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1

Inducer-Dependent Conditional-Lethal Mutant Animal Viruses  

Microsoft Academic Search

Regulatory elements of the Escherichia coli lac operon were used to construct an inducer-dependent conditional-lethal mutant animal virus. The gene encoding the repressor protein of the lac operon was integrated into the vaccinia virus genome so that it was expressed constitutively, and the lac operator was inserted next to the promoter of a gene that encodes an 11-kDa virion-associated protein

Yifan Zhang; Bernard Moss

1991-01-01

2

A nutritional conditional lethal mutant due to pyridoxine 5'-phosphate oxidase deficiency in Drosophila melanogaster.  

PubMed

The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5'-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5'-phosphate (PLP). The missense mutation (sgll(95)) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll(95) flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases. PMID:24739647

Chi, Wanhao; Zhang, Li; Du, Wei; Zhuang, Xiaoxi

2014-06-01

3

Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon-COP  

PubMed Central

The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic. PMID:8207054

1994-01-01

4

Conditional lethal mutants of adenovirus type 2-simian virus 40 hybrids. II. Ad2+ND1 host-range mutants that synthesize fragments of the Ad2+ND1 30K protein.  

PubMed Central

Adenovirus type 2 (Ad2) grows 1,000 times less well in monkey cells than in human cells. This defect can be overcome, not only upon co-infection of cells with simian virus 40 (SV40), but also when the relevant part of the SV40 genome is integrated into the adenovirus genome to form an adenovirus-SV40 hybrid virus. We have used the nondefective Ad2-SV40 hybrid virus Ad2+ND1, which contains an insertion of 17% of the SV40 genome, to isolate host-range mutants which are defective in growth on monkey cells although they grow normally on human cells. Like Ad2, these mutants are defective in the synthesis of late proteins in monkey cells. A 30,000-molecular-weight protein (30K), unique to Ad2+ND1-infected cells, can be synthesized in vitro, using Ad2+ND1 mRNA that contains SV40 sequences. 30K is not seen in cells infected with those host-range mutants that are most defective in growth on monkey cells, and translation in vitro of SV40-specific mRNA from these cells produces new unique polypeptides, instead of 30K. Genetic and biochemical analyses indicate that these mutants carry point mutations rather than deletions. Images PMID:183014

Grodzicker, T; Lewis, J B; Anderson, C W

1976-01-01

5

An Arabidopsis pex10 Null Mutant Is Embryo Lethal, Implicating Peroxisomes in an Essential Role during  

E-print Network

An Arabidopsis pex10 Null Mutant Is Embryo Lethal, Implicating Peroxisomes in an Essential Role Brookes University, Oxford OX3 0BP United Kingdom (F.B., C.H.) Peroxisomes participate in many important, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system

6

Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens  

PubMed Central

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

Dorca-Arevalo, Jonatan; Pauillac, Serge; Diaz-Hidalgo, Laura; Martin-Satue, Mireia; Popoff, Michel R.; Blasi, Juan

2014-01-01

7

An anthrax lethal factor mutant that is defective at causing pyroptosis retains pro-apoptotic activity  

PubMed Central

Summary Anthrax lethal toxin triggers death in some cell types, such as macrophages, and causes a variety of cellular dysfunctions in others. Collectively, these effects dampen the innate and adaptive immune systems to allow Bacillus anthracis to survive and proliferate in the mammalian host. The diverse effects caused by the toxin have in part been attributed to its interference with signaling pathways in target cells. Lethal factor (LF) is the proteolytic component of the toxin, which cleaves six members of the mitogen activated protein kinase kinase (MAPKK) family after being delivered to the cytosol by the cell-binding component of the toxin, protective antigen. The effect of cleaving these MAPKKs is to interfere with ERK, p38 and JNK signaling. Here we characterize an LF mutant, LF-K518E/E682G, that is defective at causing pyroptosis in RAW 264.7 cells and at activating the Nlrp1b inflammasome in a heterologous expression system. LF-K518E/E682G does not exhibit an overall impairment of function, however, because it is able to downregulate the ERK pathway, but not the p38 or JNK pathways. Furthermore, LF-K518E/E682G efficiently killed melanoma cells, which were shown previously to undergo apoptosis in response to lethal toxin or to pharmacological inhibition of the ERK pathway. Our results suggest that LF-K518E/E682G is defective at cleaving a substrate involved in the activation of the Nlrp1b inflammasome. PMID:19922472

Ngai, Stephanie; Batty, Sarah; Liao, Kuo-Chieh; Mogridge, Jeremy

2009-01-01

8

Drosophila lethal giant larvae neoplastic mutant as a genetic tool for cancer modeling.  

PubMed

Drosophila lethal giant larvae (lgl) is a tumour suppressor gene whose function in establishing apical-basal cell polarity as well as in exerting proliferation control in epithelial tissues is conserved between flies and mammals. Individuals bearing lgl null mutations show a gradual loss of tissue architecture and an extended larval life in which cell proliferation never ceases and no differentiation occurs, resulting in prepupal lethality. When tissues from those individuals are transplanted into adult normal recipients, a subset of cells, possibly the cancer stem units, are again able to proliferate and give rise to metastases which migrate to distant sites killing the host. This phenotype closely resembles that of mammalian epithelial cancers, in which loss of cell polarity is one of the hallmarks of a malignant, metastatic behaviour associated with poor prognosis. Lgl protein shares with its human counterpart Human giant larvae-1 (Hugl-1) significant stretches of sequence similarity that we demonstrated to translate into a complete functional conservation, pointing out a role in cell proliferation control and tumorigenesis also for the human homologue. The functional conservation and the power of fly genetics, that allows the researcher to manipulate the fly genome at a level of precision that exceeds that of any other multicellular genetic system, make this Drosophila mutant a very suitable model in which to investigate the mechanisms underlying epithelial tumour formation, progression and metastatisation. In this review, we will summarise the results obtained in these later years using this model for the study of cancer biology. Moreover, we will discuss how recent advances in developmental genetics techniques have succeeded in enhancing the similarities between fly and human tumorigenesis, giving Drosophila a pivotal role in the study of such a complex genetic disease. PMID:19440511

Froldi, F; Ziosi, M; Tomba, G; Parisi, F; Garoia, F; Pession, A; Grifoni, D

2008-05-01

9

C. elegans ten-1 is synthetic lethal with mutations in cytoskeleton regulators, and enhances many axon guidance defective mutants  

PubMed Central

Background Teneurins are transmembrane proteins that assist morphogenetic processes in many organisms. ten-1 is the C. elegans teneurin homolog with two transcripts, ten-1a and ten-1b, that respectively encode a long (TEN-1L) and short (TEN-1S) form of the protein. We previously isolated a C. elegans mutant where one pharyngeal neuron was frequently misplaced, and now show that it corresponds to a novel allele of ten-1. Results The novel ten-1(et5) allele is a hypomorph since its post-embryonic phenotype is weaker than the null alleles ten-1(ok641) and ten-1(tm651). ten-1 mutants have defects in all pharyngeal neurons that we examined, and in vivo reporters show that only the long form of the ten-1 gene is expressed in the pharynx, specifically in six marginal cells and the M2 neurons. Defects in the pharyngeal M2 neurons were enhanced when the ten-1(ok641) mutation was combined with mutations in the following genes: mig-14, unc-5, unc-51, unc-52 and unc-129. None of the body neurons examined show any defects in the ten-1(ok641) mutant, but genetic interaction studies reveal that ten-1(ok641) is synthetic lethal with sax-3, unc-34 and unc-73, and examination of the hypodermal cells in embryos of the ten-1(ok641) mutant point to a role of ten-1 during hypodermal cell morphogenesis. Conclusions Our results are consistent with ten-1 normally providing a function complementary to the cytoskeletal remodeling processes that occur in migrating cells or cells undergoing morphogenesis. It is possible that ten-1 influences the composition/distribution of extracellular matrix. PMID:20497576

2010-01-01

10

Natural and glucosyl flavonoids inhibit poly(ADP-ribose) polymerase activity and induce synthetic lethality in BRCA mutant cells  

PubMed Central

Poly(ADP-ribose) polymerase (PARP) inhibitors have been proven to represent superior clinical agents targeting DNA repair mechanisms in cancer therapy. We investigated PARP inhibitory effects of the natural and synthetic flavonoids (quercetin, rutin, monoglucosyl rutin and maltooligosyl rutin) and tested the synthetic lethality in BRCA2 mutated cells. In vitro ELISA assay suggested that the flavonoids have inhibitory effects on PARP activity, but glucosyl modifications reduced the inhibitory effect. Cytotoxicity tests of Chinese hamster cells defective in BRCA2 gene (V-C8) and its parental V79 cells showed BRCA2-dependent synthetic lethality when treated with the flavonoids. BRCA2 mutated cells were three times more sensitive to the flavonoids than the wild-type and gene complemented cells. Reduced toxicity was observed in a glucosyl modification-dependent manner. The present study provides support for the clinical use of new treatment drugs, and is the beginning of the potential application of flavonoids in cancer prevention and the periodic consumption of appropriate flavonoids to reduce cancer risk in individuals carrying a mutant allele of the BRCA2 gene. PMID:24317580

MAEDA, JUNKO; ROYBAL, ERICA J.; BRENTS, COLLEEN A.; UESAKA, MITSURU; AIZAWA, YASUSHI; KATO, TAKAMITSU A.

2014-01-01

11

Transgenic expression of the N525S-tuberin variant in Tsc2 mutant (Eker) rats causes dominant embryonic lethality.  

PubMed

The Tsc2 product, tuberin, negatively regulates the mTOR pathway. We have exploited the Eker (Tsc2-mutant) rat system to analyse various Tsc2 mutations. Here, we focus on the N525S-Tsc2 variant (NSM), which is known to cause distinct symptoms in patients even though normal suppression of mTOR is observed. Unexpectedly, we were repeatedly unable to generate viable rats carrying the NSM transgene. Genotypic analysis revealed that most of the embryos carrying the transgene died around embryonic day after 14.5-similar to the stage of lethality observed for Eker homozygotes. Thus, the NSM transgene appeared to have a dominant lethal effect in our rat model. Further, no significant differences were observed for various signal transduction molecules in transiently expressed NSM cells compared to WT. These results indicate that a non-mTOR pathway, critical for embryogenesis, is being regulated by tuberin, providing a link between tuberin expression and the severity of Tsc2 mutation-related pathogenesis. PMID:25088526

Shiono, Masatoshi; Kobayashi, Toshiyuki; Takahashi, Riichi; Ueda, Masatsugu; Ishioka, Chikashi; Hino, Okio

2014-01-01

12

Neonatal Lethality, Dwarfism, and Abnormal Brain Development in Dmbx1 Mutant Mice  

Microsoft Academic Search

Dmbx1 encodes a paired-like homeodomain protein that is expressed in developing neural tissues during mouse embryogenesis. To elucidate the in vivo role of Dmbx1, we generated two Dmbx1 mutant alleles. Dmbx1 lacks the homeobox and Dmbx1z is an insertion of a lacZ reporter gene. Dmbx1z appears to be a faithful reporter of Dmbx1 expression during embryogenesis and after birth. Dmbx1-lacZ

Akihira Ohtoshi; Richard R. Behringer

2004-01-01

13

A Genetic Suppressor of Two Dominant Temperature-Sensitive Lethal Proteasome Mutants of Drosophila melanogaster Is Itself a Mutated Proteasome Subunit Gene  

PubMed Central

Two dominant temperature-sensitive (DTS) lethal mutants of Drosophila melanogaster are Pros261 and Pros?21, previously known as DTS5 and DTS7. Heterozygotes for either mutant die as pupae when raised at 29°, but are normally viable and fertile at 25°. Previous studies have identified these as missense mutations in the genes encoding the ?6 and ?2 subunits of the 20S proteasome, respectively. In an effort to isolate additional proteasome-related mutants a screen for dominant suppressors of Pros261 was carried out, resulting in the identification of Pros25SuDTS [originally called Su(DTS)], a missense mutation in the gene encoding the 20S proteasome ?2 subunit. Pros25SuDTS acts in a dominant manner to rescue both Pros261 and Pros?21 from their DTS lethal phenotypes. Using an in vivo protein degradation assay it was shown that this suppression occurs by counteracting the dominant-negative effect of the DTS mutant on proteasome activity. Pros25SuDTS is a recessive polyphasic lethal at ambient temperatures. The effects of these mutants on larval neuroblast mitosis were also examined. While Pros?21 shows a modest increase in the number of defective mitotic figures, there were no defects seen with the other two mutants, other than slightly reduced mitotic indexes. PMID:16648584

Neuburger, Peter J.; Saville, Kenneth J.; Zeng, Jue; Smyth, Kerrie-Ann; Belote, John M.

2006-01-01

14

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

SciTech Connect

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.

Grider, A. Jr.

1986-01-01

15

Mutant loxP vectors for selectable marker recycle and conditional knock-outs  

PubMed Central

Background Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. Results We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. Conclusions Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved ?-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines. PMID:11591226

Arakawa, Hiroshi; Lodygin, Dimitry; Buerstedde, Jean-Marie

2001-01-01

16

An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae.  

PubMed Central

ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y. PMID:9755191

Chen, C Y; Graham, T R

1998-01-01

17

An Arabidopsis pex10 Null Mutant Is Embryo Lethal, Implicating Peroxisomes in an Essential Role during Plant Embryogenesis1  

PubMed Central

Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AtPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability. PMID:14576288

Sparkes, Imogen A.; Brandizzi, Federica; Slocombe, Stephen P.; El-Shami, Mahmoud; Hawes, Chris; Baker, Alison

2003-01-01

18

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

PubMed Central

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 ?-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 discrete subset of functions associated with virulence. SigE is the first cell envelope stress-sensing system to be described in the bordetellae. In addition to its role during lethal infection of mice deficient in adaptive immunity, our results indicate that SigE is likely to be important for survival in the face of stresses encountered in the environment between hosts. PMID:22897969

2012-01-01

19

Lethal effect of K-shell absorption of intracellular phosphorus on wild-type and radiation sensitive mutants of Escherichia coli.  

PubMed

The present study was conducted to clarify the lethality of Auger cascades induced by the K-shell photoabsorption of phosphorus in Escherichia coli. Killing of wild-type and radiation-sensitive mutants of E. coli was examined. Three x-ray energies were chosen for irradiation; at 2.153 keV: the resonance peak of K-shell photoabsorption of phosphorus; at 2.146 and 2.160 keV: off-peak. Enhancement ratio, which was defined as the ratio of the killing sensitivity of 2.153 keV to that at 2.146 keV, were 1.32 to 1.54 for examined strains. Increment of absorbed energy calculated in entire cells for 2.153 keV radiation could not explain the degree of observed enhancement of killing. Lethality of Auger cascades depended on the killing sensitivity with x-rays which did not induce Auger cascades. The lethality for wild-type was lower than that for recombination repair-deficient mutants. It was concluded that one part of damages produced by Auger cascades was repaired in wild-type strains. PMID:9004768

Maezawa, H; Furusawa, Y; Kobayashi, K; Hieda, K; Suzuki, M; Usami, N; Yokoya, A; Mori, T

1996-01-01

20

Production of viable seeds from the seedling lethal mutant ppi2-2 lacking the atToc159 chloroplast protein import receptor using plastic containers, and characterization of the homozygous mutant progeny  

PubMed Central

Biogenesis of chloroplasts is essential for plant growth and development. A number of homozygous mutants lacking a chloroplast protein exhibit an albino phenotype. In general, it is challenging to grow albino Arabidopsis plants on soil until they set seeds. Homozygous albino mutants are usually obtained as progenies of heterozygous parents. Here, we describe a method of recovering seeds from the seedling lethal Arabidopsis mutant ppi2-2, which lacks the atToc159 protein import receptor at the outer envelope membrane of chloroplast. Using plastic containers, we were able to grow homozygous ppi2-2 plants until these set seed. Although the germination rate of the harvested seeds was relatively low, it was still sufficient to allow us to further analyze the ppi2-2 progeny. Using ppi2-2 homozygous seeds, we were able to analyze the role of plastid protein import in the light-regulated induction of nuclear genes. We propose that this method be applied to other seedling lethal Arabidopsis mutants to obtain homozygous seeds, helping us further investigate the roles of plastid proteins in plant growth and development. PMID:24926298

Tada, Akari; Adachi, Fumi; Kakizaki, Tomohiro; Inaba, Takehito

2014-01-01

21

Zebrafish mutants  

NSDL National Science Digital Library

Two developing Danio mutants with slow beating two-chambered hearts. A no tail mutant shows problems with both the heart and the tail. They cannot swim to catch food making the no tail mutation lethal.

Mildred Hoover (Salem State College;Biology Department); Nancy Pelaez (Purdue University;Biological Sciences)

2008-07-19

22

Lethal, potentially lethal lesion model  

SciTech Connect

A theoretical framework to describe the formation of lethal mutations by radiation is presented. Lesions that are repaired (and misrepaired) in each type of experiment described (delayed plating and split dose) are assumed to be the same. In this model the same (potentially lethal) lesions cause both sublethal and potentially lethal damage. Potentially lethal damage is defined as damage which may be modified by alterations in postirradiation conditions. Sublethal damage is cellular damage whose accumulation may lead to lethality. A crucial consideration in the expression of the damage is the kind of medium in which the cells are placed during the repair period. Fresh or growth medium (F-medium) is assumed to cause fixation of damage after about 3 hours, while no fixation (only misrepair) occurs in conditioned medium (C-medium).

Curtis, S.B.

1983-07-01

23

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

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.

Varentsova, E.R.

1986-05-01

24

Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models.  

PubMed

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers. PMID:23245996

Corcoran, Ryan B; Cheng, Katherine A; Hata, Aaron N; Faber, Anthony C; Ebi, Hiromichi; Coffee, Erin M; Greninger, Patricia; Brown, Ronald D; Godfrey, Jason T; Cohoon, Travis J; Song, Youngchul; Lifshits, Eugene; Hung, Kenneth E; Shioda, Toshi; Dias-Santagata, Dora; Singh, Anurag; Settleman, Jeffrey; Benes, Cyril H; Mino-Kenudson, Mari; Wong, Kwok-Kin; Engelman, Jeffrey A

2013-01-14

25

Synthetic Lethal Interaction of Combined BCL-XL and MEK Inhibition Promotes Tumor Regressions in KRAS Mutant Cancer Models  

PubMed Central

SUMMARY KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers. PMID:23245996

Corcoran, Ryan B.; Cheng, Katherine A.; Hata, Aaron N.; Faber, Anthony C.; Ebi, Hiromichi; Coffee, Erin M.; Greninger, Patricia; Brown, Ronald D.; Godfrey, Jason T.; Cohoon, Travis J.; Song, Youngchul; Lifshits, Eugene; Hung, Kenneth E.; Shioda, Toshi; Dias-Santagata, Dora; Singh, Anurag; Settleman, Jeffrey; Benes, Cyril H.; Mino-Kenudson, Mari; Wong, Kwok-Kin; Engelman, Jeffrey A.

2013-01-01

26

Mechanism of Action of Nalidixic Acid on Escherichia coli III. Conditions Required for Lethality  

PubMed Central

Deitz, William H. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), Thomas M. Cook, and William A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. III. Conditions required for lethality. J. Bacteriol. 91:768–773. 1966.—Nalidixic acid selectively inhibited deoxyribonucleic acid (DNA) synthesis in cultures of Escherichia coli 15TAU. Protein and ribonucleic acid synthesis were shown to be a prerequisite for the bactericidal action of the drug. This action can be prevented by means of inhibitors at bacteriostatic concentrations. Both chloramphenicol, which inhibits protein synthesis, and dinitrophenol, which uncouples oxidative phosphorylation, effectively prevented the bactericidal action of nalidixic acid on E. coli. The lethal action of nalidixic acid also was controlled by transfer of treated cells to drug-free medium. DNA synthesis resumed immediately upon removal of the drug and was halted immediately by retreatment. These studies indicate that nalidixic acid acts directly on the replication of DNA rather than on the “initiator” of DNA synthesis. The entry of nalidixic acid into cells of E. coli was not dependent upon protein synthesis. Even in the presence of an inhibiting concentration of chloramphenicol, nalidixic acid prevented DNA synthesis by E. coli 15TAU. PMID:5327367

Deitz, William H.; Cook, Thomas M.; Goss, William A.

1966-01-01

27

Mutant Human Presenilin 1 Protects presenilin 1 Null Mouse against Embryonic Lethality and Elevates A?1–42\\/43 Expression  

Microsoft Academic Search

Mutations in presenilin 1 (PS1) are linked to early onset of familial Alzheimer's disease (FAD) and are shown to foster production of A?1–42\\/43 in FAD patients and transgenic mice. PS1 null mice are embryonic lethal and exhibit axial skeleton malformation and CNS defects. We show that transgenic mouse lines expressing either the wild-type human PS1 protein or human PS1 with

Su Qian; Ping Jiang; Xiao-Ming Guan; Gurparkash Singh; Myrna E. Trumbauer; Hong Yu; Howard Y. Chen; Hui Zheng

1998-01-01

28

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

SciTech Connect

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.

Cai, Yuping; Wolk, C.P. (Michigan State Univ., East Lansing (USA))

1990-06-01

29

Nitrate reductase deficient cell lines from diploid cell cultures and lethal mutant M2 plants of Arabidopsis thaliana.  

PubMed

Cell suspensions of diploid Arabidopsis thaliana were screened for resistance to chlorate on a medium with ammonium nitrate as the nitrogen source, and after plating on filters to increase the plating efficiency. Thirty-nine lines were selected, four of which were still resistant after two years of subculturing on non-selective medium. Of the latter lines three were nitrate reductase deficient but exhibited some residual nitrate reductase activity; the fourth line showed a high level of enzyme activity. Screening M2-seeds for callus production on selective medium with amino acids as the nitrogen source and chlorate revealed resistant calli in 17 out of 483 M2-groups. Nine well-growing lines, all but one (G3) exhibiting no detectable in vivo nitrate reductase activity, were classified as defective in the cofactor. Two lines (G1 and G3) could be analysed genetically at the plant level. Chlorate resistance was monogenic and recessive. Sucrose gradient fractionation of callus extracts of G1 revealed that a complete enzyme molecule can be assembled. Nitrate reductase activity in G1 could partly be restored by excess molybdenum. It is suggested that G1 is disturbed in the catalytic properties of the cofactor. It appeared that G1 is neither allelic with another molybdenum repairable mutant (B73) nor with another cofactor mutant (B25). Wilting of intact G1 plants could be ascribed to non-closing stomata. PMID:24247470

Scholten, H J; de Vries, S E; Nijdam, H; Feenstra, W J

1985-12-01

30

Conditional DNA repair mutants enable highly precise genome engineering.  

PubMed

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. PMID:24500200

Nyerges, Ákos; Csorg?, Bálint; Nagy, István; Latinovics, Dóra; Szamecz, Béla; Pósfai, György; Pál, Csaba

2014-04-01

31

Conditional DNA repair mutants enable highly precise genome engineering  

PubMed Central

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement. PMID:24500200

Nyerges, Akos; Csorgo, Balint; Nagy, Istvan; Latinovics, Dora; Szamecz, Bela; Posfai, Gyorgy; Pal, Csaba

2014-01-01

32

Lethal cutaneous disease in transgenic mice conditionally expressing type I human T cell leukemia virus Tax.  

PubMed

Type I human T cell leukemia virus (HTLV-I) is etiologically linked with adult T cell leukemia, an aggressive and usually fatal expansion of activated CD4+ T lymphocytes that frequently traffic to skin. T cell transformation induced by HTLV-I involves the action of the 40-kDa viral Tax transactivator protein. Tax both stimulates the HTLV-I long terminal repeat and deregulates the expression of select cellular genes by altering the activity of specific host transcription factors, including cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor, NF-kappaB/Rel, and serum response factor. To study initiating events involved in HTLV-I Tax-induced T cell transformation, we generated "Tet-off" transgenic mice conditionally expressing in a lymphocyte-restricted manner (EmuSR alpha promoter-enhancer) either wild-type Tax or mutant forms of Tax that selectively compromise the NF-kappaB (M22) or CREB/activating transcription factor (M47) activation pathways. Wild-type Tax and M47 Tax-expressing mice, but not M22-Tax expressing mice, developed progressive alopecia, hyperkeratosis, and skin lesions containing profuse activated CD4 T cell infiltrates with evidence of deregulated inflammatory cytokine production. In addition, these animals displayed systemic lymphadenopathy and splenomegaly. These findings suggest that Tax-mediated activation of NF-kappaB plays a key role in the development of this aggressive skin disease that shares several features in common with the skin disease occurring during the preleukemic stage in HTLV-I-infected patients. Of note, this skin disease completely resolved when Tax transgene expression was suppressed by administration of doxycycline, emphasizing the key role played by this viral oncoprotein in the observed pathology. PMID:16105841

Kwon, Hakju; Ogle, Louise; Benitez, Bobby; Bohuslav, Jan; Montano, Mauricio; Felsher, Dean W; Greene, Warner C

2005-10-21

33

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.  

PubMed Central

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

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

1987-01-01

34

The uses of genome-wide yeast mutant collections  

Microsoft Academic Search

We assess five years of usage of the major genome-wide collections of mutants from Saccharomyces cerevisiae: single deletion mutants, double mutants conferring 'synthetic' lethality and the 'TRIPLES' collection of mutants obtained\\u000a by random transposon insertion. Over 100 experimental conditions have been tested and more than 5,000 novel phenotypic traits\\u000a have been assigned to yeast genes using these collections.

Bart Scherens; Andre Goffeau

2004-01-01

35

Conditional Genealogies and the Age of a Neutral Mutant  

Microsoft Academic Search

This paper is concerned with the structure of the genealogy of a sample in which it is observed that some subset of chromosomes carries a particular mutation, assumed to have arisen uniquely in the history of the population. A rigorous theoretical study of this conditional genealogy is given using coalescent methods. Particular results include the mean, variance, and density of

Carsten Wiuf; Peter Donnelly

1999-01-01

36

Classical conditioning and retention in normal and mutant Drosophila melanogaster  

Microsoft Academic Search

Summary By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in type flies. About 150 flies were sequestered in a closed chamber and trained by explosing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of

Tim Tully; William G. Quinn

1985-01-01

37

Burkholderia cenocepacia conditional growth mutant library created by random promoter replacement of essential genes  

PubMed Central

Identification of essential genes by construction of conditional knockouts with inducible promoters allows the identification of essential genes and creation of conditional growth (CG) mutants that are then available as genetic tools for further studies. We used large-scale transposon delivery of the rhamnose-inducible promoter, PrhaB followed by robotic screening of rhamnose-dependent growth to construct a genomic library of 106 Burkholderia cenocepacia CG mutants. Transposon insertions were found where PrhaB was in the same orientation of widely conserved, well-characterized essential genes as well as genes with no previous records of essentiality in other microorganisms. Using previously reported global gene-expression analyses, we demonstrate that PrhaB can achieve the wide dynamic range of expression levels required for essential genes when the promoter is delivered randomly and mutants with rhamnose-dependent growth are selected. We also show specific detection of the target of an antibiotic, novobiocin, by enhanced sensitivity of the corresponding CG mutant (PrhaB controlling gyrB expression) within the library. Modulation of gene expression to achieve 30–60% of wild-type growth created conditions for specific hypersensitivity demonstrating the value of the CG mutant library for chemogenomic experiments. In summary, CG mutants can be obtained on a large scale by random delivery of a tightly regulated inducible promoter into the bacterial chromosome followed by a simple screening for the CG phenotype, without previous information on gene essentiality. PMID:23389959

Bloodworth, Ruhi A M; Gislason, April S; Cardona, Silvia T

2013-01-01

38

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

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.

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

1986-03-01

39

Mutants of Anabaena strain CA altered in their ability to grow under nitrogen-fixing conditions.  

PubMed Central

Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena. PMID:115843

Gotto, J W; Tabita, F R; Van Baalen, C

1979-01-01

40

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

PubMed Central

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

Rothstein, Steven J.

2013-01-01

41

Enhanced extinction of contextual fear conditioning in Clock?19 mutant mice.  

PubMed

Clock genes have been implicated in several disorders, such as schizophrenia, bipolar disorder, autism spectrum disorders, and drug dependence. However, few studies to date have examined the role of clock genes in fear-related behaviors. The authors used mice with the Clock?19 mutation to assess the involvement of this gene in contextual fear conditioning. Male wild-type (WT) and Clock?19 mutant mice underwent a single session of contextual fear conditioning (12 min, 4 unsignaled shocks), followed by daily 12-min retention trials. There were no differences between mutant and WT mice in the acquisition of contextual fear, and WT and mutant mice demonstrated similar freezing during the first retention session. However, extinction of contextual fear was accelerated in mutant mice across the remaining retention sessions, as compared to WT mice, suggesting a role for Clock in extinction following aversive learning. Because the Clock?19 mutation has previously been demonstrated to result in an increase in dopamine signaling, the authors confirmed the role of dopamine in extinction learning using preretention session administration of a low dose of the dopamine transport reuptake inhibitor modafinil (0.75 mg/kg), which resulted in decreased freezing across retention sessions. These findings are consistent with an emerging portrayal of the importance of Clock genes in noncircadian functions, as well as the important role of dopamine in extinction learning. PMID:24865659

Bernardi, Rick E; Spanagel, Rainer

2014-08-01

42

Studies on radiosensitive lines of Drosophila. X. Effect of 0. 8 MeV neutrons from reactor on the survival and frequency of dominant lethals in mutant line rad(2)201/sup G1/  

SciTech Connect

The frequency of dominant lethal mutations (DLM) was studied in the females of the radiosensitive mutant line rad(2)201/sup G1/ following exposure to different doses of neutrons at various stages of oogenesis. Survival of pupae after irradiation of larvae was also studied. It has been demonstrated that in respect of DLM induction, the differential sensitivity of oocytes to the action of neutrons in the control (nonmutated) line is similar to that of gamma-ray treatment. The sensitivity of oocytes at the 7th and earlier stages is higher in the mutant females than in control line. The analysis of relative biological efficiency (RBE) of the neutrons showed that they are more effective in the control line as compared to the mutant line, in respect of survival as well as frequency of DLM induction. The RBE of neutrons depended on the stage of oocyte development: the highest RBE was observed in immature sex cells of females. The possible mechanisms of higher sensitivity of mutant line rad(2)201/sup G1/ to the action of ionizing radiation are discussed.

Varentsova, E.R.; Sharygin, V.I.; Postnikov, L.N.; Efremov, O.A.

1986-04-01

43

Optimized Condition for Enhanced Soluble-Expression of Recombinant Mutant Anabaena Variabilis Phenylalanine Ammonia Lyase  

PubMed Central

Purpose: Recently discovered Anabaena variabilis phenylalanine ammonia lyase (AvPAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli. Methods: In the present study, the gene encoding mutant version of AvPAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme. Results: Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 °C and induction period of 18 hour was the highest. Conclusion: The results of this study indicated that the yield of mutant AvPAL production in E. coli can be affected mainly by culture temperature and inducer concentration. PMID:24754010

Zarei Jaliani, Hossein; Farajnia, Safar; Safdari, Yaghoub; Mohammadi, Seyyed Abolghasem; Barzegar, Abolfazl; Talebi, Saeed

2014-01-01

44

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions.  

PubMed

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration. PMID:22526328

Ruhal, Rohit; Choudhury, Bijan

2012-08-01

45

Breathing without CO(2) chemosensitivity in conditional Phox2b mutants.  

