studying the Role of Sall4 in Early Embryonic Development and Organogenesis

    PubMed Central

    Warren, Madhuri; Wang, Wei; Spiden, Sarah; Chen-Murchie, Dongrong; Tannahill, David; Steel, Karen P.; Bradley, Allan

    2008-01-01

    Summary SALL4 is a homologue of the Drosophila homeotic gene spalt, a zinc finger transcription factor, required for inner cell mass proliferation in early embryonic development. It also interacts with other transcription factors to control the development of the anorectal region, kidney, heart, limbs, and brain. Truncating mutations in SALL4 cause Okihiro syndrome, manifest as Duane anomaly, radial ray defects and sensorineural and conductive deafness. We report the characterization of a novel murine Sall4 null allele created by bacterial recombineering in ES cells. Homozygous mutant mice exhibit early embryonic lethality. Heterozygous mutant mice recapitulate phenotypic features of Okihiro syndrome including deafness, lower anogenital tract abnormalities, renal hypoplasia, anencephaly, Hirschprung’s disease, and skeletal defects. This phenotype shows important differences in cardiac and ear manifestations to previously characterized Sall4 mutant alleles and should prove useful for the investigation of the influence of modifier alleles and protein interactions on the transcriptional regulatory function of Sall4. PMID:17216607

  10. The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

    PubMed

    Melo-Silva, Carolina R; Tscharke, David C; Lobigs, Mario; Koskinen, Aulikki; Wong, Yik Chun; Buller, R Mark; Müllbacher, Arno; Regner, Matthias

    2011-11-01

    Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

  11. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis. PMID:27136067

  12. Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels.

    PubMed

    Coelho, Sónia; Oliveira, Rhaul; Pereira, Susana; Musso, Carolina; Domingues, Inês; Bhujel, Ram C; Soares, Amadeu M V M; Nogueira, António J A

    2011-06-01

    Trichlorfon (TCF) is one of the most used veterinary pharmaceuticals not only to fight infestations but also as a preventive measure worldwide. The high concentrations used generate concerns about environmental and human health. In this work we assessed the acute toxicity of this compound to non-target organisms belonging to different trophic levels: Danio rerio (early life stages and adults), Daphnia magna and algae (Pseudokirchneriella subcapitata and Chlorella vulgaris), and studied the potential of the biomarkers cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) to assess sub-lethal effects of trichlorfon in zebrafish and daphnids. The fish embryo test followed the OECD draft guideline FET and was based on the exposure of newly fertilized eggs to 0, 2.5, 5.0, 10, 20, 40, 80 and 160 mg/L of TCF for 5 days; the fish acute test followed the OECD guideline 203 and was based on the exposure of adult fish to 0, 2.5, 5, 10, 20, 40, 60 and 80 mg/L of TCF for 4 days; Daphnia sp. immobilization assay followed the OECD guideline 202 and was based on the exposure of juvenile daphnids to 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1 and 2 μg/L of TCF for 2 days and the algae growth inhibition assay followed the OECD guideline 201 and was based on the exposure of the two species to 0, 1, 3.2, 10, 32, 100 and 300 mg/L of TCF for 4 days. Biomarker levels were measured after 96 h exposure to TCF in zebrafish early life stages and adults and after 48 h exposure in D. magna. Tested organisms seem to have dissimilar sensitivities towards TCF exposure. D. magna (48 h-LC(50)=0.29 μg/L) was the most sensitive organism, followed by early life stages and adults of zebrafish (96 h-LC(50)=25.4 and 28.8 mg/L, respectively) and finally by the algae P. subcapitata (96 h-LC(50)=274.5 mg/L) and C. vulgaris (no effect observed). As daphnids are a source of food for organisms of higher trophic levels, the impairment on its population is prone to have

  13. Autopsy observations in lethal short-rib polydactyly syndromes.

    PubMed

    Okiro, Patricia; Wainwright, Helen; Spranger, Jürgen; Beighton, Peter

    2015-01-01

    The short rib-polydactyly syndromes are a heterogeneous group of lethal autosomal recessive disorders (SRP I-IV), which result from cellular ciliary dysfunction during embryogenesis. Diagnosis is conventionally based on radiographic imaging. Since 1976, postmortem investigations of 5 affected fetuses or stillbirths have been undertaken and the visceral abnormalities have been documented. These anomalies are discussed in the context of prenatal differential diagnosis and prognostication following imaging in pregnancy and at autopsy following miscarriage or stillbirth.

  14. Spondyloepiphyseal dysplasia congenita. A cause of lethal neonatal dwarfism.

    PubMed

    Macpherson, R I; Wood, B P

    1980-07-01

    Spondyloepiphyseal dysplasia congenita is a form of primary short dwarfism, that is manifest at birth generally has not been regarded as a cause of lethal neonatal dwarfism. Seven neonates with severe dwarfism are presented. The first survived the newborn period, but the other six were early neonatal deaths. All displayed the clinical and radiologic features of spondyloepiphyseal dysplasia congenita. The striking similarities between spondyloepiphyseal dysplasia congenita and achondrogenesis type 2 are discussed. PMID:6773018

  15. [Lethal achondrogenesis: a review of 56 cases (author's transl)].

    PubMed

    Schulte, M J; Lenz, W; Vogel, M

    1978-07-01

    54 cases with lethal achondrogenesis from the literature as well as two own cases are reviewed and analyzed with regard to the following characteristics: sex, hydramnios, breech presentation, duration of pregnancy, length and weight at birth, head circumference, length of upper and lower extremities, clinical and radiological data, age of mother and father at time of birth, familial occurrence and consanguinity of parents, histological, histochemical and electronmicroscopic tissue examination. PMID:353375

  16. Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cell death.

    PubMed

    Queval, Guillaume; Issakidis-Bourguet, Emmanuelle; Hoeberichts, Frank A; Vandorpe, Michaël; Gakière, Bertrand; Vanacker, Hélène; Miginiac-Maslow, Myroslawa; Van Breusegem, Frank; Noctor, Graham

    2007-11-01

    Photorespiration is a light-dependent source of H(2)O(2) in the peroxisomes, where concentrations of this signalling molecule are regulated by catalase. Growth of Arabidopsis knock-out mutants for CATALASE2 (cat2) in ambient air caused severely decreased rosette biomass, intracellular redox perturbation and activation of oxidative signalling pathways. These effects were absent when cat2 was grown at high CO(2) levels to inhibit photorespiration, but were re-established following a subsequent transfer to air. Growth of cat2 in air at different daylengths revealed that photoperiod is a critical determinant of the oxidative stress response. Decreased growth was observed in 8-h, 12-h and 16-h photoperiods, but lesion development was dependent on long days. Experiments at different light fluence rates showed that cell death in cat2 was linked to long days and not to total light exposure or the severity of oxidative stress. Perturbed intracellular redox state and oxidative signalling pathway induction were more prominent in short days than in long days, as evidenced by glutathione status and induction of defence genes and oxidative stress-responsive transcripts. Similar daylength-dependent effects were observed in the response of mature plants transferred from short days in high CO(2) conditions to ambient air conditions. Prior growth of plants with short days in air alleviated the cat2 cell-death phenotype in long days. Together, the data reveal the influence of photoperiodic events on redox signalling, and define distinct photoperiod-dependent strategies in the acclimation versus cell-death decision in stress conditions.

  17. Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.

    PubMed

    Benz, Brian A; Nandadasa, Sumeda; Takeuchi, Megumi; Grady, Richard C; Takeuchi, Hideyuki; LoPilato, Rachel K; Kakuda, Shinako; Somerville, Robert P T; Apte, Suneel S; Haltiwanger, Robert S; Holdener, Bernadette C

    2016-08-01

    Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of

  18. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  19. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  20. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  1. 28 CFR 552.25 - Use of less-than-lethal weapons, including chemical agents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Use of less-than-lethal weapons... Use of less-than-lethal weapons, including chemical agents. (a) The Warden may authorize the use of less-than-lethal weapons, including those containing chemical agents, only when the situation is...

  2. To Laugh in the Face of Death: The Games That Lethal People Play.

    ERIC Educational Resources Information Center

    Thorson, James A.; Powell, F. C.