PubMed

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O(2), CO(2), and pH homeostasis. In mammals, it is generated by a neuronal network in the lower brainstem, the respiratory rhythm generator (Feldman et al., 2003). A century-old tenet in respiratory physiology posits that the respiratory chemoreflex, the stimulation of breathing by an increase in partial pressure of CO(2) in the blood, is indispensable for rhythmic breathing. Here we have revisited this postulate with the help of mouse genetics. We have engineered a conditional mouse mutant in which the toxic PHOX2B(27Ala) mutation that causes congenital central hypoventilation syndrome in man is targeted to the retrotrapezoid nucleus, a site essential for central chemosensitivity. The mutants lack a retrotrapezoid nucleus and their breathing is not stimulated by elevated CO(2) at least up to postnatal day 9 and they barely respond as juveniles, but nevertheless survive, breathe normally beyond the first days after birth, and maintain blood PCO(2) within the normal range. Input from peripheral chemoreceptors that sense PO(2) in the blood appears to compensate for the missing CO(2) response since silencing them by high O(2) abolishes rhythmic breathing. CO(2) chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO(2) is dispensable for life-sustaining breathing and maintaining CO(2) homeostasis in the blood. PMID:21900566

Ramanantsoa, Nelina; Hirsch, Marie-Rose; Thoby-Brisson, Muriel; Dubreuil, Véronique; Bouvier, Julien; Ruffault, Pierre-Louis; Matrot, Boris; Fortin, Gilles; Brunet, Jean-François; Gallego, Jorge; Goridis, Christo

2011-09-01

46

Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions  

SciTech Connect

Mutants of Azotobacter vinelandii affected in N/sub 2/ fixation in the presence of 1 ..mu..M Na/sub 2/MoO/sub 4/ (conventional system), 50 nM V/sub 2/O/sub 5/, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants are grouped into four broad phenotypic classes. Mutants in the first class are Nif/sup -/ under Mo sufficiency but Nif/sup +/ under Mo deficiency or in the presence of V/sub 2/O/sub 5/. Mutants in the second class are Nif/sup -/ under all conditions. An FeMo-cofactor-negative mutant (NifB/sup -/) belongs to this class. The third mutant class consists of mutants incapable of N/sub 2/-dependent growth under Mo deficiency. Most of the mutants of this class are also affected in N/sub 2/ fixation in the presence of 1 ..mu..M Na/sub 2/MoO/sub 4/, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kan/sup r/ marker of mutants from this class into nifHDK or nifK deletion mutants showed N/sub 2/-dependent growth only in the presence of V/sup 2/O/sub 5/. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V/sub 2/O/sub 5/. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V/sub 2/O/sub 5/. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V/sub 2/O/sub 5/; however, the NH/sub 4//sup +/-and Mo-repressible proteins normally seen under these conditions could not be detected on two-dimensional gels.

Joerger, R.D.; Premakumar, R.; Bishop, P.E.

1986-11-01

47

Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg.  

PubMed Central

The obg gene is part of the spo0B sporulation operon and codes for a GTP-binding protein which is essential for growth. A temperature-sensitive mutant in the obg gene was isolated and found to be the result of two closely linked missense mutations in the amino domain of Obg. Temperature shift experiments revealed that the mutant was able to continue cell division for 2 to 3 generations at the nonpermissive temperature. Such experiments carried out during sporulation showed that Obg was necessary for the transition from vegetative growth to stage 0 or stage II of sporulation, but sporulation subsequent to these stages was unaffected at the nonpermissive temperature. Spores of the temperature-sensitive mutant germinated normally at the nonpermissive temperature but failed to outgrow. The primary consequence of the obg mutation may be an alteration in initiation of chromosome replication. PMID:7961486

Kok, J; Trach, K A; Hoch, J A

1994-01-01

48

Up-regulation of tubulin genes and growth phenotype of tubulin mutants in Arabidopsis under hypergravity conditions  

NASA Astrophysics Data System (ADS)

Plants resist the gravitational force by constructing a tough body via an increase in cell wall rigidity and modifications of growth anisotropy The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method has shown that a gene encoding alpha-tubulin is up-regulated by hypergravity Yoshioka et al 2003 Adv Space Res 31 2187 suggesting the involvement of microtubules in gravity resistance in plants In the present study we examined this possibility by analyzing the expression levels of each member of alpha- and beta- tubulin genes and growth behavior of tubulin mutants in Arabidopsis under hypergravity conditions produced by centrifugation Most of alpha- and beta-tubulin genes were up-regulated by hypergravity at 300 g Isolated amino acid substitution mutants tua3 D205N tua4 S178 tua6 A281T tua6 S277F showed left-handed or right-handed helical growth derived from disordered organization of cortical microtubules in hypocotyls even under 1 g conditions Such a phenotype was intensified under hypergravity conditions especially in tua3 and tua4 mutants Tubulin mutants had thicker and shorter hypocotyls than wild-type under 1 g conditions Their thickening was stimulated and elongation was suppressed by hypergravity treatment although to a lesser extent than wild-type The cell wall rigidity of wild-type hypocotyls was increased under hypergravity conditions whereas such an increase is

Matsumoto, S.; Kumasaki, S.; Soga, K.; Wakabayashi, K.; Abe, T.; Ishida, T.; Hashimoto, T.; Hoson, T.

49

Genetics Home Reference: Platyspondylic lethal skeletal dysplasia, Torrance type  

MedlinePLUS

... OMIM Genetic disorder catalog Conditions > Platyspondylic lethal skeletal dysplasia, Torrance type On this page: Description Genetic changes ... Reviewed July 2008 What is platyspondylic lethal skeletal dysplasia, Torrance type? Platyspondylic lethal skeletal dysplasia, Torrance type ...

50

Characterization of Synechocystis sp. strain PCC 6803 and ? nbl mutants under nitrogen-deficient conditions  

Microsoft Academic Search

The impact of nitrogen deficiency on the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and three nbl (non-bleaching) mutants ((nblA1, (nblA2 and (nblB) was investigated. The (nblA mutants entered a non-dividing, dormant state soon after the initiation of nitrogen starvation. The cells became larger, the membrane system was disorganized, and ribosomes were found near the membranes much less frequently. Photosystem

Hong Li; Louis A. Sherman

2002-01-01

51

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

PubMed Central

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

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

2014-01-01

52

Comparative proteomic analysis of a Candida albicans DSE1 mutant under filamentous and non-filamentous conditions.  

PubMed

Candida albicans is a common opportunistic pathogen that causes a variety of diseases in immunocompromised hosts. In a pathogen, cell wall proteins are important virulence factors. We previously characterized Dse1 as a cell wall protein necessary for virulence and resistance to cell surface-disrupting agents, such as Calcofluor white, chitin deposition, proper adhesion and biofilm formation. In the absence of decomplexation, our objectives were to investigate differential proteomic expression of a DSE1 mutant strain compared to the wild-type strain. The strains were grown under filamentous and non-filamentous conditions. The extracted cell proteome was subjected to tryptic digest, followed by generation of peptide profiles using MALDI-TOF MS. Generated peptide profiles were analysed and unique peaks for each strain and growth condition mined against a Candida database, allowing protein identification. The DSE1 mutant was shown to lack the chitin biosynthesis protein Chs5, explaining the previously observed decrease in chitin biosynthesis. The wild-type strain expressed Pra1, involved in pH response and zinc acquisition, Atg15, a lipase involved in virulence, and Sod1, required for oxidative stress tolerance, in addition to proteins involved in protein biosynthesis, explaining the increase in total protein content observed compared to the mutants strain. The mutant, on the other hand, expressed glucoamylase 1, a cell wall glycoprotein involved in carbohydrate metabolism cell wall degradation and biofilm formation. As such, MALDI-TOF MS is a reliable technique in identifying mutant-specific protein expression in C. albicans. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25231799

Zohbi, Rasha; Wex, Brigitte; Khalaf, Roy A

2014-11-01

53

Manipulating Individual Decisions and Environmental Conditions Reveal Individual Quality in Decision-Making and Non-Lethal Costs of Predation Risk  

PubMed Central

Habitat selection is a crucial decision for any organism. Selecting a high quality site will positively impact survival and reproductive output. Predation risk is an important component of habitat quality that is known to impact reproductive success and individual condition. However, separating the breeding consequences of decision-making of wild animals from individual quality is difficult. Individuals face reproductive decisions that often vary with quality such that low quality individuals invest less. This reduced reproductive performance could appear a cost of increased risk but may simply reflect lower quality. Thus, teasing apart the effects of individual quality and the effect of predation risk is vital to understand the physiological and reproductive costs of predation risk alone on breeding animals. In this study we alter the actual territory location decisions of pied flycatchers by moving active nests relative to breeding sparrowhawks, the main predators of adult flycatchers. We experimentally measure the non-lethal effects of predation on adults and offspring while controlling for effects of parental quality, individual territory choice and initiation of breeding. We found that chicks from high predation risk nests (<50 m of hawk) were significantly smaller than chicks from low risk nests (>200 m from hawk). However, in contrast to correlative results, females in manipulated high risk nests did not suffer decreased body condition or increased stress response (HSP60 and HSP70). Our results suggest that territory location decisions relative to breeding avian predators cause spatial gradients in individual quality. Small adjustments in territory location decisions have crucial consequences and our results confirm non-lethal costs of predation risk that were expressed in terms of smaller offspring produced. However, females did not show costs in physiological condition which suggests that part of the costs incurred by adults exposed to predation risk are quality determined. PMID:23272226

Thomson, Robert L.; Tomas, Gustavo; Forsman, Jukka T.; Monkkonen, Mikko

2012-01-01

54

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture  

Microsoft Academic Search

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of

Petrus S. Meyer; James C. Du Preez

1994-01-01

55

Flagellar membrane agglutination and sexual signaling in the conditional GAM1 mutant of Chlamydomonas  

Microsoft Academic Search

The temperature-sensitive gametogenesis-defective mutant, gain-1 is sex-limited, expressed only in mating type minus (rot-), and can sexually agglutinate but not fuse at the restrictive temperature (35~ with gametes of wild type (wt) mt +. Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gain-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore

CHARLENE L. FOREST; DANIEL A. GOODENOUGH

1978-01-01

56

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

PubMed Central

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

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

2011-01-01

57

Bright Mutants of Vibrio fischeri ES114 Reveal Conditions and Regulators That Control Bioluminescence and Expression of the lux Operon?  

PubMed Central

Vibrio fischeri ES114, an isolate from the Euprymna scolopes light organ, produces little bioluminescence in culture but is ?1,000-fold brighter when colonizing the host. Cell-density-dependent regulation alone cannot explain this phenomenon, because cells within colonies on solid medium are much dimmer than symbiotic cells despite their similar cell densities. To better understand this low luminescence in culture, we screened ?20,000 mini-Tn5 mutants of ES114 for increased luminescence and identified 28 independent “luminescence-up” mutants with insertions in 14 loci. Mutations affecting the Pst phosphate uptake system led to the discovery that luminescence is upregulated under low-phosphate conditions by PhoB, and we also found that ainS, which encodes an autoinducer synthase, mediates repression of luminescence during growth on plates. Other novel luminescence-up mutants had insertions in acnB, topA, tfoY, phoQ, guaB, and two specific tRNA genes. Two loci, hns and lonA, were previously described as repressors of bioluminescence in transgenic Escherichia coli carrying the light-generating lux genes, and mutations in arcA and arcB were consistent with our report that Arc represses lux. Our results reveal a complex regulatory web governing luminescence and show how certain environmental conditions are integrated into regulation of the pheromone-dependent lux system. PMID:20693328

Lyell, Noreen L.; Dunn, Anne K.; Bose, Jeffrey L.; Stabb, Eric V.

2010-01-01

58

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

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.

Iliakis, G.; Nusse, M.

1982-09-01

59

Canonical Notch signaling is not necessary for prosensory induction in the mouse cochlea: Insights from a conditional mutant of RBPj?  

PubMed Central

The mammalian organ of Corti consists of a highly organized array of hair cells and supporting cells that originate from a common population of prosensory progenitors. Proper differentiation of this complex cellular mosaic requires lateral inhibition mediated by Notch signaling. Several studies also have implicated Notch signaling in the earlier induction of the prosensory domain that lies along the length of the cochlear duct, and which forms prior to the onset of hair cell and supporting cell differentiation. To investigate the role of Notch signaling in prosensory domain formation, we conditionally inactivated the transcriptional mediator of canonical Notch signaling, RBPj?, throughout the inner ear. While RBPj? mutants have severe vestibular defects and a shortened cochlear duct, markers of the prosensory domain appear at the normal time and location in the cochlea of RBPj? mutants. Despite the lack of RBPj?, hair cell and supporting cell markers also appear at appropriate times in the cochlea, suggesting that RBPj? is dispensable for differentiation of the cochlear sensory epithelium. However, we also observed that differentiating hair cells and supporting cells rapidly die in RBPj? mutants, suggesting a requirement of RBPj? for cell survival in this tissue. Finally, in contrast to the chick basilar papilla, ectopic activation of Notch signaling did not induce ectopic sensory patches in non-sensory regions of the cochlea. Our results indicate that canonical Notch signaling is not necessary for prosensory specification in the mouse cochlea, suggesting that other signaling pathways may specify this highly derived sensory organ. PMID:21632926

Basch, Martín L.; Ohyama, Takahiro; Segil, Neil; Groves, Andrew K.

2011-01-01

60

Elemental Concentrations in the Seed of Mutants and Natural Variants of Arabidopsis thaliana Grown under Varying Soil Conditions  

PubMed Central

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 under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. Each of the modified soils resulted in a unique change to the seed ionome (the mineral nutrient content of the seeds). To help identify the genetic networks regulating the seed ionome, changes in elemental concentrations were evaluated using mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes supports an estimate that as much as 11% of the A. thaliana genome encodes proteins of functional relevance to ion homeostasis in seeds. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280 and CNGC2. In a comparison of nine different accessions to a Col-0 reference, eight accessions were observed to have reproducible differences in elemental concentrations, seven of which were dependent on specific soil conditions. These results indicate that the A. thaliana seed ionome is distinct from the vegetative ionome, and that elemental analysis is a sensitive approach to identify genes controlling ion homeostasis, including those that regulate gene expression, phospho-regulation, and ion transport. PMID:23671651

McDowell, Stephen C.; Akmakjian, Garo; Sladek, Chris; Mendoza-Cozatl, David; Morrissey, Joe B.; Saini, Nick; Mittler, Ron; Baxter, Ivan; Salt, David E.; Ward, John M.; Schroeder, Julian I.; Guerinot, Mary Lou; Harper, Jeffrey F.

2013-01-01

61

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties.  

PubMed

We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle. PMID:1837238

Dabbs, E R

1991-06-01

62

Isolation and characterization of temperature-sensitive mutants of Anabaena variabilis impaired in nitrogen fixation  

Microsoft Academic Search

Temperature-sensitive (ts) mutants of the cyanobacteriumAnabaena variabilis ATCC 29413 were isolated following mutagenesis with N-methyl-N?-nitro-N-nitrosoguanidine (MNNG) and post-treatment with metronidazole\\u000a at 40°C. Of the 8000 clones isolated and tested, six mutants were conditionally lethal at the restrictive temperature (40°C).\\u000a All the ts mutants exhibited differences in their rates of growth, chlorophyll content, pigment (phycocyanin and\\/or chlorophyll)\\u000a ratios, heterocyst frequency, oxygen

T. A. Sarma; D. P. SINGIt

1994-01-01

63

A URA3 null mutant of Candida albicans (CAI-4) causes oro-oesophageal and gastric candidiasis and is lethal for gnotobiotic, transgenic mice (Tgepsilon26) that are deficient in both natural killer and T cells.  

PubMed

Current data suggest that functional URA3 genes are necessary for the full pathogenesis of Candida albicans. Herein it is shown that a putatively avirulent URA3/URA3 null mutant of C. albicans (CAI-4) can colonize the murine alimentary tract, invade oro-oesophageal and gastric tissues with yeasts and hyphae, evoke a granulocyte-dominated inflammatory response, and kill transgenic mice that are deficient for both natural killer cells and T cells. Because C. albicans-colonized (gnotobiotic) mice lack a viable prokaryotic microbiota, this study also demonstrates that the gut microbiome is not required to supply the mutant's nutritional needs. The gnotobiotic murine model described herein can be used to assess the capacity of C. albicans mutants to colonize and infect cutaneous, mucosal and systemic tissues and kill the susceptible host via a clinically common, natural route of infection; namely the alimentary tract. PMID:19208876

Balish, Edward

2009-03-01

64

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

SciTech Connect

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.

Datta, T.; Doermer, P.

1987-12-01

65

Euglena gracilis as source of the antioxidant vitamin E. Effects of culture conditions in the wild strain and in the natural mutant WZSL  

Microsoft Academic Search

The photosynthetic wild type and the spontaneous non-photosynthetic WZSL mutant of the unicellular flagellate Euglena gracilis\\u000a were grown to investigate the influence of photoheterotrophic and heterotrophic conditions on ?-tocopherol (vitamin E) content.\\u000a HPLC analysis demonstrated a marked enhancement (almost 100%) of tocopherol content in the light in both strains, independent\\u000a of the presence of chloroplasts. These findings indicate that the

C. Kusmic; R. Barsacchi; L. Barsanti; P. Gualtieri; V. Passarelli

1998-01-01

66

Lethal familial protracted diarrhoea  

PubMed Central

24 children with severe protracted diarrhoea from 10 families, in which at least one sibling was affected, are reported. In two families the siblings were from 1st-cousin marriages, in one family both parents had unaffected children from previous marriages, and in another family the mother had a normal daughter from an earlier marriage. The onset of the diarrhoea was on the first day of life in 12 infants, some time during the first 17 days in 10, and at 13 weeks and 1 year 6 days in the remaining two. In each case the diarrhoea was `cholera-like'. Investigations failed to show any of the established causes of protracted diarrhoea and 21 (87·5%) infants died after an illness that had lasted between 12 days and 6 years 38 weeks, despite periods of prolonged intravenous feeding and the administration of a wide variety of pharmacological agents. The 2 patients who recovered appeared to do so spontaneously. 14 (58%) had associated extra-gastrointestinal or gastrointestinal-related anomalies. Steady-state perfusion studies were performed in the proximal jejunum of 2 patients, and in the colon of one. In both cases the jejunum was in a net secretory state with respect to water, glucose absorption was markedly reduced, and the transmural potential difference was also depressed; in one of these patients fructose absorption was also reduced, and in the other colonic function appeared to be normal. These studies suggest that the diarrhoea resulted from small intestinal secretion overwhelming the reabsorptive capacity of a normally-functioning colon. Although this series of lethal protracted diarrhoea does not represent a single disease entity, the familial pattern suggests an autosomal recessive mode of inheritance for at least one of the conditions. ImagesFigure PMID:7469448

Candy, D C A; Larcher, V F; Cameron, D J S; Norman, A P; Tripp, J H; Milla, P J; Pincott, J R; Harries, J T

1981-01-01

67

Metabolic Flux Analysis of Escherichia coli creB and arcA Mutants Reveals Shared Control of Carbon Catabolism under Microaerobic Growth Conditions?  

PubMed Central

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 13C-labeling experiments in chemostat cultures of a wild-type strain, ?creB and ?arcA single mutants, and a ?creB ?arcA 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 ?arcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the ?arcA 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. PMID:19561129

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

2009-01-01

68

Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. V. Sex Transformation Caused by Interactions between a Female-Specific Lethal, Sxlf#1, and the Male-Specific Lethals mle(3)132, msl-227, and mle  

PubMed Central

Interactions between a female-specific lethal mutant, Sxlf#1, and each of three male-specific lethal mutants, mle(3)132, msl-227 and mle, of Drosophila melanogaster were observed to produce morphological changes in various sexually dimorphic external characters. Chromosomal females heterozygous for Sxlf#1 and homozygous for any one of the male-specific lethals (and to a lesser degree heterozygous for male-specific lethals) sometimes had sex combs, male-type tergites, male-type sternites, male-type anal plates or male-type external genitalia. Penetrance was not high and expression was often incomplete; single individuals never had all the sexually dimorphic structures transformed. When mothers were homozygous for male-specific lethals, higher proportions of female progeny were affected than when mothers were heterozygous, suggesting a maternal effect. PMID:6818105

Uenoyama, T.; Fukunaga, A.; Ioshi, K.

1982-01-01

69

Heat shock transcriptional responses in an MC-Producing Cyanobacterium (Planktothrix agardhii) and its MC-deficient mutant under high light conditions.  

PubMed

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 ?mcyD 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

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

2013-01-01

70

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

PubMed Central

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 ?mcyD 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

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

2013-01-01

71

Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to ?-Lactams  

PubMed Central

ABSTRACT Most ?-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 ?-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem ?-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 ?-lactam resistance. The global transcriptional response of the bacterium to ?-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 ?-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 ?-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

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

2014-01-01

72

P-lacW Insertional Mutagenesis on the Second Chromosome of Drosophila melanogaster: Isolation of Lethals With Different Overgrowth Phenotypes  

Microsoft Academic Search

A single P-element insertional mutagenesis experiment was carried out for the second chromosome of Drosophila melanogaster using the P-lacW transposon. Out of 15,475 insertions on the second chromosome, 2,308 lethal and 403 semilethal mutants (altogether 2,711) were recovered. After eliminating clusters, 72% of the mutants represent independent insertions. Some of the mutants with larval, prepupal or pupal lethal phases have

Tibor Torok; Gabriella Tick; Martha Alvarado; Istvan Kiss

1993-01-01

73

Impact of acid adaptation on attachment of Listeria monocytogenes to stainless steel during long-term incubation under low or moderate temperature conditions and on subsequent recalcitrance of attached cells to lethal acid treatments.  

PubMed

This study aimed to evaluate the possible impact of acid adaptation of Listeria monocytogenes cells on their attachment to stainless steel (SS) during long-term incubation under either low or moderate temperature conditions and on the subsequent recalcitrance of attached cells to lethal acid treatments. Initially, nonadapted or acid-adapted stationary phase L. monocytogenes cells were used to inoculate (ca. 10? CFU/ml) brain-heart infusion (BHI) broth in test tubes containing vertically placed SS coupons. Incubation was carried out at either 5 or 30 °C for up to 15 days, under static conditions. On the 5th, 10th and 15th days of incubation, attached cells were subjected to lethal acid treatments by exposing them, for either 6 or 60 min, to pH 2, adjusted with either hydrochloric or lactic acid. Following the acid treatments, remaining viable cells were detached (through strong vortexing with glass beads) and enumerated by agar plating, and also indirectly quantified by conductance measurements via their metabolic activity. Results obtained from both quantification techniques, employed here in parallel, revealed that although the numbers of attached cells for nonadapted and acid-adapted ones were similar, the latter were found to present significantly (p<0.05) increased recalcitrance to all the acid treatments for both incubation temperatures and all sampling days. In addition and regardless of acid adaptation, when long (60 min) acid treatments were applied, conductance measurements revealed that the weak organic lactic acid exhibited significantly (p<0.05) stronger antilisterial activity compared to the strong inorganic hydrochloric acid (at the same pH value of 2). To conclude, present results show that acid adaptation of L. monocytogenes cells during their planktonic growth is conserved even after 15 days of incubation under both low and moderate temperature conditions, and results in the increased recalcitrance of their sessile population to otherwise lethal acid treatments. This "stress hardening" should be severely taken into account when acidic decontamination interventions are used to kill attached to equipment surfaces cells of this important pathogenic bacterium. PMID:24296256

Giaouris, Efstathios; Chorianopoulos, Nikos; Nychas, George-John

2014-02-01

74

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

PubMed

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

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

2014-11-01

75

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

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

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

2014-01-01

76

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

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

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

2013-01-01

77

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

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

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

2013-03-01

78

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

PubMed Central

Mutations in PIK3CA, the gene encoding the p110? catalytic subunit of phosphatidylinositol-3 kinase (PI3K), have been shown to transform mammary epithelial cells (MECs). Studies suggest this transforming activity requires binding of mutant p110? via p85 to phosphorylated YXXM motifs in activated receptor tyrosine kinases (RTKs) 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 PIK3CAH1047R-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 PIK3CAH1047R-expressing tumors. However, the p110?-specific inhibitor BYL719, in combination with lapatinib impaired mammary tumor growth and PI3K signaling more potently than BYL719 alone. Further, co-inhibition of p110? and ErbB3 potently suppressed proliferation and PI3K signaling in human breast cancer cells harboring PIK3CAH1047R. These data suggest that PIK3CAH1047R-driven tumor growth and PI3K signaling can occur independently of ErbB RTKs. However, simultaneous blockade of p110? 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. PMID:23633485

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

2013-01-01

79

The effects of body weight, temperature, salinity, pH, light intensity and feeding condition on lethal DO levels of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931)  

Microsoft Academic Search

Tolerance of whiteleg shrimp Litopenaeus vannamei exposed to different temperatures (14.5, 21.5, 24.8, 27.8, 30.8, and 35.0 °C), salinities (9, 15, 26, 35, and 40‰), pH (3.3, 6.5, 7.7, 8.1, and 9.2), and light intensities (strong 2100 lx and weak 60 lx) at various body weights (3.0, 3.7, 4.3, 5.7, 7.8, 9.0, 9.5, 10.7, 11.9, and 13.3 g) and feeding conditions (fed

Peidong Zhang; Xiumei Zhang; Jian Li; Guoqiang Huang

2006-01-01

80

Physiological and biochemical responses of fruit exocarp of tomato (Lycopersicon esculentum Mill.) mutants to natural photo-oxidative conditions.  

PubMed

Photo-oxidative stress was imposed under natural solar radiation on exposed and shaded sections of detached fruit of immature green tomato (Lycopersicon esculentum Miller = Solanum lycopersicum L.) mutants (anthocyanin absent, beta-carotene, Delta, and high pigment-1) and their nearly isogenic parents ('Ailsa Craig' and 'Rutgers'). After 5 h exposure to high solar irradiance, either with or without ultraviolet (UV) radiation, surface colour changes, pigment composition, photosynthetic efficiency, antioxidant metabolites and enzyme activities, and selected flavonoids and antioxidant proteins in exocarp tissue were evaluated. The imposed photo-oxidative stress reproduced the symptoms observed on attached fruit. Both high temperature and solar irradiance caused fruit surface discoloration with faster degradation of chlorophyll (Chl) than carotenoids (Car), leading to an increase in the Car/Chl ratio. Surface bleaching was mostly caused by visible light, whereas elevated temperatures were mostly responsible for the inactivation of photosynthesis, measured as decreased F(v)/F(m). Ascorbate, glutathione, and total soluble protein concentrations decreased in the exocarp as the duration of exposure increased. Specific activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase (MDHAR), glutathione reductase (GR), and catalase increased with exposure, suggesting that these proteins were conserved during the imposed stress. GR protein expression remained stable during the imposed stress, whereas, MDHAR protein expression increased. Quercetin and kaempferol concentrations increased rapidly upon exposure, but not to UV radiation, suggesting rapid photo-protection in response to visible light; however, naringenin synthesis was not induced. The apparent increased tolerance of hp-1 fruit is discussed. PMID:16698820

Torres, Carolina A; Andrews, Preston K; Davies, Neal M

2006-01-01

81

Animal Models For Disease--Knockout, Knockin And Conditional Mutant Mice David F. LePage and Ronald A. Conlon  

E-print Network

. Knockouts, knockins and conditional alleles are generated in a similar fashion using the protocols given almost twenty years since the first gene targeted mice were constructed (4, 5), not all parameters which

82

Heterogeneity of Lethals in a "Simple" Lethal Complementation Group  

PubMed Central

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster , eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua1 and pur1, which yield auxotrophic mutations.—The eight new mutations have been characterized phenotypically in double heterozygotes with gua1, pur1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2 . The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.—Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions. PMID:3080355

Janca, Frank C.; Woloshyn, Effie P.; Nash, David

1986-01-01

83

Lethality test system  

SciTech Connect

The Lethality Test System (LTS), presently under construction at Los Alamos, is an electromagnetic launcher facility designed to perform impact experiments at velocities up to 15 km/s. The launcher is a 25 mm round bore, plasma armature railgun extending 22 m in length. Preinjection is accomplished with a two-stage gas gun capable of 7 km/s. The railgun power supply utilizes traction motors, vacuum interrupters, and pulse transformers. An assembly of 28 traction motors, equipped with flywheels, stores approximately 80 MJ at 92% of full speed and energizes the primary windings of three pulse transformers at a current of 50 kA. At peak current an array of vacuum interrupters disconnects the transformer primary windings and forces the current to flow in the secondary windings. The secondary windings are connected to the railgun, and by staging the vacuum interrupter openings, a 1 MA to 1.3 MA ramped current waveform will be delivered to the railgun.

Parsons, W.M.; Sims, J.R.; Parker, J.V.

1986-01-01

84

Functional citric acid cycle in an arcA mutant of Escherichia coli during growth with nitrate under anoxic conditions  

Microsoft Academic Search

The operation of the citric acid cycle of Escherichia coli during nitrate respiration (anoxic conditions) was studied by measuring end products and enzyme activities. Excretion of\\u000a products other than CO2, such as acetate or ethanol, was taken as an indication for a non-functional cycle. From glycerol, approximately 0.3 mol\\u000a acetate was produced; the residual portion was completely oxidized, indicating the

Corinna Prohl; Birgit Wackwitz; Dorina Vlad; Gottfried Unden

1998-01-01

85

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial  

Microsoft Academic Search

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both ?134.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We

J M Markert; M D Medlock; S D Rabkin; G Y Gillespie; T Todo; W D Hunter; C A Palmer; F Feigenbaum; C Tornatore; F Tufaro; R L Martuza

2000-01-01

86

Current issues involving lethal injection  

Microsoft Academic Search

Since the reinstatement of the death penalty in 1976 by the Supreme Court in Gregg v. Georgia, over 83% of those executed have been put to death by means of lethal injection, which is intended to be more humane than other methods of capital punishment. The prevailing lethal injection procedure has been challenged by the contention that the first drug

Andrew Fulkerson; Michael Suttmoeller

2008-01-01

87

Less lethal technology: medical issues  

Microsoft Academic Search

Purpose – Less lethal weapons have become a critical tool for law enforcement when confronting dangerous, combative individuals in the field. The purpose of this paper is to review the medical aspects and implications of three different types of less lethal weapons. Design\\/methodology\\/approach – The paper conducted a comprehensive medical literature review on blunt projectiles, irritant sprays including oleoresin capsicum

Gary M. Vilke; Theodore C. Chan

2007-01-01

88

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression  

PubMed Central

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three- dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8522614

1995-01-01

89

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.  

PubMed Central

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522

O'Connell, K F; Leys, C M; White, J G

1998-01-01

90

Lethal outcome in xanthogranulomatous endometritis.  

PubMed

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

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

2006-05-01

91

Lethal and sublethal effects of cypermethrin to Hypsiboas pulchellus tadpoles  

Microsoft Academic Search

The study of the effects of the insecticide cypermethrin (CY) technical grade and its Sherpa® commercial formulation on Hypsiboas pulchellus tadpoles assessing lethality, behavior, growth, and abnormalities under standardized laboratory conditions is reported. Observed\\u000a behaviors were identified and categorized by means of a ranking system according to the loss of mobility. Results of acute\\u000a lethal effects indicate higher potency for

M. Gabriela Agostini; Guillermo S. Natale; Alicia E. Ronco

2010-01-01

92

Drosophila Embryonic Cell-Cycle Mutants  

PubMed Central

Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system. PMID:23979936

Unhavaithaya, Yingdee; Park, Eugenia A.; Royzman, Irena; Orr-Weaver, Terry L.

2013-01-01

93

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

PubMed

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. PMID:24867061

Otsuki, Hatsune; Yano, Shuichi

2014-11-01

94

Suppression Analysis of esa1 Mutants in Saccharomyces cerevisiae Links NAB3 to Transcriptional Silencing and Nucleolar Functions  

PubMed Central

The acetyltransferase Esa1 is essential in the yeast Saccharomyces cerevisiae and plays a critical role in multiple cellular processes. The most well-defined targets for Esa1 are lysine residues on histones. However, an increasing number of nonhistone proteins have recently been identified as substrates of Esa1. In this study, four genes (LYS20, LEU2, VAP1, and NAB3) were identified in a genetic screen as high-copy suppressors of the conditional temperature-sensitive lethality of an esa1 mutant. When expressed from a high-copy plasmid, each of these suppressors rescued the temperature-sensitivity of an esa1 mutant. Only NAB3 overexpression also rescued the rDNA-silencing defects of an esa1 mutant. Strengthening the connections between NAB3 and ESA1, mutants of nab3 displayed several phenotypes similar to those of esa1 mutants, including increased sensitivity to the topoisomerase I inhibitor camptothecin and defects in rDNA silencing and cell-cycle progression. In addition, nuclear localization of Nab3 was altered in the esa1 mutant. Finally, posttranslational acetylation of Nab3 was detected in vivo and found to be influenced by ESA1. PMID:23050233

Chang, Christie S.; Clarke, Astrid; Pillus, Lorraine

2012-01-01

95

A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene  

Microsoft Academic Search

SUMMARY Oncogenic mutations in the small GTPase Ras are highly prevalent in cancer, but an understanding of the vulnerabilities of these cancers is lacking. We undertook a genome-wide RNAi screen to identify synthetic lethal interactions with the KRAS onco- gene. We discovered a diverse set of proteins whose depletion selectively impaired the viability of Ras mutant cells. Among these we

Ji Luo; Michael J. Emanuele; Danan Li; Chad J. Creighton; Michael R. Schlabach; Thomas F. Westbrook; Kwok-Kin Wong; Stephen J. Elledge

2009-01-01

96

Starch mutants of Chlamydomonas  

SciTech Connect

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.

Berry-Lowe, S.L.; Schmidt, G.W. (Univ. of Georgia, Athens (USA))

1990-05-01

97

Journal of Theoretical Biology 237 (2005) 401411 Lethality and synthetic lethality in the genome-wide  

E-print Network

Journal of Theoretical Biology 237 (2005) 401­411 Lethality and synthetic lethality in the genome deletion, we investigate synthetic lethality in reaction level, where simultaneous deletion of a pair of nonlethal reactions leads to the failure of the biomass reaction. Synthetic lethals identified via flux

Kahng, Byungnam

98

Effects of oxygen and nitrogen conditions on the transformation kinetics of 1,2-dichloroethenes by Methylosinus trichosporium OB3b and its sMMO C mutant  

Microsoft Academic Search

Transformation kinetics of trans- andcis-dichloroethylenes (DCE) by Methylosinus trichosporium OB3b wild type (WT)and PP319, a mutant that expresses soluble methane monooxygenase at copper levels upto ~12 µM Cu (sMMOC), were determined to assess theeffects of O2level and N2-fixation on degradationcapabilities. Two issues were examined: (1) the influence of O2level and nitrogen-limitation on DCE degradationkinetics and toxicity in both organisms, and

Hyung J. Kim; David W. Graham

2003-01-01

99

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome  

PubMed Central

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo. PMID:17360968

Ghaboosi, Nazli

2007-01-01

100

Nonchemotactic Mutants of Escherichia coli  

PubMed Central

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

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

1967-01-01

101

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

ERIC Educational Resources Information Center

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…

Elks, Martin A.

1993-01-01

102

Cross-talk between Chk1 and Chk2 in double-mutant thymocytes  

PubMed Central

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4?CD8? double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy. PMID:17360434

Zaugg, Kathrin; Su, Yu-Wen; Reilly, Patrick T.; Moolani, Yasmin; Cheung, Carol C.; Hakem, Razquallah; Hirao, Atsushi; Liu, Quinghua; Elledge, Stephen J.; Mak, Tak W.

2007-01-01

103

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

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.

Billen, D.

1985-07-01

104

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

PubMed Central

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 ?-galactosidase (SA-?-gal) in our current study. We validated the use of embryonic SA-?-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-?-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-?-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-?-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 senescence mechanisms. PMID:18704191

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

2008-01-01

105

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

PubMed Central

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.

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

2014-01-01

106

Extinction of Hepatitis C Virus by Ribavirin in Hepatoma Cells Involves Lethal Mutagenesis  

PubMed Central

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

Ortega-Prieto, Ana M.; Sheldon, Julie; Grande-Perez, Ana; Tejero, Hector; Gregori, Josep; Quer, Josep; Esteban, Juan I.; Domingo, Esteban; Perales, Celia

2013-01-01

107

Early events of lethal action by tobramycin in Pseudomonas aeruginosa  

SciTech Connect

The immediate activities of the aminoglycoside antibiotic, tobramycin, were investigated in Pseudomonas aeruginosa PAO1. The influence of carbon growth substate and the antibiotic exposure environment in the magnitude of activity were examined. Lethality by 8 {mu}g/ml tobramycin occurred rapidly (1 to 3 minutes). The release of specific cellular components into the supernatant was associated with lethality. This material was initially detected as an increase in UV-absorbance. Magnesium in the reaction mixture provided protection against lethality and leakage, but did not reverse lethal damage after a 3 minute tobramycin treatment. Also, uptake of {sup 3}H-tobramycin was reduced in the presence of magnesium. Cells grown with glucose as a carbon source were more susceptible than organic acid grown cells as was the rapidity and amount of cell damage. Analyses of the leakage material revealed a 2-fold increase of protein in the supernatant after a 1-3 minute treatment which paralleled lethality. A prominent 29 kDa protein was observed by SDS-PAGE in the released material, which has been identified as the periplasmic enzyme, {beta}-lactamase. The immediate activities of tobramycin did not involve (i) release of overall cell protein, (ii) massive loss of total pool amino acids, (iii) cell lysis, (iv) inhibition of proline uptake, (v) release of lipopolysaccharide, or (vi) leakage of ATP. Electron microscopy showed no apparent damage after a 3 minute exposure. 40% inhibition of protein synthesis had occurred by 3 minutes of exposure, while release of UV-absorbing material and lethality were detectable after only 1 minute. Resistant cystic fibrosis isolates of P. aeruginosa did not leak under the same experimental conditions, but one of two susceptible strains examined did show increased UV-absorbance following treatment.

Raulston, J.E.

1988-01-01

108

The Maternally Expressed WRKY Transcription Factor TTG2 Controls Lethality in Interploidy Crosses of Arabidopsis  

PubMed Central

The molecular mechanisms underlying lethality of F1 hybrids between diverged parents are one target of speciation research. Crosses between diploid and tetraploid individuals of the same genotype can result in F1 lethality, and this dosage-sensitive incompatibility plays a role in polyploid speciation. We have identified variation in F1 lethality in interploidy crosses of Arabidopsis thaliana and determined the genetic architecture of the maternally expressed variation via QTL mapping. A single large-effect QTL, DR. STRANGELOVE 1 (DSL1), was identified as well as two QTL with epistatic relationships to DSL1. DSL1 affects the rate of postzygotic lethality via expression in the maternal sporophyte. Fine mapping placed DSL1 in an interval encoding the maternal effect transcription factor TTG2. Maternal parents carrying loss-of-function mutations in TTG2 suppressed the F1 lethality caused by paternal excess interploidy crosses. The frequency of cellularization in the endosperm was similarly affected by both natural variation and ttg2 loss-of-function mutants. The simple genetic basis of the natural variation and effects of single-gene mutations suggests that F1 lethality in polyploids could evolve rapidly. Furthermore, the role of the sporophytically active TTG2 gene in interploidy crosses indicates that the developmental programming of the mother regulates the viability of interploidy hybrid offspring. PMID:19071961

Dilkes, Brian P; Spielman, Melissa; Weizbauer, Renate; Watson, Brian; Burkart-Waco, Diana; Scott, Rod J; Comai, Luca

2008-01-01

109

Identification of novel attenuated Salmonella Enteritidis mutants.  

PubMed

Salmonella Enteritidis is a major food-borne pathogen that causes nontyphoidal diarrhoea in humans. Infection of adult egg-laying hens usually results in symptomless carriage but in young chicks it may cause paratyphoid disease. It is not known whether S. Enteritidis requires genes additional to known virulence genes for systemic infection of young chickens. A transposon insertion library was created using S. Enteritidis 10/02, which yielded 1246 mutants. Of 384 mutants screened in chickens for attenuation (30.8% of insertion library), 12 (3.1%) had a 50% lethal dose at least 100 times that of the parental strain. Sequencing revealed insertions in genes involved in the biosynthesis of lipopolysaccharide, cell membrane, ATP biosynthesis, transcriptional regulation of virulence and the yhbC gene, which has an unknown function. Evaluation of in vitro virulence characteristics of a Delta yhbC mutant revealed that its ability to invade HeLa cells and survive within a chicken macrophage cell line (HD11) was significantly reduced. It was also less resistant to reactive oxygen and nitrogen intermediates and had a retarded growth rate. Chickens challenged with the Delta yhbC mutant cleared the organism from the liver and spleen 1 week faster than the parental strain and were able to develop specific serum IgG antibodies against the Delta yhbC mutant. PMID:18355292

Chang, Jason; Pang, Ervinna; He, Haiqi; Kwang, Jimmy

2008-06-01

110

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

PubMed

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. PMID:25036619

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

2014-10-01

111

Effect of Anaerobic Growth on Quinolone Lethality with Escherichia coli?  

PubMed Central

Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure. PMID:17043118

Malik, Muhammad; Hussain, Syed; Drlica, Karl

2007-01-01

112

Alcohol Consumption and Nearly Lethal Suicide Attempts.  

ERIC Educational Resources Information Center

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…

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

113

Less lethal weapons: a technologist's perspective  

Microsoft Academic Search

Purpose – To provide a comprehensive picture of the wide range of technical, operational, and management issues that must be considered when developing, acquiring or using less lethal weapons for law enforcement agencies. Design\\/methodology\\/approach – The source of the insights provided in this paper come from a careful reading and critique of the less lethal technology literature and the organization

Raymond L. Downs

2007-01-01

114

Evaluation of combination treatments of sodium hydroxide and steam explosion for the production of cellulase-systems by two T. reesei mutants under solid-state fermentation conditions  

Microsoft Academic Search

A central composite orthogonal design model was used to optimize pretreatment and culture conditions for the production of cellulase-systems by T. reesei QMY-1 and MCG 80 using wheat straw as carbon source. The model was capable of predicting conditions for maximum filter paper activity (FPA) and ?-glucosidase activity (?GA). Statistical analysis showed close agreement between the experimental FPA and ?GA

V. A Awafo; D. S Chahal; B. K Simpson

2000-01-01

115

Pitfalls of the synthetic lethality screen in Saccharomyces cerevisiae: an improved design.  

PubMed

The colony color assay in yeast enables the visual identification of plasmid-loss events. In combination with a plasmid-dependence assay, it is commonly used to identify synthetic interactions between functionally related genes. Frequently, the plasmid carries the ADE3 gene and mutants are recognized as red colonies that fail to produce sectors. In these assays, a high percentage of false-positives is obtained, most of which result from synthetic lethality with the ade3 mutation. Here, we study the nature of these mutants. We report that mutations in the HIP1 and SHM1 genes exhibit synthetic lethality with ade3 deletions. A similar interaction is found between the fur1 and ura3 mutations. Lethality in the absence of the mitochondrial Shm1 and the cytoplasmic Ade3 enzymes indicates that, under certain circumstances, these cellular compartments cooperate in carrying out essential metabolic processes. In addition, we report the identification of a truncated ADE3 allele with a unique coloration phenotype and show that it can be used to improve synthetic lethal screens. PMID:12684846

Koren, Amnon; Ben-Aroya, Shay; Steinlauf, Rivka; Kupiec, Martin

2003-04-01

116

Multiple-Aminoglycoside-Resistant Mutants of Bacillus subtilis Deficient in Accumulation of Kanamycin  

PubMed Central

Three classes of spontaneous multiple-aminoglycoside-resistant (mar) mutants of Bacillus subtilis were isolated by plating on a low (1.2 ?g/ml) concentration of kanamycin sulfate and were found to be resistant also to low concentrations of paromomycin, neomycin and gentamicin. The three classes could be distinguished one from another by their degree of cytochrome deficiency, respiration deficiency, and susceptibility to kanamycin lethality. A fluctuation test showed that the mutations were spontaneous and not induced by the conditions of selection. Representative strains from two classes of mutants (mar-2 and mar-3) accumulated aminoglycoside very poorly in comparison with the parent strain, whereas a strain of the third class (mar-1) inactivated aminoglycoside present in the growth medium. The mar-3 strain studied (aroD163) had previously been shown to be a menaquinone auxotroph (Farrand and Taber, 1973) and to be deficient in amino acid uptake (Bisschop et al., 1975). Such mutants, which are resistant to low concentrations of aminoglycosides, may be of use in elucidating the biochemical and genetic bases of certain bacterial transport systems. PMID:817658

Taber, H.; Halfenger, G. M.

1976-01-01

117

Acute Lethality of Copper, Cadmium, and Zinc to Northern Squawfish  

Microsoft Academic Search

Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ?g\\/liter for copper, 1,104 ?g\\/liter for cadmium, and 3,693 ?g\\/liter for zinc in 12 C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern

James D. Andros; Ronald R. Garton

1980-01-01

118

Acute lethality of copper, cadmium, and zinc to northern squawfish  

Microsoft Academic Search

Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ..mu..g\\/liter for copper, 1104 ..mu..g\\/liter for cadmium, and 3693 ..mu..g\\/liter for zinc in 12°C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern squawfish

JAMES D. ANDROS; RONALD R. GARTON

1980-01-01

119

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

PubMed Central

FabG, ?-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of ?-ketoacyl-ACP substrates to ?-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 ?-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

Lai, Chiou-Yan; Cronan, John E.

2004-01-01

120

Lens Extrusion from Laminin Alpha 1 Mutant Zebrafish  

PubMed Central

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 ?1 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

Semina, Elena V.; Duncan, Melinda K.

2014-01-01

121

Isolation and characterization of rat skeletal myoblast mutants resistant to lectins.  

PubMed

Mutants resistant to the lethal action of the lectins phytohemagglutinin A (PHA) and wheat germ agglutinin (WGA) have been made in a line of differentiating rat skeletal myoblasts. The WGA mutants are of two types, WGArII, resistant to low concentrations of the lectin, and WGArI, resistant to high concentrations of the lectin. WGArII and PHAr mutants are unable to differentiate, whereas WGArI mutants differentiate normally. WGArII mutants are not impaired in the binding of wheat germ agglutinin, but WGArI mutants bind the lectin only to the extent of about 50% of the wild-type values. All of the mutants are cross-resistant to lectins other than those used in their selection. PMID:6689198

Gilfix, B; Rogers, J; Sanwal, B D

1983-12-01

122

BEHAVIORAL AND BIOCHEMICAL DEFECTS IN TEMPERATURE SENSITIVE ACETYLCHOLINESTERASE MUTANTS OF  

Microsoft Academic Search

Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat- sensitive and cold-sensitive mutations permited the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them

JEFFREY C. HALL; STAMATIS N. ALAHIOTIS; DAVID A. STRUMPF; KRISTIN WHITE

123

Directed Energy Non-lethal Weapons.  

National Technical Information Service (NTIS)

This basic research initiative has been an ongoing interdisciplinary effort to lay the foundation for developing, novel effective and safe non- lethal technologies that alter skeletal muscle contraction and/or neural functioning (i.e., neurosecretion) via...

G. L. Craviso, I. Chatterjee

2010-01-01

124

A Heat Shows of Two Shock-resistant Mutant of Saccharomyces Constitutive Synthesis Heat Shock Proteins and Altered Growth cerevisiae  

Microsoft Academic Search

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10 -7 from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio

HIDETOSHI IIDA; ICHIRO YAHARA

125

Differential response to intracellular stress in the skin from osteogenesis imperfecta Brtl mice with lethal and non lethal phenotype: a proteomic approach.  

PubMed

Phenotypic variability in the presence of an identical molecular defect is a recurrent feature in heritable disorders and it was also reported in osteogenesis imperfecta (OI). OI is a prototype for skeletal dysplasias mainly caused by mutations in the two genes coding for type I collagen. No definitive cure is available for this disorder, but the understanding of molecular basis in OI phenotypic modulation will have a pivotal role in identifying possible targets to develop novel drug therapy. We used a functional proteomic approach to address the study of phenotypic variability using the skin of the OI murine model Brtl. Brtl mice reproduce the molecular defect, dominant transmission and phenotypic variability of human OI patients. In the presence of a Gly349Cys substitution in ?1(I)-collagen Brtl mice can have a lethal or a moderately severe outcome. Differential expression of chaperones, proteasomal subunits, metabolic enzymes, and proteins related to cellular fate demonstrated that a different ability to adapt to cellular stress distinguished mutant from wild-type mice and mutant lethal from surviving mutant animals. Interestingly, class discovery analysis identified clusters of differentially expressed proteins associated with a specific outcome, and functional analysis contributed to a deeper investigation into biochemical and cellular pathways affected by the disease. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22846432

Bianchi, Laura; Gagliardi, Assunta; Gioia, Roberta; Besio, Roberta; Tani, Chiara; Landi, Claudia; Cipriano, Maria; Gimigliano, Anna; Rossi, Antonio; Marini, Joan C; Forlino, Antonella; Bini, Luca

2012-08-01

126

Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells.  

PubMed

An alternative to therapeutic targeting of oncogenes is to perform "synthetic lethality" screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with "undruggable" genetic alterations. PMID:19490892

Scholl, Claudia; Fröhling, Stefan; Dunn, Ian F; Schinzel, Anna C; Barbie, David A; Kim, So Young; Silver, Serena J; Tamayo, Pablo; Wadlow, Raymond C; Ramaswamy, Sridhar; Döhner, Konstanze; Bullinger, Lars; Sandy, Peter; Boehm, Jesse S; Root, David E; Jacks, Tyler; Hahn, William C; Gilliland, D Gary

2009-05-29

127

Pyrimidine requirements in deoxyuridine triphosphate nucleotidohydrolase deficient Trypanosoma brucei mutants.  

PubMed

Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and therefore strongly depend on dUDP/dUTP hydrolysis for dUMP production. Here we have addressed by gene replacement the consequences of elimination of dUTPase activity in bloodstream forms of Trypanosoma brucei. We first generated conditional DUT-knockout strains that allowed an effective decrease of dUTPase resulting in proliferation arrest, although gene repression could not be sustained long enough to cause lethality. Alternatively, DUT null mutants could be isolated in the presence of high levels of thymidine while exogenous supplementation with uracil, uridine or deoxyuridine could not complement metabolically the dUTPase deficiency. Upon thymidine removal, trypanosomes exhibited impaired proliferation and eventually died. These data establish a strict requirement for dUTPase in T. brucei viability and support a major role of the enzyme in the provision of pyrimidine nucleotides in kinetoplastids. PMID:23201394

Castillo-Acosta, Víctor M; Aguilar-Pereyra, Fernando; García-Caballero, Daniel; Vidal, Antonio E; Ruiz-Pérez, Luis M; González-Pacanowska, Dolores

2013-01-01

128

Phanerochaete mutants with enhanced ligninolytic activity  

SciTech Connect

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.

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

1993-06-01

129

Crystallographic studies of the Anthrax lethal toxin. Annual report  

SciTech Connect

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.

Frederick, C.A.

1996-07-01

130

Functional characterization of dosage-dependent lethal mutation of ubiquitin in Saccharomyces cerevisiae.  

PubMed

Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast to human. Ubiquitin plays a central role in protein homeostasis and regulation of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in relation to its function. One of the mutants generated, namely UbEP42, was a dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-deficient cells but not to ubiquitin wild-type cells. In the present study we investigated the functional reasons for the observed lethality. Expression of UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards heat stress and exposure to cycloheximide. Furthermore, its expression had a negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains. Collectively, our results establish that the effects seen with the mutant ubiquitin protein UbEP42 are not due to malfunction at the stage of polyubiquitination. PMID:25195938

Doshi, Ankita; Mishra, Pradeep; Sharma, Mrinal; Prabha, C Ratna

2014-11-01

131

Studies on the Sex-Specific Lethals of DROSOPHILA MELANOGASTER. IV. Gynandromorph Analysis of Three Male-Specific Lethals, mle, msl-227 and mle(3)132  

PubMed Central

Mutants at three male-specific lethal loci of Drosophila melanogaster (mle, msl-227 and mle(3)132) were examined by gynandromorph analysis. In all cases only a very few gynandromorphs with small X/O patches appeared. Most of these small X/O patches were in the abdomen, and the structures in these X/O regions were reduced in size. These results indicate that the primary effects of these mutants are not on any particular organs or tissues, but rather on individual cells. mle and msl-2 have been shown by Belote and Lucchesi (1980a) to be defective in dosage compensation in X/Y males. We suggest that this dosage-compensation defect results in the expression of Minute-like phenotypes in X/O cells, and hence results in the death of X/O males and flies with large X/O tissue areas. PMID:6818104

Uenoyama, T.; Uchida, S.; Fukunaga, A.; Oishi, K.

1982-01-01

132

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

PubMed Central

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

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

2013-01-01

133

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

SciTech Connect

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.

Yurchenko, N.N.; Koryakov, D.E.; Zakharov, I.K. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

1995-09-01

134

Live deaths online: internet suicide and lethality.  

PubMed

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. PMID:23233475

Klein, Carolina A

2012-01-01

135

The Drosophila Sex Determination Gene snf Is Utilized for the Establishment of the Female-Specific Splicing Pattern of Sex-lethal  

E-print Network

The Drosophila snf gene is a positive regulator of the sex determination gene Sex-lethal in both the germline and the soma. Its role in the soma is only evident when the probability of Sex-lethal activation has been reduced. For instance, in an otherwise wild-type background, females homozygous for a weak snf mutation produce both male and female progeny; however, when mated to males hemizygous for a null allele of Sex-lethal, they produce only male progeny. We demonstrate that the lack of female progeny is due to aberrant Sex-lethal regulation in late embryogenesis. In these mutant embryos, there is little accumulation of the late female-specific spliced RNAs and proteins. In contrast, in early embryogenesis, Sex-lethal regulation is not affected. The accumulation of both the early Sex-lethal transcripts and proteins is normal. These results suggest that the wild-type product of snf plays an important role in establishing the female-specific RNA splicing pattern of Sex-lethal. Whether snf influences the female-specific splice site choice directly or indirectly remains to be determined. N Drosophila melanogaster, the decision between male and female development is regulated by the binary switch gene Sex-lethal, (Sxl; recently reviewed

unknown authors

1993-01-01

136

A Saccharomyces cerevisiae Genome-Wide Mutant Screen for Altered Sensitivity to K1 Killer Toxin  

Microsoft Academic Search

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall -glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186

Nicolas Page ´; Manon Gerard-Vincent; Patrice Menard; Maude Beaulieu; Masayuki Azuma; Gerrit J. P. Dijkgraaf; Thuy Nguyen; Tim Dowse; Anne-Marie Sdicu; Howard Bussey

137

Acute lethality of copper, cadmium, and zinc to northern squawfish  

SciTech Connect

Flow-through acute toxicity tests on juvenile northern squawfish (Ptychocheilus oregonensis) were conducted with copper, cadmium, and zinc. The 96-hour median lethal concentrations were 18 ..mu..g/liter for copper, 1104 ..mu..g/liter for cadmium, and 3693 ..mu..g/liter for zinc in 12/sup 0/C water. These values, when compared to those for chinook salmon and steelhead parr tested under similar conditions, show that the northern squawfish are more tolerant than the two salmonids to zinc and cadmium but equally sensitive to copper.

Andros, J.D.; Garton, R.R.

1980-03-01

138

Uncoupler-resistant mutants of bacteria.  

PubMed Central

The chemiosmotic model of energy transduction offers a satisfying and widely confirmed understanding of the action of uncouplers on such processes as oxidative phosphorylation; the uncoupler, by facilitating the transmembrane movement of protons or other compensatory ions, reduces the electrochemical proton gradient that is posited as the energy intermediate for many kinds of bioenergetic work. In connection with this formulation, uncoupler-resistant mutants of bacteria that neither exclude nor inactivate these agents represent a bioenergetic puzzle. Uncoupler-resistant mutants of aerobic Bacillus species are, in fact, membrane lipid mutants with bioenergetic properties that are indeed challenging in connection with the chemiosmotic model. By contrast, uncoupler-resistant mutants of Escherichia coli probably exclude uncouplers, sometimes only under rather specific conditions. Related phenomena in eucaryotic and procaryotic systems, as well as various observations on uncouplers, decouplers, and certain other membrane-active agents, are also briefly considered. Images PMID:2181259

Krulwich, T A; Quirk, P G; Guffanti, A A

1990-01-01

139

Chemically Induced Conditional Rescue of the Reduced Epidermal Fluorescence8 Mutant of Arabidopsis Reveals Rapid Restoration of Growth and Selective Turnover of Secondary Metabolite Pools1[C][OPEN  

PubMed Central

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. PMID:24381065

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

2014-01-01

140

Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon.  

PubMed Central

Gram-negative septicemia elicits multiple abnormalities of the coagulation system. Although products of coagulation can lead to clot formation, thereby potentiating organ damage, recent work has shown that low concentrations of thrombin can protect animals from the shock state. Because these amounts of thrombin also lead to formation in vivo of the anticoagulant enzyme, activated protein C, we examined the role of protein C in modulation of Escherichia coli shock in baboons. First, we infused activated protein C and lethal concentrations of E. coli organisms, which prevented the coagulopathic, hepatotoxic, and lethal effects of E. coli. Second, using an antibody to protein C we blocked protein C activation in vivo to determine if this influenced the response to lethal and sublethal concentrations of E. coli organisms. Under these conditions the response to lethal concentrations of E. coli organisms was made more severe and the response to sublethal concentrations of E. coli was made lethal. The coagulopathic, hepatotoxic, and lethal responses in this latter case were prevented by infusion of exogenous protein C. PMID:3102560

Taylor, F B; Chang, A; Esmon, C T; D'Angelo, A; Vigano-D'Angelo, S; Blick, K E

1987-01-01

141

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency.  

PubMed

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast ?coq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients. PMID:24140869

Doimo, Mara; Trevisson, Eva; Airik, Rannar; Bergdoll, Marc; Santos-Ocaña, Carlos; Hildebrandt, Friedhelm; Navas, Placido; Pierrel, Fabien; Salviati, Leonardo

2014-01-01

142

Effect of vanillic acid on COQ6 mutants identified in patients with coenzyme Q10 deficiency?  

PubMed Central

Human COQ6 encodes a monooxygenase which is responsible for the C5-hydroxylation of the quinone ring of coenzyme Q (CoQ). Mutations in COQ6 cause primary CoQ deficiency, a condition responsive to oral CoQ10 supplementation. Treatment is however still problematic given the poor bioavailability of CoQ10. We employed S. cerevisiae lacking the orthologous gene to characterize the two different human COQ6 isoforms and the mutations found in patients. COQ6 isoform a can partially complement the defective yeast, while isoform b, which lacks part of the FAD-binding domain, is inactive but partially stable, and could have a regulatory/inhibitory function in CoQ10 biosynthesis. Most mutations identified in patients, including the frameshift Q461fs478X mutation, retain residual enzymatic activity, and all patients carry at least one hypomorphic allele, confirming that the complete block of CoQ biosynthesis is lethal. These mutants are also partially stable and allow the assembly of the CoQ biosynthetic complex. In fact treatment with two hydroxylated analogues of 4-hydroxybenzoic acid, namely, vanillic acid or 3-4-hydroxybenzoic acid, restored the respiratory growth of yeast ?coq6 cells expressing the mutant huCOQ6-isoa proteins. These compounds, and particularly vanillic acid, could therefore represent an interesting therapeutic option for COQ6 patients. PMID:24140869

Doimo, Mara; Trevisson, Eva; Airik, Rannar; Bergdoll, Marc; Santos-Ocana, Carlos; Hildebrandt, Friedhelm; Navas, Placido; Pierrel, Fabien; Salviati, Leonardo

2014-01-01

143

Potentially lethal damage repair in drug arrested g2-phase cells after radiation exposure.  

PubMed

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2. PMID:25251700

Maeda, Junko; Bell, Justin J; Genet, Stefan C; Fujii, Yoshihiro; Genet, Matthew D; Brents, Colleen A; Genik, Paula C; Kato, Takamitsu A

2014-10-01

144

Deadly Lessons: Understanding Lethal School Violence.  

ERIC Educational Resources Information Center

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"…

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

145

Lethal Malaria: Marchiafava and Bignami Were Right  

PubMed Central

One hundred and twenty years ago, the Italian malariologists Marchiafava and Bignami proposed that the fundamental pathological process underlying lethal falciparum malaria was microvascular obstruction. Since then, several alternative hypotheses have been proposed. These formed the basis for adjunctive interventions, which have either been ineffective or harmful. Recent evidence strongly suggests that Marchiafava and Bignami were right. PMID:23585685

White, Nicholas J.; Turner, Gareth D. H.; Day, Nicholas P. J.; Dondorp, Arjen M.

2013-01-01

146

Sarcocystis Species Lethal for Domestic Pigeons  

PubMed Central

A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible. PMID:20202429

Gruber, Achim D.; Kohls, Andrea; Hafez, Hafez M.; Heydorn, Alfred Otto; Mehlhorn, Heinz; Lierz, Michael

2010-01-01

147

Chemistry & Biology Synthetic Lethal Screening Identifies Compounds  

E-print Network

Chemistry & Biology Article Synthetic Lethal Screening Identifies Compounds Activating IronDepartment of Biological Sciences 2Department of Chemistry Columbia University, Fairchild Center, MC2406, 1212 Amsterdam Avenue, New York, NY 10027, USA *Correspondence: stockwell@biology.columbia.edu DOI 10

Stockwell, Brent R.

148

Non-lethal technologies—an overview  

Microsoft Academic Search

hilst the focus for this issue of Disarmament Forum is on chemical and biological weapons, sight should not be lost of the spectrum of non-lethal technologies that are being deployed or under development. These technologies will have an increasing impact on war fighting, peace support operations, civil policing and prison control. It is our purpose here to briefly review the

Nick LEWER; Neil DAVISON

149

The Streptococcus pyogenes Capsule Is Required for Adhesion of Bacteria to Virus-Infected Alveolar Epithelial Cells and Lethal Bacterial-Viral Superinfection  

Microsoft Academic Search

An apparent worldwide resurgence of invasive group A Streptococcus (GAS) infections remains unexplained. However, we recently demonstrated in mice that when an otherwise nonlethal intranasal GAS infection is preceded by a nonlethal influenza A virus (IAV) infection, induction of lethal invasive GAS infections is often the result. In the present study, we established several isogenic mutants from a GAS isolate

Shigefumi Okamoto; Shigetada Kawabata; Yutaka Terao; Hideaki Fujitaka; Yoshinobu Okuno; Shigeyuki Hamada

2004-01-01

150

Unexpected double lethal oleander poisoning.  

PubMed

Nerium oleander is a very popular urban ornamental plant in Europe, but it is also extremely dangerous because it contains several types of glycosides, accidental ingestion of which can cause cardiac arrhythmias and even deaths. The rarity of such cases makes it difficult to think of oleander poisoning without evidences that suggest this possibility as the cause of the unexpected death. This report concerns the discovery of the bodies of 2 young people, a man and a woman, in a forest in conditions of extreme malnutrition. Medicolegal investigations showed neither pathologic nor traumatic causes of death, but the presence of vegetal remains in the stomach was noticed. A common toxicological analysis resulted negative, but the implementation of more detailed investigations showed the presence of digoxin in the blood of both cadavers, excluding the possibility of a pharmaceutical provenience of digoxin, this laboratory result was interpreted as evidence of ingestion of oleander, which contains oleandrine, the cross reaction of which with digoxin is widely described in the literature. Identification of the 2 subjects, which occurred after 4 years, strengthened the hypothesis of accidental poisoning by oleander because it was ascertained that the 2 young people were vegans--extreme vegetarians who reject the ingestion of foods of animal origin and live by eating only what they find in nature. PMID:21926903

Papi, Luigi; Luciani, Alessandro Bassi; Forni, David; Giusiani, Mario

2012-03-01

151

40 CFR 798.5450 - Rodent dominant lethal assay.  

Code of Federal Regulations, 2011 CFR

...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) HEALTH EFFECTS TESTING GUIDELINES Genetic Toxicity § 798.5450 Rodent dominant lethal assay. (a) Purpose. Dominant lethal (DL) effects cause...