    1990-01-01

    A total of 399 individuals completed a lethal behaviors scale and a measure of death anxiety, which were found to have no significant correlation. Predictors of lethalness included doing dangerous things for the fun of it and having ever driven a motorcycle. The most lethal individuals were young, male, and less educated. (Author/ABL)

  3. 28 CFR 552.25 - Use of chemical agents or non-lethal weapons.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Use of chemical agents or non-lethal weapons. 552.25 Section 552.25 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE... agents or non-lethal weapons. The Warden may authorize the use of chemical agents or non-lethal...

  4. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  5. Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei).

    PubMed

    Fritz, D L; Vogel, P; Brown, D R; Deshazer, D; Waag, D M

    2000-11-01

    Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation. Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation. In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3. These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver. Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens. At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow. The incidence and severity of these changes were maximal at day 2. In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study. The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice. Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice. Our findings indicate that mice are susceptible to B. mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings. Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B. mallei.

  6. Lethality of First Contact Dysentery Epidemics on Pacific Islands.

    PubMed

    Shanks, G Dennis

    2016-08-01

    Infectious diseases depopulated many isolated Pacific islands when they were first exposed to global pathogen circulation from the 18th century. Although the mortality was great, the lack of medical observers makes determination of what happened during these historical epidemics largely speculative. Bacillary dysentery caused by Shigella is the most likely infection causing some of the most lethal island epidemics. The fragmentary historical record is reviewed to gain insight into the possible causes of the extreme lethality that was observed during first-contact epidemics in the Pacific. Immune aspects of the early dysentery epidemics and postmeasles infection resulting in subacute inflammatory enteric disease suggest that epidemiologic isolation was the major lethality risk factor on Pacific islands in the 19th century. Other possible risk factors include human leukocyte antigen homogeneity from a founder effect and pathogen-induced derangement of immune tolerance to gut flora. If this analysis is correct, then Pacific islands are currently at no greater risk of emerging disease epidemics than other developing countries despite their dark history. PMID:27185765

  7. Intact alternation performance in high lethality suicide attempters.

    PubMed

    Keilp, John G; Wyatt, Gwinne; Gorlyn, Marianne; Oquendo, Maria A; Burke, Ainsley K; John Mann, J

    2014-09-30

    Suicide attempters often perform poorly on tasks linked to ventral prefrontal cortical (VPFC) function. Object Alternation (OA) - a VPFC probe - has not been used in these studies. In this study, currently depressed medication-free past suicide attempters whose most severe attempt was of high (n=31) vs. low (n=64) lethality, 114 medication-free depressed non-attempters, and 86 non-patients completed a computerized OA task. Participants also completed comparison tasks assessing the discriminant validity of OA (Wisconsin Card Sort), its concurrent validity relative to tasks associated with past attempt status (computerized Stroop task, Buschke Selective Reminding Test), and its construct validity as a VPFC measure (Go-No Go and Iowa Gambling Task). Against expectations, high lethality suicide attempters - the majority of whom used non-violent methods in their attempts with some planning - outperformed other depressed groups on OA, with no group differences observed on Wisconsin Card Sort. Despite intact performance on OA, past attempters exhibited deficits on the Stroop and Buschke. OA performance was associated with performance on Go-No Go and Iowa Gambling, confirming that OA measures a similar construct. VPFC dysfunction may not be a characteristic of all suicide attempters, especially those who make more carefully planned, non-violent - though potentially lethal - attempts. PMID:24878299

  8. Lethal injection, autonomy and the proper ends of medicine.

    PubMed

    Silver, David

    2003-04-01

    Gerald Dworkin has argued that it is inconsistent with the proper ends of medicine for a physician to participate in an execution by lethal injection. He does this by proposing a principle by which we are to judge whether an action is consistent with the proper ends of medicine. I argue: (a) that this principle, if valid, does not show that it is inconsistent with the proper ends of medicine for a physician to participate in an execution by lethal injection; and (b) that this principle is not valid, and this is because it mistakenly views the promotion of patient autonomy as one of the proper ends of medicine. Rather, I propose, we should view respect for a patient's autonomy as a constraint on the pursuit of the proper ends of medicine, rather than as one of the proper ends itself. With this revised understanding of the proper ends of medicine, we can conclude that it is inconsistent with the proper ends of medicine for a physician to participate in an execution by lethal injection.

  9. Lethality of First Contact Dysentery Epidemics on Pacific Islands.

    PubMed

    Shanks, G Dennis

    2016-08-01

    Infectious diseases depopulated many isolated Pacific islands when they were first exposed to global pathogen circulation from the 18th century. Although the mortality was great, the lack of medical observers makes determination of what happened during these historical epidemics largely speculative. Bacillary dysentery caused by Shigella is the most likely infection causing some of the most lethal island epidemics. The fragmentary historical record is reviewed to gain insight into the possible causes of the extreme lethality that was observed during first-contact epidemics in the Pacific. Immune aspects of the early dysentery epidemics and postmeasles infection resulting in subacute inflammatory enteric disease suggest that epidemiologic isolation was the major lethality risk factor on Pacific islands in the 19th century. Other possible risk factors include human leukocyte antigen homogeneity from a founder effect and pathogen-induced derangement of immune tolerance to gut flora. If this analysis is correct, then Pacific islands are currently at no greater risk of emerging disease epidemics than other developing countries despite their dark history.

  10. Inactivating Mutations in MFSD2A, Required for Omega-3 Fatty Acid Transport in Brain, Cause a Lethal Microcephaly Syndrome

    PubMed Central

    Guemez-Gamboa, Alicia; Nguyen, Long N.; Yang, Hongbo; Zaki, Maha S.; Kara, Majdi; Ben-Omran, Tawfeg; Akizu, Naiara; Rosti, Rasim Ozgur; Rosti, Basak; Scott, Eric; Schroth, Jana; Copeland, Brett; Vaux, Keith K.; Cazenave-Gassiot, Amaury; Quek, Debra Q.Y.; Wong, Bernice H.; Tan, Bryan C.; Wenk, Markus R.; Gunel, Murat; Gabriel, Stacey; Chi, Neil C.; Silver, David L.; Gleeson, Joseph G.

    2015-01-01

    Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and although considered essential, deficiency has not been linked to disease1,2. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the Major Facilitator Superfamily Domain 2a (Mfsd2a)3. Mfsd2a transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Patients exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa zebrafish morphants, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans. PMID:26005868

  11. Screening of Bacillus subtilis transposon mutants with altered riboflavin production.

    PubMed

    Tännler, Simon; Zamboni, Nicola; Kiraly, Csilla; Aymerich, Stéphane; Sauer, Uwe

    2008-09-01

    To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture. PMID:18582593

  12. Towards an informative mutant phenotype for every bacterial gene

    DOE PAGES

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, inmore » Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.« less

  13. Towards an informative mutant phenotype for every bacterial gene

    SciTech Connect

    Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjen; Leon, Dacia; Arkin, Adam P.; Skerker, Jeffrey M.

    2014-08-11

    Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

  14. Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

    PubMed Central

    Roufa, Donald J.; Reed, Susan J.

    1975-01-01

    Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxy-uridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39° and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39° to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115–47 and 115–53) also stop incorporating radioactive amino acids and uridine within six hours at 39°. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33° after 8 hours at 39°, both resumed DNA synthesis immediately (< 1 hour). Reversal of defective DNA synthesis in both mutants was sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication. PMID:1232024

  15. Global gene expression of a murein (Braun) lipoprotein mutant of Salmonella enterica serovar Typhimurium by microarray analysis.

    PubMed

    Fadl, A A; Galindo, C L; Sha, J; Klimpel, G R; Popov, V L; Chopra, A K

    2006-06-01

    Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.

  16. Mutants resistant to anti-microtubule herbicides map to a locus on the uni linkage group in Chlamydomonas reinhardtii

    SciTech Connect

    James, S.W.; Ranum, L.P.W.; Silflow, C.D.; Lefebvre, P.A.

    1988-01-01

    The authors have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzaline (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33/sup 0/) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between amp2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts/sup -/ and 2-fold resistant to the herbicides. From the results described the authors suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.

  17. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  18. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa.