2011-07-01

152

40 CFR 798.5450 - Rodent dominant lethal assay.  

Code of Federal Regulations, 2010 CFR

...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) HEALTH EFFECTS TESTING GUIDELINES Genetic Toxicity § 798.5450 Rodent dominant lethal assay. (a) Purpose. Dominant lethal (DL) effects cause...

2010-07-01

153

Lethal Time of Centruroides Sculpturatus Venom in Rats.  

National Technical Information Service (NTIS)

The effects of sex, weight, and dosage on the lethal time of scorpion Centruroides sculpturatus venom was studied. Sex and weight of the rats did not significantly alter lethal time whereas increased dosages of venom did.

H. L. Stahnke

1967-01-01

154

Generation of conditional Cited2 null alleles.  

PubMed

Cited2 is a transcriptional co-factor that is widely expressed in both embryonic and extraembryonic cells during early development. It is essential for embryonic development with Cited2 null embryos showing abnormal development of organs including heart, neural tube, adrenal glands, and placenta (both in trophoblast derivatives and invading fetal vasculature), as well as having defects in the establishment of the left-right body axis. We report the generation of two conditional null alleles allowing Cre-recombinase-mediated somatic cell gene inactivation. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing conditional mutants with CMV-Cre transgenic mice produces an embryonic-lethal phenotype in the offspring indistinguishable from germline null mutants. We also demonstrate that conditional deletion results in lacZ expression under the control of the Cited2 promoter. These alleles are therefore useful genetic tools for dissecting the functions of Cited2 in the formation of different organs and patterning of the developing embryo. genesis PMID:17133411

Preis, Jost I; Wise, Natalie; Solloway, Mark J; Harvey, Richard P; Sparrow, Duncan B; Dunwoodie, Sally L

2006-12-01

155

Endocrine Perturbation Increases Susceptibility of Mice to Anthrax Lethal Toxin  

Microsoft Academic Search

Bacillus anthracis lethal toxin (LT) causes vascular collapse and high lethality in BALB\\/cJ mice, intermediate lethality in C57BL\\/6J mice, and no lethality in DBA\\/2J mice. We found that adrenalectomized (ADX) mice of all three strains had increased susceptibility to LT. The increased susceptibility of ADX-DBA\\/2J mice was not accompanied by changes in their macrophage sensitivity or cytokine response to LT.

Mahtab Moayeri; Jeanette I. Webster; Jason F. Wiggins; Stephen H. Leppla; Esther M. Sternberg

2005-01-01

156

Induction of a high-yield lovastatin mutant of Aspergillus terreus by ¹²C?? heavy-ion beam irradiation and the influence of culture conditions on lovastatin production under submerged fermentation.  

PubMed

Heavy-ion beams, possessing a wide mutation spectrum and increased mutation frequency, have been used effectively as a breeding method. In this study, the heavy-ion beams generated by the Heavy-Ion Research Facility in Lanzhou were used to mutagenize Aspergillus terreus CA99 for screening high-yield lovastatin strains. Furthermore, the main growth conditions as well as the influences of carbon and nitrogen sources on the growth and the lovastatin production of the mutant and the original strains were investigated comparatively. The spores of A. terreus CA99 were irradiated by 15, 20, 25, and 30 Gy of 80 MeV/u (12)C(6+) heavy-ion beams. Based on the lovastatin contents in the fermentation broth, a strain designated as A. terreus Z15-7 has been selected from the clone irradiated by the heavy-ion beam. When compared with the original strain, the content of lovastatin in the fermentation broth of A. terreus Z15-7 increased 4-fold. Moreover, A. terreus Z15-7 efficiently used the carbon and nitrogen sources for the growth and production of lovastatin when compared to the original strain. The maximum yield of lovastatin, 916.7 ?g/ml, was obtained as A. terreus Z15-7 was submerged cultured in the chemically defined medium supplemented with 3% glycerol as a carbon source, 1% corn meal as an organic nitrogen source, and 0.2% sodium nitrate as an inorganic nitrogen source at 30 °C in the shake flask. The result shows that heavy-ion beam irradiation is an effective method for the mutation breeding of lovastatin production of A. terreus. PMID:21710210

Li, Shi-Weng; Li, Mei; Song, Hong-Ping; Feng, Jia-Li; Tai, Xi-Sheng

2011-10-01

157

Cytochrome c oxidase activity in T\\/t 6 (balanced lethal) mutant mice  

Microsoft Academic Search

R-1 (1450g) and R-2 (25,000g) liver fractions from T\\/t6 and B6CBAF1 hybrid mice were analyzed for their protein content, mitochondria concentrations, and activities of three respiratory-chain enzymes of the mitochondrial inner membrane: cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, E.C. 1.9.3.1), a-glycerophosphate dehydrogenase [l-glycerol-3-phosphate: (acceptor) oxidoreductase, E.C. 1.1.99.5], and succinate-cytochrome c reductase. Only cytochrome c oxidase activity, calculated as units

Robert L. Blake

1977-01-01

158

Defective cranial skeletal development, larval lethality and haploinsufficiency in Myod mutant zebrafish  

PubMed Central

Summary Myogenic regulatory factors of the myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. Here we show that a mutation in the zebrafish myod gene delays and reduces early somitic and pectoral fin myogenesis, reduces miR-206 expression, and leads to a persistent reduction in somite size until at least the independent feeding stage. A mutation in myog, encoding a second MRF, has little obvious phenotype at early stages, but exacerbates the loss of somitic muscle caused by lack of Myod. Mutation of both myod and myf5 ablates all skeletal muscle. Haploinsufficiency of myod leads to reduced embryonic somite muscle bulk. Lack of Myod causes a severe reduction in cranial musculature, ablating most muscles including the protractor pectoralis, a putative cucullaris homologue. This phenotype is accompanied by a severe dysmorphology of the cartilaginous skeleton and failure of maturation of several cranial bones, including the opercle. As myod expression is restricted to myogenic cells, the data show that myogenesis is essential for proper skeletogenesis in the head. PMID:21798255

Hinits, Yaniv; Williams, Victoria C.; Sweetman, Dylan; Donn, Thomas M.; Ma, Taylur P.; Moens, Cecilia B.; Hughes, Simon M.

2012-01-01

159

Genome-wide P-element screen for Drosophila synaptogenesis mutants.  

PubMed

A molecular understanding of synaptogenesis is a critical step toward the goal of understanding how brains "wire themselves up," and then "rewire" during development and experience. Recent genomic and molecular advances have made it possible to study synaptogenesis on a genomic scale. Here, we describe the results of a screen for genes involved in formation and development of the glutamatergic Drosophila neuromuscular junction (NMJ). We screened 2185 P-element transposon mutants representing insertions in approximately 16% of the entire Drosophila genome. We first identified recessive lethal mutants, based on the hypothesis that mutations causing severe disruptions in synaptogenesis are likely to be lethal. Two hundred twenty (10%) of all insertions were homozygous lethal. Two hundred five (93%) of these lethal mutants developed at least through late embryogenesis and formed neuromusculature. We examined embryonic/larval NMJs in 202 of these homozygous mutants using immunocytochemistry and confocal microscopy. We identified and classified 88 mutants with altered NMJ morphology. Insertion loci in these mutants encode several different types of proteins, including ATP- and GTPases, cytoskeletal regulators, cell adhesion molecules, kinases, phosphatases, RNA regulators, regulators of protein formation, transcription factors, and transporters. Thirteen percent of insertions are in genes that encode proteins of novel or unknown function. Complementation tests and RT-PCR assays suggest that approximately 51% of the insertion lines carry background mutations. Our results reveal that synaptogenesis requires the coordinated action of many different types of proteins--perhaps as much as 44% of the entire genome--and that transposon mutageneses carry important caveats that must be respected when interpreting results generated using this method. PMID:16408305

Liebl, Faith L W; Werner, Kristen M; Sheng, Qi; Karr, Julie E; McCabe, Brian D; Featherstone, David E

2006-03-01

160

Lethal recurrent hemorrhages of a brainstem cavernoma  

Microsoft Academic Search

Hemorrhages of brainstem cavernomas may cause severe neurological deficits. Surgical strategies are frequently described,\\u000a and advanced neuromonitoring with intraoperative imaging can help neurosurgeons to achieve good results. However, patients\\u000a are often confronted with significant therapeutic risks by the primary doctor before talking to an experienced brainstem neurosurgeon.\\u000a On the other hand, lethal progression with repeated hemorrhages is rarely described, although

Alexandru Vlad Ciurea; Cristian Nastase; Alexandru Tascu; Felix Mircea Brehar

2007-01-01

161

Lethality of Multiple Endocrine Neoplasia Type I  

Microsoft Academic Search

. The lethality of the endocrine tumors associated with multiple endocrine neoplasia type I (MEN-I), particularly\\u000a the pancreatic islet cell tumors, has been controversial. We evaluated the cause and age of death in MEN-I kindreds. Our database\\u000a contains 34 distinct kindreds with 1838 members. Reliable death data are available for 103 people (excluding accidents and\\u000a age < 18 years). We

Gerard M. Doherty; John A. Olson; Margaret M. Frisella; Terry C. Lairmore; Jeffrey A. Norton

1998-01-01

162

Bullying and Lethal Acts of School Violence  

Microsoft Academic Search

\\u000a In Chap. 3, we address a topic that has received considerable research attention over the past decade and has been implicated as a causal\\u000a factor in LSV. It has been reported that many lethal school shootings were in part in retaliation for being bullied. Within\\u000a this chapter, we address prevalence of bullying in the USA and then discuss different forms

Jeffrey A. Daniels; Mary C. Bradley

163

Functional Genomics Reveals the Induction of Inflammatory Response and Metalloproteinase Gene Expression during Lethal Ebola Virus Infection?†  

PubMed Central

Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NPma, ZEBOV-VP24ma, and ZEBOV-NP/VP24ma) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24ma) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24ma was nearly the same as that to ZEBOV-NP/VP24ma, the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality. PMID:21734050

Cilloniz, Cristian; Ebihara, Hideki; Ni, Chester; Neumann, Gabriele; Korth, Marcus J.; Kelly, Sara M.; Kawaoka, Yoshihiro; Feldmann, Heinz; Katze, Michael G.

2011-01-01

164

Phenotypic disruption to orofacial movement topography in conditional mutants with generalized CamKIIa/Cre D1Tox versus striatal-specific DARPP-32/Cre D1Tox ablation of D1 dopamine receptor-expressing cells.  

PubMed

Orofacial movements were quantified in (a) DARPP-32/Cre D1Tox mutants, having progressive loss of D1 dopamine receptor expressing striatal medium spiny neurons and (b) CamKIIa/Cre D1Tox mutants, having progressive, generalized loss of forebrain D1 receptor expressing cells. Horizontal jaw movements and tongue protrusions were reduced in DARPP-32/Cre but not in CamKIIa/Cre mutants; head and vibrissae movements were increased in DARPP-32/Cre but decreased in CamKIIa/Cre mutants. In drug challenge studies, tongue protrusions were increased in CamKIIa/Cre mutants following vehicle, suggesting a stress-related phenotype. These findings indicate that mice with progressive loss of striatal-specific D1 receptor expressing cells have an orofacial phenotype that may be modulated by the loss of extrastriatal D1 receptor expressing cells. As progressive loss of D1 dopamine receptor-expressing cells is a hallmark feature of Huntington's disease (HD), these findings may inform the functional role of loss of this cell population in the overall pathobiology of HD. PMID:21308794

Tomiyama, Katsunori; Kim, Hyun Ah; Kinsella, Anthony; Ehrlich, Michelle E; Schütz, Günter; Koshikawa, Noriaki; Lawrence, Andrew J; Waddington, John L; Drago, John

2011-09-01

165

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

PubMed Central

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 photoautotrophic conditions. These results can pave the way towards a more feasible microalgae-driven TAG production platform. PMID:24920957

2014-01-01

166

Rapid Antibiotic Resistance Evolution of GASP Mutants  

NASA Astrophysics Data System (ADS)

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.

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

2012-02-01

167

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity  

PubMed Central

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1? and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1? strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1? mutants in comparison with the pkc1? mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. PMID:23573236

Silveira, Wagner da Silva; Valentini, Sandro Roberto; Zanelli, Cleslei Fernando

2013-01-01

168

A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine.  

PubMed

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function. PMID:10673025

Yeh, L C; Lee, J C

1999-12-23

169

FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model  

Microsoft Academic Search

model. The phenotype of FLT3-TKD in mice has not yet been investigated. We transduced murine bone marrow with ret- rovirus-expressing FLT3-TKD mutants or FLT3-ITD and transplanted these cells into lethally irradiated mice. Mice that received a transplant of FLT3-ITD devel- oped an oligoclonal myeloproliferative disease as previously described. In con- trast, FLT3-TKD mutants induced an oligoclonal lymphoid disorder with longer

Rebekka Grundler; Cornelius Miething; Christian Thiede; Christian Peschel; Justus Duyster

2005-01-01

170

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project  

SciTech Connect

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.

Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

2007-01-01

171

Mutant maize variety containing the glt1-1 allele  

DOEpatents

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.

Nelson, O.E.; Pan, D.

1994-07-19

172

Essential Plasticity and Redundancy of Metabolism Unveiled by Synthetic Lethality Analysis  

PubMed Central

We unravel how functional plasticity and redundancy are essential mechanisms underlying the ability to survive of metabolic networks. We perform an exhaustive computational screening of synthetic lethal reaction pairs in Escherichia coli in a minimal medium and we find that synthetic lethal pairs divide in two different groups depending on whether the synthetic lethal interaction works as a backup or as a parallel use mechanism, the first corresponding to essential plasticity and the second to essential redundancy. In E. coli, the analysis of pathways entanglement through essential redundancy supports the view that synthetic lethality affects preferentially a single function or pathway. In contrast, essential plasticity, the dominant class, tends to be inter-pathway but strongly localized and unveils Cell Envelope Biosynthesis as an essential backup for Membrane Lipid Metabolism. When comparing E. coli and Mycoplasma pneumoniae, we find that the metabolic networks of the two organisms exhibit a large difference in the relative importance of plasticity and redundancy which is consistent with the conjecture that plasticity is a sophisticated mechanism that requires a complex organization. Finally, coessential reaction pairs are explored in different environmental conditions to uncover the interplay between the two mechanisms. We find that synthetic lethal interactions and their classification in plasticity and redundancy are basically insensitive to medium composition, and are highly conserved even when the environment is enriched with nonessential compounds or overconstrained to decrease maximum biomass formation. PMID:24854166

2014-01-01

173

Studying synthetic lethal interactions in the zebrafish system: insight into disease genes and mechanisms  

PubMed Central

The post-genomic era is marked by a pressing need to functionally characterize genes through understanding gene-gene interactions, as well as interactions between biological pathways. Exploiting a phenomenon known as synthetic lethality, in which simultaneous loss of two interacting genes leads to loss of viability, aids in the investigation of these interactions. Although synthetic lethal screening is a powerful technique that has been used with great success in many model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans, this approach has not yet been applied in the zebrafish, Danio rerio. Recently, the zebrafish has emerged as a valuable system to model many human disease conditions; thus, the ability to conduct synthetic lethal screening using zebrafish should help to uncover many unknown disease-gene interactions. In this article, we discuss the concept of synthetic lethality and provide examples of its use in other model systems. We further discuss experimental approaches by which the concept of synthetic lethality can be applied to the zebrafish to understand the functions of specific genes. PMID:22107871

Hajeri, Vinita A.; Amatruda, James F.

2012-01-01

174

Lethal Mutagenesis of Foot-and-Mouth Disease Virus Involves Shifts in Sequence Space?†  

PubMed Central

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. PMID:21917974

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

2011-01-01

175

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

PubMed

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

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

2010-10-01

176

Lethal mobilization of DDT by cowbirds  

USGS Publications Warehouse

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 than concentrations considered to be lethal. In contrast, carcass levels of DDT and DDD were significantly lower, and DDE was only slightly higher, in the birds that died in the second period of food restriction. It is concluded that stored DDT residues present a hazard to birds, which utilize stored fat during periods of stress due to reproduction, cold weather, disease, injury, limited food supply, or migration.

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

1972-01-01

177

Issues surrounding lethal injection as a means of capital punishment.  

PubMed

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. PMID:19025423

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

2008-12-01

178

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

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

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

2004-01-01

179

Expression of Nlrp1b Inflammasome Components in Human Fibroblasts Confers Susceptibility to Anthrax Lethal Toxin ?  

PubMed Central

Anthrax lethal toxin causes macrophages and dendritic cells from some mouse strains to undergo caspase-1-dependent cell death. Central to this process is the NOD-like receptor Nlrp1b (Nalp1b), which detects intoxication and then self-associates to form a complex, termed an inflammasome, that is capable of activating the procaspase-1 zymogen. The nature of the signal detected directly by Nlrp1b is not known, and the mechanisms of inflammasome assembly are poorly understood. Here, we demonstrate that transfection of human fibroblasts with plasmids encoding murine Nlrp1b and procaspase-1 was sufficient to confer susceptibility to lethal toxin-mediated death on the cells. As has been observed in murine macrophages, the enzymatic activities of lethal toxin and the proteasome were both required for activation of the Nlrp1b inflammasome and this activation led to prointerleukin-1? processing. Release of interleukin-1? from cells was not dependent on cell lysis, as its secretion was not affected by an osmoprotectant that prevented the appearance of lactate dehydrogenase in the culture medium. We generated constitutively active mutants of Nlrp1b by making amino-terminal deletions to the protein and observed that the ability to activate procaspase-1 was dependent on the CARD domain, which bound procaspase-1, and a region adjacent to the CARD domain that promoted self-association. Our results demonstrate that lethal toxin can activate Nlrp1b in a nonmyeloid cell line and are consistent with work that suggests that activation induces proximity of procaspase-1. PMID:19651869

Liao, Kuo-Chieh; Mogridge, Jeremy

2009-01-01

180

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

SciTech Connect

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.

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

1988-11-01

181

Mutation Induced Extinction in Finite Populations: Lethal Mutagenesis and Lethal Isolation  

Microsoft Academic Search

Reproduction is inherently risky, in part because genomic replication can introduce new mutations that are usually deleterious toward fitness. This risk is especially severe for organisms whose genomes replicate “semi-conservatively,” e.g. viruses and bacteria, where no master copy of the genome is preserved. Lethal mutagenesis refers to extinction of populations due to an unbearably high mutation rate (U), and is

C. Scott Wylie; Eugene I. Shakhnovich

2012-01-01

182

Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis.  

PubMed

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotope-coded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions. PMID:18633119

Rutschow, Heidi; Ytterberg, A Jimmy; Friso, Giulia; Nilsson, Robert; van Wijk, Klaas J

2008-09-01

183

Dissecting the pathways that destabilize mutant p53  

PubMed Central

One fundamental feature of mutant forms of p53 consists in their accumulation at high levels in tumors. At least in the case of neomorphic p53 mutations, which acquire oncogenic activity, stabilization is a driving force for tumor progression. It is well documented that p53 mutants are resistant to proteasome-dependent degradation compared with wild-type p53, but the exact identity of the pathways that affect mutant p53 stability is still debated. We have recently shown that macroautophagy (autophagy) provides a route for p53 mutant degradation during restriction of glucose. Here we further show that in basal conditions of growth, inhibition of autophagy with chemical inhibitors or by downregulation of the essential autophagic genes ATG1/Ulk1, Beclin-1 or ATG5, results in p53 mutant stabilization. Conversely, overexpression of Beclin-1 or ATG1/Ulk1 leads to p53 mutant depletion. Furthermore, we found that in many cell lines, prolonged inhibition of the proteasome does not stabilize mutant p53 but leads to its autophagic-mediated degradation. Therefore, we conclude that autophagy is a key mechanism for regulating the stability of several p53 mutants. We discuss plausible mechanisms involved in this newly identified degradation pathway as well as the possible role played by autophagy during tumor evolution driven by mutant p53. PMID:23466706

Choudhury, Sujata; Kolukula, Vamsi K.; Preet, Anju; Albanese, Chris; Avantaggiati, Maria Laura

2013-01-01

184

Characterization of a Phosphate-Accumulator Mutant of Arabidopsis thaliana.  

PubMed Central

We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis. PMID:12228355

Delhaize, E.; Randall, P. J.

1995-01-01

185

Cocaethylene-induced lethality in mice is potentiated by alcohol  

Microsoft Academic Search

Mice of the heterogeneously bred HS line were concurrently administered intraperitoneal injections of either 95, 75, 60, or 48 mg\\/kg Ccaehylene or 48, 38, or 30 mg\\/kg cocaethylene in conjunction with the non-lethal dose of 6.0 g\\/kg (20% w\\/v) alcohol. Results indicate that alcohol administration significantly potentiated cocaethylene-induced lethality. This observation suggests that alcohol is capable of enhancing the lethal

Susanne M. Meehan; Martin D. Schechter

1995-01-01

186

Riot Control Agents, Tasers, and Other Less Lethal Weapons  

Microsoft Academic Search

Less lethal weapons have become increasingly popular for law enforcement use when confronting dangerous, combative individuals\\u000a in the field. On the use-of-force continuum, these technologies occupy an intermediate level between verbal and physical control\\u000a methods and lethal force such as actual firearms. Less lethal weapons include riot control agents, electric stun devices such\\u000a as tasers, and other blunt projectile weapons.

Christian Sloane; Gary M. Vilke

187

Neurodegeneration in Drop-Dead Mutant Drosophila melanogaster Is Associated with the Respiratory System but Not with Hypoxia  

PubMed Central

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia. PMID:23874488

Sansone, Christine Lynn; Blumenthal, Edward M.

2013-01-01

188

Lethal photosensitization of biofilm-grown bacteria  

NASA Astrophysics Data System (ADS)

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.

Wilson, Michael

1997-12-01

189

Ants defend aphids against lethal disease.  

PubMed

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. PMID:19923138

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

2010-04-23

190

A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.  

PubMed Central

To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. PMID:10511542

Merrill, B J; Holm, C

1999-01-01

191

Leafy Cotyledon Mutants of Arabidopsis.  

PubMed

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis. PMID:12244265

Meinke, D. W.; Franzmann, L. H.; Nickle, T. C.; Yeung, E. C.

1994-08-01

192

Sequence characterization of ENU-induced mutants of glucose phosphate isomerase in mouse  

Microsoft Academic Search

Four of five mutations producing GPI1 null lethal phenotypes in the homozygous state, which were previously identified from the offspring of male mice, spermatogonially treated with N-ethyl N-nitrosourea (ENU), have been characterized at the nucleotide level by reverse transcription of RNA from heterozygotes for mutant and wild-type alleles and cycle sequencing with cDNA-derived primers. In three of the mutations studied,

S. R. Pearce; J. Peters; S. Ball; M. J. Morgan; J. I. H. Walker; P. Faik

1995-01-01

193

ECB deacylase mutants  

DOEpatents

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.

Arnold, Frances H. (Pasadena, CA); Shao, Zhixin (Penzberg, DE); Zhao, Huimin (San Diego, CA); Giver, Lorraine J. (Sunnyvale, CA)

2002-01-01

194

Vitellogenin Receptor Mutation Leads to the Oogenesis Mutant Phenotype "scanty vitellin" of the Silkworm, Bombyx mori*  

PubMed Central

In insects, the vitellogenin receptor (VgR) mediates the uptake of vitellogenin (Vg) from the hemolymph by developing oocytes. The oogenesis mutant scanty vitellin (vit) of Bombyx mori (Bm) lacks vitellin and 30-kDa proteins, but B. mori egg-specific protein and BmVg are normal. The vit eggs are white and smaller compared with the pale yellow eggs of the wild type and are embryonic lethal. This study found that a mutation in the B. mori VgR gene (BmVgR) is responsible for the vit phenotype. We cloned the cDNA sequences encoding WT and vit BmVgR. The functional domains of BmVgR are similar to those of other low-density lipoprotein receptors. When compared with the wild type, a 235-bp genomic sequence in vit BmVgR is substituted for a 7-bp sequence. This mutation has resulted in a 50-amino acid deletion in the third Class B region of the first epidermal growth factor (EGF1) domain. BmVgR is expressed specifically in oocytes, and the transcriptional level is changed dramatically and consistently with maturation of oocytes during the previtellogenic periods. Linkage analysis confirmed that BmVgR is mutated in the vit mutant. The coimmunoprecipitation assay confirmed that mutated BmVgR is able to bind BmVg but that BmVg cannot be dissociated under acidic conditions. The WT phenotype determined by RNA interference was similar to that of the vit phenotype for nutritional deficiency, such as BmVg and 30-kDa proteins. These results showed that BmVgR has an important role in transporting proteins for egg formation and embryonic development in B. mori. PMID:23515308

Lin, Ying; Meng, Yan; Wang, Yan-Xia; Luo, Juan; Katsuma, Susumu; Yang, Cong-Wen; Banno, Yutaka; Kusakabe, Takahiro; Shimada, Toru; Xia, Qing-You

2013-01-01

195

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

PubMed Central

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

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

2012-01-01

196

Communication A novel poxvirus lethal to red squirrels  

E-print Network

Short Communication A novel poxvirus lethal to red squirrels (Sciurus vulgaris) Kathryn Thomas,1 has been implicated in the decline of the red squirrel in the United Kingdom. Virus was isolated from an outbreak of lethal disease in red squirrels in the north-east of England. Experimental infection of captive

197

Non-Lethal Weapons: The Potential and the Pitfalls  

Microsoft Academic Search

Use of non-lethal weapons by the police could significantly reduce the number of episodes involving deadly force. This paper discusses reasons why more effective nonlethal weapons have not been produced, noting in particular the priorities of military research. It also examines logistic barriers to more widespread adoption of non-lethal weapons, including the awkwardness of having the correct weapon on hand

Gilbert Geis; Arnold Binder

1990-01-01

198

Maize Mutants Lacking Chloroplast FtsY Exhibit Pleiotropic Defects in the Biogenesis of Thylakoid MembranesW?  

PubMed Central

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a “leaky” allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus. PMID:14688289

Asakura, Yukari; Hirohashi, Toshiya; Kikuchi, Shingo; Belcher, Susan; Osborne, Erin; Yano, Satoshi; Terashima, Ichiro; Barkan, Alice; Nakai, Masato

2004-01-01

199

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

PubMed Central

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

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

200

Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress  

PubMed Central

Activating K-RAS mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream signaling pathways have been shown to be deregulated by oncogenic K-ras. However, to date there are still no effective targeted therapies for this genetically defined subset of patients. Here we report the results of a small molecule, synthetic lethal screen using mouse embryonic fibroblasts derived from a mouse model harboring a conditional oncogenic K-rasG12D allele. Among the >50,000 compounds screened, we identified a class of drugs with selective activity against oncogenic K-ras–expressing cells. The most potent member of this class, lanperisone, acts by inducing nonapoptotic cell death in a cell cycle- and translation-independent manner. The mechanism of cell killing involves the induction of reactive oxygen species that are inefficiently scavenged in K-ras mutant cells, leading to oxidative stress and cell death. In mice, treatment with lanperisone suppresses the growth of K-ras–driven tumors without overt toxicity. Our findings establish the specific antitumor activity of lanperisone and reveal oxidative stress pathways as potential targets in Ras-mediated malignancies. PMID:21555567

Shaw, Alice T.; Winslow, Monte M.; Magendantz, Margaret; Ouyang, Chensi; Dowdle, James; Subramanian, Aravind; Lewis, Timothy A.; Maglathin, Rebecca L.; Tolliday, Nicola; Jacks, Tyler

2011-01-01

201

Mutants of Alfalfa Mosaic Virus.  

National Technical Information Service (NTIS)

In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can...

J. Roosien

1983-01-01

202

Characterization of mutations that are synthetic lethal with pol3-13 , a mutated allele of DNA polymerase delta in Saccharomyces cerevisiae  

Microsoft Academic Search

The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase d, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain. To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13. Mutations in genes encoding the two other subunits of DNA

Roland Chanet; Martine Heude

2003-01-01

203

Structural characterization of plancitoxin I, a deoxyribonuclease II-like lethal factor from the crown-of-thorns starfish Acanthaster planci , by expression in Chinese hamster ovary cells  

Microsoft Academic Search

Plancitoxin I, the major lethal factor from the spines of crown-of-thorns starfish Acanthaster planci, is a 37 kDa protein composed of two different subunits, and it has potent hepatotoxicity. It is homologous with mammalian\\u000a deoxyribonuclease II (DNase II) and exhibits DNase activity responsible for the hepatotoxicity. To obtain information on the\\u000a structure–activity relationship of plancitoxin I, various mutants were expressed in

Ai Watanabe; Hiroshi Nagai; Yuji Nagashima; Kazuo Shiomi

2009-01-01

204

Transgenic Rescue from Embryonic Lethality and Renal Carcinogenesis in the Eker Rat Model by Introduction of a Wild-Type Tsc2 Gene  

Microsoft Academic Search

We recently reported that a germ-line insertion in the rat homologue of the human tuberous sclerosis gene (TSC2) gives rise to dominantly inherited cancer in the Eker rat model. In this study, we constructed transgenic Eker rats with introduction of a wild-type Tsc2 gene to ascertain whether suppression of the Eker phenotype is possible. Rescue from embryonic lethality of mutant

Toshiyuki Kobayashi; Hiroaki Mitani; Ri-Ichi Takahashi; Masumi Hirabayashi; Masatsugu Ueda; Hiroshi Tamura; Okio Hino

1997-01-01

205

Pyramiding resistances based on translation initiation factors in Arabidopsis is impaired by male gametophyte lethality.  

PubMed

In eukaryotes, eIF4E translation initiation factors are essential proteins encoded by a small multigene family. In plants, they are a major source of host plant resistance to potyviruses that require specific 4E factors to infect cells. Combining mutations in different eIF4E genes could be a way of broadening the spectrum of plant resistance to viruses. We attempted to combine null mutations affecting the two main Arabidopsis thaliana 4E factors eIF4E1 and eIFiso4E but discovered that this combination is lethal. Transmission through the male gametophyte is completely abolished in the eif4e1 eifiso4e double mutant. This shows that eIF4E1 and eIFiso4E are essential for male gametophyte development and act redundantly. These results may have implications for eIF4E-based pyramiding strategies to improve crop resistance. PMID:24492391

Callot, Caroline; Gallois, Jean Luc

2014-01-01

206

Lethal body burdens of polar narcotics: Chlorophenols  

SciTech Connect

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.

Wezel, A.P. van; Punte, S.S. [Utrecht Univ. (Netherlands). Environmental Chemistry Group; Opperhuizen, A. [Ministry of Transport, Public Works and Water Management, The Hague (Netherlands). National Institute for Coastal and Marine Management

1995-09-01

207

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

PubMed Central

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

Garrido-Allepuz, Carlos; Gonzalez-Lamuno, Domingo; Ros, Maria A.

2012-01-01

208

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

PubMed Central

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

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

209

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

PubMed

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

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

2013-05-01

210

Conformational properties of nine purified cystathionine beta-synthase mutants  

PubMed Central

Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine beta-synthase (CBS) deficiency. In our previous studies, we have successfully expressed, purified and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence and second derivative-UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to the wild-type although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible towards cleavage suggesting their increased local flexibility or propensity to local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the cleavage rate of wild-type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of wild-type and the other mutants. Taken together, the proteolytic data show that the conformation of pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are a useful tool for the assessment of the biochemical penalty of missense mutations in CBS. PMID:22612060

Hnizda, Ales; Majtan, Tomas; Liu, Lu; Pey, Angel L.; Carpenter, John F.; Kodicek, Milan; Kozich, Viktor; Kraus, Jan P.

2012-01-01

211

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

SciTech Connect

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.

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

2006-01-09

212

ZBTB42 mutation defines a novel lethal congenital contracture syndrome (LCCS6).  

PubMed

Lethal congenital contracture syndrome (LCCS) is a lethal autosomal recessive form of arthrogryposis multiplex congenita (AMC). LCCS is genetically heterogeneous with mutations in five genes identified to date, all with a role in the innervation or contractile apparatus of skeletal muscles. In a consanguineous Saudi family with multiple stillbirths presenting with LCCS, we excluded linkage to all known LCCS loci and combined autozygome analysis and whole-exome sequencing to identify a novel homozygous variant in ZBTB42, which had been shown to be enriched in skeletal muscles, especially at the neuromuscular junction. Knockdown experiments of zbtb42 in zebrafish consistently resulted in grossly abnormal skeletal muscle development and myofibrillar disorganization at the microscopic level. This severe muscular phenotype is successfully rescued with overexpression of the human wild-type ZBTB42 gene, but not with the mutant form of ZBTB42 that models the human missense change. Our data assign a novel muscular developmental phenotype to ZBTB42 in vertebrates and establish a new LCCS6 type caused by ZBTB42 mutation. PMID:25055871

Patel, Nisha; Smith, Laura L; Faqeih, Eissa; Mohamed, Jawahir; Gupta, Vandana A; Alkuraya, Fowzan S

2014-12-15

213

Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli.  

PubMed

Lethal toxin (LT) that composed by protective antigen and lethal factor (LF) is the major virulence factor of Bacillus anthracis. The treatments of LT in animals could reproduce most manifestations of B. anthracis infections that greatly improves our knowledge in LT-mediated pathogenesis and facilitates anthrax-related researches without having to directly contact the hazardous bacterium B. anthracis. The recombinant protein of LF (rLF), however, still lacks a simple purification method. Herein, we developed single-step nickel affinity purification of rLF with yield up to 3mg/l. By fusion to the leader sequence of outer membrane protein OmpA, rLF could easily be purified from the periplasm of Escherichia coli. To investigate whether the rLT is functional in our system, both wild type rLF and the catalytic mutant rLF that contains a single amino acid substitution at zinc-binding site (LF(E687A)), were subjected to macrophage cytotoxicity analysis. Our data showed that the rLT is fully functional, while the LF(E687A) fail to induce cell death of tested macrophage cells. These findings suggested that the purification protocol herein is a user-friendly method that allows researchers to obtain the functional rLF by single-step purification. PMID:16797097

Chang, Hsin-Hou; Tsai, Mei-Fang; Chung, Chia-Pei; Chen, Po-Kong; Hu, Hsin-I; Kau, Jyh-Hwa; Huang, Hsin-Hsien; Lin, Hung-Chi; Sun, Der-Shan

2006-11-10

214

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

PubMed

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. PMID:24336460

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

2014-03-01

215

Synthetic lethal interactions for the development of cancer therapeutics: biological and methodological advancements  

Microsoft Academic Search

Synthetic lethal interaction is defined as a combination of two mutations that is lethal when present in the same cell; each\\u000a individual mutation is non-lethal. Synthetic lethal interactions attract attention in cancer research fields since the discovery\\u000a of synthetic lethal genes with either oncogenes or tumor suppressor genes (TSGs) provides novel cancer therapeutic targets.\\u000a Due to the selective lethal effect

Shinji Mizuarai; Hidehito Kotani

2010-01-01

216

Mutant prevention concentrations of pradofloxacin for susceptible and mutant strains of Escherichia coli with reduced fluoroquinolone susceptibility.  

PubMed

Pharmacodynamic and mutant prevention properties of the fluoroquinolone pradofloxacin (PRA) were measured against a set of 17 Escherichia coli strains carrying no, one or two known mutations conferring reduced fluoroquinolone susceptibility. The strains included susceptible wild-types, isogenic constructed mutants, isogenic selected mutants and clinical isolates. The effectiveness of PRA was determined with regard to preventing the selection of resistant mutants, using static and changing concentrations of drug. Ciprofloxacin was used as a reference drug. Minimum inhibitory concentrations (MICs) and mutant prevention concentrations (MPCs) of PRA for the susceptible wild-type strains were in the range 0.012-0.016mg/L and 0.2-0.3mg/L, respectively, giving a mean±standard deviation mutant prevention index (MPI=MPC/MIC) of 17.7±1.1. The mean MPI PRA of the 14 mutant strains was 19.2±12, and the mean MPI across all 17 strains was 18.9±10.8. In an in vitro kinetic model in which PRA was diluted with a half-life of 7h to mimic in vivo conditions, an initial concentration of PRA of 1.6-2.4mg/L (8-10× MPC), giving a PRA AUC/MPC ratio of 73-92, and a T>MPC of 21-23h was sufficient to prevent the selection of resistant mutants from the three susceptible wild-type strains. Dosing to reduce selection for antibiotic resistance in veterinary therapy has a role in reducing the reservoir of resistant mutants. We conclude that a level of dosing that prevents the selection of resistant mutants during therapy should be achievable in vivo. PMID:25129317

Marcusson, Linda L; Komp Lindgren, Patricia; Olofsson, Sara K; Hughes, Diarmaid; Cars, Otto

2014-10-01

217

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli  

SciTech Connect

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.

Billen, D.

1987-08-01

218

The genealogy, site frequency spectrum and ages of two nested mutant alleles.  

PubMed

In this paper we consider the genealogy of two nested mutant alleles, assuming the constant-size neutral coalescent model with infinite sites mutation. We study the conditional genealogy and derive explicit formulas for the joint and marginal site frequency spectra for the double, single and zero mutant allele. In addition, we find the mean ages of the two mutations. We show that the age of the youngest mutation does not depend on the frequency of the single mutant allele and that the frequency spectra for the single mutant allele and the zero mutant allele are the same. PMID:19249321

Hobolth, Asger; Wiuf, Carsten

2009-06-01

219

Phytohormone mutants in plant research  

Microsoft Academic Search

The techniques used for the production and identification of plant hormone mutants are described. The properties used to classify\\u000a these mutants into the broad synthesis and sensitivity categories are discussed, and the genetic considerations needed to\\u000a allow their effective use in plant hormone research examined. A brief outline of significant recent work on the gibberellin\\u000a (GA), abscisic acid (ABA), auxin,

James B. Reid

1990-01-01

220

Internet suicide: communities of affirmation and the lethality of communication.  

PubMed

As a tool of instant information dissemination and social networking, the Internet has made possible the formation and affirmation of public identities based on personality traits that are usually characterized by clinicians as pathological. The wide variety of online communities of affirmation reveals new conditions for permissiveness and inclusiveness in expressions of these socially marginal and clinically pathologized identities. Much the same kind of discourse common to these online communities is evident in some suicide forums. Web sites with suicide as their central raison d'être, taken together, encompass a wide range of ideas and commitments, including many that provide collective affirmation outside of (and often with hostility toward) professional intervention. The paradox of a potentially life-affirming effect of such forums runs counter to a stark dualism between online therapy versus "prochoice" forums and, by extension, to simple models of the influence of ideas on the lethality of suicide. Different forums either intensify or mitigate self-destructive tendencies in ways that are significant for understanding the place of communication in the occurrence of suicide and for therapeutic practice. PMID:23315147

Niezen, Ronald

2013-04-01

221

Ebola virus-like particles prevent lethal Ebola virus infection  

E-print Network

... successfully immunized mice against Ebola virus using virus-like particles ... exposed to lethal doses of Ebola . The work could serve as ... basis for developing countermeasures to Ebola , which causes hemorrhagic fever with ...

222

Diel Variations in Sensitivity of Fishes to Potentially Lethal Stimuli  

Microsoft Academic Search

Fathead minnows (Pimephales promelas) and golden shiners (Notemigonus crysoleucas) exhibited differences in sensitivity to potentially lethal levels of chlorine, formalin, or heat, depending on the time of day of treatment.

Richard E. Spieler; Teresa A. Noeske; Gregory L. Seegert

1977-01-01

223

Exploiting synthetic lethal interactions for targeted cancer therapy  

E-print Network

Emerging data suggests that synthetic lethal interactions between mutated oncogenes/tumor suppressor genes and molecules involved in DNA damage signaling and repair can be therapeutically exploited to preferentially kill ...

Jiang, Hai

224

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue  

E-print Network

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety ...

Graci, Jason D.; Harki, Daniel A.; Korneeva, Victoria S.; Edathil, Jocelyn P.; Too, Kathleen; Franco, David; Smidansky, Eric D.; Paul, Aniko V.; Peterson, Blake R.; Brown, Daniel M.; Loakes, David; Cameron, Craig E.

2007-10-01

225

Tetrahymena mutants with short telomeres.  

PubMed Central

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation. PMID:9755196

Ahmed, S; Sheng, H; Niu, L; Henderson, E

1998-01-01

226

Brine shrimp lethality of the compounds from Phryma leptostachya L  

Microsoft Academic Search

Brine shrimp assay-guided fractionation and isolation of the EtOAc soluble fraction ofPhryma leptostachya L. (Phrymacaceae) gave two active compounds, phrymarolin II (1) and ursolic acid (2), which were identified by physicochemical and spectroscopic methods. Compound 1 exhibited potent lethality with LD50 value of 0.0013 ?g\\/ml, whereas2 showed moderate lethality with LD50 value of 27.0 ?g\\/ml against brine shrimp. The cytotoxic

SangMyung Lee; ByungSun Min; YungHee Kho

2002-01-01

227

Mechanism by Which Caffeine Potentiates Lethality of Nitrogen Mustard  

Microsoft Academic Search

Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 mu M for 1 hr) caused

Ching C. Lau; Arthur B. Pardee

1982-01-01

228

Characterization and mapping of novel chlorophyll deficient mutant genes in durum wheat  

PubMed Central

The yellow-green leaf mutant has a non-lethal chlorophyll-deficient mutation that can be exploited in photosynthesis and plant development research. A novel yellow-green mutant derived from Triticum durum var. Cappelli displays a yellow-green leaf color from the seedling stage to the mature stage. Examination of the mutant chloroplasts with transmission electron microscopy revealed that the shape of chloroplast changed, grana stacks in the stroma were highly variable in size and disorganized. The pigment content, including chlorophyll a, chlorophyll b, total chlorophyll and carotene, was decreased in the mutant. In contrast, the chla/chlb ratio of the mutants was increased in comparison with the normal green leaves. We also found a reduction in the photosynthetic rate, fluorescence kinetic parameters and yield-related agronomic traits of the mutant. A genetic analysis revealed that two nuclear recessive genes controlled the expression of this trait. The genes were designated ygld1 and ygld2. Two molecular markers co-segregated with these genes. ygld 1 co-segregated with the SSR marker wmc110 on chromosome 5AL and ygld 2 co-segregated with the SSR marker wmc28 on chromosome 5BL. These results will contribute to the gene cloning and the understanding of the mechanisms underlying chlorophyll metabolism and chloroplast development in wheat. PMID:23853511

Li, Ning; Jia, Jizeng; Xia, Chuan; Liu, Xu; Kong, Xiuying

2013-01-01

229

Lethal Injection as a Component of a Therapeutics Toxicology Module  

PubMed Central

Objective. To create and implement a required module that addresses both the clinical and ethical issues surrounding the use of lethal injection as a means of capital punishment. Design. As a component of a pharmacotherapeutics module in toxicology, pharmacy students were introduced to ethical and clinical considerations and controversies with the use of drugs as a means of capital punishment. Basic information was provided on the history of capital punishment and the origins of lethal injection. Pharmacotherapeutic limitations and challenges were presented in the context of clinical and ethical dilemmas. Assessment. Instructed material was assessed using block course examinations that had both objective and subjective components. Students were asked to synthesize information by both purposing a lethal injection reversal protocol and by acting as consults in the fictional design of more effective lethal injection protocols. Students provided formative and summative evaluations of the instruction through regular student liaison meetings and summative course evaluations. Conclusion. Lethal injection as a means of capital punishment in the United States is a controversial and ethically challenging topic on which pharmacists may be consulted and therefore should be knowledgeable about. Students positively evaluated this lethal injection module, which covered multiple clinical and ethical issues. PMID:21931455

2011-01-01

230

Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen  

PubMed Central

Assembly of anthrax toxin complexes at the mammalian cell surface involves competitive binding of the edema factor (EF) and lethal factor (LF) to heptameric oligomers and lower order intermediates of PA63, the activated carboxyl-terminal 63-kDa fragment of protective antigen (PA). We used sequence differences between PA63 and homologous PA-like proteins to delineate a region within domain 1? of PA that may represent the binding site for these ligands. Substitution of alanine for any of seven residues in or near this region (R178, K197, R200, P205, I207, I210, and K214) strongly inhibited ligand binding. Selected mutations from this set were introduced into two oligomerization-deficient PA mutants, and the mutants were used in various combinations to map the single ligand site within dimeric PA63. The site was found to span the interface between two adjacent subunits, explaining the dependence of ligand binding on PA oligomerization. The locations of residues comprising the site suggest that a single ligand molecule sterically occludes two adjacent sites, consistent with the finding that the PA63 heptamer binds a maximum of three ligand molecules. These results elucidate the process by which the components of anthrax toxin, and perhaps other binary bacterial toxins, assemble into toxic complexes. PMID:11997439

Cunningham, Kristina; Lacy, D. Borden; Mogridge, Jeremy; Collier, R. John

2002-01-01

231

RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency.  

PubMed

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (?F/?F), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ?F/?F mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (?F/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ?F/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ?F/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ?F/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus. PMID:23212600

Volta, Viviana; Beugnet, Anne; Gallo, Simone; Magri, Laura; Brina, Daniela; Pesce, Elisa; Calamita, Piera; Sanvito, Francesca; Biffo, Stefano

2013-04-01

232

Connexin mutant embryonic stem cells and human diseases  

PubMed Central

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.

Nishii, Kiyomasa; Shibata, Yosaburo; Kobayashi, Yasushi

2014-01-01

233

A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (A{sup y}) mutation  

SciTech Connect

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.

Michaud, E.J.; Klebig, M.L.; Stubbs, L.J.; Russell, L.B.; Woychik, R.P. [Oak Ridge National Lab., TN (United States); Bultman, S.J. [Oak Ridge National Lab., TN (United States)]|[Univ. of Tennessee, Oak Ridge, TN (United States); Vugt, M.J. van [Oak Ridge National Lab., TN (United States)]|[Agricultural Univ. of Wageningen (Netherlands)

1994-03-29

234

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.  

PubMed

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (-)-cocaine but also certain selectivity for (-)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (-)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (-)-cocaine but also the desirable selectivity for (-)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. PMID:20971807

Xue, Liu; Ko, Mei-Chuan; Tong, Min; Yang, Wenchao; Hou, Shurong; Fang, Lei; Liu, Junjun; Zheng, Fang; Woods, James H; Tai, Hsin-Hsiung; Zhan, Chang-Guo

2011-02-01

235

Embryonic lethality in mice lacking mismatch-specific thymine DNA glycosylase is partially prevented by DOPS, a precursor of noradrenaline.  

PubMed

Thymine DNA glycosylase (TDG) is involved in the repair of G:T and G:U mismatches caused by hydrolytic deamination of 5-methylcytosine and cytosine, respectively. Recent studies have shown that TDG not only has G-T/U glycosylase activities but also acts in the maintaining proper epigenetic status. In order to investigate the function of TDG in vivo, mice lacking Tdg, Tdg (-/-), were generated. Tdg mutant mice died in utero by 11.5 days post coitum (dpc), although there were no significant differences in the spontaneous mutant frequencies between wild type and Tdg (-/-) embryos. On the other hand, the levels of noradrenaline in 10.5 dpc whole embryos, which is necessary for normal embryogenesis, were dramatically reduced in Tdg (-/-) embryos. Consequently, we tested the effect of D, L-threo-3, 4-dihydroxyphenylserine (DOPS), a synthetic precursor of noradrenaline, on the survival of the Tdg (-/-) embryos. DOPS was given to pregnant Tdg (+/-) mice from 6.5 dpc through drinking water. Most of the Tdg (-/-) embryos were alive at 11.5 dpc, and they were partially rescued up to 14.5 dpc by the administration of DOPS. In contrast, the administration of L-3, 4-dihydroxyphenylalanine (L-DOPA) had marginal effects on Tdg (-/-) embryonic lethality. No embryo was alive without DOPS beyond 11.5 dpc, suggesting that the lethality in (-/-) embryos is partially due to the reduction of noradrenaline. These results suggest that embryonic lethality in Tdg (-/-) embryos is due, in part, to the reduction of noradrenaline levels. PMID:22200605

Saito, Yusuke; Ono, Tetsuya; Takeda, Naoki; Nohmi, Takehiko; Seki, Masayuki; Enomoto, Takemi; Noda, Tetsuo; Uehara, Yoshihiko

2012-01-01

236

Mutagenesis-Mediated Virus Extinction: Virus-Dependent Effect of Viral Load on Sensitivity to Lethal Defection  

PubMed Central

Background Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis. Results The effect of the mutagenic base analogue 5-fluorouracil (FU) on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV) can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI), or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV), but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV). The increase in mutation frequency and Shannon entropy (mutant spectrum complexity) as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. Conclusions (i) Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii) This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii) The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv) LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v) The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development. PMID:22442668

Moreno, Hector; Tejero, Hector; de la Torre, Juan Carlos; Domingo, Esteban; Martin, Veronica

2012-01-01

237

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.  

PubMed Central

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle. Images PMID:3031463

Shin, D Y; Matsumoto, K; Iida, H; Uno, I; Ishikawa, T

1987-01-01

238

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.  

PubMed

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2? mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2? now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2? mutants. Interaction of dna2? and DNA damage checkpoint mutations provides insight as to why dna2? is lethal but rad27? is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset. PMID:21508669

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

239

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.  

PubMed Central

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division. PMID:2656638

Ogura, T; Bouloc, P; Niki, H; D'Ari, R; Hiraga, S; Jaffe, A

1989-01-01

240

D-2-hydroxyglutarate metabolism is linked to photorespiration in the shm1-1 mutant.  

PubMed

The Arabidopsis mutant shm1-1 is defective in mitochondrial serine hydroxymethyltransferase 1 activity and displays a lethal photorespiratory phenotype at ambient CO2 concentration but grows normally at high CO2 . After transferring high CO2 -grown shm1-1 plants to ambient CO2 , the younger leaves remain photosynthetically active while developed leaves display increased yellowing and decreased FV /FM values. Metabolite analysis of plants transferred from high CO2 to ambient air indicates a massive light-dependent (photorespiratory) accumulation of glycine, 2-oxoglutarate (2OG) and D-2-hydroxyglutarate (D-2HG). Amino acid markers of senescence accumulated in ambient air in wild-type and shm1-1 plants maintained in darkness and also build up in shm1-1 in the light. This, together with an enhanced transcription of the senescence marker SAG12 in shm1-1, suggests the initiation of senescence in shm1-1 under photorespiratory conditions. Mitochondrial D-2HG dehydrogenase (D-2HGDH) converts D-2HG into 2OG. In vitro studies indicate that 2OG exerts competitive inhibition on D-2HGDH with a Ki of 1.96 mm. 2OG is therefore a suitable candidate as inhibitor of the in vivo D-2HGDH activity, as 2OG is produced and accumulates in mitochondria. Inhibition of the D-2HGDH by 2OG is likely a mechanism by which D-2HG accumulates in shm1-1, however it cannot be ruled out that D-2HG may also accumulate due to an active senescence programme that is initiated in these plants after transfer to photorespiratory conditions. Thus, a novel interaction of the photorespiratory pathway with cellular processes involving D-2HG has been identified. PMID:23551974

Kuhn, A; Engqvist, M K M; Jansen, E E W; Weber, A P M; Jakobs, C; Maurino, V G

2013-07-01

241

Towards an Informative Mutant Phenotype for Every Bacterial Gene  

PubMed Central

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. PMID:25112473

Deutschbauer, Adam; Price, Morgan N.; Wetmore, Kelly M.; Tarjan, Daniel R.; Xu, Zhuchen; Shao, Wenjun; Leon, Dacia

2014-01-01

242

Fitness of transgenic mosquito Aedes aegypti males carrying a dominant lethal genetic system.  

PubMed

OX513A is a transgenic strain of Aedes aegypti engineered to carry a dominant, non-sex-specific, late-acting lethal genetic system that is repressed in the presence of tetracycline. It was designed for use in a sterile-insect (SIT) pest control system called RIDL® (Release of Insects carrying a Dominant Lethal gene) by which transgenic males are released in the field to mate with wild females; in the absence of tetracycline, the progeny from such matings will not survive. We investigated the mating fitness of OX513A in the laboratory. Male OX513A were as effective as Rockefeller (ROCK) males at inducing refractoriness to further mating in wild type females and there was no reduction in their ability to inseminate multiple females. They had a lower mating success but yielded more progeny than the wild-type comparator strain (ROCK) when one male of each strain was caged with a ROCK female. Mating success and fertility of groups of 10 males-with different ratios of RIDL to ROCK-competing for five ROCK females was similar, but the median longevity of RIDL males was somewhat (18%) lower. We conclude that the fitness under laboratory conditions of OX513A males carrying a tetracycline repressible lethal gene is comparable to that of males of the wild-type comparator strain. PMID:23690948

Massonnet-Bruneel, Blandine; Corre-Catelin, Nicole; Lacroix, Renaud; Lees, Rosemary S; Hoang, Kim Phuc; Nimmo, Derric; Alphey, Luke; Reiter, Paul

2013-01-01

243

The Population Genetics of X-Autosome Synthetic Lethals and Steriles  

PubMed Central

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. PMID:21900269

Lachance, Joseph; Johnson, Norman A.; True, John R.

2011-01-01

244

Overexpression of dilp2 causes nutrient-dependent semi-lethality in Drosophila.  

PubMed

Insulin/insulin-like growth factor (IGF) plays an important role as a systemic regulator of metabolism in multicellular organisms. Hyperinsulinemia, a high level of blood insulin, is often associated with impaired physiological conditions such as hypoglycemia, insulin resistance, and diabetes. However, due to the complex pathophysiology of hyperinsulinemia, the causative role of excess insulin/IGF signaling has remained elusive. To investigate the biological effects of a high level of insulin in metabolic homeostasis and physiology, we generated flies overexpressing Drosophila insulin-like peptide 2 (Dilp2), which has the highest potential of promoting tissue growth among the Ilp genes in Drosophila. In this model, a UAS-Dilp2 transgene was overexpressed under control of sd-Gal4 that drives expression predominantly in developing imaginal wing discs. Overexpression of Dilp2 caused semi-lethality, which was partially suppressed by mutations in the insulin receptor (InR) or Akt1, suggesting that dilp2-induced semi-lethality is mediated by the PI3K/Akt1 signaling. We found that dilp2-overexpressing flies exhibited intensive autophagy in fat body cells. Interestingly, the dilp2-induced autophagy as well as the semi-lethality was partially rescued by increasing the protein content relative to glucose in the media. Our results suggest that excess insulin/IGF signaling impairs the physiology of animals, which can be ameliorated by controlling the nutritional balance between proteins and carbohydrates, at least in flies. PMID:24795642

Sato-Miyata, Yukiko; Muramatsu, Keigo; Funakoshi, Masabumi; Tsuda, Manabu; Aigaki, Toshiro

2014-01-01

245

Green tea extract-induced lethal toxicity in fasted but not in nonfasted dogs.  

PubMed

Recent chronic toxicity studies performed on green tea extracts in fasted dogs have revealed some unique dose-limiting lethal liver, gastrointestinal, and renal toxicities. Key findings included necrosis of hepatic cells, gastrointestinal epithelia and renal tubules, atrophy of reproductive organs, atrophy and necrosis of hematopoietic tissues, and associated hematological changes. The polyphenol cachetins (a mixture of primarily epigallocatechin gallate [?55%]; plus up to 10% each of epigallocatechin, epicatechin, and epigallocatechin gallate) appeared to be the causative agents for the observed toxicities because they are the active ingredients of green tea extract studied. Conduct of the study in nonfasted dogs under the same testing conditions and dose levels showed unremarkable results. Assuming both studies were valid, at the identified no observed adverse effect levels (NOAEL) of each study, systemic exposures (based on area under the curve [AUC]) were actually lower in fasted than nonfasted dogs, suggesting that fasting may have rendered the target organ systems potentially more vulnerable to the effects of green tea extract. The toxicity mechanisms that produced lethality are not known, but the results are scientifically intriguing. Because tea drinking has become more popular in the United States and abroad, the mode of action and site of action of green tea extract-induced lethal toxicities during fasting and the role of other phytochemical components of Folia Camellia sinensis (including nonpolyphenol fractions, which are often consumed when whole-leaf products are presented) warrant further investigation. PMID:21098339

Wu, Kuei-Meng; Yao, Jiaqin; Boring, Daniel

2011-02-01

246

A double mutant between fission yeast telomerase and RecQ helicase is sensitive to thiabendazole, an anti-microtubule drug.  

PubMed

In the fission yeast Schizosaccharomyces pombe, deletion of trt1(+) causes gradual telomere shortening, while deletion of pot1(+) causes rapid telomere loss. The double mutant between pot1 and RecQ helicase rqh1 is synthetically lethal. We found that the trt1 rqh1 double mutant was not synthetically lethal. The chromosome end fragments in both the trt1? rqh1? and the trt1? rqh1-hd (helicase dead) double mutants did not enter a pulsed-field electrophoresis gel. Both the trt1? rqh1? and the trt1? rqh1-hd double mutants were sensitive to the anti-microtubule drug thiabendazole. Moreover, the trt1? rqh1-hd double mutant displayed RPA foci on the chromosome bridge at high frequency in M phase cells. These phenotypes are very similar to that of the pot1? rqh1-hd double mutant, in which recombination intermediates accumulate at the chromosme ends in the M phase. These results suggest that the entangled chromosome ends, most likely recombination intermediates, are present in the M phase in the trt1? rqh1-hd double mutant. PMID:22313747

Ukimori, Shinobu; Kawabata, Naoki; Shimada, Hideki; Imano, Ryota; Takahashi, Katsunori; Yukawa, Masashi; Tsuchiya, Eiko; Ueno, Masaru

2012-01-01

247

The Rorschach Suicide Constellation: assessing various degrees of lethality.  

PubMed

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

Fowler, J C; Piers, C; Hilsenroth, M J; Holdwick, D J; Padawer, J R

2001-04-01

248

Are High-Lethality Suicide Attempters With Bipolar Disorder a Distinct Phenotype?  

Microsoft Academic Search

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

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

2009-01-01

249

Electrophysiological evidence suggests a defective Ca 2+ control mechanism in a new Paramecium mutant  

Microsoft Academic Search

Summary A new mutant ofParamecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca2+-regulated currents were altered in the mutant. (i) The voltage-dependent Ca2+ current recovered from Ca2+-dependent inactivation two- to

Thomas C. Evans; Todd Hennessey; David L. Nelson

1987-01-01

250

Evaluation of lethal and non-lethal sampling methods for the detection of white sturgeon iridovirus infection in white sturgeon, Acipenser transmontanus (Richardson).  

PubMed

Pectoral fin tissue of white sturgeon was investigated as a potential non-lethal sample source for the detection of white sturgeon iridovirus (WSIV) infection. Histopathology and polymerase chain reaction (PCR) results using fin tissue were compared with the standard lethal histopathology sampling method that utilizes head tissue. Tissues for each of the three sampling methods were collected weekly for 8 weeks from individual sturgeon undergoing an experimental cohabitation challenge with fish infected with the Abernathy isolate of WSIV. Non-lethal fin histopathological evaluation did not reveal infection during the first 3 weeks of sampling, while non-lethal PCR and the lethal method were variable. However, all three sampling methods were equally capable of identifying infection from 4 to 8 weeks post-exposure. Of the survivors tested, all were negative by PCR and the lethal method, and only one fish was identified as being positive by non-lethal fin histopathology. In another experiment, all three sampling methods were applied to asymptomatic WSIV carriers in a case study conducted at the Kootenai Tribal Sturgeon Conservation Hatchery. Results showed that both lethal and non-lethal fin histopathology were equally effective in detecting infection, but PCR was unable to identify this strain of WSIV. Depending on the virus isolate, these results suggest that non-lethal sampling of fin tissue (histopathology or PCR) is comparable with the lethal sampling method at identifying WSIV infection once infection is established, and under certain circumstances may provide an alternative to lethal sampling. PMID:17498180

Drennan, J D; Lapatra, S E; Samson, C A; Ireland, S; Eversman, K F; Cain, K D

2007-06-01

251

[Bart syndrome associated to lethal junctional epidermolysis bullosa (Herlitz form)].  

PubMed

We present the case of a newborn with congenital absence of skin in the anterior part of the left leg that shortly after developed bulla and erosions in hands, feet, ears, buttocks and mouth. The cutaneous biopsy and ultrastructural and immunohistochemical studies showed a subepidermal bulla in the lamina lucida, absence of hemidesmosomes and marked decrease of laminin 5, thus establishing the diagnosis of Bart syndrome associated to the Herlitz form of lethal junctional epidermolysis bullosa. Bart syndrome consists of congenital and localized absence of skin, nail abnormalities and mucoc-cutaneous bullae. It is usually associated to dystrophic epidermolysis bullosa. The Herlitz form of junctional epidermolysis bullosa is a rare variant, usually lethal that is produced by mutations in the genes coding for the anchor protein laminin 5. To our knowledge this is the second case that reports an association between Bart syndrome and lethal junctional epidermolysis bullosa and the first in which the results of immunofluorescence mapping are published. PMID:17173830

Casanova, J M; Martí, R M; Baradad, M; Egido, R; Mascaró, J M

2006-12-01

252

Proteinase Mutants of SACCHAROMYCES CEREVISIAE  

PubMed Central

Fifty-nine mutants with reduced ability to cleave the chymotrypsin substrate N-acetyl-DL-phenylalanine ?-naphthyl ester have been isolated in S. cerevisiae. All have reduced levels of one or more of the three well-characterized proteinases in yeast. All have reduced levels of proteinase C (carboxy-peptidase Y). These mutations define 16 complementation groups. PMID:320092

Jones, Elizabeth W.

1977-01-01

253

GLYCOLYSIS MUTANTS IN SACCHAROMYCES CEREVISIAE  

Microsoft Academic Search

Mutants have been isolated in S. cereuisiae with the phenotype of growth on pyruvate but not on glucose, or growth on rich medium with pyruvate but inhibition by glucose. Screening of mutagenized cultures was either without an enrichment step, or after enrichment using the antibiotic netropsin (YOUNG et al. 1976) or inositol starvation (HENRY, DONAHUE and CULBERTSON 1975). One class

SHELLEY B. WEINSTOCK; DAN G. FRAENKEL

254

Tetrahymena Mutants With Short Telomeres  

Microsoft Academic Search

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo .I n Tetrahymena thermophila, telomeres become long at 308, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres

Shawn Ahmed; Hong Sheng; Luming Niu; Eric Henderson

1998-01-01

255

A new mouse mutant, skijumper  

Microsoft Academic Search

Low blood sugar levels are a well-known cause of severe illness and often death in newborn humans, especially those that are small for age. Few of the causes of neonatal hypoglycemia are known, and many remain to be found. We describe a novel mouse mutant, 'skijumper' (skimp), in which pups, despite feeding well, have low levels of glucose and develop

Majid Hafezparast; Simon Ball; Sharon J. Nicholson; Abi Witherden; Demet Arac; Neil Broadway; David Saggerson; Edwin Cooper; Mahmoud Naase; Stephen Gokhale; Patti Quant; Carol Lascelles; Carole Nickols; Cathy S. Baker; Josephine Peters; Joanne E. Martin; Elizabeth M. C. Fisher

2002-01-01

256

MutMap+: Genetic Mapping and Mutant Identification without Crossing in Rice  

PubMed Central

Advances in genome sequencing technologies have enabled researchers and breeders to rapidly associate phenotypic variation to genome sequence differences. We recently took advantage of next-generation sequencing technology to develop MutMap, a method that allows rapid identification of causal nucleotide changes of rice mutants by whole genome resequencing of pooled DNA of mutant F2 progeny derived from crosses made between candidate mutants and the parental line. Here we describe MutMap+, a versatile extension of MutMap, that identifies causal mutations by comparing SNP frequencies of bulked DNA of mutant and wild-type progeny of M3 generation derived from selfing of an M2 heterozygous individual. Notably, MutMap+ does not necessitate artificial crossing between mutants and the wild-type parental line. This method is therefore suitable for identifying mutations that cause early development lethality, sterility, or generally hamper crossing. Furthermore, MutMap+ is potentially useful for gene isolation in crops that are recalcitrant to artificial crosses. PMID:23874658

Abe, Akira; Natsume, Satoshi; Yaegashi, Hiroki; Sharma, Shailendra; Sharma, Shiveta; Kanzaki, Hiroyuki; Matsumura, Hideo; Saitoh, Hiromasa; Mitsuoka, Chikako; Utsushi, Hiroe; Uemura, Aiko; Kanzaki, Eiko; Kosugi, Shunichi; Yoshida, Kentaro; Cano, Liliana; Kamoun, Sophien; Terauchi, Ryohei

2013-01-01

257

Conserved Role of unc-79 in Ethanol Responses in Lightweight Mutant Mice  

PubMed Central

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans. PMID:20714347

Speca, David J.; Chihara, Daisuke; Ashique, Amir M.; Bowers, M. Scott; Pierce-Shimomura, Jonathan T.; Lee, Jungsoo; Rabbee, Nusrat; Speed, Terence P.; Gularte, Rodrigo J.; Chitwood, James; Medrano, Juan F.; Liao, Mark; Sonner, James M.; Eger, Edmond I.