    PubMed

    Kolyadko, Vladimir N; Lushchekina, Sofya V; Vuimo, Tatiana A; Surov, Stepan S; Ovsepyan, Ruzanna A; Korneeva, Vera A; Vorobiev, Ivan I; Orlova, Nadezhda A; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V; Ataullakhanov, Fazoil I; Panteleev, Mikhail A

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  19. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

    PubMed

    Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  20. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

    PubMed

    Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  1. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

    PubMed Central

    Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  2. Intraguild relationships between sympatric predators exposed to lethal control: predator manipulation experiments

    PubMed Central

    2013-01-01

    populations of native prey fauna at lower trophic levels are unlikely to be negatively affected by contemporary dingo control practices through the release of mesopredators. We conclude that contemporary lethal control practices used on some top-predator populations do not produce the conditions required to generate positive responses from mesopredators. Functional relationships between sympatric terrestrial predators may not be altered by exposure to spatially and temporally sporadic application of non-selective lethal control. PMID:23842144

  3. Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model.

    PubMed

    Yin, Yuelan; Tian, Debin; Jiao, Hongmei; Zhang, Chenju; Pan, Zhiming; Zhang, Xiaoming; Wang, Xiaobo; Jiao, Xinan

    2011-03-01

    Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.

  4. An Allele of Sequoia Dominantly Enhances a Trio Mutant Phenotype to Influence Drosophila Larval Behavior

    PubMed Central

    Liebl, Eric C.

    2013-01-01

    The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency. PMID:24376789

  5. Construction of mouse adenovirus type 1 mutants.

    PubMed

    Cauthen, Angela N; Welton, Amanda R; Spindler, Katherine R

    2007-01-01

    Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wild-type mouse adenovirus, production of viral DNA and viral DNA-protein complex, and transfection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of the mutant viruses are also presented.

  6. Preventing neurodegeneration in the Drosophila mutant bubblegum.

    PubMed

    Min, K T; Benzer, S

    1999-06-18

    The Drosophila melanogaster recessive mutant bubblegum (bgm) exhibits adult neurodegeneration, with marked dilation of photoreceptor axons. The bubblegum mutant shows elevated levels of very long chain fatty acids (VLCFAs), as seen in the human disease adrenoleukodystrophy (ALD). In ALD, the excess can be lowered by dietary treatment with "Lorenzo's oil," a mixture of unsaturated fatty acids. Feeding the fly mutant one of the components, glyceryl trioleate oil, blocked the accumulation of excess VLCFAs as well as development of the pathology. Mutant flies thus provide a potential model system for studying mechanisms of neurodegenerative disease and screening drugs for treatment.

  7. Melanin-deficient mutants of Cryptococcus neoformans.

    PubMed

    Torres-Guererro, H; Edman, J C

    1994-01-01

    Cryptococcus neoformans is a significant fungal pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin has been correlated with virulence. The role of melanin in promoting virulence is unclear, although an anti-oxidant function has been suggested. To begin to define the genetic mechanisms responsible for melanin production in C. neoformans, we describe the isolation of seven melanin-deficient mutant classes. Some of the mutants can be suppressed by addition of Cu2+ to media, suggesting that the phenoloxidase of C. neoformans, like other fungal phenoloxidases, contains copper. Other mutants display a recessive sterile phenotype. A genetic and phenotypic characterisation of these mutants is presented. PMID:7983575

  8. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2.

    PubMed Central

    Maier, R J

    1981-01-01

    Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity. PMID:6780521

  9. Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata

    PubMed Central

    Tscherner, Michael; Kuchler, Karl

    2016-01-01

    Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014). PMID:27774498

  10. Pseudomonas aeruginosa UV-A-induced lethal effect: influence of salts, nutritional stress and pyocyanine.

    PubMed

    Fernández, R O; Pizarro, R A

    1999-05-01

    The presence of NaCl in plating media shows an important protection against the Pseudomonas aeruginosa UV-A-induced lethal effect, contrasting with the known sensitizing action of salts on UV-A-irradiated Escherichia coli cells. MgSO4 exhibits a similar protection, but lower concentrations than for NaCl are needed to achieve the same effect. NaCl protection from lethal effects involves an osmotic mechanism, while MgSO4 could act by a different process. On the other hand, when cells grown in a complete medium are then incubated for 20 min in a synthetic medium and irradiated with UV-A, a very marked protection is obtained. This protection is dependent on protein synthesis, since treatment with tetracycline, during the nutritional stress, blocks its induction. These results offer a new example of cross-protection among different stressing agents. In our experimental conditions, natural phenazines of P. aeruginosa are not present in the cells, ruling out the possibility that these pigments act as photosensitizers. Conversely, pyocyanine (the major phenazine produced by this microorganism) prevents the UV-A killing effect in a concentration-dependent way when present in the irradiation media. Finally, UV-A irradiation induces, as in E. coli, the accumulation of guanosine tetraphosphate and guanosine pentaphosphate, although the physiological meaning of this finding has yet to be determined. PMID:10443032

  11. Survival of Escherichia coli under lethal heat stress by L-form conversion

    PubMed Central

    Markova, Nadya; Slavchev, Georgi; Michailova, Lilia; Jourdanova, Mimi

    2010-01-01

    Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria. PMID:20582223

  12. Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

    SciTech Connect

    Mendonca, M.S.; Rodriguez, A.; Alpen, E.L. )

    1990-04-01

    The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.

  13. Severe hypoglycemia-induced lethal cardiac arrhythmias are mediated by sympathoadrenal activation.

    PubMed

    Reno, Candace M; Daphna-Iken, Dorit; Chen, Y Stefanie; VanderWeele, Jennifer; Jethi, Krishan; Fisher, Simon J

    2013-10-01

    For people with insulin-treated diabetes, severe hypoglycemia can be lethal, though potential mechanisms involved are poorly understood. To investigate how severe hypoglycemia can be fatal, hyperinsulinemic, severe hypoglycemic (10-15 mg/dL) clamps were performed in Sprague-Dawley rats with simultaneous electrocardiogram monitoring. With goals of reducing hypoglycemia-induced mortality, the hypotheses tested were that: 1) antecedent glycemic control impacts mortality associated with severe hypoglycemia; 2) with limitation of hypokalemia, potassium supplementation could limit hypoglycemia-associated deaths; 3) with prevention of central neuroglycopenia, brain glucose infusion could prevent hypoglycemia-associated arrhythmias and deaths; and 4) with limitation of sympathoadrenal activation, adrenergic blockers could prevent hypoglycemia-induced arrhythmic deaths. Severe hypoglycemia-induced mortality was noted to be worsened by diabetes, but recurrent antecedent hypoglycemia markedly improved the ability to survive an episode of severe hypoglycemia. Potassium supplementation tended to reduce mortality. Severe hypoglycemia caused numerous cardiac arrhythmias including premature ventricular contractions, tachycardia, and high-degree heart block. Intracerebroventricular glucose infusion reduced severe hypoglycemia-induced arrhythmias and overall mortality. β-Adrenergic blockade markedly reduced cardiac arrhythmias and completely abrogated deaths due to severe hypoglycemia. Under conditions studied, sudden deaths caused by insulin-induced severe hypoglycemia were mediated by lethal cardiac arrhythmias triggered by brain neuroglycopenia and the marked sympathoadrenal response.