2010-01-01

258

Lethal toxicity of cadmium to Cyprinus carpio and Tilapia aurea  

SciTech Connect

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.

Not Available

1986-09-01

259

Isolation and characterization of type III group B streptococcal mutants defective in biosynthesis of the type-specific antigen.  

PubMed Central

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:6352490

Yeung, M K; Mattingly, S J

1983-01-01

260

Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants  

SciTech Connect

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.

Winkelman, J.W.; Clark, D.P.

1984-11-01

261

A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana  

Microsoft Academic Search

Monogenic mutants of the early ecotype Landsberg erecta were selected on the basis of late flowering under long day (LD) conditions after treatment with ethyl methanesulphonate or irradiation. In addition to later flowering the number of rosette and cauline leaves is proportionally higher in all mutants, although the correlation coefficient between the two parameters is not the same for all

M. Koornneef; C. J. Hanhart; J. H. Veen

1991-01-01

262

Growth of Arabidopsis flavonoid mutants under solar radiation and UV filters  

Microsoft Academic Search

Growth of the chalcone isomerase defective tt-5 mutant of Arabidopsis thaliana and its Landsberg erecta progenitor were compared under a variety of full spectrum solar radiation conditions to determine if the tt-5 mutant could serve as an adequate subject for studies of the mechanisms of damage by UV-B radiation. An experiment was conducted in the fall of 1995 under open

Edwin L. Fiscus; Robert Philbeck; Anne B. Britt; Fitzgerald L. Booker

1999-01-01

263

Genetic and Phenotypic Analysis of Flagellar Assembly Mutants in Chlamydomonas reinhardtii  

Microsoft Academic Search

Conditional mutants for flagellar assembly (fla) provide a useful tool to study intraflagellar transport (IFT) at the molecular level, and provide a unique set of tools to analyze cilia. The analysis of IFT phenotypes of fla mutants at the permissive temperature by a quantitative image analysis approach identified four distinct phases of the IFT cycle and directly demonstrated structural and

Carlo Iomini; Jacob E. Till; Susan K. Dutcher

2009-01-01

264

Determination of Lethality Rate Constants and D-Values for Bacillus atrophaeus (ATCC 9372) Spores Exposed to Dry Heat from 115°C to 170°C  

NASA Astrophysics Data System (ADS)

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°C and 125°C for up to 50 hours, while controlling the humidity to very low values. Collection of lethality data at temperatures above 125°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°C. The goal of this research is to determine the survival rates of Bacillus atrophaeus (ATCC 9372) spores subjected to temperatures higher than 125°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°C to 170°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°C and 125°C, the controlled humidity lethality rate constant was faster than th:e ambient humidity lethality rate constant. At 135°C, the ambient and controlled humidity lethality rate constants were statistically identical. At 150°C and 170°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.

Kempf, M. J.; Schubert, W. W.; Beaudet, R. A.

2008-12-01

265

Dominant membrane uncoupling by mutant adenine nucleotide translocase in mitochondrial diseases  

PubMed Central

Adenine nucleotide translocase (Ant) is the most abundant protein on the mitochondrial inner membrane (MIM) primarily involved in ADP/ATP exchange. Ant also possesses a discrete membrane uncoupling activity. Specific mis-sense mutations in the human Ant1 cause autosomal dominant Progressive External Ophthalmoplegia (adPEO), mitochondrial myopathy and cardiomyopathy, which are commonly manifested by fractional mitochondrial DNA (mtDNA) deletions. It is currently thought that the pathogenic mutations alter substrate preference (e.g. ATP versus ADP) thereby dominantly disturbing adenine nucleotide homeostasis in mitochondria. This may interfere with mtDNA replication, consequently affecting mtDNA stability and oxidative phosphorylation. Here, we showed that the adPEO-type A128P, A106D and M114P mutations in the yeast Aac2p share the following common dominant phenotypes: electron transport chain damage, intolerance to moderate over-expression, synthetic lethality with low ??m conditions, hypersensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mtDNA instability. More interestingly, the aac2A137D allele mimicking ant1A123D in mitochondrial myopathy and cardiomyopathy exhibits similar dominant phenotypes. Because Aac2A137D is known to completely lack transport activity, it is strongly argued that the dominant mitochondrial damages are not caused by aberrant nucleotide transport. The four pathogenic mutations occur in a structurally dynamic gating region on the cytosolic side. We provided direct evidence that the mutant alleles uncouple mitochondrial respiration. The pathogenic mutations likely enhance the intrinsic proton-conducting activity of Ant, which excessively uncouples the MIM thereby affecting energy transduction and mitochondrial biogenesis. mtDNA disintegration is a phenotype co-lateral to mitochondrial damages. These findings provide mechanistic insights into the pathogenesis of the Ant1-induced diseases. PMID:18809618

Wang, Xiaowen; Salinas, Kelly; Zuo, Xiaoming; Kucejova, Blanka; Chen, Xin Jie

2008-01-01

266

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana  

Microsoft Academic Search

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain “Columbia”, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and

J. D. Heath; R. Weldon; C. Monnot; D. W. Meinke

1986-01-01

267

Switching devices used in the Lethality Test System  

Microsoft Academic Search

The Lethality Test System (LTS) is an experiment which will utilize a light gas gun-railgun combination to accelerate projectiles for impact studies. The power supply for the railgun consists of an 80 MJ (kinetic) generator-flywheel array used to charge the primary windings of three cryogenic pulse transformers. The secondary windings of these transformers will supply up to 50 MJ of

W. M. Parsons; R. D. Ford; P. Wildi; P. Chowdhuri

1987-01-01

268

Brine shrimp lethality bioassay of selected Indian medicinal plants  

Microsoft Academic Search

Ethanolic extracts of six Indian medicinal plants, piperine, guggulsterone E and guggulsterone Z were tested for cytotoxicity using brine shrimp lethality test. Piper longum showed most potent cytotoxic activity. Piperine, guggulsterone E and guggulsterone Z showed potent activity with LC50 2.4, 8.9 and 4.9, respectively.

R Padmaja; P. C Arun; D Prashanth; M Deepak; A Amit; M Anjana

2002-01-01

269

Antioxidant, icthyotoxicity and brine shrimp lethality tests of Magonia glabrata  

Microsoft Academic Search

The ethanolic extract of the fruit bark from Magonia glabrata yielded shikimic acid, scopoletin, sitosterol glycoside and 2-O-methyl-l-inositol. Antioxidant, icthyotoxicity and brine shrimp lethality activities were observed in this extract. The major constituent, 2-O-methyl-l-inositol, was found to be inactive in two assays but showed moderate activity as a radical scavenger.

Telma L. G. Lemos; Luciana L. Machado; João S. N. Souza; Aluisio M. Fonseca; Juliana L. Maia; Otilia D. L. Pessoa

2006-01-01

270

Evaluation of Brine Shrimp Lethality of Cinnamomum Species  

Microsoft Academic Search

Cinnamomum species have long been used as spices. The preliminary bioactive constituent’s identification and brine shrimp lethality activities of ethanolic extracts of seven Cinnamomum species, viz., C. travancoricum, C. walaiwarense, C. wightii, C. verum, C. sulphuratum, C. riparium, and C. perrottetii were evaluated in this study. The results of cytotoxic activity of the bark extracts of seven Cinnamomum species were

Muthiah Maridass

2008-01-01

271

Dominant-lethal mutations and heritable translocations in mice  

SciTech Connect

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.

Generoso, W.M.

1983-01-01

272

Subcutaneous wounding postirradiation reduces radiation lethality in mice.  

PubMed

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

Garrett, Joy; Orschell, Christie M; Mendonca, Marc S; Bigsby, Robert M; Dynlacht, Joseph R

2014-06-01

273

Physicians' Attitudes About Involvement in Lethal Injection for Capital Punishment  

Microsoft Academic Search

Background: Physicians could play various roles in car- rying out capital punishment via lethal injection. Medi- cal societies like the American Medical Association (AMA) and American College of Physicians have established which roles are acceptable and which are disallowed. No one has explored physicians' attitudes toward their po- tential roles in this process. Methods: We surveyed physicians about how accept-

Neil Farber; Elizabeth B. Davis; Joan Weiner; Janine Jordan; E. Gil Boyer; Peter A. Ubel

2000-01-01

274

Pathophysiological Manifestations in Mice Exposed to Anthrax Lethal Toxin  

PubMed Central

Pathophysiological changes associated with anthrax lethal toxin included loss of plasma proteins, decreased platelet count, slower clotting times, fibrin deposits in tissue sections, and gross and histopathological evidence of hemorrhage. These findings suggest that blood vessel leakage and hemorrhage lead to disseminating intravascular coagulation and/or circulatory shock as an underlying pathophysiological mechanism. PMID:16177381

Culley, Nathan C.; Pinson, David M.; Chakrabarty, Anuradha; Mayo, Matthew S.; LeVine, Steven M.

2005-01-01

275

Help-Seeking Behavior Prior to Nearly Lethal Suicide Attempts.  

ERIC Educational Resources Information Center

The association between help-seeking and nearly lethal suicide attempts was evaluated using data from a population-based, case-control study. Measures of help-seeking included type of consultant contacted, and whether suicide was discussed. Findings suggest efforts to better understand the role of help-seeking in suicide prevention deserves…

Barnes, Lauren Seymour; Ikeda, Robin M.; Kresnow, Marcie-jo

2002-01-01

276

Non-Lethality of the Mid Factor in Lythrum Salicaria  

Microsoft Academic Search

IN 1927 East1 proposed a theory of the inheritance of style-length in tristylic Lythrum Salicaria which involved three factors. One of these, S, was epistatic to the others, and determined Short style as opposed to Mid and Long. The other two, each of which was supposed to give Mid style as opposed to Long, were lethal when homozygous and were

B. A. Fisher; K. Mather

1940-01-01

277

Analysis of Ebola Glycoprotein Sequences from Strains of Varying Lethality  

E-print Network

Analysis of Ebola Glycoprotein Sequences from Strains of Varying Lethality Biochem 218 Spring 2002 Tammy Doukas tdoukas@stanford.edu I. Background and Significance Ebola hemorrhagic fever is a disease in humans, chimpanzees, and monkeys, caused by infection with Ebola virus, and associated with high

278

Gene family encoding the major toxins of lethal Amanita mushrooms  

E-print Network

, 2007 (received for review August 6, 2007) Amatoxins, the lethal constituents of poisonous mushrooms -amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a phallotoxin, indicating phalloidin phallotoxin amatoxin Mushrooms in the genus Amanita section Phalloideae ac- count for 90% of all

Bruns, Tom

279

The Prevalence, Lethality and Intent of Suicide Attempts among Adolescents.  

ERIC Educational Resources Information Center

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…

Andrews, Judy A.; Lewinsohn, Peter M.

280

High-Lethality Status in Patients with Borderline Personality Disorder  

Microsoft Academic Search

Recurrent suicidal behaviors in patients with Borderline Personality Disorder (BPD) are often considered communicative gestures; however, 10% complete suicide. This study seeks to identify risk factors for sui- cide within a BPD sample by comparing patients with High- and Low- Lethality attempts. BPD attempters (n = 113) were assessed on dem- ographic, diagnostic, and personality variables: clinical symptoms, suicidal behaviors;

Paul H. Soloff; Anthony Fabio; Thomas M. Kelly; Kevin M. Malone; J. John Mann

2005-01-01

281

Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles  

E-print Network

Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles Jaan-Olle Andressoo1¤a sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic, Jans J, de Wit J, Coin F, Hoogstraten D, et al. (2006) Rescue of progeria in trichothiodystrophy

Boyer, Edmond

282

Escherichia coli genes that reduce the lethal effects of stress  

Microsoft Academic Search

BACKGROUND: The continuing emergence of antimicrobial resistance requires the development of new compounds and\\/or enhancers of existing compounds. Genes that protect against the lethal effects of antibiotic stress are potential targets of enhancers. To distinguish such genes from those involved in drug uptake and efflux, a new susceptibility screen is required. RESULTS: Transposon (Tn5)-mediated mutagenesis was used to create a

Xiulin Han; Angella Dorsey-Oresto; Muhammad Malik; Jian-Ying Wang; Karl Drlica; Xilin Zhao; Tao Lu

2010-01-01

283

Regulation of apoptosis by lethal cytokines in human mesothelial cells  

Microsoft Academic Search

Regulation of apoptosis by lethal cytokines in human mesothelial cells.BackgroundDysregulation of peritoneal cell death may contribute to the complications of peritoneal dialysis (PD). Chronic peritoneal dialysis and acute peritonitis are both associated with loss of mesothelial cells. In addition, acute peritonitis is characterized by sudden changes in the number of peritoneal leukocytes. However, the factors regulating peritoneal cell survival are

Marina Penélope Catalan; Dolores Subirá; Ana Reyero; Rafael Selgas; Arturo Ortiz-Gonzalez; Jesús Egido; Alberto Ortiz

2003-01-01

284

The Discrete Character of High-Lethality Youth Violence  

Microsoft Academic Search

The purpose of this paper is to derive lessons about high-lethality adolescent violence from study of the extraordinary increase, and then decrease, in youth homicide arrests over the period 1985-2001 in the United States. This effort continues a long tradition of exploring the possible causes of shifts in homicide rates by examining patterns of violence to see whether there are

Franklin E. Zimring

2005-01-01

285

The Discrete Character of High-Lethality Youth Violence  

Microsoft Academic Search

The purpose of this paper is to derive lessons about high-lethality adolescent violence from study of the extraordinary increase, and then decrease, in youth homicide arrests over the period 1985-2001 in the United States. This effort continues a long tradition of exploring the possible causes of shifts in homicide rates by examining patterns of violence to see whether there are

Franklin E. Zimring

2004-01-01

286

Standards for lethal response to problem urban wildlife  

Microsoft Academic Search

Managers face limited options when dealing with problems created by urban wildlife. Destroying an animal that is perceived to be a nuisance is sometimes acceptable; at other times destroying the animal may be controversial. This paper uses the structural norm approach to develop standards for an agency's use of lethal response to problem urban wildlife. The paper describes three structural

Karin Wittmann; Jerry J. Vaske; Michael J. Manfredo; Harry C. Zinn

1998-01-01

287

Contributed Paper Lethal Effects of Water Quality on Threatened  

E-print Network

Contributed Paper Lethal Effects of Water Quality on Threatened California Salamanders but Not on Co-Occurring Hybrid Salamanders MAUREEN E. RYAN, JARRETT R. JOHNSON, BENJAMIN M. FITZPATRICK, LINDA, and Center for Population Biology, University of California, Davis, One Shields Avenue, Davis, CA 95616, U

Shaffer, H. Bradley

288

RESEARCH ARTICLE Lethal Intergroup Aggression by Chimpanzees in Kibale  

E-print Network

relations throughout most or all of their geographic range. Hostilities include aggressive encountersRESEARCH ARTICLE Lethal Intergroup Aggression by Chimpanzees in Kibale National Park, Uganda DAVID intergroup coalitionary aggression is highest for adult males and infants, and lowest for sexually swollen

289

Galactosamine-Induced Sensitization to the Lethal Effects of Endotoxin  

Microsoft Academic Search

Treatment of rabbits, rats, and mice with D-galactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysacharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before

Chris Galanos; Marina A. Freudenberg; Werner Reutter

1979-01-01

290

Analysis of NSF mutants reveals residues involved in SNAP binding and ATPase stimulation.  

PubMed

N-Ethylmaleimide-sensitive fusion protein (NSF) and its yeast orthologue, Sec18, are cytoplasmic AAA(+) ATPases required for most intracellular membrane fusion events. The primary function of NSF is thought to be the disassembly of cis-SNARE complexes, thus allowing trans-SNARE complex formation and subsequent membrane fusion. The importance of NSF/Sec18 in intracellular membrane traffic in vivo is highlighted by the inhibition of neurotransmission in Drosophila comatose (NSF) mutants and of constitutive secretion in yeast sec18 mutants. However, the underlying biochemical defects in these mutant proteins are largely unknown. Here, we identify the sec18-1 mutation as a G89D substitution in the N domain of Sec18p. This mutation results in an inhibition of the mutant protein's ability to bind to Sec17p (yeast alpha-SNAP). In contrast, engineering the comatose(st53)() mutation (S483L) into mammalian NSF (S491L) has no effect on alpha-SNAP binding. Instead, the stimulation of ATPase activity by alpha-SNAP required for wild-type NSF to disassemble SNARE complexes does not occur in the mutant NSF(st53) protein. This biochemical phenotype predicts a dominant negative effect, which was confirmed by engineering the st53 mutation into Sec18 (A505L), resulting in a dominant lethal phenotype in vivo. These findings suggest a biochemical basis for the block in membrane fusion observed in the mutant organisms. Furthermore, the mutants characterized here define key residues involved in two essential, but mechanistically distinct, biochemical functions of NSF: SNAP binding and SNAP-dependent ATPase stimulation. PMID:11955072

Horsnell, William G C; Steel, Gregor J; Morgan, Alan

2002-04-23

291

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.  

PubMed

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes. PMID:2668746

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

292

Discovery and identification of a novel Ligon lintless-like mutant (Lix) similar to the Ligon lintless (Li1) in allotetraploid cotton.  

PubMed

Mutants are a powerful resource for studying gene structure, function, and evolution. In this present study, a novel Ligon lintless-like mutant (Lix), that has short fibers and deformed leaves and stems, was isolated from the progeny of transgenic cottons. The Lix mutant is similar in morphology to the Ligon lintless (Li1) mutant. Genetic analysis and molecular mapping were performed for the Lix and Li1 mutants. These two mutants are monogenic dominant mutants with distorted growth of vegetative and reproductive structures. Seedlings of the dominant homozygote Li 1 Li 1 genotype are lethal, while LixLix plants are viable but show no reproductive growth. Molecular tagging showed that the Lix gene is located on Chr. 04 in a 30.9-cM region spanned by NAU8376 and NAU3469. In a previous study, the Li 1 gene was mapped to Chr. 22, and Chr. 04 and Chr. 22 are homoelogous chromosomes in tetraploid cotton. So, we propose that Lix and Li1 mutants have similar mutated morphology, and Lix is mapped to a homoelogous chromosome carrying Li 1 . The identification and genetic mapping of Lix/Li 1 genes using mutants provides a foundation for isolating these genes. In turn, this will permit studies to elucidate the functional and evolutionary roles for these genes in cotton growth and development. PMID:23397278

Cai, Caiping; Tong, Xiangchao; Liu, Fengju; Lv, Fenni; Wang, Haihai; Zhang, Tianzhen; Guo, Wangzhen

2013-04-01

293

Altered lipid composition in Streptococcus pneumoniae cpoA mutants  

PubMed Central

Background Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. Results We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as ?-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg2+ at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. Conclusions The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic. PMID:24443834

2014-01-01

294

Problem-Solving Test: Tryptophan Operon Mutants  

ERIC Educational Resources Information Center

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…

Szeberenyi, Jozsef

2010-01-01

295

A Multivariate Model of Stakeholder Preference for Lethal Cat Management  

PubMed Central

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

Wald, Dara M.; Jacobson, Susan K.

2014-01-01

296

Burkholderia pseudomallei Known Siderophores and Hemin Uptake Are Dispensable for Lethal Murine Melioidosis  

PubMed Central

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 a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth. PMID:22745846

Kvitko, Brian H.; Goodyear, Andrew; Propst, Katie L.; Dow, Steven W.; Schweizer, Herbert P.

2012-01-01

297

The repair of sub-lethal damage and the stimulated repair of potentially lethal damage in Saintpaulia.  

PubMed

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

Leenhouts, H P; Sijsma, M J; Litwiniszyn, M; Chadwick, K H

1981-10-01

298

Development of a non-lethal method for evaluating transcriptomic endpoints in Arctic grayling (Thymallus arcticus).  

PubMed

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. PMID:24780232

Veldhoen, Nik; Beckerton, Jean E; Mackenzie-Grieve, Jody; Stevenson, Mitchel R; Truelson, Robert L; Helbing, Caren C

2014-07-01

299

Disruption of TTDA Results in Complete Nucleotide Excision Repair Deficiency and Embryonic Lethality  

PubMed Central

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda?/?) mouse-model resembling TTD-A patients. Unexpectedly, Ttda?/? mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda?/? cells was not affected. Surprisingly, Ttda?/? cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda?/? cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda?/? cells as a unique class of TFIIH mutants. PMID:23637614

Theil, Arjan F.; Nonnekens, Julie; Steurer, Barbara; Mari, Pierre-Olivier; de Wit, Jan; Lemaitre, Charlene; Marteijn, Jurgen A.; Raams, Anja; Maas, Alex; Vermeij, Marcel; Essers, Jeroen; Hoeijmakers, Jan H. J.; Giglia-Mari, Giuseppina; Vermeulen, Wim

2013-01-01

300

Interaction of the sex-lethal RNA binding domains with RNA.  

PubMed Central

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo. Images PMID:7556096

Kanaar, R; Lee, A L; Rudner, D Z; Wemmer, D E; Rio, D C

1995-01-01

301

Adenovirus-Mediated Gene Delivery Rescues a Neonatal Lethal Murine Model of mut0 Methylmalonic Acidemia  

PubMed Central

Methylmalonic acidemia (MMA), an autosomal recessive metabolic disorder, is most often caused by mutations in methylmalonyl-CoA mutase (MUT). Severely affected patients typically present with metabolic crisis in the early neonatal period and can perish despite intervention. Survivors follow an unstable course and can require elective liver transplantation to prevent life-threatening metabolic decompensation. Therapeutic alternatives to liver transplantation such as hepatocyte-directed gene and cell therapies lack experimental validation. We have used a murine model of mut0 MMA to assess the efficacy of virus-mediated gene therapy to rescue the neonatal lethality seen in the Mut?/? mice. Affected pups and control littermates received either intramuscular or intrahepatic injections of adenovirus carrying the Mut gene expressed under the control of the cytomegalovirus promoter. All of the Mut?/? pups injected via the intramuscular route perished within the first 48 hr of birth. However, more than 50% of the Mut?/? pups that received intrahepatic injections survived beyond weaning (day 15). The treated mutants expressed methylmalonyl-CoA mutase mRNA and protein, and displayed decreased metabolite levels compared with uninjected Mut?/? mice. The results demonstrate that adenovirus-mediated, hepatic methylmalonyl-CoA mutase expression can rescue Mut?/? pups from neonatal mortality and provide proof-of-principle evidence for the efficacy of liver-directed gene delivery in methylmalonic acidemia. PMID:18052792

CHANDLER, RANDY J.; VENDITTI, CHARLES P.

2009-01-01

302

Suppression of a Lethal Trisomic Phenotype in Drosophila melanogaster by Increased Dosage of an Unlinked Locus  

Microsoft Academic Search

One of the most extreme examples of gene dosage sensitivity is the Triplo-lethal locus (Tpl) on the third chromosome of Drosophila melanogaster, which is lethal when present in either one or three copies. Increased dosage of an unlinked locus, Zsis, suppresses the triplo-lethal phenotype of Tpl, but not the haplo-lethal phenotype. We have mapped Zsis to the X chromosome region

Douglas R. Dorer; Marilyn A. Cadden; Beth Gordesky-Gold; Garth Harries; Alan C. Christensen

1993-01-01

303

9 CFR 430.4 - Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products.  

... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products...hazard that an establishment producing post-lethality exposed RTE products...

2014-01-01

304

9 CFR 430.4 - Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products.  

Code of Federal Regulations, 2010 CFR

... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products...hazard that an establishment producing post-lethality exposed RTE products...

2010-01-01

305

9 CFR 430.4 - Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products.  

Code of Federal Regulations, 2011 CFR

... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products... Control of Listeria monocytogenes in post-lethality exposed ready-to-eat products...hazard that an establishment producing post-lethality exposed RTE products...

2011-01-01

306

Characterization of an avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phospholipases.  

PubMed Central

An avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis subsp. gelechiae, isolated by Heierson et al. (A. Heierson, I. Sidén, A. Kivaisi, and H. G. Boman, J. Bacteriol. 167:18-24, 1986) as a spontaneous phage-resistant mutant, was further characterized and found to lack the expression of phosphatidylcholine- and phosphatidylinositol-degrading phospholipase C, beta-lactamase, and flagellin because of the absence of corresponding mRNAs. The avirulent mutant was also found to be less efficient in killing insect cells in vitro than the wild type and to have altered behavior in vivo; wild-type B. thuringiensis does not circulate in the insect hemolymph after injection, whereas the avirulent mutant and nonpathogenic control bacteria remain in circulation. Flagella and motility may be important for virulence in the early stages of an infection; mutants with decreased motility appear less virulent when fed to Trichoplusia ni but not when injected. The 50% lethal doses of wild-type strain Bt13 and avirulent mutant strain Bt1302 were estimated to be 0.52 +/- 0.25 and 2,600 +/- 1,300 CFU per injected larva, respectively. Images PMID:7693592

Zhang, M Y; Lovgren, A; Low, M G; Landen, R

1993-01-01

307

Lethal and sublethal effects of spinosad on Spodoptera exigua (Lepidoptera: Noctuidae).  

PubMed

As a selective biological insecticide, spinosad has been used widely for the control of pests including beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). To form effective pest control strategies, lethal and sublethal effects should be considered for a complete analysis of spinosad impact. However, few studies have been reported to investigate sublethal effects of spinosad on S. exigua. This study attempts to evaluate the lethal and sublethal effects of spinosad on this pest by recording and analyzing various toxicological and physiological parameters. The toxicity of spinosad against S. exigua was determined under laboratory conditions by oral exposure of late second-instar larvae to the compound. The LC50 values of spinosad to S. exigua at 48 and 72 h after treatment were 0.317 and 0.293 mg x kg(-1), respectively. Spinosad at sublethal concentrations significantly extended the developmental period of survivor larvae, and reduced larval wet weight. Postexposure effects were indicated by decreased pupation ratio and pupal weight, by prolonged prepupal and pupal periods and by decreased emergence ratio, fecundity and longevity of adults. The net replacement rate (Ro) tended to be lower in the exposed spinosad groups than those in the unexposed spinosad group. Intrinsic rate of population increase (r(m)) for the high-dose group (0.365) was significantly lower than the control (0.521) and the low-dose group (0.521), but the latter two were not significantly difference. These results suggest that the combination of lethal and sublethal effects of spinosad might affect S. exigua population dynamics significantly by decreasing its survival and reproduction, and by delaying its development. PMID:24020299

Wang, Dong; Wang, Yong-Ming; Liu, Hui-Yuan; Xin, Zheng; Xue, Ming

2013-08-01

308

Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants  

E-print Network

and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant

Oregon, University of

309

Rat survival to anthrax lethal toxin is likely controlled by a single gene  

Microsoft Academic Search

We examined whether survival of different rat strains administered anthrax lethal toxin is genetically determined. A reproducible test population of first filial generation hybrid rats was bred based on the susceptibility of progenitors to anthrax lethal toxin and to maximize genetic diversity across the strains. These rats were then tested with varying doses of anthrax lethal toxin. We found that

S H Nye; A L Wittenburg; D L Evans; J A O'Connor; R J Roman; H J Jacob

2008-01-01

310

Assessment of Cocaethylene Lethality in Long–Evans Female and Male Rats  

Microsoft Academic Search

Cocaethylene, the metabolite of cocaine produced only in the presence of alcohol, produces a number of pharmacological, physiological, and behavioral effects. It also has a range of toxicological consequences, the most severe being lethality. Given that the assessments of cocaethylene lethality have been limited to mice, the present study assessed the lethality of cocaethylene in rats. Further, because of within-species

B.-F. X. Sobel; A. C. Hutchinson; H. F. Diamond; S. A. Etkind; S. D. Ziervogel; C. M. Ferrari; A. L. Riley

1998-01-01

311

The Concept of Synthetic Lethality in the Context of Anticancer Therapy  

Microsoft Academic Search

Two genes are synthetic lethal if mutation of either alone is compatible with viability but mutation of both leads to death. So, targeting a gene that is synthetic lethal to a cancer-relevant mutation should kill only cancer cells and spare normal cells. Synthetic lethality therefore provides a conceptual framework for the development of cancer-specific cytotoxic agents. This paradigm has not

William G. Kaelin Jr

2005-01-01

312

Mutants resistant to anti-microtubule herbicides map to a locus on the uni linkage group in Chlamydomonas reinhardtii  

SciTech Connect

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.

James, S.W.; Ranum, L.P.W.; Silflow, C.D.; Lefebvre, P.A.

1988-01-01

313

Chromosomal localization, hematologic characterization, and iron metabolism of the hereditary erythroblastic anemia (hea) mutant mouse.  

PubMed

Understanding iron metabolism has been enhanced by identification of genes for iron deficiency mouse mutants. We characterized the genetics and iron metabolism of the severe anemia mutant hea (hereditary erythroblastic anemia), which is lethal at 5 to 7 days. The hea mutation results in reduced red blood cell number, hematocrit, and hemoglobin. The hea mice also have elevated Zn protoporphyrin and serum iron. Blood smears from hea mice are abnormal with elevated numbers of smudge cells. Aspects of the hea anemia can be transferred by hematopoietic stem cell transplantation. Neonatal hea mice show a similar hematologic phenotype to the flaky skin (fsn) mutant. We mapped the hea gene near the fsn locus on mouse chromosome 17 and show that the mutants are allelic. Both tissue iron overloading and elevated serum iron are also found in hea and fsn neonates. There is a shift from iron overloading to iron deficiency as fsn mice age. The fsn anemia is cured by an iron-supplemented diet, suggesting an iron utilization defect. When this diet is removed there is reversion to anemia with concomitant loss of overloaded iron stores. We speculate that the hea/fsn gene is required for iron uptake into erythropoietic cells and for kidney iron reabsorption. PMID:15155459

White, Robert A; McNulty, Steven G; Roman, Shelly; Garg, Uttam; Wirtz, Eric; Kohlbrecher, Deanna; Nsumu, Ndona N; Pinson, David; Gaedigk, Roger; Blackmore, Krista; Copple, Angela; Rasul, Sidrah; Watanabe, Masayo; Shimizu, Koji

2004-09-01

314

A Saccharomyces cerevisiae genome-wide mutant screen for altered sensitivity to K1 killer toxin.  

PubMed Central

Using the set of Saccharomyces cerevisiae mutants individually deleted for 5718 yeast genes, we screened for altered sensitivity to the antifungal protein, K1 killer toxin, that binds to a cell wall beta-glucan receptor and subsequently forms lethal pores in the plasma membrane. Mutations in 268 genes, including 42 in genes of unknown function, had a phenotype, often mild, with 186 showing resistance and 82 hypersensitivity compared to wild type. Only 15 of these genes were previously known to cause a toxin phenotype when mutated. Mutants for 144 genes were analyzed for alkali-soluble beta-glucan levels; 63 showed alterations. Further, mutants for 118 genes with altered toxin sensitivity were screened for SDS, hygromycin B, and calcofluor white sensitivity as indicators of cell surface defects; 88 showed some additional defect. There is a markedly nonrandom functional distribution of the mutants. Many genes affect specific areas of cellular activity, including cell wall glucan and mannoprotein synthesis, secretory pathway trafficking, lipid and sterol biosynthesis, and cell surface signal transduction, and offer new insights into these processes and their integration. PMID:12663529

Page, Nicolas; Gerard-Vincent, Manon; Menard, Patrice; Beaulieu, Maude; Azuma, Masayuki; Dijkgraaf, Gerrit J P; Li, Huijuan; Marcoux, Jose; Nguyen, Thuy; Dowse, Tim; Sdicu, Anne-Marie; Bussey, Howard

2003-01-01

315

Systematic Triple Mutant Analysis Uncovers Functional Connectivity Between Pathways Involved in Chromosome Regulation  

PubMed Central

Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed Triple Mutant Analysis (TMA). We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1? cac1? to TMA, we found that the Swi/Snf Rdh54 protein, compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1? strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered using traditional double mutant analyses. PMID:23746449

Haber, James E.; Braberg, Hannes; Wu, Qiuqin; Alexander, Richard; Haase, Julian; Ryan, Colm; Lipkin-Moore, Zach; Franks-Skiba, Kathleen E.; Johnson, Tasha; Shales, Michael; Lenstra, Tineke L.; Holstege, Frank C. P.; Johnson, Jeffrey R.; Bloom, Kerry; Krogan, Nevan J.

2013-01-01

316

Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically.  

PubMed Central

Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed. PMID:369552

Giordano, G; Grillet, L; Rosset, R; Dou, J H; Azoulay, E; Haddock, B A

1978-01-01

317

Intact alternation performance in high lethality suicide attempters.  

PubMed

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

Keilp, John G; Wyatt, Gwinne; Gorlyn, Marianne; Oquendo, Maria A; Burke, Ainsley K; John Mann, J

2014-09-30

318

5-Lipoxygenase Deficiency Reduces Acetaminophen-Induced Hepatotoxicity and Lethality  

PubMed Central

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-1?, TNF-?, IFN-?, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-?-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

Hohmann, Miriam S. N.; Cardoso, Renato D. R.; Pinho-Ribeiro, Felipe A.; Crespigio, Jefferson; Cunha, Thiago M.; Alves-Filho, Jose C.; da Silva, Rosiane V.; Pinge-Filho, Phileno; Ferreira, Sergio H.; Cunha, Fernando Q.; Casagrande, Rubia; Verri, Waldiceu A.

2013-01-01

319

Lethality of the morphinan isomers levorphanol and dextrorphan  

PubMed Central

Significantly different (P<0·05) LD50 values were found in Swiss-Webster mice for levorphanol (73 mg/kg, i.p.) and dextrorphan (120 mg/kg, i.p.). A subcutaneous injection of naloxone 15 min before challenge prevented the lethal effect of an LD98 of levorphanol, with ED50 value of 1·36 mg/kg. Naloxone, in doses from 2 to 100 mg/kg, did not prevent death caused by 150 mg/kg of either dextrorphan or levorphanol. Levorphanol was lethal for mice pretreated with 10 mg/kg of naloxone, a dose sufficient to block opiate-specific lethal effects, but the LD50 value was 109 mg/kg, in contrast to 73 mg/kg in the absence of naloxone. By the criteria of stereospecificity and naloxone blockade, levorphanol-induced mortality in mice is a typical opiate effect in the lower of the two dose ranges studied. At higher doses of levorphanol a non-specific effect supervenes, with an LD50 value virtually the same as that of dextrorphan. PMID:4788214

Dingledine, R.; Goldstein, A.

1973-01-01

320

Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels.  

PubMed

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 ?g/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 ?g/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 consequences in the entire ecosystem. The biomarker activities measured in daphnids and fish seemed to be useful tools in the assessment of trichlorfon effects, especially ChE activity which was the most sensitive biomarker tested for all organisms. Trichlorfon was teratogenic for zebrafish embryos leading to anomalies in the absorption of the yolk sac, spine bending and pericardial oedemas. The present research suggests that further work is urgently needed in order to monitor environmental concentrations of trichlorfon and to test the long term effects of environmentally realistic concentrations of this compound. PMID:21473847

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

321

Intraguild relationships between sympatric predators exposed to lethal control: predator manipulation experiments  

PubMed Central

Introduction Terrestrial top-predators are expected to regulate and stabilise food webs through their consumptive and non-consumptive effects on sympatric mesopredators and prey. The lethal control of top-predators has therefore been predicted to inhibit top-predator function, generate the release of mesopredators and indirectly harm native fauna through trophic cascade effects. Understanding the outcomes of lethal control on interactions within terrestrial predator guilds is important for zoologists, conservation biologists and wildlife managers. However, few studies have the capacity to test these predictions experimentally, and no such studies have previously been conducted on the eclectic suite of native and exotic, mammalian and reptilian taxa we simultaneously assess. We conducted a series of landscape-scale, multi-year, manipulative experiments at nine sites spanning five ecosystem types across the Australian continental rangelands to investigate the responses of mesopredators (red foxes, feral cats and goannas) to contemporary poison-baiting programs intended to control top-predators (dingoes) for livestock protection. Result Short-term behavioural releases of mesopredators were not apparent, and in almost all cases, the three mesopredators we assessed were in similar or greater abundance in unbaited areas relative to baited areas, with mesopredator abundance trends typically either uncorrelated or positively correlated with top-predator abundance trends over time. The exotic mammals and native reptile we assessed responded similarly (poorly) to top-predator population manipulation. This is because poison baits were taken by multiple target and non-target predators and top-predator populations quickly recovered to pre-control levels, thus reducing the overall impact of baiting on top-predators and averting a trophic cascade. Conclusions These results are in accord with other predator manipulation experiments conducted worldwide, and suggest that Australian 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

2013-01-01

322

The occurrence of new mutants in the X-linked recessive Lesch-Nyhan disease.  

PubMed Central

In a population at equilibrium for a sex-linked lethal, one-third of the genes for that lethal must arise anew each generation. Therefore, one-third of all cases of Lesch-Nyhan disease, a severe X-linked recessive lethal disorder, should be new mutants. To test this hypothesis, we have collected 47 families, 20 with a single proband and 27 with multiple affected males in which the patients' mothers and other female relatives had been studied for heterozygosity. Available carrier detection tests identify heterozygous for HPRT deficiency in hair roots and skin fibroblasts. Only four mothers were found not to be carriers. This result deviates significantly from expected (P less than .001). Statistical tests for ascertainment effects indicated absence of bias for multiple proband families but strong bias in favor of families with many heterozygous females. When the analysis was limited to single proband families, the deviation from expected was still significant (P less than .01). The incidence of new mutants among the heterozygous mothers, as determined by the ratio of +/+ to +/- maternal grandmothers, should be one-half (see Appendix). Of all 20 maternal grandmothers studied, five were +/+ and 15 were +/- (P less than .05). Considering only the single proband families, the ratio of 5 +/+ to 8 +/- was not significantly different from expected. In four of the five cases in which the heterozygous mother of an affected individual was a new mutation, the age of her parents was considerably higher than the mean parental age in the population. This raises the possibility of a paternal age effect on X-linked mutations. There appears to be a true deficiency of new mutatnts among males but not among females. Data on additional Lesch-Nyhan families are needed before conclusions regarding a possible higher mutation rate in males can be drawn. PMID:1266847

Francke, U; Felsenstein, J; Gartler, S M; Migeon, B R; Dancis, J; Seegmiller, J E; Bakay, F; Nyhan, W L

1976-01-01

323

Electromagnetic pulse (EMP) coupling codes for use with the vulnerability/lethality (VIL) taxonomy. Final report, June-October 1984  

SciTech Connect

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.

Mar, M.H.

1995-07-01

324

Reduction of renal mass is lethal in mice lacking vimentin. Role of endothelin-nitric oxide imbalance.  

PubMed Central

Modulation of vascular tone by chemical and mechanical stimuli is a crucial adaptive phenomenon which involves cytoskeleton elements. Disruption, by homologous recombination, of the gene encoding vimentin, a class III intermediate filament protein mainly expressed in vascular cells, was reported to result in apparently normal phenotype under physiological conditions. In this study, we evaluated whether the lack of vimentin affects vascular adaptation to pathological situations, such as reduction of renal mass, a pathological condition which usually results in immediate and sustained vasodilation of the renal vascular bed. Ablation of 3/4 of renal mass was constantly lethal within 72 h in mice lacking vimentin (Vim-/-), whereas no lethality was observed in wild-type littermates. Death in Vim-/- mice resulted from end-stage renal failure. Kidneys from Vim-/- mice synthesized more endothelin, but less nitric oxide (NO), than kidneys from normal animals. In vitro, renal resistance arteries from Vim-/- mice were selectively more sensitive to endothelin, less responsive to NO-dependent vasodilators, and exhibited an impaired flow (shear stress)- induced vasodilation, which is NO dependent, as compared with those from normal littermates. Finally, in vivo administration of bosentan, an endothelin receptor antagonist, totally prevented lethality in Vim-/- mice. These results suggest that vimentin plays a key role in the modulation of vascular tone, possibly via the tuning of endothelin-nitric oxide balance. PMID:9294120

Terzi, F; Henrion, D; Colucci-Guyon, E; Federici, P; Babinet, C; Levy, B I; Briand, P; Friedlander, G

1997-01-01

325

Analyzing the control of mosquito-borne diseases by a dominant lethal genetic system  

PubMed Central

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 ?1 year is feasible for affected human populations on the order of 105 to 106, 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. PMID:17519336

Atkinson, Michael P.; Su, Zheng; Alphey, Nina; Alphey, Luke S.; Coleman, Paul G.; Wein, Lawrence M.

2007-01-01

326

Severe Hypoglycemia-Induced Lethal Cardiac Arrhythmias Are Mediated by Sympathoadrenal Activation  

PubMed Central

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. ?-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. PMID:23835337

Reno, Candace M.; Daphna-Iken, Dorit; Chen, Y. Stefanie; VanderWeele, Jennifer; Jethi, Krishan; Fisher, Simon J.

2013-01-01

327

Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells.  

PubMed

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

Yip, Shirley S M; Zhou, Meixia; Joly, John; Snedecor, Bradley; Shen, Amy; Crawford, Yongping

2014-09-01

328

Experimental ecology of selected vertebrate species. Final report. [Effects of sub-lethal. gamma. radiation on survival of chipmunks and pocket gophers  

Microsoft Academic Search

This report summarizes the results of a long term (1960 to 1973) study designed to determine the suitability of various vertebrate species for experimental radioecology, to determine their individual and population characteristics under natural conditions, and to utilize these characteristics to gauge the effects of sub-lethal doses of gamma radiation. The study focused on free ranging populations of Tamias striatus

R. T. Hartman; D. L. Graybill

1976-01-01

329

Ubiquitination and Degradation of Mutant p53  

Microsoft Academic Search

While wild-type p53 is normally a rapidly degraded protein, mutant forms of p53 are stabilized and accumulate to high levels in tumor cells. In this study, we show that mutant and wild-type p53 proteins are ubiquitinated and degraded through overlapping but distinct pathways. While Mdm2 can drive the degrada- tion of both mutant and wild-type p53, our data suggest that

Natalia Lukashchuk; Karen H. Vousden

2007-01-01

330

Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras-Mediated Transformation  

Microsoft Academic Search

The oncogenic function of mutant ras in mammalian cells has been extensively investigated using multiple human and animal models. These systems include overexpression of exogenous mutant ras transgenes, conditionally expressed knock-in mouse models, and somatic cell knockout of mutant and wild-type ras genes in human cancer cell lines. However, phenotypic discrepancies between knock-in mice and trans- genic mutant ras overexpression

Hiroyuki Konishi; Bedri Karakas; Abde M. Abukhdeir; Josh Lauring; John P. Gustin; Joseph P. Garay; Yuko Konishi; Eike Gallmeier; Kurtis E. Bachman

2007-01-01

331

A Genetic Screen for High Copy Number Suppressors of the Synthetic Lethality Between elg1? and srs2? in Yeast  

PubMed Central

Elg1 and Srs2 are two proteins involved in maintaining genome stability in yeast. After DNA damage, the homotrimeric clamp PCNA, which provides stability and processivity to DNA polymerases and serves as a docking platform for DNA repair enzymes, undergoes modification by the ubiquitin-like molecule SUMO. PCNA SUMOylation helps recruit Srs2 and Elg1 to the replication fork. In the absence of Elg1, both SUMOylated PCNA and Srs2 accumulate at the chromatin fraction, indicating that Elg1 is required for removing SUMOylated PCNA and Srs2 from DNA. Despite this interaction, which suggests that the two proteins work together, double mutants elg1? srs2? have severely impaired growth as haploids and exhibit synergistic sensitivity to DNA damage and a synergistic increase in gene conversion. In addition, diploid elg1? srs2? double mutants are dead, which implies that an essential function in the cell requires at least one of the two gene products for survival. To gain information about this essential function, we have carried out a high copy number suppressor screen to search for genes that, when overexpressed, suppress the synthetic lethality between elg1? and srs2?. We report the identification of 36 such genes, which are enriched for functions related to DNA- and chromatin-binding, chromatin packaging and modification, and mRNA export from the nucleus. PMID:23704284

Gazy, Inbal; Liefshitz, Batia; Bronstein, Alex; Parnas, Oren; Atias, Nir; Sharan, Roded; Kupiec, Martin

2013-01-01

332

The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen  

PubMed Central

The three proteins that comprise anthrax toxin, edema factor (EF), lethal factor (LF), and protective antigen (PA), assemble at the mammalian cell surface into toxic complexes. After binding to its receptor, PA is proteolytically activated, yielding a carboxyl-terminal 63-kDa fragment (PA63) that coordinates assembly of the complexes, promotes their endocytosis, and translocates EF and LF to the cytosol. PA63 spontaneously oligomerizes to form symmetric ring-shaped heptamers that are capable of binding three molecules of EF and/or LF as competing ligands. To determine whether binding of these ligands depends on oligomerization of PA63, we prepared two oligomerization-deficient forms of this protein, each mutated on a different PA63–PA63 contact face. In solution or when bound to receptors on Chinese hamster ovary K1 cells, neither mutant alone bound ligand, but a mixture of them did. After the two mutants were proteolytically activated and mixed with ligand in solution, a ternary complex was isolated containing one molecule of each protein. Thus EF and LF bind stably only to PA63 dimers or higher order oligomers. These findings are relevant to the kinetics and pathways of assembly of anthrax toxin complexes. PMID:11997437

Mogridge, Jeremy; Cunningham, Kristina; Lacy, D. Borden; Mourez, Michael; Collier, R. John

2002-01-01

333

Loss of vps54 function leads to vesicle traffic impairment, protein mis-sorting and embryonic lethality.  

PubMed

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. PMID:23708095

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

334

Loss of Vps54 Function Leads to Vesicle Traffic Impairment, Protein Mis-Sorting and Embryonic Lethality  

PubMed Central

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. PMID:23708095

Karlsson, Pall; Droce, Aida; Moser, Jakob M.; Cuhlmann, Simon; Padilla, Carolina Ortiz; Heimann, Peter; Bartsch, Jorg W.; Fuchtbauer, Annette; Fuchtbauer, Ernst-Martin; Schmitt-John, Thomas

2013-01-01

335

High throughput RNAi screening identifies ID1 as a synthetic sick/lethal gene interacting with the common TP53 mutation R175H  

PubMed Central

The TP53 mutation (R175H) is one of the most common mutations in human cancer. It is a highly attractive strategy for cancer therapy to find the genes that lead the R175H-expressing cancer cells. The aim of this study was to identify the synthetic sick/lethal gene interacting with R175H. Using lentiviral bar-coded comprehensive shRNA library and a tetracycline-inducible R175H expressed in the SF126 human glioblastoma cell line (SF126-tet-R175H), we conducted high-throughput screening to identify the candidate genes that induce synthetic sickness/lethality in R175H-expressing cells. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition in the presence of R175H. Inhibitor of differentiation 1 (ID1) was one of the candidate genes, and its suppression by siRNA resulted in the acceleration of growth inhibition in cell lines both transiently and endogenously expressing R175H but not in TP53-null cell lines or other common p53 mutants (such as R273H). Flow cytometry analysis showed that ID1 suppression resulted in G1 arrest, and the arrest was accelerated by the expression of R175H. ID1 is a synthetic sick/lethal gene that interacts with R175H and is considered to be a novel molecular target for cancer therapy in R175H-expressing cells. PMID:24378760

IMAI, HIROO; KATO, SHUNSUKE; SAKAMOTO, YASUHIRO; KAKUDO, YUICHI; SHIMODAIRA, HIDEKI; ISHIOKA, CHIKASHI

2014-01-01

336

Control of Free Methionine Production in Wild Type and Ethionine-resistant Mutants of Chlorella sorokiniana  

PubMed Central

Mutants of Chlorella sorokiniana selected for resistance to the methionine analogue ethionine took up ethionine at the same rate as did the wild type strain. Cells of two ethionine-resistant mutants produced severalfold higher levels of free methionine and cysteine than did wild type cells. Exogenous methionine had no apparent effect on free methionine production in a mutant that produces excessive levels of free methionine. Under the same conditions, production of free methionine was relatively inhibited in wild type cells and in a mutant that produces wild type levels of free methionine. The results suggest that free methionine production in the wild type strain is subject to endproduct control, and that this control is lacking in one class of ethionine-resistant mutants. PMID:16658726

Sloger, Marcia; Owens, Lowell D.

1974-01-01

337

Genome stability in the uvh6 mutant of Arabidopsis thaliana.  

PubMed

Plant XPD homolog UVH6 is the protein involved in the repair of strand breaks, and the excision repair and uvh6 mutant is not impaired in transgenerational increase in HRF. While analyzing the transgenerational response to stress in plants, we found that the promoter and gene body of Arabidopsis thaliana (Arabidopsis) XPD homolog UVH6 underwent hypomethylation and showed an increase in the level of transcript. Here, we analyzed the mutant of this gene, uvh6-1, by crossing it to two different reporter lines: one which allows for analysis of homologous recombination frequency (HRF) and another which makes it possible to analyze the frequency of point mutations. We observed that uvh6-1 plants exhibited lower rate of spontaneous homologous recombination but higher frequencies of spontaneous point mutations. The analysis of strand breaks using ROPS and Comet assays showed that the mutant had a much higher level of strand breaks at non-induced conditions. Exposure to stresses such as UVC, heat, cold, flood and drought showed that the mutant was not impaired in an increase in somatic HRF. The analysis of spontaneous HRF in the progeny of control plants compared to that of the progeny of stressed plants demonstrated that uvh6-1 was mildly affected in response to temperature, UV and drought. Our data suggest that UVH6 may be involved in the repair of strand breaks and excision repair, but it is unlikely that UVH6 is required for transgenerational increase in HRF. PMID:24553752

Bilichak, Andriy; Yao, Youli; Titov, Viktor; Golubov, Andrey; Kovalchuk, Igor

2014-06-01

338

The Alternative Oxidase AOX Does Not Rescue the Phenotype of tko25t Mutant Flies  

PubMed Central

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

Kemppainen, Kia K.; Kemppainen, Esko; Jacobs, Howard T.

2014-01-01

339

Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants  

PubMed Central

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1? cells. In searching for spontaneous suppressors of synthetic lethality of tsa1? rad51? double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1? mutant displayed a CanR mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1? mutant reduced substantially the CanR mutation rate of tsa1? strain (33-fold), and to a lesser extent, of rad51? strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1? -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1? trx2? cells, Yap1 was only moderately activated; consistently, the trx1? trx2? double deletion failed to efficiently rescue the viability of tsa1? rad51?. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1? and trx1? trx2? strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations. PMID:25247923

Ragu, Sandrine; Dardalhon, Michele; Sharma, Sushma; Iraqui, Ismail; Buhagiar-Labarchede, Geraldine; Grondin, Virginie; Kienda, Guy; Vernis, Laurence; Chanet, Roland; Kolodner, Richard D.; Huang, Meng-Er; Faye, Gerard

2014-01-01

340

Preparation and characterization of cobalt-substituted anthrax lethal factor  

SciTech Connect

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.

Saebel, Crystal E.; Carbone, Ryan; Dabous, John R.; Lo, Suet Y. [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)] [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada); Siemann, Stefan, E-mail: ssiemann@laurentian.ca [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)] [Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Rd., Sudbury, Ontario, Canada P3E 2C6 (Canada)

2011-12-09

341

Left ventricular function during lethal and sublethal endotoxemia in swine  

SciTech Connect

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.

Goldfarb, R.D.; Nightingale, L.M.; Kish, P.; Weber, P.B.; Loegering, D.J.

1986-08-01

342

Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles  

Microsoft Academic Search

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

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

2006-01-01

343

EXCALIBIR - A space experiment in orbital debris lethality  

NASA Astrophysics Data System (ADS)

The study proposes a space experiment using extended Space Shuttle external tanks to test the impact of orbital debris. The External Tank Calibrated Impact Response test, EXCALIBIR, is a low-cost low-risk, high-payoff approach to investigating the threat to resident space objects posed by untrackable orbital debris, to provide lethality data to the kinetic energy weapons community, and to aid in the testing of space and missile interceptor technology. This experiment is a feasible use of existing assets - the external tank, observation and data collection facilities, launch facilities, and interceptor technology and tests planned for other programs.

Culp, Robert D.; Dickey, Michael R.

344

Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome.  

PubMed Central

A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants. PMID:9724628

Rubin, D M; Glickman, M H; Larsen, C N; Dhruvakumar, S; Finley, D

1998-01-01

345

X-ray survival characteristics and genetic analysis for nine saccharomyces deletion mutants that show altered radiation sensitivity  

SciTech Connect

The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X-rays, we are screening these mutants to identify additional genes that show increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype co-segregates with the deletion allele and are obtaining multipoint survival-versus-dose assays in at least two haploid and one homozygous diploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1 and VID21/EAF1, and discuss their potential roles in repair. Eight of these genes have a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, has at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultra-violet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino-acids are also X-ray sensitive, seeming to confirm that methylation of the lysine-79 residue is required for effective repair of radiation damage.

Game, John C.; Williamson, Marsha S.; Baccari, Clelia

2004-01-07

346

Suppression of autolysis and cell wall turnover in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.  

PubMed Central

Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation in buffer and for cell wall turnover during growth. The normal (relatively fast) autolysis and turnover rates of the parent strain were retained in a Tn551 mutant in which the insert was located within the mec gene and which produced undetectable levels of penicillin-binding protein 2A. On the other hand, autolysis and cell wall turnover rates were greatly reduced in auxiliary mutants, i.e., mutants in which the transposon caused conversion of the high-level and uniform resistance of the parent strain to a variety of distinct heterogeneous expression types and greatly decreased resistance levels. All of these mutants contained an intact mec gene and produced normal amounts of penicillin-binding protein 2A, and one of the mutations was located in the femA region of the staphylococcal chromosome (B. Berger-Bachi, L. Barberis-Maino, A. Strassle, and F. H. Kayser, Mol. Gen. Genet. 219:263-269, 1989). Autolysis rates were related to the degree of residual methicillin resistance and to the sites of Tn551 insertion. Fast cell wall turnover may help expression of high-level methicillin resistance by providing a mechanism for the excision of abnormal (and potentially lethal) structural elements of the cell wall synthesized by the bacteria in the presence of methicillin. Images PMID:1846855

de Jonge, B L; de Lencastre, H; Tomasz, A

1991-01-01

347

Nebulin binding impedes mutant desmin filament assembly  

PubMed Central

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle. PMID:23615443

Baker, Laura K.; Gillis, David C.; Sharma, Sarika; Ambrus, Andy; Herrmann, Harald; Conover, Gloria M.

2013-01-01

348

Red Mutant Hunt with Saccharomyces cerevisiae  

NSDL National Science Digital Library

In this laboratory exercise, haploid yeast are exposed to UV radiation (UV-C) to induce mutations. Survivors are screened for red adenie mutants when characterized for nutritional requirements. Mutants produced in both mating types are crossed to determine inheritance patterns.

Brad Williamson (Olathe East High School;)

1999-01-01

349

Conditional Deletion of Jak2 Reveals an Essential Role in Hematopoiesis throughout Mouse Ontogeny: Implications for Jak2 Inhibition in Humans  

PubMed Central

Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at ?2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans. PMID:23544085

Park, Sung O.; Wamsley, Heather L.; Bae, Kyungmi; Hu, Zhongbo; Li, Xiaomiao; Choe, Se-woon; Slayton, William B.; Oh, S. Paul; Wagner, Kay-Uwe; Sayeski, Peter P.

2013-01-01

350

Stress Granule-Defective Mutants Deregulate Stress Responsive Transcripts  

PubMed Central

To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants. Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress. PMID:25375155

Yang, Xiaoxue; Hao, Xinxin; Krumlinde, Daniel; Cvijovi?, Marija; Arens, Christina; Nyström, Thomas; Liu, Beidong; Sunnerhagen, Per

2014-01-01

351

The effect of sexual harassment on lethal mutation rate in female Drosophila melanogaster.  

PubMed

The rate by which new mutations are introduced into a population may have far-reaching implications for processes at the population level. Theory assumes that all individuals within a population have the same mutation rate, but this assumption may not be true. Compared with individuals in high condition, those in poor condition may have fewer resources available to invest in DNA repair, resulting in elevated mutation rates. Alternatively, environmentally induced stress can result in increased investment in DNA repair at the expense of reproduction. Here, we directly test whether sexual harassment by males, known to reduce female condition, affects female capacity to alleviate DNA damage in Drosophila melanogaster fruitflies. Female gametes can repair double-strand DNA breaks in sperm, which allows manipulating mutation rate independently from female condition. We show that male harassment strongly not only reduces female fecundity, but also reduces the yield of dominant lethal mutations, supporting the hypothesis that stressed organisms invest relatively more in repair mechanisms. We discuss our results in the light of previous research and suggest that social effects such as density and courtship can play an important and underappreciated role in mediating condition-dependent mutation rate. PMID:23173200

Maklakov, Alexei A; Immler, Simone; Løvlie, Hanne; Flis, Ilona; Friberg, Urban

2013-01-01

352

THE PROPORTION OF TERRAMYCIN-RESISTANT MUTANTS IN B. MEGATHERIUM CULTURES  

PubMed Central

The number of terramycin-resistant mutants in Bacillus megatherium cultures, their mutation rate, and the growth rate of the wild and mutant cells have been determined under various conditions. These values are in agreement with the following equations (Northrop and Kunitz, 1957):— See PDF for Equation ? = mutation rate, A = growth rate constant of wild cells, B = growth rate constant of mutants, See PDF for Equation equilibrium. The value of the mutation rate as determined from equation (6) agrees with that found by the null fraction method. PMID:13463274

Northrop, John H.

1957-01-01

353

Overexpression of inactive arylsulphatase mutants and in vitro activation by light-dependent oxidation with vanadate  

PubMed Central

Arylsulphatases B (ASB) and A (ASA) are subject to a unique post-translational modification that is required for their function. The modification reaction, conversion of an active-site cysteine into a formylglycine, becomes saturated when these enzymes are overexpressed. We have removed the possibility of in vivo modification by expressing mutants of ASB and ASA in which the active-site cysteine is substituted with a serine. These mutants are expressed much more efficiently when compared with the native enzymes under identical conditions. The purified ASB mutant can then be converted into catalytically active ASB in vitro using vanadate and light. PMID:15175008

2004-01-01

354

Concomitant Lethal Mutagenesis of Human Immunodeficiency Virus Type 1  

PubMed Central

RNA virus population dynamics is complex, and sophisticated approaches are needed in many cases for therapeutic intervention. One such approach, termed lethal mutagenesis, is directed at targeting the virus population structure for extinction or error catastrophe. Previous studies have demonstrated the concept of this approach with human immunodeficiency virus type 1 (HIV-1) by use of chemical mutagens (i.e., 5-azacytidine) as well as by host factors with mutagenic properties (i.e., APOBEC3G). In this study, these two unrelated mutagenic agents were used concomitantly to investigate the interplay of these distinct mutagenic mechanisms. Specifically, an HIV-1 was produced from APOBEC3G (A3G)-expressing cells and used to infect permissive target cells treated with 5-azacytidine (5-AZC). Reduced viral infectivity and increased viral mutagenesis was observed with both the viral mutagen (i.e., G-to-C mutations) and the host restriction factor (i.e., G-to-A mutations); however, when combined, had complex interactions. Intriguingly, nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-AZC and A3G resulted in an increase of G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. A3G catalytic activity was required for the diminution in G-to-C mutagenesis. Taken together, our findings provide the first demonstration for potentiation of the mutagenic effect of a cytosine analog by A3G expression, resulting in concomitant HIV-1 lethal mutagenesis. PMID:22426127

Dapp, Michael J.; Holtz, Colleen M.; Mansky, Louis M.

2012-01-01

355

Effectiveness of lethal, directed wolf-depredation control in Minnesota  

USGS Publications Warehouse

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.

Harper, E. K.; Paul, W. J.; Mech, L. D.; Weisberg, S.

2008-01-01

356

An outbreak of lethal adenovirus infection among different otariid species.  

PubMed

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

Inoshima, Yasuo; Murakami, Tomoaki; Ishiguro, Naotaka; Hasegawa, Kazuhiro; Kasamatsu, Masahiko

2013-08-30

357

Injury Risk Assessment of Non-Lethal Projectile Head Impacts  

PubMed Central

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.

Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

2014-01-01

358

Injury risk assessment of non-lethal projectile head impacts.  

PubMed

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

Oukara, Amar; Nsiampa, Nestor; Robbe, Cyril; Papy, Alexandre

2014-01-01

359

Loss of Desmoplakin Tail Causes Lethal Acantholytic Epidermolysis Bullosa*  

PubMed Central

The cytoplasmic plaque protein desmoplakin (DP), which is located in desmosomes, plays a major role in epithelial and muscle cell adhesion by linking the transmembrane cadherins to the cytoplasmic intermediate filament network. Mutations of DP may cause striate palmoplantar keratoderma, arrhythmogenic right ventricular dysplasia, skin fragility/woolly hair syndrome, Naxos-like disease, and Carvajal syndrome. DP must be indispensable, because DP-/- mice are early abortive. Here, we report a patient with severe fragility of skin and mucous membranes caused by genetic truncation of the DP tail. The new phenotype is lethal in the neonatal period because of immense transcutaneous fluid loss. The phenotype also comprised universal alopecia, neonatal teeth, and nail loss. Histology showed suprabasal clefting and acantholysis throughout the spinous layer, mimicking pemphigus. Electron microscopy revealed disconnection of keratin intermediate filaments from desmosomes. Immunofluorescence staining of DP showed a distinct punctate intercellular pattern in the patient’s skin. Protein analysis revealed expression of truncated DP polypeptides. Mutational analysis of the patient demonstrated compound heterozygosity for two DP mutations, 6079C?T (R1934X) and 6370delTT, respectively. Aberrant mRNA transcripts that predict premature termination of translation with loss of the three intermediate filament-binding subdomains in the DP tail were detected by RT-PCR. The new dramatic phenotype, which we named “lethal acantholytic epidermolysis bullosa,” underscores the paramount role of DP in epidermal integrity. PMID:16175511

Jonkman, Marcel F.; Pasmooij, Anna M. G.; Pasmans, Suzanne G. M. A.; van den Berg, Maarten P.; ter Horst, Henk J.; Timmer, Albertus; Pas, Hendri H.

2005-01-01

360

Suppressive Effects of Anthrax Lethal Toxin on Megakaryopoiesis  

PubMed Central

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34+ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax. PMID:23555687

Lin, Guan-Ling; Wang, Tsung-Pao; Lai, Yi-Ling; Lin, Ting-Kai; Hsieh, Ming-Chun; Kau, Jyh-Hwa; Huang, Hsin-Hsien; Hsu, Hui-Ling; Liao, Chi-Yuan; Sun, Der-Shan

2013-01-01

361

Suppressive effects of anthrax lethal toxin on megakaryopoiesis.  