  14. Genotoxicity of sub-lethal di-n-butyl phthalate (DBP) in Nile tilapia (Oreochromis niloticus).

    PubMed

    Benli, Aysel Çağlan Karasu; Erkmen, Belda; Erkoç, Figen

    2016-03-01

    This study aimed to assess genotoxicity in Nile tilapia (Oreochromis niloticus) erythrocytes after exposure to a sub-lethal concentration of 10 mg L(-1) di-n-butyl phthalate (DBP) for 24 and 96 h. The results showed that mean MN frequencies in both DBP and ethyl methane-sulfonate (EMS, positive control for MN bioassay) groups were significantly different (p<0.01) with respect to control and solvent control groups, in both exposure scenarios. When analysing nuclear abnormalities, the frequency of notched nuclei was significantly different (p<0.05) but the frequencies of other subtypes did not change. The 96-h exposure led to an increase in the mean frequencies of notched nuclei, and also caused significant differences between MN frequencies in all groups (p<0.01). Our findings indicate that sub-lethal DBP concentrations when tested in controlled laboratory conditions have genotoxic potential towards Nile tilapia. Further detailed studies should be done for the determination of the environmental risk assessment for aquatic life since DBP is a high risk contaminant of freshwater and marine ecosystems. PMID:27092636

  15. Analyzing the control of mosquito-borne diseases by a dominant lethal genetic system.

    PubMed

    Atkinson, Michael P; Su, Zheng; Alphey, Nina; Alphey, Luke S; Coleman, Paul G; Wein, Lawrence M

    2007-05-29

    Motivated by the failure of current methods to control dengue fever, we formulate a mathematical model to assess the impact on the spread of a mosquito-borne viral disease of a strategy that releases adult male insects homozygous for a dominant, repressible, lethal genetic trait. A dynamic model for the female adult mosquito population, which incorporates the competition for female mating between released mosquitoes and wild mosquitoes, density-dependent competition during the larval stage, and realization of the lethal trait either before or after the larval stage, is embedded into a susceptible-exposed-infectious-susceptible human-vector epidemic model for the spread of the disease. For the special case in which the number of released mosquitoes is maintained in a fixed proportion to the number of adult female mosquitoes at each point in time, we derive mathematical formulas for the disease eradication condition and the approximate number of released mosquitoes necessary for eradication. Numerical results using data for dengue fever suggest that the proportional policy outperforms a release policy in which the released mosquito population is held constant, and that eradication in approximately 1 year is feasible for affected human populations on the order of 10(5) to 10(6), although the logistical considerations are daunting. We also construct a policy that achieves an exponential decay in the female mosquito population; this policy releases approximately the same number of mosquitoes as the proportional policy but achieves eradication nearly twice as fast.

  16. [Bladder tumor lethality. Results in the autonomous community of Rioja between 1975-1991].

    PubMed

    Fernández Fernández, A; Gil Fabra, J; Fernández Ruíz, M; Angulo Castellanos, M G; Blanco Martín, E; Otero Mauricio, G

    1998-01-01

    Between 1975-1991, a total of 557 cases of bladder carcinoma were identified in the Autonomous Community of La Rioja (CAR) which were followed up to December 1994. The overall lethality was 21.9%. 492 cases with 22.35% lethality were identified in males. In females, however, there was 65 cases with 18.46% lethality. The comparison of males and females lethality resulted in p = 0.525. Lethality between cases diagnosed within each 5-year period analyzed is: 1975-1981: 177 cases, lethality 23.72%. 1982-1986: 168 cases, lethality 30.95%. 1987-1991: 212 cases, lethality 13.20%. Between the first and the second 5-year periods, p = 0.132; between the first and third 5-year periods p = 0.007 and between the second and third 5-year periods p < 0.000. Bladder tumours accounts in CAR for a 22.35% lethality. Lethality is higher in males that in females but the difference is not statistically significant. In the last 5-year period assessed, 1987-1991, a reduction of lethality from bladder neoplasms has been documented.

  17. Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions

    NASA Technical Reports Server (NTRS)

    Waldren, C.; Vannais, D.; Drabek, R.; Gustafson, D.; Kraemer, S.; Lenarczyk, M.; Kronenberg, A.; Hei, T.; Ueno, A.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

  18. Electromagnetic pulse (EMP) coupling codes for use with the vulnerability/lethality (VIL) taxonomy. Final report, June-October 1984

    SciTech Connect

    Mar, M.H.

    1995-07-01

    Based on the vulnerability Lethality (V/L) taxonomy developed by the Ballistic Vulnerability Lethality Division (BVLD) of the Survivability Lethality Analysis Directorate (SLAD), a nuclear electromagnetic pulse (EMP) coupling V/L analysis taxonomy has been developed. A nuclear EMP threat to a military system can be divided into two levels: (1) coupling to a system level through a cable, antenna, or aperture; and (2) the component level. This report will focus on the initial condition, which includes threat definition and target description, as well as the mapping process from the initial condition to damaged components state. EMP coupling analysis at a system level is used to accomplish this. This report introduces the nature of EMP threat, interaction between the threat and target, and how the output of EMP coupling analysis at a system level becomes the input to the component level analysis. Many different tools (EMP coupling codes) will be discussed for the mapping process, which correponds to the physics of phenomenology. This EMP coupling V/L taxonomy and the models identified in this report will provide the tools necessary to conduct basic V/L analysis of EMP coupling.

  19. Deficiency of Suppressor Enhancer Lin12 1 Like (SEL1L) in Mice Leads to Systemic Endoplasmic Reticulum Stress and Embryonic Lethality*

    PubMed Central

    Francisco, Adam B.; Singh, Rajni; Li, Shuai; Vani, Anish K.; Yang, Liu; Munroe, Robert J.; Diaferia, Giuseppe; Cardano, Marina; Biunno, Ida; Qi, Ling; Schimenti, John C.; Long, Qiaoming

    2010-01-01

    Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development. PMID:20197277

  20. Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.

    PubMed

    Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

    2014-09-01

    Accumulation of high level of lactate can negatively impact cell growth during fed-batch culture process. In this study, we attempted to knockout the lactate dehydrogenase A (LDHA) gene in CHO cells in order to attenuate the lactate level. To prevent the potential deleterious effect of pyruvate accumulation, consequent to LDHA knockout, on cell culture, we chose a pyruvate dehydrogenase kinase 1, 2, and 3 (PDHK1, 2, and 3) knockdown cell line in which to knock out LDHA alleles. Around 3,000 clones were screened to obtain 152 mutants. Only heterozygous mutants were identified. An attempt to knockout the remaining wild-type allele from one such heterozygote yielded only two mutants after screening 567 clones. One had an extra valine. Another evidenced a duplication event, possessing at lease one wild-type and two different frameshifted alleles. Both mutants still retained LDH activity. Together, our data strongly suggest that a complete knockout of LDHA is lethal in CHO cells, despite simultaneous down-regulation of PDHK1, 2, and 3. PMID:24841241

  1. Filgrastim Improves Survival in Lethally Irradiated Nonhuman Primates

    PubMed Central

    Farese, Ann M.; Cohen, Melanie V.; Katz, Barry P.; Smith, Cassandra P.; Gibbs, Allison; Cohen, Daniel M.; MacVittie, Thomas J.

    2015-01-01

    Treatment of individuals exposed to potentially lethal doses of radiation is of paramount concern to health professionals and government agencies. We evaluated the efficacy of filgrastim to increase survival of nonhuman primates (NHP) exposed to an approximate mid-lethal dose (LD50/60) (7.50 Gy) of LINAC-derived photon radiation. Prior to total-body irradiation (TBI), nonhuman primates were randomized to either a control (n =22) or filgrastim-treated (n =24) cohorts. Filgrastim (10 μg/kg/d) was administered beginning 1 day after TBI and continued daily until the absolute neutrophil count (ANC) was >1,000/μL for 3 consecutive days. All nonhuman primates received medical management as per protocol. The primary end point was all cause overall mortality over the 60 day in-life study. Secondary end points included mean survival time of decedents and all hematologic-related parameters. Filgrastim significantly (P < 0.004) reduced 60 day overall mortality [20.8% (5/24)] compared to the controls [59.1% (13/22)]. Filgrastim significantly decreased the duration of neutropenia, but did not affect the absolute neutrophil count nadir. Febrile neutropenia (ANC <500/μL and body temperature ≥103°F) was experienced by 90.9% (20/22) of controls compared to 79.2% (19/24) of filgrastim-treated animals (P = 0.418). Survival was significantly increased by 38.3% over controls. Filgrastim, administered at this dose and schedule, effectively mitigated the lethality of the hematopoietic subsyndrome of the acute radiation syndrome. PMID:23210705

  2. Left ventricular function during lethal and sublethal endotoxemia in swine

    SciTech Connect

    Goldfarb, R.D.; Nightingale, L.M.; Kish, P.; Weber, P.B.; Loegering, D.J.

    1986-08-01

    Previous studies suggested that after a median lethal dose (LD50) of endotoxin, cardiac contractility was depressed in nonsurviving dogs. The canine cardiovascular system is unlike humans in that dogs have a hepatic vein sphincter that is susceptible to adrenergic stimulation capable of raising hepatic and splanchnic venous pressures. The authors retested the hypothesis that lethality after endotoxin administration is associated with cardiac contractile depression in pigs, because of the hepatic circulation in this species is similar to that of humans. They compared cardiac mechanical function of pigs administered a high dose (250 g/kg) or a low dose (100 g/kg) endotoxin by use of the slope of the end-systolic pressure-diameter relationship (ESPDR) as well as other measurements of cardiac performance. In all the pigs administered a high dose, ESPDR demonstrated a marked, time-dependent depression whereas we observed no significant ESPDR changes after low endotoxin doses. The other cardiodynamic variables were uninterpretable, due to the significant changes in heart rate, end-diastolic diameter (preload), and aortic diastolic pressure (afterload). Plasma myocardia depressant factor activity accumulated in all endotoxin-administered animals, tending to be greater in the high-dose group. In this group, both subendocardial blood flow and global function were depressed, whereas pigs administered the low dose endotoxin demonstrated slight, but nonsignificant, increases in flow and function. These observations indicate that myocardial contractile depression is associated with a lethal outcome to high doses of endotoxin. Myocardial perfusion was measured using radiolabeled microspheres infused into the left atria.

  3. Preparation and characterization of cobalt-substituted anthrax lethal factor

    SciTech Connect

    Saebel, Crystal E.; Carbone, Ryan; Dabous, John R.; Lo, Suet Y.; Siemann, Stefan

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Cobalt-substituted anthrax lethal factor (CoLF) is highly active. Black-Right-Pointing-Pointer CoLF can be prepared by bio-assimilation and direct exchange. Black-Right-Pointing-Pointer Lethal factor binds cobalt tightly. Black-Right-Pointing-Pointer The electronic spectrum of CoLF reveals penta-coordination. Black-Right-Pointing-Pointer Interaction of CoLF with thioglycolic acid follows a 2-step mechanism. -- Abstract: Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl{sub 2}, and (ii) direct exchange by treatment of zinc-LF with CoCl{sub 2}. Independent of the method employed, the protein was found to contain one Co{sup 2+} per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co{sup 2+} ion to be five-coordinate, an observation similar to that reported for other Co{sup 2+}-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co{sup 2+}:TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF's active site, which may be utilized in future studies to gain further insight into the enzyme's mechanism and inhibitor interactions.

  4. 5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced Hepatotoxicity and Lethality

    PubMed Central

    Hohmann, Miriam S. N.; Cardoso, Renato D. R.; Pinho-Ribeiro, Felipe A.; Crespigio, Jefferson; Cunha, Thiago M.; Alves-Filho, José C.; da Silva, Rosiane V.; Pinge-Filho, Phileno; Ferreira, Sergio H.; Cunha, Fernando Q.; Casagrande, Rubia; Verri, Waldiceu A.

    2013-01-01

    5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO−/−) mice and background wild type mice were challenged with APAP (0.3–6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO−/− mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1β, TNF-α, IFN-γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-β-D-glucosaminidase activity, Nrf2 and gp91phox mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO−/− mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage. PMID:24288682

  5. Loss of vps54 function leads to vesicle traffic impairment, protein mis-sorting and embryonic lethality.

    PubMed

    Karlsson, Páll; Droce, Aida; Moser, Jakob M; Cuhlmann, Simon; Padilla, Carolina Ortiz; Heimann, Peter; Bartsch, Jörg W; Füchtbauer, Annette; Füchtbauer, Ernst-Martin; Schmitt-John, Thomas

    2013-01-01

    The identification of the mutation causing the phenotype of the amyotrophic lateral sclerosis (ALS) model mouse, wobbler, has linked motor neuron degeneration with retrograde vesicle traffic. The wobbler mutation affects protein stability of Vps54, a ubiquitously expressed vesicle-tethering factor and leads to partial loss of Vps54 function. Moreover, the Vps54 null mutation causes embryonic lethality, which is associated with extensive membrane blebbing in the neural tube and is most likely a consequence of impaired vesicle transport. Investigation of cells derived from wobbler and Vps54 null mutant embryos demonstrates impaired retrograde transport of the Cholera-toxin B subunit to the trans-Golgi network and mis-sorting of mannose-6-phosphate receptors and cargo proteins dependent on retrograde vesicle transport. Endocytosis assays demonstrate no difference between wobbler and wild type cells, indicating that the retrograde vesicle traffic to the trans-Golgi network, but not endocytosis, is affected in Vps54 mutant cells. The results obtained on wobbler cells were extended to test the use of cultured skin fibroblasts from human ALS patients to investigate the retrograde vesicle traffic. Analysis of skin fibroblasts of ALS patients will support the investigation of the critical role of the retrograde vesicle transport in ALS pathogenesis and might yield a diagnostic prospect.

  6. Screening for gene regulation mutants by bioluminescence imaging.

    PubMed

    Chinnusamy, Viswanathan; Stevenson, Becky; Lee, Byeong-ha; Zhu, Jian-Kang

    2002-07-09

    Because plants cannot move, they have evolved complex sensing and response systems to cope with the physical environment. Adverse environmental conditions, such as those causing abiotic stress, often cause significant losses in crop productivity and quality. Because of a paucity of well-defined visible phenotypes, conventional genetic screens have not been very successful in isolating abiotic stress signal transduction mutants of plants. Here, we describe a reporter gene-based strategy to screen for mutants affected in abiotic stress-regulated gene transcription. Our genetic screen uses the firefly luciferase reporter gene driven by the cold, drought, salt, and abscisic acid (ABA)-responsive RD29A promoter (RD29A::LUC). Arabidopsis plants transformed with the RD29A::LUC reporter emit bioluminescence in response to cold, drought, salt, or ABA treatment. After mutagenesis of these plants with ethyl methanesulfonate (EMS), mutants can be screened from the M2 population by monitoring the level of stress-inducible bioluminescence with a high-throughput, low-light imaging system. This protocol describes in detail the procedures for this luciferase reporter-based genetic screen for Arabidopsis mutants defective in abiotic stress signaling.

  7. Mutant IDH is sufficient to initiate enchondromatosis in mice.

    PubMed

    Hirata, Makoto; Sasaki, Masato; Cairns, Rob A; Inoue, Satoshi; Puviindran, Vijitha; Li, Wanda Y; Snow, Bryan E; Jones, Lisa D; Wei, Qingxia; Sato, Shingo; Tang, Yuning J; Nadesan, Puviindran; Rockel, Jason; Whetstone, Heather; Poon, Raymond; Weng, Angela; Gross, Stefan; Straley, Kimberly; Gliser, Camelia; Xu, Yingxia; Wunder, Jay; Mak, Tak W; Alman, Benjamin A

    2015-03-01

    Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas. PMID:25730874

  8. An Escherichia coli Mutant That Makes Exceptionally Long Cells

    PubMed Central

    Newman, Elaine B.

    2015-01-01

    ABSTRACT Although Escherichia coli is a very small (1- to 2-μm) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division. IMPORTANCE Escherichia coli is usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells. PMID:25691528

  9. Differential Effects of Sex-Lethal Mutations on Dosage Compensation Early in Drosophila Development

    PubMed Central

    Bernstein, M.; Cline, T. W.

    1994-01-01

    In response to the primary sex determination signal, X chromosome dose, the Sex-lethal gene controls all aspects of somatic sex determination and differentiation, including X chromosome dosage compensation. Two complementary classes of mutations have been identified that differentially affect Sxl somatic functions: (1) those impairing the ``early'' function used to set developmental pathway choice in response to the sex determination signal and (2) those impairing ``late'' functions involved in maintaining the pathway choice independent of the initiating signal and/or in directing differentiation. This ``early vs. late'' distinction correlates with a switch in promoter utilization from Sxl(Pe) to Sxl(Pm) at the blastoderm stage and a corresponding switch from transcriptional to RNA splicing control. Here we characterize five partial-loss-of-function Sxl alleles to explore a distinction between ``early vs. late'' functioning of Sxl in dosage compensation. Assaying for dosage compensation during the blastoderm stage, we find that the earliest phase of the dosage compensation process is controlled by products of the early Sxl promoter, Sxl(Pe). Hence, in addition to triggering the sexual pathway decision of cells, products derived from Sxl(Pe) also control early dosage compensation, the first manifestation of sexually dimorphic differentiation. The effects of mutant Sxl alleles on early dosage compensation are consistent with their previous categorization as early vs. late defective with respect to their effects on pathway initiation. Results reported here suggest that the dosage compensation regulatory genes currently known to function downstream of Sxl, genes known as the ``male-specific lethals,'' do not control all aspects of dosage compensation either at the blastoderm stage or later in development. In the course of this study, we also discovered that the canonical early defective allele, Sxl(f9), which is impaired in its ability to establish the female

  10. Perinatal lethal type II osteogenesis imperfecta: a case report

    PubMed Central

    Ayadi, Imene Dahmane; Hamida, Emira Ben; Rebeh, Rania Ben; Chaouachi, Sihem; Marrakchi, Zahra

    2015-01-01

    We report a new case of osteogenesis imperfecta (OI) type II which is a perinatal lethal form. First trimester ultrasound didn't identified abnormalities. Second trimester ultrasound showed incurved limbs, narrow chest, with hypomineralization and multiple fractures of ribs and long bones. Parents refused pregnancy termination; they felt that the diagnosis was late. At birth, the newborn presented immediate respiratory distress. Postnatal examination and bone radiography confirmed the diagnosis of OI type IIA. Death occurred on day 25 of life related to respiratory failure. PMID:26401205

  11. Intimate partner homicide: new insights for understanding lethality and risks.

    PubMed

    Sheehan, Brynn E; Murphy, Sharon B; Moynihan, Mary M; Dudley-Fennessey, Erin; Stapleton, Jane G

    2015-02-01

    Research on covictims, family members, and close friends who have lost loved ones to intimate partner homicide (IPH) is a neglected area of study. We conducted phenomenological interviews with covictims to gain insights into risk and lethality, examined affidavits from criminal case files, and reviewed news releases. The data uncovered acute risk factors prior to the homicide, identified changes in the perpetrators' behavior and the perpetrators' perceived loss of control over the victim, and described barriers that victims faced when attempting to gain safety. Findings suggest that recognizing acute risk factors is an important area for future IPH research.

  12. Potent inhibitors of anthrax lethal factor from green tea

    PubMed Central

    Dell'Aica, Isabella; Donà, Massimo; Tonello, Fiorella; Piris, Alejandro; Mock, Michèle; Montecucco, Cesare; Garbisa, Spiridione

    2004-01-01

    The anthrax lethal factor (LF) has a major role in the development of anthrax. LF is delivered by the protective antigen (PA) inside the cell, where it exerts its metalloprotease activity on the N-terminus of MAPK-kinases. PA+LF are cytotoxic to macrophages in culture and kill the Fischer 344 rat when injected intravenously. We describe here the properties of some polyphenols contained in green tea as powerful inhibitors of LF metalloproteolytic activity, and how the main catechin of green tea, (−)epigallocatechin-3-gallate, prevents the LF-induced death of macrophages and Fischer 344 rats. PMID:15031715

  13. Neonatal Carnitine Palmitoyltransferase II Deficiency: A Lethal Entity.

    PubMed

    Malik, Sushma; Paldiwal, Ashutosh Abhimanyu; Korday, Charusheela Sujit; Jadhav, Shruti Sudhir

    2015-10-01

    Carnitine palmitoyltransferase II (CPTII) deficiency is a rare disorder of mitochondrial fatty acid oxidation with autosomal recessive mode of inheritance. Three classic forms of CPT II deficiency have been described namely the lethal neonatal form, severe infantile hepatocardiomuscular form and the myopathic form. We present a three-day-old female child, admitted to us for lethargy, icterus, low sugars and convulsions. Persistent non ketotic hypoglycaemia, hyperammonemia, raised liver enzymes with hepatomegaly and cardiomyopathy led to the suspicion of fatty acid oxidation defect. Tandem mass spectrometry helped to clinch the diagnosis of CPT II Deficiency in the present case. PMID:26557586

  14. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

    PubMed Central

    Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L.; Grainger, Robert M.

    2014-01-01

    SUMMARY The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant. PMID:25224223

  15. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

    PubMed

    Fish, Margaret B; Nakayama, Takuya; Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L; Grainger, Robert M

    2014-11-15

    The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

  16. CMPD: cancer mutant proteome database.

    PubMed

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes.

  17. CMPD: cancer mutant proteome database

    PubMed Central

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes. PMID:25398898

  18. Mutants of thermotaxis in Dictyostelium discoideum

    SciTech Connect

    Schneider, M.J.; Fontana, D.R.; Poff, K.L.

    1982-08-01

    Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the 'wild type' (HL50).The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.

  19. The Alternative Oxidase AOX Does Not Rescue the Phenotype of tko25t Mutant Flies

    PubMed Central

    Kemppainen, Kia K.; Kemppainen, Esko; Jacobs, Howard T.

    2014-01-01

    A point mutation [technical knockout25t (tko25t)] in the Drosophila gene coding for mitoribosomal protein S12 generates a phenotype of developmental delay and bang sensitivity. tko25t has been intensively studied as an animal model for human mitochondrial diseases associated with deficiency of mitochondrial protein synthesis and consequent multiple respiratory chain defects. Transgenic expression in Drosophila of the alternative oxidase (AOX) derived from Ciona intestinalis has previously been shown to mitigate the toxicity of respiratory chain inhibitors and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of tko25t using the GeneSwitch system to activate expression at different times in development. The developmental delay of tko25t was not mitigated by expression of AOX throughout development. AOX expression for 1 d after eclosion, or continuously throughout development, had no effect on the bang sensitivity of tko25t adults, and continued expression in adults older than 30 d also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with tko25t and was lethal when combined with both AOX and tko25t. We conclude that AOX does not rescue tko25t and that the mutant phenotype is not solely due to limitations on electron flow in the respiratory chain, but rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value. PMID:25147191

  20. Lethal, potentially lethal, and nonlethal damage induction by heavy ions in cultured human cells

    SciTech Connect

    Todd, P.; Wood, J.C.; Walker, J.T.; Weiss, S.J.

    1985-01-01

    In the fields of high-LET radiotherapy and space radiation safety it is important to know the relative probabilities with which a cell whose nucleus is struck by a heavy ion will be damaged or killed. Experiments were performed in which synchronous cultured human T-1 cells (presumptive HeLa) were irradiated with natural alpha particles of energy approximately 3.5 MeV at various times after mitotic selection up to the middle of S phase. Nuclear-area histograms were determined as a function of time after mitosis under conditions identical to those used for irradiation. The efficiency with which one particle passing through the nucleus killed a cell was found to be 0.14-0.20. This value was extrapolated to experimental cell survival data obtained when asynchronous cultured human cells were irradiated with He, Li, B, C, N, O, Ne, Ar ions of energy 6.58 or 5.5 MeV/amu, and the cell killing efficiency was found to be in the broad range of 0.5-1.0 under single-hit conditions. Similarly irradiated cells were examined for colony-size distribution by an image analysis technique, and it was found that the loss of large colonies was dose and LET-dependent in a systematic way. Dose-response data suggest two predominant subpopulations, resistant and sensitive cells, and it appears that the sensitive population is affected by single-hit kinetics. The single-hit coefficient for the induction of inherited slow growth varied with LET in a similar way to that for survival. The action cross section for this form of heritable damage appears to be comparable to the geometric cross section of the cell nucleus.

  1. Lethal and sub-lethal effects of elevated CO2 concentrations on marine benthic invertebrates and fish.

    PubMed

    Lee, Changkeun; Hong, Seongjin; Kwon, Bong-Oh; Lee, Jung-Ho; Ryu, Jongseong; Park, Young-Gyu; Kang, Seong-Gil; Khim, Jong Seong

    2016-08-01

    Concern about leakage of carbon dioxide (CO2) from deep-sea storage in geological reservoirs is increasing because of its possible adverse effects on marine organisms locally or at nearby coastal areas both in sediment and water column. In the present study, we examined how elevated CO2 affects various intertidal epibenthic (benthic copepod), intertidal endobenthic (Manila clam and Venus clam), sub-tidal benthic (brittle starfish), and free-living (marine medaka) organisms in areas expected to be impacted by leakage. Acute lethal and sub-lethal effects were detected in the adult stage of all test organisms exposed to varying concentrations of CO2, due to the associated decline in pH (8.3 to 5.2) during 96-h exposure. However, intertidal organisms (such as benthic copepods and clams) showed remarkable resistance to elevated CO2, with the Venus clam being the most tolerant (LpH50 = 5.45). Sub-tidal species (such as brittle starfish [LpH50 = 6.16] and marine medaka [LpH50 = 5.91]) were more sensitive to elevated CO2 compared to intertidal species, possibly because they have fewer defensive capabilities. Of note, the exposure duration might regulate the degree of acute sub-lethal effects, as evidenced by the Venus clam, which showed a time-dependent effect to elevated CO2. Finally, copper was chosen as a model toxic element to find out the synergistic or antagonistic effects between ocean acidification and metal pollution. Combination of CO2 and Cu exposure enhances the adverse effects to organisms, generally supporting a synergistic effect scenario. Overall, the significant variation in the degree to which CO2 adversely affected organisms (viz., working range and strength) was clearly observed, supporting the general concept of species-dependent effects of elevated CO2.

  2. Lethal and sub-lethal effects of elevated CO2 concentrations on marine benthic invertebrates and fish.

    PubMed

    Lee, Changkeun; Hong, Seongjin; Kwon, Bong-Oh; Lee, Jung-Ho; Ryu, Jongseong; Park, Young-Gyu; Kang, Seong-Gil; Khim, Jong Seong

    2016-08-01

    Concern about leakage of carbon dioxide (CO2) from deep-sea storage in geological reservoirs is increasing because of its possible adverse effects on marine organisms locally or at nearby coastal areas both in sediment and water column. In the present study, we examined how elevated CO2 affects various intertidal epibenthic (benthic copepod), intertidal endobenthic (Manila clam and Venus clam), sub-tidal benthic (brittle starfish), and free-living (marine medaka) organisms in areas expected to be impacted by leakage. Acute lethal and sub-lethal effects were detected in the adult stage of all test organisms exposed to varying concentrations of CO2, due to the associated decline in pH (8.3 to 5.2) during 96-h exposure. However, intertidal organisms (such as benthic copepods and clams) showed remarkable resistance to elevated CO2, with the Venus clam being the most tolerant (LpH50 = 5.45). Sub-tidal species (such as brittle starfish [LpH50 = 6.16] and marine medaka [LpH50 = 5.91]) were more sensitive to elevated CO2 compared to intertidal species, possibly because they have fewer defensive capabilities. Of note, the exposure duration might regulate the degree of acute sub-lethal effects, as evidenced by the Venus clam, which showed a time-dependent effect to elevated CO2. Finally, copper was chosen as a model toxic element to find out the synergistic or antagonistic effects between ocean acidification and metal pollution. Combination of CO2 and Cu exposure enhances the adverse effects to organisms, generally supporting a synergistic effect scenario. Overall, the significant variation in the degree to which CO2 adversely affected organisms (viz., working range and strength) was clearly observed, supporting the general concept of species-dependent effects of elevated CO2. PMID:27074931

  3. Lethal body burdens of polar narcotic chemicals: Chlorophenols

    SciTech Connect

    Wezel, A. van; Punte, S.; Opperhuizen, A.

    1994-12-31

    Lethal body burdens (LBBs) were measured of low chlorinated phenols to obtain insight in the intrinsic toxicity of these compounds and in the origins of differences in toxicity between polar and nonpolar narcotic chemicals. Fathead minnows were exposed until death to 2-chlorophenol, 4-chlorophenol or 2,4-chlorophenol at fixed pH. LBBs of chlorophenols were shown to be significantly lower than LBBs of the nonpolar chlorobenzenes. The pH of the exposure doesn`t influence the LBB of chlorophenols, whereas it does influence chemical speciation and thus the uptake and the LC50 of the chlorophenols. Octanol-water partition coefficients were measured at different pH, to gain insight in the distribution of the chlorophenols between aqueous and fatty compartments inside the organism. It is shown that at a physiological pH only a fraction of the total amount of chlorophenols inside the organism is accumulated in the fatty parts of the organism. These fatty parts, including the membranes, are usually considered as the target site for narcotic chemicals. It is concluded that the concentration at the membrane needed for lethality is lower for polar narcotic chemicals than for nonpolar narcotic chemicals, implying either a different mode of action at the same target site or another target site.

  4. Protection against both lethal and behavioral effects of soman.

    PubMed

    Harris, L W; McDonough, J H; Stitcher, D L; Lennox, W J

    1984-01-01

    This work developed two drug mixtures which alone had no effect on performance of a criterion behavior but when given as a pretreatment would protect against organophosphate-induced lethality and incapacitation. Candidate drugs (alone and together) were given to rats trained to respond on a two-component Fixed Ratio 10 - Extinction (FR10-EXT) schedule. After generating dose response curves for each cholinolytic drug, mixtures of atropine (A) + mecamylamine (M) + pyridostigmine (Py) or physostigmine (Ph) were prepared and a combination of doses that produced no effects on operant performance was determined (Mix I:A = .78, M = .78, Py = .056 mg/kg; Mix II:A = .78, M = .78, Ph = .026 mg/kg). Both pretreatment mixtures provided equivalent protection against the lethal effects of the organophosphate soman; however only Mix II was capable of reversing soman-induced physical incapacitation (PI) as assessed by performance on an accelerating rotarod or FR10 responding. Pretreatment of animals with Mix II resulted in significantly higher levels of brain acetylcholinesterase (AChE) than Mix I pretreated subjects 4 hrs after 1.3 LD50 soman, although peripheral AChE levels were not different. The results indicate organophosphate-induced PI can be attenuated by pretreatment with tertiary carbamates which protect significant amounts of brain AChE from irreversible inhibition.

  5. Molecular markers in prostate cancer. Part I: predicting lethality

    PubMed Central

    Agrawal, Sachin; Dunsmuir, William D.

    2009-01-01

    Assessing the lethality of 'early,' potentially organ-confined prostate cancer (PCa) is one of the central controversies in modern-day urological clinical practice. Such cases are often considered for radical 'curative' treatment, although active surveillance may be equally appropriate for many men. Moreover, the balance between judicious intervention and overtreatment can be difficult to judge. The patient's age, comorbidities, family history and philosophy of self-health care can be weighed against clinical features such as the palpability of disease, the number and percentage of biopsy cores involved with the disease, histological grade, presenting prostate-specific antigen (PSA) and possible previous PSA kinetics. For many years, scientists and physicians have sought additional molecular factors that may be predictive for disease stage, progression and lethality. Usually, claims for a 'new' unique marker fall short of true clinical value. More often than not, such molecular markers are useful only in multivariate models. This review summarizes relevant molecular markers and models reported up to and including 2008. PMID:19050690

  6. Equation of state and fragmentation issues in computational lethality analysis

    SciTech Connect

    Trucano, T.G.

    1993-07-01

    The purpose of this report is to summarize the status of computational analysis of hypervelocity impact lethality in relatively nontechnical terms from the perspective of the author. It is not intended to be a review of the technical literature on the problems of concern. The discussion is focused by concentrating on two phenomenology areas which are of particular concern in computational impact studies. First, the material`s equation of state, specifically the treatment of expanded states of metals undergoing shock vaporization, is discussed. Second, the process of dynamic fragmentation is addressed. In both cases, the context of the discussion deals with inaccuracies and difficulties associated with numerical hypervelocity impact simulations. Laboratory experimental capabilities in hypervelocity impact for impact velocities greater than 10.0 km/s are becoming increasingly viable. This paper also gives recommendations for experimental thrusts which utilize these capabilities that will help to resolve the uncertainties in the numerical lethality studies that are pointed out in the present report.

  7. Effectiveness of lethal, directed wolf-depredation control in Minnesota

    USGS Publications Warehouse

    Harper, E.K.; Paul, W.J.; Mech, L.D.; Weisberg, S.

    2008-01-01

    Wolf (Canis lupus) depredations on livestock in Minnesota, USA, are an economic problem for many livestock producers, and depredating wolves are lethally controlled. We sought to determine the effectiveness of lethal control through the analysis of data from 923 government-verified wolf depredations from 1979 to 1998. We analyzed the data by 1) assessing the correlations between the number of wolves killed in response to depredations with number of depredations the following year at state and local levels, and 2) the time to the next depredation. No analysis indicated that trapping wolves substantially reduced the following year's depredations at state or local levels. However, more specific analyses indicated that in certain situations, killing wolves was more effective than no action (i.e., not trapping). For example, trapping and killing adult males decreased the re-depredation risk. At sheep farms, killing wolves was generally effective. Attempting to trap, regardless of the results, seemed more effective at reducing depredations than not trapping, suggesting that mere human activity near depredation sites might deter future depredations.

  8. An outbreak of lethal adenovirus infection among different otariid species.

    PubMed

    Inoshima, Yasuo; Murakami, Tomoaki; Ishiguro, Naotaka; Hasegawa, Kazuhiro; Kasamatsu, Masahiko

    2013-08-30

    An outbreak of fatal fulminant hepatitis at a Japanese aquarium involved 3 otariids: a California sea lion (Zalophus californianus), a South African fur seal (Arctocephalus pusillus) and a South American sea lion (Otaria flavescens). In a span of about a week in February 2012, 3 otariids showed diarrhea and were acutely low-spirited; subsequently, all three animals died within a period of 3 days. Markedly increased aspartate amino transferase and alanine amino transferase activities were observed. Necrotic hepatitis and eosinophilic intranuclear inclusion bodies in liver hepatocytes and intestinal epithelial cells were observed in the South American sea lion on histological examination. Otarine adenovirus DNA was detected from the livers of all three animals by polymerase chain reaction and determination of the sequences showed that all were identical. These results suggest that a single otarine adenovirus strain may have been the etiological agent of this outbreak of fatal fulminant hepatitis among the different otariid species, and it may be a lethal threat to wild and captive otariids. This is the first evidence of an outbreak of lethal adenovirus infection among different otariid species. PMID:23643878

  9. Fluorescence resonance energy transfer studies on anthrax lethal toxin.

    PubMed

    Croney, John C; Cunningham, Kristina M; Collier, R John; Jameson, David M

    2003-08-28

    Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA(63) heptamer and for one residue (E126) on LF(N), the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF(N) was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF(N), whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF(N) binds near the top of the ring-shaped (PA(63))(7) heptamer.

  10. Injury risk assessment of non-lethal projectile head impacts.

    PubMed

    Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

    2014-01-01

    Kinetic energy non-lethal projectiles are used to impart sufficient effect onto a person in order to deter uncivil or hazardous behavior with a low probability of permanent injury. Since their first use, real cases indicate that the injuries inflicted by such projectiles may be irreversible and sometimes lead to death, especially for the head impacts. Given the high velocities and the low masses involved in such impacts, the assessment approaches proposed in automotive crash tests and sports may not be appropriate. Therefore, there is a need of a specific approach to assess the lethality of these projectiles. In this framework, some recent research data referred in this article as "force wall approach" suggest the use of three lesional thresholds (unconsciousness, meningeal damages and bone damages) that depend on the intracranial pressure. Three corresponding critical impact forces are determined for a reference projectile. Based on the principle that equal rigid wall maximal impact forces will produce equal damage on the head, these limits can be determined for any other projectile. In order to validate the consistence of this innovative method, it is necessary to compare the results with other existing assessment methods. This paper proposes a comparison between the "force wall approach" and two different head models. The first one is a numerical model (Strasbourg University Finite Element Head Model-SUFEHM) from Strasbourg University; the second one is a mechanical surrogate (Ballistics Load Sensing Headform-BLSH) from Biokinetics. PMID:25400712

  11. The lethal effects of Cyperus iria on Aedes aegypti.

    PubMed

    Schwartz, A M; Paskewitz, S M; Orth, A P; Tesch, M J; Toong, Y C; Goodman, W G

    1998-03-01

    The sedge Cyperus iria, a common weed in rice, contains large amounts of the insect hormone (10R) juvenile hormone III (JH III). Given its widespread distribution in Asia and Africa, we examined the possibility that C. iria could be used as a safe, inexpensive, and readily available mosquito larvicide. Plants of varying ages were harvested and leaves tested for lethal effects on larvae of the yellow fever mosquito, Aedes aegypti. The median lethal doses (LD50s) for frozen leaves from 1- and 2-month-old plants were 267 and 427 mg/100 ml of water, respectively. Leaves from 1-month-old C. iria contained 193 micrograms JH III/g fresh weight, whereas leaves from 2-month-old plants contained 143 micrograms JH III/g fresh weight. Larval sensitivity to the plant differed with age; 4-day-old larvae displayed the greatest mortality followed in decreasing sensitivity by larvae 5, 6, 3, and 2 days old. Six Cyperus species (C. albostriatus, C. alternifolius, C. esculentus, C. iria, C. miliifolius, and C. papyrus) of similar developmental stage were assayed for JH III content. Only C. iria was found to contain significant levels of JH III. PMID:9599328

  12. Non-lethal sampling for mercury evaluation in crocodilians.

    PubMed

    Lázaro, Wilkinson L; de Oliveira, Robson F; dos Santos-Filho, Manoel; da Silva, Carolina J; Malm, Olaf; Ignácio, Áurea R A; Díez, Sergi

    2015-11-01

    Mercury (Hg) is a ubiquitous environmental contaminant that poses potential threats to ecosystems due to its toxicity to humans and wildlife. The development of non-lethal sampling techniques is a critical step for evaluation of Hg in threatened species in tropical floodplain environments, where most of Hg found is the result of land use and gold mining activities, and more methylation sites are available. We evaluated the spatial and seasonal effectiveness of caudal scutes and claws to estimate Hg bioaccumulation in crocodilians (Caiman yacare), in the scarcely documented Pantanal. Hence, we investigated the potential for Hg bioaccumulation in top predators according to its proximity to mining sites, and in water bodies with different hydrological characteristics and connectivity with the main river during two phases of the flood pulse (dry and flood). The highest Hg concentrations were detected in caimans captured close to mining activities, in claws (2176 ng g(-1) ww) and caudal scutes (388 ng g(-1) ww). THg concentration in claws was related to the flood season and its mean concentration was thirteen fold higher than Hg concentration in scutes during whole year. Both tissues were found to be effective as non-lethal sampling techniques for measuring Hg bioaccumulation in reptiles over time. Nevertheless, claw tissue seems to have a more consistent result, since its constitutional chemical characteristics makes it a better indicator of spatial patterns that influence on Hg exposure. PMID:26026900

  13. Lethal Nipah virus infection induces rapid overexpression of CXCL10.

    PubMed

    Mathieu, Cyrille; Guillaume, Vanessa; Sabine, Amélie; Ong, Kien Chai; Wong, Kum Thong; Legras-Lachuer, Catherine; Horvat, Branka

    2012-01-01

    Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis.

  14. Injury Risk Assessment of Non-Lethal Projectile Head Impacts

    PubMed Central

    Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

    2014-01-01

    Kinetic energy non-lethal projectiles are used to impart sufficient effect onto a person in order to deter uncivil or hazardous behavior with a low probability of permanent injury. Since their first use, real cases indicate that the injuries inflicted by such projectiles may be irreversible and sometimes lead to death, especially for the head impacts. Given the high velocities and the low masses involved in such impacts, the assessment approaches proposed in automotive crash tests and sports may not be appropriate. Therefore, there is a need of a specific approach to assess the lethality of these projectiles. In this framework, some recent research data referred in this article as “force wall approach” suggest the use of three lesional thresholds (unconsciousness, meningeal damages and bone damages) that depend on the intracranial pressure. Three corresponding critical impact forces are determined for a reference projectile. Based on the principle that equal rigid wall maximal impact forces will produce equal damage on the head, these limits can be determined for any other projectile. In order to validate the consistence of this innovative method, it is necessary to compare the results with other existing assessment methods. This paper proposes a comparison between the “force wall approach” and two different head models. The first one is a numerical model (Strasbourg University Finite Element Head Model-SUFEHM) from Strasbourg University; the second one is a mechanical surrogate (Ballistics Load Sensing Headform-BLSH) from Biokinetics. PMID:25400712

  15. Radiation-induced cell lethality of samonella typhimurium ATCC 14028: Cooperative effect of hydroxyl radical and oxygen

    SciTech Connect

    Kim,