PubMed

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34(+) cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax. PMID:23555687

Chen, Po-Kong; Chang, Hsin-Hou; Lin, Guan-Ling; Wang, Tsung-Pao; Lai, Yi-Ling; Lin, Ting-Kai; Hsieh, Ming-Chun; Kau, Jyh-Hwa; Huang, Hsin-Hsien; Hsu, Hui-Ling; Liao, Chi-Yuan; Sun, Der-Shan

2013-01-01

362

Lethal Nipah Virus Infection Induces Rapid Overexpression of CXCL10  

PubMed Central

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. PMID:22393386

Mathieu, Cyrille; Guillaume, Vanessa; Sabine, Amelie; Ong, Kien Chai; Wong, Kum Thong; Legras-Lachuer, Catherine; Horvat, Branka

2012-01-01

363

HLA-A2-Restricted Protection against Lethal Lymphocytic Choriomeningitis? †  

PubMed Central

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8+ T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides—nucleoprotein NP69-77, glycoprotein precursor GPC10-18, GPC447-455, and zinc-binding protein Z49-58—that displayed high-affinity binding (?275 nM) to HLA-A*0201, induced CD8+ T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC447-455), after peptide immunization of HLA-A*0201 mice, induced CD8+ T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV. PMID:17166907

Botten, Jason; Whitton, J. Lindsay; Barrowman, Polly; Sidney, John; Whitmire, Jason K.; Alexander, Jeff; Ting, Joey P. C.; Bui, Huynh-Hoa; Sette, Alessandro; Buchmeier, Michael J.

2007-01-01

364

Genetic and phenotypic analysis of flagellar assembly mutants in Chlamydomonas reinhardtii.  

PubMed

Conditional mutants for flagellar assembly (fla) provide a useful tool to study intraflagellar transport (IFT) at the molecular level, and provide a unique set of tools to analyze cilia. The analysis of IFT phenotypes of fla mutants at the permissive temperature by a quantitative image analysis approach identified four distinct phases of the IFT cycle and directly demonstrated structural and functional remodeling of IFT particles at both axonemal extremities. In addition, the genetic analysis of fla mutants reveal interesting interactions among genes involved in flagellar assembly that help to provide information about the structure and function of IFT particles and their motors. This chapter provides protocols to isolate, characterize, and identify conditional Chlamydomonas flagellar assembly mutants and their genes and to test genetic interactions among proteins encoded by these genes. PMID:20409815

Iomini, Carlo; Till, Jacob E; Dutcher, Susan K

2009-01-01

365

Analysis of Escherichia coli Mutants with a Linear Respiratory Chain  

PubMed Central

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important. PMID:24475268

Steinsiek, Sonja; Stagge, Stefan; Bettenbrock, Katja

2014-01-01

366

[Locus Adh and adaptation of cn and vg mutants in the experimental populations of Drosophila melanogaster Meig].  

PubMed

Complex study of adaptation and allozyme belonging of alcoholdehydrogenase (ADH) in cn and vg mutants has been carried out in the initial pure lines, in their panmixia populations and in condition of substitution of the mutant genotype by saturating crossings. It was shown that the high level of adaptation of cn mutants and the low level of adaptation of vg mutants was combined with the presence of different ADH allozymes. During the saturating crossings the reliable coadaptation of the genes cn and Adh(S) as well as vg and Adh(F) was detected that confirmes the postulated earlier conception of gene adaptation complexes. PMID:17494340

Belokon', S V; Khaystova, N D; Totski?, V N

2007-01-01

367

Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L. ) deficient in chloroplast phosphoglucomutase activity  

Microsoft Academic Search

A mutant of Arabidopsis thaliana (L.) Heynh. which lacks leaf starch was isolated by screening for plants which did not stain with iodine. When grown in a 12-h photoperiod, leaves of the wild-type accumulated substantial amounts of starch but lower levels of soluble sugars. Under these conditions, the mutant accumulated relatively high levels of soluble sugars. Rates of growth and

T. Caspar; S. C. Huber; C. Somerville

1985-01-01

368

How many loci on the X-chromosome of Drosophila melanogaster can mutate to recessive lethals  

SciTech Connect

The sensitivity of the sex-linked recessive lethal test is due to the fact that a very large number of loci are included in the mutation study. From extensive studies on the spontaneous sex-linked recessive lethal frequency and spontaneous specific locus mutation rates, it is possible to derive an estimate of the number of loci included in the recessive lethal test. The average number derived from three estimates on male and female germ cells in 563 loci. A second independent approach derives from published data which analyzed short regions of the genome and the proportion of loci within these regions which mutate to lethality. This analysis suggests that 830 loci are potentially lethal mutables. We describe the reasons for concluding that 600 to 800 loci of the approximately 1000 loci on the X-chromosome are involved in the X-linked recessive lethal test.

Abrahamson, S. (Univ. of Wisconsin, Madison); Wuergler, F.E.; DeJongh, C.; Meyer, H.U.

1980-01-01

369

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer  

PubMed Central

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. PMID:24711431

McFadden, David G.; Vernon, Amanda; Santiago, Philip M.; Martinez-McFaline, Raul; Bhutkar, Arjun; Crowley, Denise M.; McMahon, Martin; Sadow, Peter M.; Jacks, Tyler

2014-01-01

370

Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity.  

PubMed

A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities. PMID:4008442

Expert, D; Toussaint, A

1985-07-01

371

A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth  

PubMed Central

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced. These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56). Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control. PMID:6384238

1984-01-01

372

The Effect of Non-Lethal Weapons on Police Officer Safety  

Microsoft Academic Search

Between 1990 and 2000, there was an increase in the use of non-lethal weapons and a decline in the number and severity of attacks on police officers. Using longitudinal data on several hundred U.S. police agencies, I investigate the relationship between police officer safety and the adoption of non-lethal weapons. I find that the adoption of non-lethal chemical weapons had

Alex Yuskavage

373

Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila.  

PubMed

A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf 01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours. PMID:25116613

Seki, Yuuichi; Tanimura, Teiichi

2014-09-01

374

A Suitable Streptomycin-Resistant Mutant for Constructing Unmarked In-Frame Gene Deletions Using rpsL as a Counter-Selection Marker  

PubMed Central

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD50 (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ?1,000-fold less lethal (?2×105 CFU) and the K87R mutant was ?50-fold less lethal (?1×104 CFU) than the parental strain (?2×102 CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P<0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker. PMID:25268958

Tsai, Yu-Kuo; Liou, Ci-Hong; Lin, Jung-Chung; Ma, Ling; Fung, Chang-Phone; Chang, Feng-Yee; Siu, L. Kristopher

2014-01-01

375

Deficits in memory and motor performance in synaptotagmin IV mutant mice  

PubMed Central

Synaptotagmin (Syt) IV is a synaptic vesicle protein. Syt IV expression is induced in the rat hippocampus after systemic kainic acid treatment. To examine the functional role of this protein in vivo, we derived Syt IV null [Syt IV(?/?)] mutant mice. Studies with the rotorod revealed that the Syt IV mutants have impaired motor coordination, a result consistent with constitutive Syt IV expression in the cerebellum. Because Syt IV is thought to modulate synaptic function, we also have examined Syt IV mutant mice in learning and memory tests. Our studies show that the Syt IV mutation disrupts contextual fear conditioning, a learning task sensitive to hippocampal and amygdala lesions. In contrast, cued fear conditioning is normal in the Syt IV mutants, suggesting that this mutation did not disrupt amygdala function. Conditioned taste aversion, which also depends on the amygdala, is normal in the Syt IV mutants. Consistent with the idea that the Syt IV mutation preferentially affects hippocampal function, Syt IV mutant mice also display impaired social transmission of food preference. These studies demonstrate that Syt IV is critical for brain function and suggest that the Syt IV mutation affects hippocampal-dependent learning and memory, as well as motor coordination. PMID:10792055

Ferguson, Gregory D.; Anagnostaras, Stephan G.; Silva, Alcino J.; Herschman, Harvey R.

2000-01-01

376

Impulsivity, aggression and brain structure in high and low lethality suicide attempters with borderline personality disorder.  

PubMed

Impulsivity and aggressiveness are trait dispositions associated with the vulnerability to suicidal behavior across diagnoses. They are associated with structural and functional abnormalities in brain networks involved in regulation of mood, impulse and behavior. They are also core characteristics of borderline personality disorder (BPD), a disorder defined, in part, by recurrent suicidal behavior. We assessed the relationships between personality traits, brain structure and lethality of suicide attempts in 51 BPD attempters using multiple regression analyses on structural MRI data. BPD was diagnosed by the Diagnostic Interview for Borderline Patients-revised, impulsivity by the Barratt Impulsiveness Scale (BIS), aggression by the Brown-Goodwin Lifetime History of Aggression (LHA), and high lethality by a score of 4 or more on the Lethality Rating Scale (LRS). Sixteen High Lethality attempters were compared to 35 Low Lethality attempters, with no significant differences noted in gender, co-morbidity, childhood abuse, BIS or LHA scores. Degree of medical lethality (LRS) was negatively related to gray matter volumes across multiple fronto-temporal-limbic regions. Effects of impulsivity and aggression on gray matter volumes discriminated High from Low Lethality attempters and differed markedly within lethality groups. Lethality of suicide attempts in BPD may be related to the mediation of these personality traits by specific neural networks. PMID:24656768

Soloff, Paul; White, Richard; Diwadkar, Vaibhav A

2014-06-30

377

Risks of non-lethal weapon use: case studies of three French victims of stinger grenades.  

PubMed

The development of non-lethal weapons started in the 1960s. In France, they have been used by the police for about 10 years. We relate the cases of three French women, victims of stinger grenades, non-lethal weapons recently adopted by the French law enforcement to distract and disperse crowds. The three victims presented serious injuries requiring emergency surgical care. One lost her eye. Based on these cases, we discuss the lethal character of these weapons and propose measures to be taken to prevent their dramatic consequences. Although the danger is obviously less than for firearms, stinger grenades are nonetheless potentially lethal and cause serious physical injuries. PMID:22981215

Scolan, V; Herry, C; Carreta, M; Stahl, C; Barret, L; Romanet, J P; Paysant, F

2012-11-30

378

Loss of ceramide synthase 3 causes lethal skin barrier disruption.  

PubMed

The stratum corneum as the outermost epidermal layer protects against exsiccation and infection. Both the underlying cornified envelope (CE) and the intercellular lipid matrix contribute essentially to these two main protective barriers. Epidermis-unique ceramides with ultra-long-chain acyl moities (ULC-Cers) are key components of extracellular lipid lamellae (ELL) and are bound to CE proteins, thereby contributing to the cornified lipid envelope (CLE). Here, we identified human and mouse ceramide synthase 3 (CerS3), among CerS1-6, to be exclusively required for the ULC-Cer synthesis in vitro and of mouse CerS3 in vivo. Deficiency of CerS3 in mice results in complete loss of ULC-Cers (?C26), lack of continuous ELL and a non-functional CLE. Consequently, newborn mutant mice die shortly after birth from transepidermal water loss. Mutant skin is prone to Candida albicans infection highlighting ULC-Cers to be pivotal for both barrier functions. Persistent periderm, hyperkeratosis and deficient cornification are hallmarks of mutant skin demonstrating loss of Cers to trigger a keratinocyte maturation arrest at an embryonic pre-barrier stage. PMID:22038835

Jennemann, Richard; Rabionet, Mariona; Gorgas, Karin; Epstein, Sharon; Dalpke, Alexander; Rothermel, Ulrike; Bayerle, Aline; van der Hoeven, Franciscus; Imgrund, Silke; Kirsch, Joachim; Nickel, Walter; Willecke, Klaus; Riezman, Howard; Gröne, Hermann-Josef; Sandhoff, Roger

2012-02-01

379

Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS*  

PubMed Central

Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression. PMID:23161513

Demory Beckler, Michelle; Higginbotham, James N.; Franklin, Jeffrey L.; Ham, Amy-Joan; Halvey, Patrick J.; Imasuen, Imade E.; Whitwell, Corbin; Li, Ming; Liebler, Daniel C.; Coffey, Robert J.

2013-01-01

380

CSNK1E/CTNNB1 Are Synthetic Lethal To TP53 in Colorectal Cancer and Are Markers for Prognosis  

PubMed Central

Two genes are called synthetic lethal (SL) if their simultaneous mutations lead to cell death, but each individual mutation does not. Targeting SL partners of mutated cancer genes can kill cancer cells specifically, but leave normal cells intact. We present an integrated approach to uncovering SL pairs in colorectal cancer (CRC). Screening verified SL pairs using microarray gene expression data of cancerous and normal tissues, we first identified potential functionally relevant (simultaneously differentially expressed) gene pairs. From the top-ranked pairs, ~ 20 genes were chosen for immunohistochemistry (IHC) staining in 171 CRC patients. To find novel SL pairs, all 169 combined pairs from the individual IHC were synergistically correlated to five clinicopathological features, e.g. overall survival. Of the 11 predicted SL pairs, MSH2-POLB and CSNK1E-MYC were consistent with literature, and we validated the top two pairs, CSNK1E-TP53 and CTNNB1-TP53 using RNAi knockdown and small molecule inhibitors of CSNK1E in isogenic HCT-116 and RKO cells. Furthermore, synthetic lethality of CSNK1E and TP53 was verified in mouse model. Importantly, multivariate analysis revealed that CSNK1E-P53, CTNNB1-P53, MSH2-RB1, and BRCA1-WNT5A were independent prognosis markers from stage, with CSNK1E-P53 applicable to early-stage and the remaining three throughout all stages. Our findings suggest that CSNK1E is a promising target for TP53-mutant CRC patients which constitute ~ 40% to 50% of patients, while to date safety regarding inhibition of TP53 is controversial. Thus the integrated approach is useful in finding novel SL pairs for cancer therapeutics, and it is readily accessible and applicable to other cancers. PMID:24947187

Tiong, Khong-Loon; Chang, Kuo-Ching; Yeh, Kun-Tu; Liu, Ting-Yuan; Wu, Jia-Hong; Hsieh, Ping-Heng; Lin, Shu-Hui; Lai, Wei-Yun; Hsu, Yu-Chin; Chen, Jeou-Yuan; Chang, Jan-Gowth; Shieh, Grace S.

2014-01-01

381

Lethal sex: conditions of disclosure in counseling sexually active clients with HIV.  

PubMed

Confidentiality in psychological counseling is necessary if clients are to feel comfortable in revealing their darkest secrets. But this bond of trust has its moral limits. These limits are crossed in some cases in which HIV positive clients are sexually active with unsuspecting third parties. Distinguishing between Type 1 and Type 2 cases, the author shows how he has used applied ethics in drafting and defending a model rule for the American Counseling Association's Code of Ethics that permits, and sometimes morally requires disclosure in the former cases, but not in the latter. PMID:15462041

Cohen, Elliot D

2003-01-01

382

Combining Inhibitor Resistance-conferring Mutations in Cytochrome b Creates Conditional Synthetic Lethality in  

E-print Network

inhibitors. Inhibitor titrations of bc1 complex activities in mitochondrial membranes from the various yeast for the utilization of center N as a potential drug target. The mitochondrial cytochrome bc1 complex is an essential mitochondrial membrane, thereby forming the electrochemical gradient needed for the production of ATP (1

Trumpower, Bernard L.

383

Dominant lethal test in rats treated with some plant extracts.  

PubMed

The present study was undertaken to investigate the toxic effect of aqueous extracts of Aegle marmelos (AM), Stevia rebaudiana (SR), Pouteria cambodiana (PC) and Clausena excavata (CE) on rats by dominant lethal test. The data of 8-week treatment suggested that none of the extracts adversely affected male body and testicular weights as well as cauda epididymal sperm counts. No notable changes in sperm morphology and motility were observed. On the other hand, sperm count in the CE group was significantly higher as compared to both control and other treatment groups. There were no abnormal changes in the number of implantation sites, number of viable fetuses and number of dead fetuses in females mated with plant extract-treated males relative to controls. Based on these results, it could be concluded that all the investigated plant extracts have no toxic effect on male rat reproduction and progeny outcome. PMID:11414451

Aritajat, S; Kaweewat, K; Manosroi, J; Manosroi, A

2000-01-01

384

Aroclor 1254 residues in birds: Lethal levels and loss rates  

USGS Publications Warehouse

Lethal residues of polychlorinated biphenyls (PCBs) were determined experimentally in four species of wild birds (male common grackles (Quiscalus quiscula ), immature female red-winged blackbirds (Agelaius phoeniceus ), adult male brown-headed cowbirds (Molothrus ater ) and immature female starlings (Sturnus vulgaris)) given dietary dosage of 1,500 ppm of Aroclor 1254) until one-half had died, sacrificing the survivors, chemically analyzing the tissues, and comparing results in dead birds and survivors. For all species, residues of 310 ppm or higher in the brain showed increasing likelihood of death from PCB poisoning. Residues in dead birds did not differ among species except for starlings (Sturnus vulgaris ), which averaged slightly lower than the others. However, the species differed in the length of time to 50% mortality and in the levels of PCBs in brains at sacrifice.

Stickel, W.H.; Stickel, L.F.; Dyrland, R.A.; Hughes, D.L.

1984-01-01

385

Non-lethal laser dazzling as a personnel countermeasure  

NASA Astrophysics Data System (ADS)

Optical distraction is likely one of the original and simpler optical countermeasure concepts with a technology history dating back to the 1800's. The objective is to distract or suppress either equipment or personnel with optical radiation from a safe distance. This paper is intended to review and expand on the concepts presented at the 2012 SPIE Security and Defense meeting; "Non-Lethal Optical Interruption (Dazzling): Technology, Devices, and Scenarios". The information that follows will focus primarily on the technology and techniques associated with the safe laser dazzling of personnel. Key product design guidelines will be highlighted and reviewed. Recent advances in laser technology and their associated impact on hand-held devices will also be discussed. Finally, the author will offer his opinion on the growth rate of military and non-military markets for laser dazzlers.

Shannon, David C.

2013-10-01

386

Clinical Effects and Lethal and Forensic Aspects of Propofol*  

PubMed Central

Propofol is a potent intravenous anesthetic agent that rapidly induces sedation and unconsciousness. The potential for propofol dependency, recreational use and abuse has only recently been recognized and several cases of accidental overdose and suicide have emerged. In addition, the first documented case of murder using propofol was reported a few months ago and a high profile case of suspected homicide with propofol is currently under investigation. A number of analytical methods have been employed to detect and quantify propofol concentrations in biological specimens. The reported propofol related deaths and post-mortem blood and tissue levels are reviewed. Importantly, limitations of propofol detection are discussed and future considerations are presented. Because propofol has the potential for diversion with lethal consequences, the forensic scientist must have a basic understanding of its clinical indications and uses, pharmacologic properties, and detection methods. In addition, medical institutions should develop systems to prevent and detect diversion of this potential drug of abuse. PMID:20950316

Levy, Richard J.

2010-01-01

387

Batrachochytrium dendrobatidis infection and lethal chytridiomycosis in caecilian amphibians (Gymnophiona).  

PubMed

Batrachochytrium dendrobatidis (Bd) is commonly termed the 'amphibian chytrid fungus' but thus far has been documented to be a pathogen of only batrachian amphibians (anurans and caudatans). It is not proven to infect the limbless, generally poorly known, and mostly soil-dwelling caecilians (Gymnophiona). We conducted the largest qPCR survey of Bd in caecilians to date, for more than 200 field-swabbed specimens from five countries in Africa and South America, representing nearly 20 species, 12 genera, and 8 families. Positive results were recovered for 58 specimens from Tanzania and Cameroon (4 families, 6 genera, 6+ species). Quantities of Bd were not exceptionally high, with genomic equivalent (GE) values of 0.052-17.339. In addition, we report the first evidence of lethal chytridiomycosis in caecilians. Mortality in captive (wild-caught, commercial pet trade) Geotrypetes seraphini was associated with GE scores similar to those we detected for field-swabbed, wild animals. PMID:23677560

Gower, David J; Doherty-Bone, Thomas; Loader, Simon P; Wilkinson, Mark; Kouete, Marcel T; Tapley, Benjamin; Orton, Frances; Daniel, Olivia Z; Wynne, Felicity; Flach, Edmund; Müller, Hendrik; Menegon, Michele; Stephen, Ian; Browne, Robert K; Fisher, Mathew C; Cunningham, Andrew A; Garner, Trenton W J

2013-06-01

388

Case of Desbuquois dysplasia type 1: potentially lethal skeletal dysplasia.  

PubMed

We report a boy with Desbuquois dysplasia type 1. He had the typical skeletal changes: a "Swedish key" appearance of the proximal femora; advanced carpal ossification and other distinctive features of the hand, including an extra-ossification center at the base of the proximal phalanx of the index and middle fingers; dislocation of the metacarpophalangeal joint of the index finger; and bifid distal phalanx of the thumb. In addition, he presented with very severe prenatal growth failure, respiratory distress as a neonate, subsequent failure to thrive and susceptibility to airway infection, and sudden death in early childhood. Molecular analysis identified homozygous 1 bp deletion in the Calcium-Activated Nucleotidase 1 gene (CANT1). To our knowledge, this is the first report of Desbuquois dysplasia type 1 in Japan. Our experience suggests potential lethality in the disorder. PMID:25252066

Inoue, Shinkai; Ishii, Atsushi; Shirotani, Goro; Tsutsumi, Makoto; Ohta, Eiji; Nakamura, Masatoshi; Mori, Toshiko; Inoue, Takahito; Nishimura, Gen; Ogawa, Atsushi; Hirose, Shinichi

2014-08-01

389

Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt  

PubMed Central

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ?-Km interposon within the hrp gene cluster of each strain. The resulting Hrp- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants. Images PMID:16349373

Frey, Pascal; Prior, Philippe; Marie, Corinne; Kotoujansky, Alain; Trigalet-Demery, Daniele; Trigalet, Andre

1994-01-01

390

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium  

SciTech Connect

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study the authors isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity ({sup 14}C-labeled synthetic lignin {yields}{sup 14}CO{sub 2}) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidases, a key H{sub 2}O{sub 2}-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.

Boominathan, K.; Dass, S.B.; Randall, T.A.; Kelley, R.L.; Reddy, C.A. (Michigan State Univ., East Lansing (USA))

1990-01-01

391

Lysosome and Phagosome Stability in Lethal Cell Injury  

PubMed Central

In two types of cell injury in a tissue culture system, the possibility was tested that lysosome rupture may be a lethal cellular reaction to injury, and thus an important general cause of irreversibility of damage in injured tissue. Prior labeling of secondary lysosomes with the fluorochrome acridine orange, or with ferritin, was used to trace changes in lysosomes after applying an injury. The metabolic inhibitors iodoacetate and cyanide were used together to block the cell's energy supply, or attachment of antiserum and subsequent complement attack were used to damage the surface membrane, producing rapid loss of cell volume control. Living cells were studied by time-lapse phase-contrast cinemicrography and fluorescence microscopy, and samples were fixed at intervals for electron microscopy. The cytolytic action of complement was lethal to sensitized cells within 2 hours, but results showed that lysosomes did not rupture for approximately 4 hours and in fact did not release the fluorescent dye until after reaching the postmortem necrotic phase of injury. Cells treated with metabolic inhibitors also showed irreversible alterations, while lysosomes remained intact and retained the ferritin marker. The fluorochrome marker, acridine orange, escaped from lysosomes early after metabolic injury, but the significance of this observation is not clear. The results are interpreted as evidence against the concept that lysosome rupture threatens the survival of injured cells. The original suicide bag mechanism of cell damage thus is apparently not operative in the systems studied. Lysosomes appear to be relatively stable organelles which, following injury of the types studied, burst only after cell death, acting then as scavengers which help to clear cellular debris. ImagesFigs 5-7Fig 18Fig 19Fig 20Figs 21-23Fig 8Fig 9Fig 10Fig 11Figs 24-27Fig 12Figs 13 and 14Fig 1Fig 2Fig 3Fig 4Fig 15Fig 16Fig 17 PMID:4340333

Hawkins, Hal K.; Ericsson, Jan L. E.; Biberfeld, Peter; Trump, Benjamin F.

1972-01-01

392

Recombinant Thrombomodulin Protects Mice against Histone-Induced Lethal Thromboembolism  

PubMed Central

Introduction Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. Methods Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. Results Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. Conclusions Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC. PMID:24098750

Kawahara, Ko-ichi; Yamamoto, Mika; Nagasato, Tomoka; Shrestha, Binita; Yamada, Shingo; Miyauchi, Takahiro; Higuchi, Koji; Takenaka, Toshihiro; Yasuda, Tomotsugu; Matsunaga, Akira; Kakihana, Yasuyuki; Hashiguchi, Teruto; Kanmura, Yuichi; Maruyama, Ikuro

2013-01-01

393

Antibiotics induce redox-related physiological alterations as part of their lethality.  

PubMed

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. PMID:24803433

Dwyer, Daniel J; Belenky, Peter A; Yang, Jason H; MacDonald, I Cody; Martell, Jeffrey D; Takahashi, Noriko; Chan, Clement T Y; Lobritz, Michael A; Braff, Dana; Schwarz, Eric G; Ye, Jonathan D; Pati, Mekhala; Vercruysse, Maarten; Ralifo, Paul S; Allison, Kyle R; Khalil, Ahmad S; Ting, Alice Y; Walker, Graham C; Collins, James J

2014-05-20

394

Mutant p53: one name, many proteins  

PubMed Central

There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function. PMID:22713868

Freed-Pastor, William A.; Prives, Carol

2012-01-01

395

A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12.  

PubMed

The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart. PMID:16677308

Boonsombat, Ruethairat; Yeh, Su-Ping; Milne, Amy; Sandler, Steven J

2006-05-01

396

Evaluating the lethal and pre-lethal effects of a range of fungi against adult Anopheles stephensi mosquitoes  

PubMed Central

Background Insecticide resistance is seriously undermining efforts to eliminate malaria. In response, research on alternatives to the use of chemical insecticides against adult mosquito vectors has been increasing. Fungal entomopathogens formulated as biopesticides have received much attention and have shown considerable potential. This research has necessarily focused on relatively few fungal isolates in order to ‘prove concept’. Further, most attention has been paid to examining fungal virulence (lethality) and not the other properties of fungal infection that might also contribute to reducing transmission potential. Here, a range of fungal isolates were screened to examine variation in virulence and how this relates to additional pre-lethal reductions in feeding propensity. Methods The Asian malaria vector, Anopheles stephensi was exposed to 17 different isolates of entomopathogenic fungi belonging to species of Beauveria bassiana, Metarhizium anisopliae, Metarhizium acridum and Isaria farinosus. Each isolate was applied to a test substrate at a standard dose rate of 1×109 spores ml-1 and the mosquitoes exposed for six hours. Subsequently the insects were removed to mesh cages where survival was monitored over the next 14 days. During this incubation period the mosquitoes’ propensity to feed was assayed for each isolate by offering a feeding stimulant at the side of the cage and recording the number probing. Results and conclusions Fungal isolates showed a range of virulence to A. stephensi with some causing >80% mortality within 7 days, while others caused little increase in mortality relative to controls over the study period. Similarly, some isolates had a large impact on feeding propensity, causing >50% pre-lethal reductions in feeding rate, whereas other isolates had very little impact. There was clear correlation between fungal virulence and feeding reduction with virulence explaining nearly 70% of the variation in feeding reduction. However, there were some isolates where either feeding decline was not associated with high virulence, or virulence did not automatically prompt large declines in feeding. These results are discussed in the context of choosing optimum fungal isolates for biopesticide development. PMID:23126549

2012-01-01

397

Gravitropism in a starchless mutant of Arabidopsis  

Microsoft Academic Search

The starch-statolith theory of gravity reception has been tested with a mutant of Arabidopsis thaliana (L.) Heynh. which, lacking plastid phosphoglucomutase (EC 2.7.5.1) activity, does not synthesize starch. The hypocotyls and seedling roots of the mutant were examined by light and electron microscopy to confirm that they did not contain starch. In upright wild-type (WT) seedlings, starch-filled plastids in the

Timothy Caspar; Barbara G. Pickard

1989-01-01

398

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster  

Microsoft Academic Search

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation . At 6 h after the removal of purines from the growth medium, there is a decrease in semicon- servative DNA replication . Alkaline velocity centrifugation of the DNA synthe- sized during a 1-min pulse under conditions of

MARIA ZANNIS-HADJOPOULOS; MILTON W. TAYLOR; ROGER HAND

1980-01-01

399

Isolation of Cyanide Hydratase Mutants from Gloeocerospora Sorghi at alkaline pH  

E-print Network

random mutagenesis of the target fungal gene using error-prone polymerase chain reaction and an in vivo picric acid assay that tests the activity of the mutant enzymes at target conditions. Experimentation was used to determine the ideal conditions for a...

Lessen, Henry Joseph

2013-02-04

400

Pollen embryogenesis to induce, detect, and analyze mutants.  

PubMed Central

The development of fully differentiated plants from individual pollen grains through a series of developmental phases that resemble embryogenesis beginning with the zygote was demonstrated during the mid-1960's. This technology opened the door to the use of haploid plants (sporophytes with the gametic number of chromosomes) for plant breeding and genetic studies, biochemical and metabolic studies, and the selection of mutations. Although pollen embryogenesis has been demonstrated successfully in numerous plant genera, the procedure cannot as yet be used routinely to generate large populations of plants for experiments. Practical results from use of the technology in genetic toxicology research to detect mutations have failed to fully realize the theoretical potential; further developments of the technology could overcome the limitations. Pollen embryogenesis could be used to develop plants from mutant pollen grains to verify that genetic changes are involved. Through either spontaneous or induced chromosome doubling, these plants can be made homozygous and used to analyze genetically the mutants involved. The success of this approach will depend on the mutant frequency relative to the fraction of pollen grains that undergo embryogenesis; these two factors will dictate population size needed for success. Research effort is needed to further develop pollen embryogenesis for use in the detection of genotoxins under both laboratory and in situ conditions. PMID:7460882

Constantin, M J

1981-01-01

401

Genetic analysis of salt-tolerant mutants in Arabidopsis thaliana.  

PubMed Central

Stress caused by the increased salinity of irrigated fields impairs plant growth and is one of the major constraints that limits crop productivity in many important agricultural areas. As a contribution to solving such agronomic problems, we have carried out a large-scale screening for Arabidopsis thaliana mutants induced on different genetic backgrounds by EMS treatment, fast neutron bombardment, or T-DNA insertions. From the 675,500 seeds we screened, 17 mutant lines were isolated, all but one of which yielded 25-70% germination levels on 250 mm NaCl medium, a condition in which their ancestor ecotypes are unable to germinate. Monogenic recessive inheritance of NaCl-tolerant germination was displayed with incomplete penetrance by all the selected mutants, which fell into five complementation groups. These were named SALOBRENO (SAN) and mapped relative to polymorphic microsatellites, the map positions of three of them suggesting that they are novel genes. Strains carrying mutations in the SAN1-SAN4 genes display similar responses to both ionic effects and osmotic pressure, their germination being NaCl and mannitol tolerant but KCl and Na(2)SO(4) sensitive. In addition, NaCl-, KCl-, and mannitol-tolerant as well as abscisic-acid-insensitive germination was displayed by sañ5, whose genetic and molecular characterization indicates that it carries an extremely hypomorphic or null allele of the ABI4 gene, its deduced protein product lacking the APETALA2 DNA binding domain. PMID:10629000

Quesada, V; Ponce, M R; Micol, J L

2000-01-01

402

Light-Induced Acclimation of the Arabidopsis chlorina1 Mutant to Singlet Oxygen[C][W  

PubMed Central

Singlet oxygen (1O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1O2-induced transcriptomic changes result in acclimation to 1O2. Here, using a chlorophyll b–less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1O2 formation, indicating acclimation to 1O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient