Science.gov

Sample records for a53t mutant form

  1. Controlling aggregation propensity in A53T mutant of alpha-synuclein causing Parkinson's disease

    SciTech Connect

    Kumar, Sonu; Sarkar, Anita; Sundar, Durai

    2009-09-18

    Understanding {alpha}-synuclein in terms of fibrillization, aggregation, solubility and stability is fundamental in Parkinson's disease (PD). The three familial mutations, namely, A30P, E46K and A53T cause PD because the hydrophobic regions in {alpha}-synuclein acquire {beta}-sheet configuration, and have a propensity to fibrillize and form amyloids that cause cytotoxicity and neurodegeneration. On simulating the native form and mutants (A30P, E46K and A53T) of {alpha}-synuclein in water solvent, clear deviations are observed in comparison to the all-helical 1XQ8 PDB structure. We have identified two crucial residues, {sup 40}Val and {sup 74}Val, which play key roles in {beta}-sheet aggregation in the hydrophobic regions 36-41 and 68-78, respectively, leading to fibrillization and amyloidosis in familial (A53T) PD. We have also identified V40D{sub V}74D, a double mutant of A53T (the most amyloidogenic mutant). The simultaneous introduction of these two mutations in A53T nearly ends its aggregation propensity, increases its solubility and positively enhances its thermodynamic stability.

  2. Mutant A53T α-Synuclein Improves Rotarod Performance Before Motor Deficits and Affects Metabolic Pathways.

    PubMed

    Guerreiro, Patrícia S; Coelho, Joana E; Sousa-Lima, Inês; Macedo, Paula; Lopes, Luísa V; Outeiro, Tiago F; Pais, Teresa F

    2017-03-01

    The protein α-synuclein (α-Syn) interferes with glucose and lipid uptake and also activates innate immune cells. However, it remains unclear whether α-Syn or its familial mutant forms contribute to metabolic alterations and inflammation in synucleinopathies, such as Parkinson's disease (PD). Here, we address this issue in transgenic mice for the mutant A53T human α-Syn (α-SynA53T), a mouse model of synucleinopathies. At 9.5 months of age, mice overexpressing α-SynA53T (homozygous) had a significant reduction in weight, exhibited improved locomotion and did not show major motor deficits compared with control transgenic mice (heterozygous). At 17 months of age, α-SynA53T overexpression promoted general reduction in grip strength and deficient hindlimb reflex and resulted in severe disease and mortality in 50 % of the mice. Analysis of serum metabolites further revealed decreased levels of cholesterol, triglycerides and non-esterified fatty acids (NEFA) in α-SynA53T-overexpressing mice. In fed conditions, these mice also showed a significant decrease in serum insulin without alterations in blood glucose. In addition, assessment of inflammatory gene expression in the brain showed a significant increase in TNF-α mRNA but not of IL-1β induced by α-SynA53T overexpression. Interestingly, the brain mRNA levels of Sirtuin 2 (Sirt2), a deacetylase involved in both metabolic and inflammatory pathways, were significantly reduced. Our findings highlight the relevance of the mechanisms underlying initial weight loss and hyperactivity as early markers of synucleinopathies. Moreover, we found that changes in blood metabolites and decreased brain Sirt2 gene expression are associated with motor deficits.

  3. Metabolic abnormalities and hypoleptinemia in α-synuclein A53T mutant mice.

    PubMed

    Rothman, Sarah M; Griffioen, Kathleen J; Fishbein, Kenneth W; Spencer, Richard G; Makrogiannis, Sokratis; Cong, Wei-Na; Martin, Bronwen; Mattson, Mark P

    2014-05-01

    Parkinson's disease (PD) patients frequently display loss of body fat mass and increased energy expenditure, and several studies have outlined a relationship between these metabolic abnormalities and disease severity, yet energy metabolism is largely unstudied in mouse models of PD. Here we characterize metabolic and physiologic responses to a high calorie diet (HCD) in mice expressing in neurons a mutant form of human α-synuclein (A53T) that causes dominantly inherited familial forms of the disease. A53T (SNCA) and wild type (WT) littermate mice were placed on a HCD for 12 weeks and evaluated for weight gain, food intake, body fat, blood plasma leptin, hunger, glucose tolerance, and energy expenditure. Results were compared with both SNCA and WT mice on a control diet. Despite consuming similar amounts of food, WT mice gained up to 66% of their original body weight on a HCD, whereas SNCA mice gained only 17%. Further, after 12 weeks on a HCD, magnetic resonance imaging analysis revealed that WT mice had significantly greater total and visceral body fat compared with SNCA mice (p < 0.007). At the age of 24 weeks SNCA mice displayed significantly increased hunger compared with WT (p < 0.03). At the age of 36 weeks, SNCA mice displayed significant hypoleptinemia compared with WT, both on a normal diet and a HCD (p < 0.03). The HCD induced insulin insensitivity in WT, but not SNCA mice, as indicated by an oral glucose tolerance test. Finally, SNCA mice displayed greater energy expenditure compared with WT, as measured in a Comprehensive Laboratory Animal Monitoring System, after 12 weeks on a HCD. Thus, SNCA mice are resistant to HCD-induced obesity and insulin resistance and display reduced body fat, increased hunger, hypoleptinemia and increased energy expenditure. Our findings reveal a profile of metabolic dysfunction in a mouse model of PD that is similar to that of human PD patients, thus providing evidence that α-synuclein pathology is sufficient to drive such

  4. Metabolic abnormalities and hypoleptinemia in α-synuclein A53T mutant mice

    PubMed Central

    Rothman, Sarah M.; Griffioen, Kathleen J.; Fishbein, Kenneth W.; Spencer, Richard G.; Makrogiannis, Sokratis; Cong, Wei-na; Martin, Bronwen; Mattson, Mark P.

    2013-01-01

    Parkinson’s disease (PD) patients frequently display loss of body fat mass and increased energy expenditure, and several studies have outlined a relationship between these metabolic abnormalities and disease severity, yet energy metabolism is largely unstudied in mouse models of PD. Here we characterize metabolic and physiologic responses to a high calorie diet (HCD) in mice expressing in neurons a mutant form of human α-synuclein (A53T) that causes dominantly inherited familial forms of the disease. A53T (SNCA) and wild type (WT) littermate mice were placed on a HCD for 12 weeks and evaluated for weight gain, food intake, body fat, blood plasma leptin, hunger, glucose tolerance, and energy expenditure. Results were compared to both SNCA and WT mice on a control diet. Despite consuming similar amounts of food, WT mice gained up to 66% of their original body weight on a HCD whereas SNCA mice gained only 17%. Further, after 12 weeks on a HCD, MRI analysis revealed that WT mice had significantly greater total and visceral body fat compared to SNCA mice (p<0.007). At 24 weeks of age SNCA mice displayed significantly increased hunger compared to WT (p<0.03). At 36 weeks of age, SNCA mice displayed significant hypoleptinemia compared to WT, both on a normal diet and a HCD (p<0.03). The HCD induced insulin insensitivity in WT, but not SNCA mice, as indicated by an oral glucose tolerance test. Finally, SNCA mice displayed greater energy expenditure compared to WT, as measured in a Comprehensive Lab Animal Monitoring System, after 12 weeks on a HCD. Thus, SNCA mice are resistant to HCD-induced obesity and insulin resistance and display reduced body fat, increased hunger, hypoleptinemia and increased energy expenditure. Our findings reveal a profile of metabolic dysfunction in a mouse model of PD that is similar to that of human PD patients, thus providing evidence that α-synuclein pathology is sufficient to drive such metabolic abnormalities and providing an animal

  5. Structures and free energy landscapes of the A53T mutant-type α-synuclein protein and impact of A53T mutation on the structures of the wild-type α-synuclein protein with dynamics.

    PubMed

    Coskuner, Orkid; Wise-Scira, Olivia

    2013-07-17

    utilizing our new theoretical strategy show that the residual secondary structure conversion stabilities resulting in α-helix formation are not significantly affected by the mutation. Interestingly, the residual secondary structure conversion stabilities show that secondary structure conversions resulting in β-sheet formation are influenced by the A53T mutation and the most stable residual transition yielding β-sheet occurs directly from the coil structure. Long-range interactions detected between the NAC region and the N- or C-terminal regions of the wild-type αS disappear upon A53T mutation. The A53T mutant-type αS structures are thermodynamically more stable than those of the wild-type αS protein structures in aqueous solution. Overall, the higher propensity of the A53T mutant-type αS protein to aggregate in comparison to the wild-type αS protein is related to the increased β-sheet formation and lack of strong intramolecular long-range interactions in the N-terminal region in comparison to its wild-type form. The specific residual secondary structure component stabilities reported herein provide information helpful for designing and synthesizing small organic molecules that can block the β-sheet forming residues, which are reactive toward aggregation.

  6. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in Microglia: Stronger Effects of the A53T Mutant

    PubMed Central

    Hoenen, Claire; Gustin, Audrey; Birck, Cindy; Kirchmeyer, Mélanie; Beaume, Nicolas; Felten, Paul; Grandbarbe, Luc; Heuschling, Paul; Heurtaux, Tony

    2016-01-01

    Parkinson’s disease (PD) is histologically described by the deposition of α-synuclein, whose accumulation in Lewy bodies causes dopaminergic neuronal death. Although most of PD cases are sporadic, point mutations of the gene encoding the α-synuclein protein cause inherited forms of PD. There are currently six known point mutations that result in familial PD. Oxidative stress and neuroinflammation have also been described as early events associated with dopaminergic neuronal degeneration in PD. Though it is known that microglia are activated by wild-type α-synuclein, little is known about its mutated forms and the signaling cascades responsible for this microglial activation. The present study was designed to investigate consequences of wild-type and mutant α-synuclein (A53T, A30P and E46K) exposure on microglial reactivity. Interestingly, we described that α-synuclein-induced microglial reactivity appeared to be peptide-dependent. Indeed, the A53T protein activated more strongly microglia than the wild-type α-synuclein and other mutants. This A53T-induced microglial reactivity mechanism was found to depend on phosphorylation mechanisms mediated by MAPKs and on successive NFkB/AP-1/Nrf2 pathways activation. These results suggest that the microgliosis intensity during PD might depend on the type of α-synuclein protein implicated. Indeed, mutated forms are more potent microglial stimulators than wild-type α-synuclein. Based on these data, anti-inflammatory and antioxidant therapeutic strategies may be valid in order to reduce microgliosis but also to subsequently slow down PD progression, especially in familial cases. PMID:27622765

  7. Enhanced Autophagy from Chronic Toxicity of Iron and Mutant A53T α-Synuclein

    PubMed Central

    Chew, Katherine C. M.; Ang, Eng-Tat; Tai, Yee Kit; Tsang, Fai; Lo, Shun Qiang; Ong, Elijah; Ong, Wei-Yi; Shen, Han-Ming; Lim, Kah-Leong; Dawson, Valina L.; Dawson, Ted M.; Soong, Tuck Wah

    2011-01-01

    Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe2+ uptake, which concomitantly promotes cell death. This Fe2+-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe2+-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe2+ overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe2+-mediated cell death. PMID:21795716

  8. Differential effects of wild-type and A53T mutant isoform of alpha-synuclein on the mitochondrial proteome of differentiated SH-SY5Y cells.

    PubMed

    Pennington, Kyla; Peng, Jianhe; Hung, Chao-Chun; Banks, Rosamonde E; Robinson, Philip A

    2010-05-07

    Increased levels of wild-type (WT) alpha-synuclein (alpha-syn) and mutant A53T alpha-syn are associated with Parkinson's disease (PD), a disease linked to abnormal mitochondrial function. This study compared mitochondria prepared from differentiated SH-SY5Y cells overexpressing WT or A53T alpha-syn with control cells, using 2-D difference in-gel electrophoresis. Statistical analysis was carried out primarily using ANOVA (p < 0.01; Host:WT:A53T) and subsequently using independent t tests (host vs WT, host vs A53T). Of the protein spots found to be differentially expressed (n = 71; p < 0.01, >1.8/<-1.8 fold change), 63 proteins were identified by LC-MS/MS, with the majority (77%) significantly altered in WT samples only. Twenty-three proteins known to be integral components of the mitochondria were abnormally expressed including those with roles in ATP synthesis, oxidoreduction, motor activity, carbohydrate metabolism, protein transcription, and protein folding. Thirteen forms of cytoskeletal proteins were also found to be overexpressed in the mitochondrial preparations from WT alpha-syn cells, suggesting an increased interaction of mitochondria with the cytoskeletal network. Altered levels of four mitochondrial proteins (HSPA9 (mortalin), NDUFS1, DLAT, ATP5A1) were confirmed using Western blot analysis. Furthermore, a significant reduction in OXPHOS 1 activity was observed in the WT alpha-syn cells, suggesting that there are functional consequences of the observed altered protein expression changes in the mitochondria.

  9. Potentiation of neurotoxicity in double-mutant mice with Pink1 ablation and A53T-SNCA overexpression

    PubMed Central

    Gispert, Suzana; Brehm, Nadine; Weil, Jonas; Seidel, Kay; Rüb, Udo; Kern, Beatrice; Walter, Michael; Roeper, Jochen; Auburger, Georg

    2015-01-01

    The common age-related neurodegeneration of Parkinson's disease can result from dominant causes like increased dosage of vesicle-associated alpha-synuclein (SNCA) or recessive causes like deficiency of mitophagy factor PINK1. Interactions between these triggers and their convergence onto shared pathways are crucial, but currently conflicting evidence exists. Here, we crossed previously characterized mice with A53T-SNCA overexpression and with Pink1 deletion to generate double mutants (DMs). We studied their lifespan and behavior, histological and molecular anomalies at late and early ages. DM animals showed potentiated phenotypes in comparison with both single mutants (SMs), with reduced survival and strongly reduced spontaneous movements from the age of 3 months onwards. In contrast to SMs, a quarter of DM animals manifested progressive paralysis at ages >1 year and exhibited protein aggregates immunopositive for pSer129-SNCA, p62 and ubiquitin in spinal cord and basal brain. Brain proteome quantifications of ubiquitination sites documented altered degradation of SNCA and the DNA-damage marker H2AX at the age of 18 months. Global brain transcriptome profiles and qPCR validation experiments identified many consistent transcriptional dysregulations already at the age of 6 weeks, which were absent from SMs. The observed downregulations for Dapk1, Dcaf17, Rab42 and the novel SNCA-marker Lect1 as well as the upregulations for Dctn5, Mrpl9, Tmem181a, Xaf1 and H2afx reflect changes in ubiquitination, mitochondrial/synaptic/microtubular/cell adhesion dynamics and DNA damage. Thus, our study confirmed that SNCA-triggered neurotoxicity is exacerbated by the absence of PINK1 and identified a novel molecular signature that is detectable early in the course of this double pathology. PMID:25296918

  10. MicroRNA expressing profiles in A53T mutant alpha-synuclein transgenic mice and Parkinsonian

    PubMed Central

    Mo, Mingshu; Xiao, Yousheng; Huang, Shuxuan; Cen, Luan; Chen, Xiang; Zhang, Limin; Luo, Qin; Li, Shaomin; Yang, Xinling; Lin, Xian; Xu, Pingyi

    2017-01-01

    α-synuclein gene mutations can cause α-synuclein protein aggregation in the midbrain of Parkinson's disease (PD) patients. MicroRNAs (miRNAs) play a key role in the metabolism of α-synuclein but the mechanism involved in synucleinopathy remains unclear. In this study, we investigated the miRNA profiles in A53T-α-synuclein transgenic mice and analyzed the candidate miRNAs in the cerebrospinal fluid (CSF) of PD patients. The 12-month A53T-transgenic mouse displayed hyperactive movement and anxiolytic-like behaviors with α-synuclein aggregation in midbrain. A total of 317,759 total and 289,207 unique small RNA sequences in the midbrain of mice were identified by high-throughput deep sequencing. We found 644 miRNAs were significantly changed in the transgenic mice. Based on the conserved characteristic of miRNAs, we selected 11 candidates from the 40 remarkably expressed miRNAs and explored their expression in 44 CSF samples collected from PD patients. The results revealed that 11 microRNAs were differently expressed in CSF, emphatically as miR-144-5p, miR-200a-3p and miR-542-3p, which were dramatically up-regulated in both A53T-transgenic mice and PD patients, and had a helpful accuracy for the PD prediction. The ordered logistic regression analysis showed that the severity of PD has strong correlation with an up-expression of miR-144-5p, miR-200a-3p and miR-542-3p in CSF. Taken together, our data suggested that miRNAs in CSF, such as miR-144-5p, miR-200a-3p and miR-542-3p, may be useful to the PD diagnosis as potential biomarkers. PMID:27965467

  11. A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T α-Synuclein Toxicity.

    PubMed

    Xiong, Rui; Zhou, Wenbo; Siegel, David; Kitson, Russell R A; Freed, Curt R; Moody, Christopher J; Ross, David

    2015-12-01

    A potential cause of neurodegenerative diseases, including Parkinson's disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  12. A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T α-Synuclein Toxicity

    PubMed Central

    Xiong, Rui; Zhou, Wenbo; Siegel, David; Kitson, Russell R. A.; Freed, Curt R.; Moody, Christopher J.

    2015-01-01

    A potential cause of neurodegenerative diseases, including Parkinson’s disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein. PMID:26405178

  13. Microglial PHOX and Mac-1 are essential to the enhanced dopaminergic neurodegeneration elicited by A30P and A53T mutant alpha-synuclein.

    PubMed

    Zhang, Wei; Dallas, Shannon; Zhang, Dan; Guo, Jian-Ping; Pang, Hao; Wilson, Belinda; Miller, David S; Chen, Biao; Zhang, Wanqin; McGeer, Patrick L; Hong, Jau-Shyong; Zhang, Jing

    2007-08-15

    alpha-Synuclein, a gene whose mutations, duplication, and triplication has been linked to autosomal dominant familial Parkinson's disease (fPD), appears to play a central role in the pathogenesis of sporadic PD (sPD) as well. Enhancement of neurodegeneration induced by mutant alpha-synuclein has been attributed to date largely to faster formation of alpha-synuclein aggregates in neurons. Recently, we reported that microglial activation enhances wild type (WT) alpha-synuclein-elicited dopaminergic neurodegeneration. In the present study, using a primary mesencephalic culture system, we tested whether mutated alpha-synuclein could activate microglia more powerfully than WT alpha-synuclein, thereby contributing to the accelerated neurodegeneration observed in fPD. The results showed that alpha-synuclein with the A30P or A53T mutations caused greater microglial activation than WT alpha-synuclein. Furthermore, the extent of microglial activation paralleled the degree of dopaminergic neurotoxicity induced by WT and mutant alpha-synuclein. Mutant alpha-synuclein also induced greater production of reactive oxygen species than WT alpha-synuclein by NADPH oxidase (PHOX), and PHOX activation was linked to direct activation of macrophage antigen-1 (Mac-1) receptor, rather than alpha-synuclein internalization via scavenger receptors. These results have, for the first time, demonstrated that microglia are also critical in enhanced neurotoxicity induced by mutant alpha-synuclein. (c) 2007 Wiley-Liss, Inc.

  14. Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function.

    PubMed

    Oaks, Adam W; Frankfurt, Maya; Finkelstein, David I; Sidhu, Anita

    2013-01-01

    Expression of A53T mutant human alpha-synuclein under the mouse prion promoter is among the most successful transgenic models of Parkinson's disease. Accumulation of A53T alpha-synuclein causes adult mice to develop severe motor impairment resulting in early death at 8-12 months of age. In younger, pre-symptomatic animals, altered motor activity and anxiety-like behaviors have also been reported. These behavioral changes, which precede severe neuropathology, may stem from non-pathological functions of alpha-synuclein, including modulation of monoamine neurotransmission. Our analysis over the adult life-span of motor activity, anxiety-like, and depressive-like behaviors identifies perturbations both before and after the onset of disease. Young A53T mice had increased distribution of the dopamine transporter (DAT) to the membrane that was associated with increased striatal re-uptake function. DAT function decreased with aging, and was associated with neurochemical alterations that included increased expression of beta-synuclein and gamma synuclein. Prior to normalization of dopamine uptake, transient activation of Tau kinases and hyperphosphorylation of Tau in the striatum were also observed. Aged A53T mice had reduced neuron counts in the substantia nigra pars compacta, yet striatal medium spiny neuron dendritic spine density was largely maintained. These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway.

  15. A53T in a parkinsonian family: a clinical update of the SNCA phenotypes.

    PubMed

    Tambasco, Nicola; Nigro, Pasquale; Romoli, Michele; Prontera, Paolo; Simoni, Simone; Calabresi, Paolo

    2016-11-01

    Approximately 15 % of PD patients with Parkinson Disease (PD) have the familial type and 5-10 % of these are known to have monogenic forms with either an autosomal dominant or a recessive inheritance pattern. Here, we report on a family carrying the A53T SNCA mutation and we review SNCA mutation phenotypes by comparing point mutations within each other as well as with duplication and triplication.

  16. Longitudinal Metabolomics Profiling of Parkinson’s Disease-Related α-Synuclein A53T Transgenic Mice

    PubMed Central

    Chen, Xi; Xie, Chengsong; Sun, Lixin; Ding, Jinhui; Cai, Huaibin

    2015-01-01

    Metabolic homeostasis is critical for all biological processes in the brain. The metabolites are considered the best indicators of cell states and their rapid fluxes are extremely sensitive to cellular changes. While there are a few studies on the metabolomics of Parkinson’s disease, it lacks longitudinal studies of the brain metabolic pathways affected by aging and the disease. Using ultra-high performance liquid chromatography and tandem mass spectroscopy (UPLC/MS), we generated the metabolomics profiling data from the brains of young and aged male PD-related α-synuclein A53T transgenic mice as well as the age- and gender-matched non-transgenic (nTg) controls. Principal component and unsupervised hierarchical clustering analyses identified distinctive metabolites influenced by aging and the A53T mutation. The following metabolite set enrichment classification revealed the alanine metabolism, redox and acetyl-CoA biosynthesis pathways were substantially disturbed in the aged mouse brains regardless of the genotypes, suggesting that aging plays a more prominent role in the alterations of brain metabolism. Further examination showed that the interaction effect of aging and genotype only disturbed the guanosine levels. The young A53T mice exhibited lower levels of guanosine compared to the age-matched nTg controls. The guanosine levels remained constant between the young and aged nTg mice, whereas the aged A53T mice showed substantially increased guanosine levels compared to the young mutant ones. In light of the neuroprotective function of guanosine, our findings suggest that the increase of guanosine metabolism in aged A53T mice likely represents a protective mechanism against neurodegeneration, while monitoring guanosine levels could be applicable to the early diagnosis of the disease. PMID:26317866

  17. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice

    PubMed Central

    Vidal-Martínez, Guadalupe; Vargas-Medrano, Javier; Gil-Tommee, Carolina; Medina, David; Garza, Nathan T.; Yang, Barbara; Segura-Ulate, Ismael; Dominguez, Samantha J.; Perez, Ruth G.

    2016-01-01

    Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS. PMID:27528608

  18. Impairment of mitochondria dynamics by human A53T α-synuclein and rescue by NAP (davunetide) in a cell model for Parkinson's disease.

    PubMed

    Melo, T Q; van Zomeren, K C; Ferrari, M F R; Boddeke, H W G M; Copray, J C V M

    2017-03-01

    The formation of oligomers and aggregates of overexpressed or mutant α-synuclein play a role in the degeneration of dopaminergic neurons in Parkinson's disease by causing dysfunction of mitochondria, reflected in their disturbed mobility and production of ROS. The mode of action and mechanisms underlying this mitochondrial impairment is still unclear. We have induced stable expression of wild-type, A30P or A53T α-synuclein in neuronally differentiated SH-SY5Y neuroblastoma cells and studied anterograde and retrograde mitochondrial trafficking in this cell model for Parkinson's disease. In contrast to wild-type and A30P, A53T α-synuclein significantly inhibited mitochondrial trafficking, at first retrogradely and in a later stage anterogradely. Accordingly, A53T α-synuclein also caused the highest increase in ROS production in the dysmobilized mitochondria in comparison to wild-type or A30P α-synuclein. Treatment with NAP, the eight amino acid peptide identified as the active component of activity-dependent neuroprotective protein (ADNP), completely annihilated the adverse effects of A53T on mitochondrial dynamics. Our results reveal that A53T α-synuclein (oligomers or aggregates) leads to the inhibition of mitochondrial trafficking, which can be rescued by NAP, suggesting the involvement of microtubule disruption in the pathophysiology of Parkinson's disease.

  19. Neurodegenerative phenotypes in an A53T α-synuclein transgenic mouse model are independent of LRRK2

    PubMed Central

    Daher, João Paulo L.; Pletnikova, Olga; Biskup, Saskia; Musso, Alessandra; Gellhaar, Sandra; Galter, Dagmar; Troncoso, Juan C.; Lee, Michael K.; Dawson, Ted M.; Dawson, Valina L.; Moore, Darren J.

    2012-01-01

    Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain. PMID:22357653

  20. Non-motor parkinsonian pathology in aging A53T α-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function.

    PubMed

    Farrell, Kaitlin F; Krishnamachari, Sesha; Villanueva, Ernesto; Lou, Haiyan; Alerte, Tshianda N M; Peet, Eloise; Drolet, Robert E; Perez, Ruth G

    2014-02-01

    Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α-synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α-synuclein for behavior and α-synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α-synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α-synuclein. Low adrenal PP2A activity co-occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α-synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α-synuclein pathology associated with pre-motor stages of PD.

  1. Non-motor parkinsonian pathology in aging A53T α-Synuclein mice is associated with progressive synucleinopathy and altered enzymatic function

    PubMed Central

    Farrell, Kaitlin F; Krishnamachari, Sesha; Villanueva, Ernesto; Lou, Haiyan; Alerte, Tshianda N M; Peet, Eloise; Drolet, Robert E; Perez, Ruth G

    2014-01-01

    Aging, the main risk factor for Parkinson's disease (PD), is associated with increased α–synuclein levels in substantia nigra pars compacta (SNc). Excess α-synuclein spurs Lewy-like pathology and dysregulates the activity of protein phosphatase 2A (PP2A). PP2A dephosphorylates many neuroproteins, including the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH). A loss of nigral dopaminergic neurons induces PD movement problems, but before those abnormalities occur, behaviors such as olfactory loss, anxiety, and constipation often manifest. Identifying mouse models with early PD behavioral changes could provide a model in which to test emerging therapeutic compounds. To this end, we evaluated mice expressing A53T mutant human (A53T) α–synuclein for behavior and α–synuclein pathology in olfactory bulb, adrenal gland, and gut. Aging A53T mice exhibited olfactory loss and anxiety that paralleled olfactory and adrenal α-synuclein aggregation. PP2A activity was also diminished in olfactory and adrenal tissues harboring insoluble α-synuclein. Low adrenal PP2A activity co-occurred with TH hyperactivity, making this the first study to link adrenal synucleinopathy to anxiety and catecholamine dysregulation. Aggregated A53T α–synuclein recombinant protein also had impaired stimulatory effects on soluble recombinant PP2A. Collectively, the data identify an excellent model in which to screen compounds for their ability to block the spread of α-synuclein pathology associated with pre-motor stages of PD. PMID:24117685

  2. Astrocytic expression of Parkinson's disease-related A53T alpha-synuclein causes neurodegeneration in mice.

    PubMed

    Gu, Xing-Long; Long, Cai-Xia; Sun, Lixin; Xie, Chengsong; Lin, Xian; Cai, Huaibin

    2010-04-21

    Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of alpha-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, alpha-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of alpha-synuclein, however, is unclear. Here we show that PD-related A53T mutant alpha-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of alpha-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T alpha-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons. Our findings demonstrate a critical involvement of astrocytic alpha-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases.

  3. Proteinase K-resistant alpha-synuclein is deposited in presynapses in human Lewy body disease and A53T alpha-synuclein transgenic mice.

    PubMed

    Tanji, Kunikazu; Mori, Fumiaki; Mimura, Junsei; Itoh, Ken; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2010-08-01

    Abnormally modified alpha-synuclein is a pathological hallmark of Parkinson's disease and the other alpha-synucleinopathies. Since proteinase K (PK) treatment is known to enhance the immunoreactivity of abnormal alpha-synuclein, we immunohistochemically examined the brain of transgenic (Tg) mice expressing human mutant A53T alpha-synuclein using this retrieval method. PK treatment abolished the immunoreactivity of alpha-synuclein in abnormal inclusions as well as of endogenous alpha-synuclein in Tg mice, whereas PK-resistant alpha-synuclein was found in the presynaptic nerve terminals, especially in the hippocampus and temporal cortex. In human Lewy body disease, PK-resistant alpha-synuclein was deposited in Lewy bodies and Lewy neurites, as well as in the presynapses in distinct brain regions, including the hippocampus, temporal cortex and substantia nigra. Biochemical analysis revealed that PK-resistant alpha-synuclein was detected in the presynaptic fraction in Tg mice and human Lewy body disease. Although PK-resistant alpha-synuclein was found in the presynapse in Tg mice even at 1 week of age, it was not phosphorylated until at least 8 months of age. Moreover, PK-resistant alpha-synuclein in the presynapse was not phosphorylated in human Lewy body disease. These findings suggest that phosphorylation is not necessary to cause the conversion of soluble form to PK-resistant alpha-synuclein. Considering that native alpha-synuclein is a soluble protein localized to the presynaptic terminals, our findings suggest that PK-resistant alpha-synuclein may disturb the neurotransmission in alpha-synucleinopathies.

  4. Disposal of iron by a mutant form of lipocalin 2

    PubMed Central

    Barasch, Jonathan; Hollmen, Maria; Deng, Rong; Hod, Eldad A.; Rupert, Peter B.; Abergel, Rebecca J.; Allred, Benjamin E.; Xu, Katherine; Darrah, Shaun F.; Tekabe, Yared; Perlstein, Alan; Wax, Rebecca; Bruck, Efrat; Stauber, Jacob; Corbin, Kaitlyn A.; Buchen, Charles; Slavkovich, Vesna; Graziano, Joseph; Spitalnik, Steven L.; Bao, Guanhu; Strong, Roland K.; Qiu, Andong

    2016-01-01

    Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe−/−) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway. PMID:27796299

  5. Neuronal expression of familial Parkinson's disease A53T α-synuclein causes early motor impairment, reduced anxiety and potential sleep disturbances in mice.

    PubMed

    Rothman, Sarah M; Griffioen, Kathleen J; Vranis, Neil; Ladenheim, Bruce; Cong, Wei-na; Cadet, Jean-Lud; Haran, Jamie; Martin, Bronwen; Mattson, Mark P

    2013-01-01

    Mutations in the human α-synuclein gene lead to early-onset Parkinson's disease (PD); however, phenotypes of α-synuclein mutant mice vary depending upon the promoter driving transgene expression. The goal of this study was to characterize behavior and neurochemical alterations in mice expressing mutant (A53T) human α-synuclein, controlled by a neuron-specific Thy-1 promoter. Our data provide important additional phenotypic and biochemical characterization of a previously generated model of PD. A53T (SNCA) and wild type (WT) littermate mice were evaluated for motor function (rotarod and stride length) and anxiety (elevated plus maze and open field) every 2 weeks. At 24 weeks mice were evaluated in a Comprehensive Lab Animal Monitoring System (CLAMS). A separate cohort of mice were euthanized at 12, 24 and 36 weeks for immunoblot analysis of α-synuclein, dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the striatum, and hypothalamic serotonin and metabolites were measured. SNCA mice display significant motor deficits at 14-18 weeks of age compared to WT mice, which progress over time. CLAMS analysis revealed an increase in activity during the dark phase and a reduction in overall estimated sleep time for SNCA mice compared to WT consistent with clinical reports of sleep abnormalities in PD. A transient change in the levels of DAT appeared at 12 weeks in the striatum and serotonin levels were also altered in the hypothalamus at this time point. This PD model displays consistent and clinically relevant motor and sleep phenotypes. Anxiety phenotypes are consistent with other α-synuclein based PD models yet incongruous with typical clinical symptoms. Early increases in serotonin levels potentially explain reductions in anxiety behaviors and sleep.

  6. FTY720/Fingolimod Reduces Synucleinopathy and Improves Gut Motility in A53T Mice: CONTRIBUTIONS OF PRO-BRAIN-DERIVED NEUROTROPHIC FACTOR (PRO-BDNF) AND MATURE BDNF.

    PubMed

    Vidal-Martínez, Guadalupe; Vargas-Medrano, Javier; Gil-Tommee, Carolina; Medina, David; Garza, Nathan T; Yang, Barbara; Segura-Ulate, Ismael; Dominguez, Samantha J; Perez, Ruth G

    2016-09-23

    Patients with Parkinson's disease (PD) often have aggregated α-synuclein (aSyn) in enteric nervous system (ENS) neurons, which may be associated with the development of constipation. This occurs well before the onset of classic PD motor symptoms. We previously found that aging A53T transgenic (Tg) mice closely model PD-like ENS aSyn pathology, making them appropriate for testing potential PD therapies. Here we show that Tg mice overexpressing mutant human aSyn develop ENS pathology by 4 months. We then evaluated the responses of Tg mice and their WT littermates to the Food and Drug Administration-approved drug FTY720 (fingolimod, Gilenya) or vehicle control solution from 5 months of age. Long term oral FTY720 in Tg mice reduced ENS aSyn aggregation and constipation, enhanced gut motility, and increased levels of brain-derived neurotrophic factor (BDNF) but produced no significant change in WT littermates. A role for BDNF was directly assessed in a cohort of young A53T mice given vehicle, FTY720, the Trk-B receptor inhibitor ANA-12, or FTY720 + ANA-12 from 1 to 4 months of age. ANA-12-treated Tg mice developed more gut aSyn aggregation as well as constipation, whereas FTY720-treated Tg mice had reduced aSyn aggregation and less constipation, occurring in part by increasing both pro-BDNF and mature BDNF levels. The data from young and old Tg mice revealed FTY720-associated neuroprotection and reduced aSyn pathology, suggesting that FTY720 may also benefit PD patients and others with synucleinopathy. Another finding was a loss of tyrosine hydroxylase immunoreactivity in gut neurons with aggregated aSyn, comparable with our prior findings in the CNS.

  7. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  8. Clioquinol Improves Cognitive, Motor Function, and Microanatomy of the Alpha-Synuclein hA53T Transgenic Mice.

    PubMed

    Finkelstein, David I; Hare, Dominic J; Billings, Jessica L; Sedjahtera, Amelia; Nurjono, Milawaty; Arthofer, Elisa; George, Sonia; Culvenor, Janetta G; Bush, Ashley I; Adlard, Paul A

    2016-01-20

    The abnormal accumulation of alpha-synuclein (α-syn) has been linked to a number of neurodegenerative disorders, the most noteworthy of which is Parkinson's disease. Alpha-synuclein itself is not toxic and fulfills various physiological roles in the central nervous system. However, specific types of aggregates have been shown to be toxic, and metals have been linked to the assembly of these toxic aggregates. In this paper, we have characterized a transgenic mouse that overexpresses the A53T mutation of human α-syn, specifically assessing cognition, motor performance, and subtle anatomical markers that have all been observed in synucleinopathies in humans. We hypothesized that treatment with the moderate-affinity metal chelator, clioquinol (CQ), would reduce the interaction between metals and α-syn to subsequently improve the phenotype of the A53T animal model. We showed that CQ prevents an iron-synuclein interaction, the formation of urea-soluble α-syn aggregates, α-syn-related substantia nigra pars compacta cell loss, reduction in dendritic spine density of hippocampal and caudate putamen medium spiny neurons, and the decline in motor and cognitive function. In conclusion, our data suggests that CQ is capable of mitigating the pathological metal/α-syn interactions, suggesting that the modulation of metal ions warrants further study as a therapeutic approach for the synucleinopathies.

  9. Isolation and preliminary characterization of auxotrophic and morphological mutants of the yeastlike form of Paracoccidioides brasiliensis.

    PubMed Central

    San Blas, F; Centeno, S

    1977-01-01

    N-methyl-N'-nitro-N-nitrosoguanidine, which is known to be a very effective mutagen in many systems, was used to induce mutants in the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9, an imperfect fungus. Forty-three auxotrophic and 27 prototrophic morphological mutants were isolated after treatment with 50 mug of nitrosoguanidine per ml in 0.1 M citrate buffer, pH 5.0. Auxotrophic mutants required primarily either amino acids, purines, or pyrimidines. Some auxotrophs were also morphological mutants. The main morphological difference from the parental strain was the texture or the color of the yeast-like colonies. Only one prototrophic morphological mutant differed in the size and form of the yeastlike cells when compared with the parental strain. Suxotrophic mutants were used in pairwise combination to attempt heterokaryon formation without success. Images PMID:830638

  10. Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration

    PubMed Central

    Tsika, Elpida; Moysidou, Maria; Guo, Jing; Cushman, Mimi; Gannon, Patrick; Sandaltzopoulos, Raphael; Giasson, Benoit I.; Krainc, Dimitri; Ischiropoulos, Harry; Mazzulli, Joseph R.

    2010-01-01

    Aggregation of α-synuclein (α-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric α-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we employed two-dimensional analytical separation and an array of biochemical and cell based assays to characterize α-syn oligomers that are present in the nervous system of A53T α-syn transgenic mice. The most prominent species identified were 53 Å detergent soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing α-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea, but were sensitive to proteinase-K digestion. Even though the oligomers shared similar basic biochemical properties, those obtained from inclusion bearing regions were prominently reactive to antibodies that recognize oxidized α-syn oligomers, significantly accelerated aggregation of α-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion bearing regions were not toxic and delayed the in vitro formation of α-syn fibrils. These data indicate that specific conformations of α-syn oligomers are present in distinct brain regions of A53T α-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties, but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region specific intrinsic factors. PMID:20203200

  11. Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

    PubMed Central

    McFall, E

    1975-01-01

    A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis. PMID:1090587

  12. Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

    PubMed

    McFall, E

    1975-03-01

    A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.

  13. Mutant Strains of Escherichia coli K-12 Unable to Form Ubiquinone

    PubMed Central

    Cox, G. B.; Gibson, F.; Pittard, James

    1968-01-01

    A strain of Escherichia coli was isolated which was unable to form ubiquinone. This mutant was obtained by selecting strains unable to grow on malate as sole source of carbon. Such strains were further screened by examination of the quinone content of cells grown on a glucose medium. A mutant unable to form vitamin K was also isolated by this procedure. A genetic analysis of the ubiquinoneless strain showed that it possessed two mutations affecting ubiquinone biosynthesis. Images PMID:4870277

  14. A53T Human α-Synuclein Overexpression in Transgenic Mice Induces Pervasive Mitochondria Macroautophagy Defects Preceding Dopamine Neuron Degeneration

    PubMed Central

    Xie, Zhiguo; Turkson, Susie

    2015-01-01

    In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies. PMID:25609609

  15. Subthalamic nucleus deep brain stimulation is neuroprotective in the A53T α‐synuclein Parkinson's disease rat model

    PubMed Central

    Musacchio, Thomas; Rebenstorff, Maike; Fluri, Felix; Brotchie, Jonathan M.; Volkmann, Jens; Koprich, James B.

    2017-01-01

    Objective Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a highly effective symptomatic therapy for motor deficits in Parkinson's disease (PD). An additional, disease‐modifying effect has been suspected from studies in toxin‐based PD animal models, but these models do not reflect the molecular pathology and progressive nature of PD that would be required to evaluate a disease‐modifying action. Defining a disease‐modifying effect could radically change the way in which DBS is used in PD. Methods We applied STN‐DBS in an adeno‐associated virus (AAV) 1/2‐driven human mutated A53T α‐synuclein (aSyn)‐overexpressing PD rat model (AAV1/2‐A53T‐aSyn). Rats were injected unilaterally, in the substantia nigra (SN), with AAV1/2‐A53T‐aSyn or control vector. Three weeks later, after behavioral and nigrostriatal dopaminergic deficits had developed, rats underwent STN‐DBS electrode implantation ipsilateral to the vector‐injected SN. Stimulation lasted for 3 weeks. Control groups remained OFF stimulation. Animals were sacrificed at 6 weeks. Results Motor performance in the single pellet reaching task was impaired in the AAV1/2‐A53T‐aSyn–injected stim‐OFF group, 6 weeks after AAV1/2‐A53T‐aSyn injection, compared to preoperative levels (–82%; p < 0.01). Deficits were reversed in AAV1/2‐A53T‐aSyn, stim‐ON rats after 3 weeks of active stimulation, compared to the AAV1/2‐A53T‐aSyn stim‐OFF rats (an increase of ∼400%; p < 0.05), demonstrating a beneficial effect of DBS. This motor improvement was maintained when the stimulation was turned off and was accompanied by a higher number of tyrosine hydroxylase+ SN neurons (increase of ∼29%), compared to AAV1/2‐A53T‐aSyn stim‐OFF rats (p < 0.05). Interpretation Our data support the putative neuroprotective and disease‐modifying effect of STN‐DBS in a mechanistically relevant model of PD. Ann Neurol 2017;81:825–836 PMID:28470693

  16. Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

    PubMed Central

    Jaffé, A; D'Ari, R; Hiraga, S

    1988-01-01

    The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution. Images PMID:2838458

  17. Synthetic analogs of rhamnolipids modulate structured biofilms formed by rhamnolipid-nonproducing mutant of Pseudomonas aeruginosa.

    PubMed

    Zheng, Hewen; Singh, Nischal; Shetye, Gauri S; Jin, Yucheng; Li, Diana; Luk, Yan-Yeung

    2017-03-15

    Rhamnolipids secreted by Pseudomonas aeruginosa are required for the bacteria to form biofilm efficiently and form biofilm with internal structures including pores and channels. In this work, we explore the effect of a class of synthetic analogs of rhamnolipids at controlling (promoting and inhibiting) the biofilm formation activities of a non-rhamnolipid-producing strain - rhlA - of P. aeruginosa. This class of rhamnolipid analogs is known to modulate the swarming motilities of wild-type PAO1 and rhlA mutant, but its effect on biofilm formation of rhlA mutant is unknown. We show that small structural details of these molecules are important for the bioactivities, but do not affect the general physical properties of the molecules. The bioactive synthetic analogs of rhamnolipids promote biofilm formation by rhlA mutant at low concentrations, but inhibit the biofilm formation at high concentrations. To explore the internal structures formed by the biofilms, we first demonstrate that wild-type biofilms are formed with substantial topography (hills and valleys) when the sample is under shaking conditions. Using this observation as a comparison, we found that synthetic analogs of rhamnolipids promoted structured (porous) biofilm of rhlA mutant, at intermediate concentrations between the low ones that promoted biofilm formation and the high ones that inhibited biofilm formation. This study suggests a potential chemical signaling approach to control multiple bacterial activities.

  18. Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis †

    PubMed Central

    Freudenthal, Bret D.; Ramaswamy, S.; Hingorani, Manju M.; Washington, M. Todd

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a sliding clamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesis have been identified in genetic studies in yeast. One such mutant protein (encoded by the rev6-1 allele) is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To better understand how this substitution interferes with translesion synthesis, we have determined the X-ray crystal structure of the G178S PCNA mutant protein. This substitution has little effect on the structure of the domain in which the substitution occurs. Instead, significant, local structural changes are observed in the adjacent subunit. The most notable difference between mutant and wild-type structures is in a single, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutant protein structure, loop J adopts a very different conformation in which the atoms of the protein backbone have moved by as much as 6.5 Å from their positions in the wild-type structure. To better understand the functional consequences of this structural change, we have examined the ability of this mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady state kinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These results show that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis. PMID:19053247

  19. Characterization of inclusion bodies with cytoprotective properties formed by seipinopathy-linked mutant seipin.

    PubMed

    Ito, Daisuke; Yagi, Takuya; Ikawa, Masahito; Suzuki, Norihiro

    2012-02-01

    Gain-of-toxic mutations in the N-glycosylation motif of the seipin/BSCL2 gene (namely, the N88S and S90L mutations) cause autosomal dominant motor neuron diseases, termed 'seipinopathy'. Expressed mutant seipin is improperly folded and accumulates in the endoplasmic reticulum (ER), leading to an unfolded protein response (UPR). Furthermore, cells expressing mutant seipin contain unique cytoplasmic inclusion bodies (IB) that form via a different mechanism from that of ubiquitinated inclusions, or aggresomes. Whether the formation of these IB is pathogenic or protective in neurodegenerative diseases remains unclear. Here, we determined that mutant seipin IB are negative for two well-established ER markers, immunoglobulin-heavy-chain-binding protein and calnexin, indicating a distinct compartmentalization from the main ER, and that mutant seipin IB are formed via a mechanism that is independent of major UPR transducers and ER chaperons. Electron microscopy and coexpression study with variant α1-antitrypsin cDNA showed that seipin IB are compatible with unique cytoplasmic vesicles known as ER-derived protective organelles (ERPO). We also obtained evidence that seipin IB exhibit a cytoprotective property via the attenuation of ER stress. These findings suggest that ERPO, such as seipin IB, are a novel adaptation machinery against the accumulation of unfolded proteins in the ER.

  20. A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan.

    PubMed Central

    Geremia, R A; Cavaignac, S; Zorreguieta, A; Toro, N; Olivares, J; Ugalde, R A

    1987-01-01

    A mutant of Rhizobium meliloti that elicited the formation of inactive nodules in alfalfa was found not to form beta-(1----2) glucan in vivo or in vitro. It was nonmotile because it lacks flagella. The 235-kilodalton protein which acts as an intermediate in beta-(1----2) glucan synthesis was undetectable in the mutant. These properties of the mutant are common to those of chvB mutants of Agrobacterium tumefaciens. Exopolysaccharide formation by the R. meliloti mutant was about double that by the wild type. Images PMID:3804979

  1. Electron transfer quenching in light adapted and mutant forms of the AppA BLUF domain.

    PubMed

    Laptenok, Sergey P; Lukacs, Andras; Brust, Richard; Haigney, Allison; Gil, Agnieszka; Towrie, Michael; Greetham, Gregory M; Tonge, Peter J; Meech, Stephen R

    2015-01-01

    The Blue Light Using Flavin (BLUF) domain proteins are an important family of photoreceptors controlling a range of responses in a wide variety of organisms. The details of the primary photochemical mechanism, by which light absorption in the isoalloxazine ring of the flavin is converted into a structure change to form the signalling state of the protein, is unresolved. In this work we apply ultrafast time resolved infra-red (TRIR) spectroscopy to investigate the primary photophysics of the BLUF domain of the protein AppA (AppABLUF) a light activated antirepressor. Here a number of mutations at Y21 and W104 in AppABLUF are investigated. The Y21 mutants are known to be photoinactive, while W104 mutants show the characteristic spectral red-shift associated with BLUF domain activity. Using TRIR we observed separately the decay of the excited state and the recovery of the ground state. In both cases the kinetics are found to be non-single exponential for all the proteins studied, suggesting a range of ground state structures. In the Y21 mutants an intermediate state was also observed, assigned to formation of the radical of the isoalloxazine (flavin) ring. The electron donor is the W104 residue. In contrast, no radical intermediates were detected in the studies of the photoactive dark adapted proteins, dAppABLUF and the dW104 mutants, suggesting a structure change in the Y21 mutants which favours W104 to isoalloxazine electron transfer. In contrast, in the light adapted form of the proteins (lAppABLUF, lW104) a radical intermediate was detected and the kinetics were greatly accelerated. In this case the electron donor was Y21 and major structural changes are associated with the enhanced quenching. In AppABLUF and the seven mutants studied radical intermediates are readily observed by TRIR spectroscopy, but there is no correlation with photoactivity. This suggests that if a charge separated state has a role in the BLUF photocycle it is only as a very short lived

  2. Isolation and characterization of mutants of Salmonella typhimurium with a disturbed process of generation of nonculturable forms

    SciTech Connect

    Romanova, Y.M.; Terekhov, A.A.; Gintsburg, A.L.

    1995-08-01

    A laboratory model of the induction of nonculturable forms in Salmonella typhimurium has been developed. Mutants of S. typhimurium were obtained using insertion mutagenesis via the TnPhoA transposon. These mutants were impaired in the cell transition from the vegetative to the nonculturable state assayed in this model. Mutants have various phenotypes and are located in different regions of the chromosome, as shown by the data obtained using pulsed-field electrophoresis of genomic DNA. 11 refs., 3 figs., 1 tab.

  3. Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43.

    PubMed

    Tanaka, Yoshinori; Hasegawa, Masato

    2016-07-03

    Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition.

  4. 6-Oxooxazolidine-quinazolines as noncovalent inhibitors with the potential to target mutant forms of EGFR.

    PubMed

    Shao, Jiaan; Chen, En; Shu, Ke; Chen, Wenteng; Zhang, Guolin; Yu, Yongping

    2016-08-15

    Despite the remarkable benefits of gefitinib, the clinical efficacy is eventually diminished due to the acquired point mutations in the EGFR (T790M). To address this unmet medical need, we demonstrated a strategy to prepare a hybrid analogue consisting of the oxooxazolidine ring and the quinazoline scaffold and provided alternative noncovalent inhibitors targeting mutant forms of EGFR. Most of the derivatives displayed moderate to good anti-proliferative activity against gefitinib-resistant NCI-H1975. Some of them exhibited potent EGFR kinase inhibitory activities, especially on EGFR(T790M) and EGFR(L858R) kinases. SAR studies led to the identification of a hit 9a that can target both of the most common EGFR mutants: L858R and T790M. Also, 9a displayed weaker inhibitory against cancer cell lines with low level of EGFR expression and good chemical stability under different pH conditions. The work presented herein showed the potential for developing noncovalent inhibitors targeting EGFR mutants. Copyright © 2016. Published by Elsevier Ltd.

  5. Photocycle in the M-form in bacteriorhodopsin mutants devoid of primary proton acceptor Asp-85.

    PubMed

    Lukashev, E P; Kolodner, P

    2001-01-01

    Photoinduced changes in absorption of the deprotonated M-form in the mutant bacteriorhodopsin without primary proton acceptor Asp-85 were studied and additional evidence in support of the complete transmembrane proton transfer in photocycle was obtained. Measurements of the absorption spectrum were carried out at various pH, temperature, and humidity. The direction of proton transfer was the same as in the normal photocycle of the wild-type bacteriorhodopsin: from the internal to the external side of the membrane. The effect on this process of a terminal acceptor Glu-204 was shown.

  6. Heterotetrameric forms of human phenylalanine hydroxylase: co-expression of wild-type and mutant forms in a bicistronic system.

    PubMed

    Leandro, João; Leandro, Paula; Flatmark, Torgeir

    2011-05-01

    Hybrid forms of human phenylalanine hydroxylase (hPAH) mutants have been found to present catalytic activities lower than predicted from the individual recombinant forms, indicating that interallelic complementation could be a major determinant of the metabolic phenotype of compound heterozygous phenylketonuric (PKU) patients. To provide a molecular explanation for interallelic complementation we have here developed a bicistronic expression system and a purification strategy to obtain isolated hPAH heteromeric forms. On co-expression of WT-hPAH (~50% tetramer; ~10% dimer) and the N- and C-terminally truncated form ΔN102/ΔC24-hPAH (~80% dimer) no heterodimers were recovered. Moreover, by co-expression of WT-hPAH and the N-terminally truncated form ΔN102-hPAH (~95% tetramer), heterotetramers, as a result of an assembly of two different homodimers, were isolated. The recovered (WT)/(ΔN102)-hPAH heterotetramers revealed a catalytic activity deviating significantly from that calculated by averaging the respective recombinant homotetrameric forms. The heterotetramer assembly also results in conformational changes in the WT-hPAH protomer, as detected by trypsin limited proteolysis. The finding that the presence of two homodimers with different kinetic parameters influences the properties of the resulting heterotetrameric protein indicates that the dimers exhibit interactions which are transmitted across the assembled tetramer. The bicistronic expression system developed here allowed the isolation of hybrid forms that exhibit negative interallelic complementation, and may represent a model system for studying the molecular pathogenic mechanisms of PAH gene mutations in compound heterozygous PKU patients, providing the rationale to understand the observed inconsistencies both in genotype/phenotype correlations and in the response to BH(4) supplementation.

  7. Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion.

    PubMed

    Richardson, I W; Anthony, C

    1992-11-01

    Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a 'chaperone' function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein.

  8. Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185----phenylalanine.

    PubMed

    Duñach, M; Berkowitz, S; Marti, T; He, Y W; Subramaniam, S; Khorana, H G; Rothschild, K J

    1990-10-05

    The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

  9. [Suppression of telomerase activity leukemic cells by mutant forms of Rhodospirillum rubrum L-asparaginase].

    PubMed

    Pokrovskaya, M V; Zhdanov, D D; Eldarov, M A; Aleksandrova, S S; Veselovskiy, A V; Pokrovskiy, V S; Grishin, D V; Gladilina, Ju A; Sokolov, N N

    2017-01-01

    The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.

  10. Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion.

    PubMed Central

    Richardson, I W; Anthony, C

    1992-01-01

    Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a 'chaperone' function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein. PMID:1332681

  11. The heterozygous A53T mutation in the alpha-synuclein gene in a Chinese Han patient with Parkinson disease: case report and literature review.

    PubMed

    Xiong, Wei-Xi; Sun, Yi-Min; Guan, Rong-Yuan; Luo, Su-Shan; Chen, Chen; An, Yu; Wang, Jian; Wu, Jian-Jun

    2016-10-01

    The missense mutation A53T of alpha-synuclein gene (SNCA) was reported to be a rare but definite cause of sporadic and familial Parkinson disease (PD). It seemed to be restricted geographically in Greece and Italy. We aimed to identify the SNCA mutations in a Chinese PD cohort. Ninety-one early onset PD patients or familial PD probands were collected consecutively for the screening of PD-related genes. The genetic analysis was carried out by target sequencing of the exons and the corresponding flanking regions of the PD-related genes using Illumina HiSeq 2000 sequencer and further confirmed by Sanger sequencing or restriction fragment length polymorphism. Dosage mutations of exons in these genes were carried out by multiple ligation-dependent probe amplification. Among the 91 patients, we found only one heterozygous mutation of SNCA A53T, in a 23-year-old male patient with negative family history. The [(11)C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) PET and PD-related spatial covariance pattern (PDRP) via [(18)F]-fluorodeoxyglucos (FDG) PET confirmed a typical pattern of PD. After examining his parents, we found his mother was an asymptomatic carrier, with declined hand dexterity detected by quantitative motor tests. Reduced dopamine transporter uptake of his mother was identified by CFT PET, and abnormal PDRP pattern was found by FDG PET. Our investigation expanded the clinical and genetic spectrum of Chinese PD patients, and we suggested SNCA mutations to be screened in familial and early onset Chinese PD patients.

  12. Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts.

    PubMed

    Suzuki, Hiroyuki; Suda, Naoto; Shiga, Momotoshi; Kobayashi, Yukiho; Nakamura, Masataka; Iseki, Sachiko; Moriyama, Keiji

    2012-09-01

    Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with β-tricalcium phosphate (β-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome. Copyright © 2011

  13. A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum.

    PubMed

    Fasana, Elisa; Fossati, Matteo; Ruggiano, Annamaria; Brambillasca, Silvia; Hoogenraad, Casper C; Navone, Francesca; Francolini, Maura; Borgese, Nica

    2010-05-01

    VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a approximately 30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons.

  14. Familial Parkinson mutant alpha-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.

    PubMed

    Kuwahara, Tomoki; Koyama, Akihiko; Gengyo-Ando, Keiko; Masuda, Mayumi; Kowa, Hisatomo; Tsunoda, Makoto; Mitani, Shohei; Iwatsubo, Takeshi

    2006-01-06

    Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

  15. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character

    PubMed Central

    Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L.; Grainger, Robert M.

    2014-01-01

    SUMMARY The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant. PMID:25224223

  16. Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

    PubMed

    Fish, Margaret B; Nakayama, Takuya; Fisher, Marilyn; Hirsch, Nicolas; Cox, Amanda; Reeder, Rollin; Carruthers, Samantha; Hall, Amanda; Stemple, Derek L; Grainger, Robert M

    2014-11-15

    The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.

  17. Huntingtin Fragments and SOD1 Mutants Form Soluble Oligomers in the Cell

    PubMed Central

    Norton, Mark; Taylor, J. Paul; Eisenberg, Evan; Greene, Lois E.

    2012-01-01

    Diffusion coefficients of huntingtin (Htt) fragments and SOD1 mutants expressed in cells were measured using fluorescence correlation spectroscopy. The diffusion mobilities of both non-pathological Htt fragments (25 polyQs) and pathological Htt fragments (103 polyQs) were much slower than expected for monomers suggesting that they oligomerize. The mobility of these fragments was unaffected by duration of expression or by over-expression of Hsp70 and Hsp40. However in cells with HttQ103 inclusions, diffusion measurements showed that the residual cytosolic HttQ103 was monomeric. These results suggest that both non-pathological and pathological Htt fragments form soluble oligomers in the cytosol with the properties of the oligomers determining whether they cause pathology. SOD1 with point mutations (A4V, G37R, and G85R) also had slower diffusional mobility than the wild-type protein whose mobility was consistent with that of a dimer. However, the decrease in mobility of the different SOD1 mutantsdid not correlate with their known pathology. Therefore, while soluble oligomers always seem to be present under conditions where cell pathology occurs, the presence of the oligomers, in itself, does not determine the extent of neuropathology. PMID:22768276

  18. Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

    PubMed Central

    Kim, Bongi; Shin, Jun-Seop

    2013-01-01

    CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation. PMID:23559896

  19. Allergenic characterization of new mutant forms of Pru p 3 as new immunotherapy vaccines.

    PubMed

    Gómez-Casado, C; Garrido-Arandia, M; Gamboa, P; Blanca-López, N; Canto, G; Varela, J; Cuesta-Herranz, J; Pacios, L F; Díaz-Perales, A; Tordesillas, L

    2013-01-01

    Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

  20. Wildtype and A30P Mutant Alpha-Synuclein Form Different Fibril Structures

    PubMed Central

    Langkilde, Annette Eva; Giehm, Lise; Kyrsting, Anders; Svane, Anna Sigrid Pii; Manno, Mauro; Christiansen, Gunna; Nielsen, Niels Christian; Oddershede, Lene; Vestergaard, Bente; Otzen, Daniel Erik

    2013-01-01

    Parkinson’s Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied. PMID:23861789

  1. Wildtype and A30P mutant alpha-synuclein form different fibril structures.

    PubMed

    Nielsen, Søren Bang; Macchi, Francesca; Raccosta, Samuele; Langkilde, Annette Eva; Giehm, Lise; Kyrsting, Anders; Svane, Anna Sigrid Pii; Manno, Mauro; Christiansen, Gunna; Nielsen, Niels Christian; Oddershede, Lene; Vestergaard, Bente; Otzen, Daniel Erik

    2013-01-01

    Parkinson's Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied.

  2. Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis.

    PubMed

    Zhong, Ruiqin; Morrison, W Herbert; Freshour, Glenn D; Hahn, Michael G; Ye, Zheng-Hua

    2003-06-01

    Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.

  3. Correlation of mutant menin stability with clinical expression of multiple endocrine neoplasia type 1 and its incomplete forms.

    PubMed

    Shimazu, Satoko; Nagamura, Yuko; Yaguchi, Hiroko; Ohkura, Naganari; Tsukada, Toshihiko

    2011-11-01

    Germline mutations of the tumor suppressor gene MEN1 are found not only in typical multiple endocrine neoplasia type 1 (MEN1) but also in its incomplete forms such as familial isolated hyperparathyroidism (FIHP) and apparently sporadic parathyroid tumor (ASPT). No definitive genotype-phenotype correlation has been established between these clinical forms and MEN1 gene mutations. We previously demonstrated that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. To examine whether the intracellular stability of mutant menin is correlated with clinical phenotypes, we developed a method of evaluating menin stability and examined 20 mutants associated with typical MEN1 (17 missense, two in-frame deletion, one nonsense) and 21 mutants associated with FIHP or ASPT (19 missense, two in-frame deletion). All tested mutants associated with typical MEN1 showed reduced stability. Some missense and in-frame deletion mutants (G28A, R171W, T197I, E255K, E274A, Y353del and E366D) associated with FIHP or ASPT were almost as stable as or only slightly less stable than wild-type menin, while others were as unstable as those associated with typical MEN1. Some stable mutants exhibited substantial biological activities when tested by JunD-dependent transactivation assay. These findings suggest that certain missense and in-frame mutations are fairly stable and retain intrinsic biological activity, and might be specifically associated with incomplete clinical phenotypes. The menin stability test will provide useful information for the management of patients carrying germline MEN1 mutations especially when they have missense or in-frame variants of ambiguous clinical significance.

  4. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore.

    PubMed

    Mishin, Alexander S; Subach, Fedor V; Yampolsky, Ilia V; King, William; Lukyanov, Konstantin A; Verkhusha, Vladislav V

    2008-04-22

    Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags.

  5. The First Mutant of the Aequorea victoria Green Fluorescent Protein That Forms a Red Chromophore†

    PubMed Central

    Mishin, Alexander S.; Subach, Fedor V.; Yampolsky, Ilia V.; King, William; Lukyanov, Konstantin A.; Verkhusha, Vladislav V.

    2010-01-01

    Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags. PMID:18366185

  6. Mutant p53 forms a complex with Sp1 on HIV-LTR DNA.

    PubMed

    Chicas, A; Molina, P; Bargonetti, J

    2000-12-20

    Many mutants of p53 activate HIV-LTR driven transcription and promote HIV replication. The region of the HIV-LTR containing Sp1-binding sites is important for this effect. In this study we test the hypothesis that mutant p53 interacts with DNA-bound Sp1 and in this way can increase transcription from Sp1-dependent promoters. We have used the breast cancer cell line MDA-MB-468 that expresses endogenous mutant p53(His273) as our source of p53 protein. First, we demonstrated that this mutant p53 participates in activating transcription from the HIV-LTR by showing that HIV-LTR-directed transcription in MDA-MB-468 cells is inhibited in a dominant-negative manner by p53(Val135). Using HIV-LTR DNA affinity chromatography, we detected coelution of p53(His273) and Sp1. We also demonstrated that this mutant p53 binds sequence specifically to the super consensus sequence (SCS) and that Sp1 coeluted with p53(His273) from a column containing this site. These data indicate that p53(His273) can associate with DNA-bound Sp1 suggesting that activated HIV-LTR transcription associated with mutant p53 occurs through a DNA driven multi-protein complex.

  7. Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

    PubMed Central

    Gómez-Casado, C.; Garrido-Arandia, M.; Gamboa, P.; Blanca-López, N.; Canto, G.; Varela, J.; Cuesta-Herranz, J.; Pacios, L. F.; Díaz-Perales, A.; Tordesillas, L.

    2013-01-01

    Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients. PMID:24324505

  8. nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response.

    PubMed

    Veereshlingam, Harita; Haynes, Janine G; Penmetsa, R Varma; Cook, Douglas R; Sherrier, D Janine; Dickstein, Rebecca

    2004-11-01

    To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses

  9. Initial characterization of 17 viruses harboring mutant forms of the immediate-early gene of equine herpesvirus 1.

    PubMed

    Buczynski, Kimberly A; Kim, Seong K; O'Callaghan, Dennis J

    2005-10-01

    The sole immediate-early (IE) gene of equine herpesvirus 1 (EHV-1) encodes a major regulatory protein of 1487 amino acids (aa) capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Using a specially designed recombination system and a library of IE linker-insertion, deletion, point, and nonsense mutant constructs that encode forms of the IE protein (IEP) harboring mutations within all five regions, 17 mutant viruses were generated and characterized. Ribonuclease protection analyses revealed that all 17 mutants synthesize the IE mRNA in RK-13 cells, whereas those that failed to replicate on non-complementing RK-13 cells displayed a defect in the transcription of either an important early gene (EICP0) and/or an essential late gene (glycoprotein D). Western blot analyses showed that the IEP was synthesized and detectable in cells infected with each mutant virus, including those mutants that failed to replicate on non-complementing RK-13 cells. Eleven of the 17 mutants were capable of growth on non-complementing RK-13 cells, whereas mutant viruses with deletions within the serine-rich tract (SRT), nucleus localization signal (NLS), or DNA-binding domain (DBD) were capable of growth only on the IEP-producing cell line (IE13.1). Lastly, temperature shift experiments revealed that mutant viruses containing deletions within the C-terminus (KyAn1029 and KyAn1411) or within the SRT (KyADeltaSRT2) of the IEP exhibited a temperature-sensitive phenotype in that these viruses, in contrast to the parent KyA, failed to replicate at 39 degrees C. Overall, these results indicate that the C-terminus of the IEP is not essential for IEP function in cell culture, but this region contains elements that enhance the function(s) of the IEP. The initial characterization of these 17 EHV-1 mutants has shown that sequences totaling at least 43% of the IEP are not essential for virus replication in cell culture.

  10. Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

    PubMed

    Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

    2017-08-23

    Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. Curvature Dynamics of α-Synuclein Familial Parkinson Disease Mutants

    PubMed Central

    Perlmutter, Jason D.; Braun, Anthony R.; Sachs, Jonathan N.

    2009-01-01

    α-Synuclein remains a protein of interest due to its propensity to form fibrillar aggregates in neurodegenerative disease and its putative function in synaptic vesicle regulation. Herein, we present a series of atomistic molecular dynamics simulations of wild-type α-synuclein and three Parkinson disease familial mutants (A30P, A53T, and E46K) in two distinct environments. First, in order to match recent NMR experiments, we have simulated each protein bound to an SDS detergent micelle. Second, in order to connect more closely to the true biological environment, we have simulated the proteins bound to a 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid bilayer. In the micelle-bound case, we find that the wild type and all of the variants of α-synuclein flatten the underlying micelle, decreasing its surface area. A30P is known to lessen α-synuclein/membrane affinity and, consistent with experiment, destabilizes the simulated secondary structure. In the case of A53T, our simulations reveal a range of stabilizing hydrogen bonds that form with the threonine. In both environments, the E46K mutation, which is known to increase bilayer affinity, leads to an additional hydrogen bond between the protein and either the detergent or lipid. Simulations indicate that αS and its variants are less dynamic in the bilayer than in the micelle. Furthermore, the simulations of the mutants suggest how changes in the structure and dynamics of α-synuclein may affect its biological role. PMID:19126542

  12. Crystal Structure of Arachidonic Acid Bound to a Mutant of Prostaglandin Endoperoxide Synthase-1 that Forms Predominantly 11-HPETE

    SciTech Connect

    Harman, C.; Rieke, C.J.; Garavito, R.M.; Smith, W.L.

    2010-03-05

    Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G{sub 2} (PGG{sub 2}), 11Rhydroperoxyeicosatetraenoic acid (HPETE), and 15R,SHPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11RHPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co{sup 3+}-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 {angstrom}. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val{sup 349} and Trp{sup 387} help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG{sub 2}. In the V349A/W387F oPGHS-1 {center_dot} AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG{sub 2}. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG{sub 2}.

  13. NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

    SciTech Connect

    Birdsall, B.; Andrews, J.; Ostler, G.; Tendler, S.J.B.; Feeney, J.; Roberts, G.C.K.; Davies, R.W.; Cheung, H.T.A. )

    1989-02-07

    Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21 {yields} Leu and Asp 26 {yields} Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. {sup 1}H, {sup 13}C, and {sup 31}P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. {sup 1}H NMR studies of the Trp 21 {yields} Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 {angstrom} away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21 {yields} Leu mutant indicate that the delicately poised equilibria can be perturbed. Some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim.

  14. Characterization of a Rhodobacter capsulatus reaction center mutant that enhances the distinction between spectral forms of the initial electron donor.

    PubMed

    Eastman, J E; Taguchi, A K; Lin, S; Jackson, J A; Woodbury, N W

    2000-12-05

    A large scale mutation of the Rhodobacter capsulatus reaction center M-subunit gene, sym2-1, has been constructed in which amino acid residues M205-M210 have been changed to the corresponding L subunit amino acids. Two interconvertable spectral forms of the initial electron donor are observed in isolated reaction centers from this mutant. Which conformation dominates depends on ionic strength, the nature of the detergent used, and the temperature. Reaction centers from this mutant have a ground-state absorbance spectrum that is very similar to wild-type when measured immediately after purification in the presence of high salt. However, upon subsequent dialysis against a low ionic strength buffer or the addition of positively charged detergents, the near-infrared spectral band of P (the initial electron donor) in sym2-1 reaction centers is shifted by over 30 nm to the blue, from 852 to 820 nm. Systematically varying either the ionic strength or the amount of charged detergent reveals an isobestic point in the absorbance spectrum at 845 nm. The wild-type spectrum also shifts with ionic strength or detergent with an isobestic point at 860 nm. The large spectral separation between the two dominant conformational forms of the sym2-1 reaction center makes detailed measurements of each state possible. Both of the spectral forms of P bleach in the presence of light. Electrochemical measurements of the P/P+ midpoint potential of sym2-1 reaction centers show an increase of about 30 mV upon conversion from the long-wavelength form to the short-wavelength form of the mutant. The rate constant of initial electron transfer in both forms of the mutant reaction centers is essentially the same, suggesting that the spectral characteristics of P are not critical for charge separation. The short-wavelength form of P in this mutant also converts to the long-wavelength form as a function of temperature between room temperature and 130 K, again giving rise to an isobestic point, in this

  15. A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants.

    PubMed

    Frigeri, Claudia; Tsao, Jennivine; Cordova, Martha; Schimmer, Bernard P

    2002-10-01

    ACTH resistance in mutant derivatives of the Y1 mouse adrenocortical tumor cell line results from a defect that affects the activity of steroidogenic factor-1 (SF1), thereby preventing the expression of the melanocortin-2 receptor. In this report, we show that the SF1 genes in ACTH-resistant mutants differ from the gene in ACTH-responsive Y1 cells by two base changes-one that changes an Ala to Ser at codon 172, and one in the third position of codon 3 that does not affect the protein sequence. Furthermore, several of the mutants contain multiple copies of this alternate SF1 gene (SF1(S172)) on acentric chromosome fragments. The SF1(S172) allele represents a polymorphism rather than a spontaneous mutation because the two SF1 alleles can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(A172) allele also is found in C57Bl/6J and C57Bl/10J mice, whereas the SF1(S172) allele also is found in C3H/HeJ and DBA/2J mice. The two forms of SF1 had only modest differences in activity suggesting that the SF1 polymorphism per se is not directly responsible for ACTH resistance. Our results indicate that the SF1(S172) allele is a marker of ACTH resistance in this family of adrenocortical tumor cells.

  16. A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

    PubMed Central

    González-Rivera, Christian; Campbell, Anne M.; Rutherford, Stacey A.; Pyburn, Tasia M.; Foegeding, Nora J.; Barke, Theresa L.; Spiller, Benjamin W.; McClain, Mark S.; Ohi, Melanie D.

    2016-01-01

    Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. PMID:27382020

  17. A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain.

    PubMed

    González-Rivera, Christian; Campbell, Anne M; Rutherford, Stacey A; Pyburn, Tasia M; Foegeding, Nora J; Barke, Theresa L; Spiller, Benjamin W; McClain, Mark S; Ohi, Melanie D; Lacy, D Borden; Cover, Timothy L

    2016-09-01

    Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.

  18. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form.

    PubMed

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme.

  19. Molecular Dynamics Approach in the Comparison of Wild-Type and Mutant Paraoxonase-1 Apoenzyme Form

    PubMed Central

    Amine, Khadija; Miri, Lamia; Naimi, Adil; Saile, Rachid; El Kharrim, Abderrahmane; Mikou, Afaf; Kettani, Anass

    2015-01-01

    There is some evidence linking the mammalian paraoxonase-1 (PON1) loops (L1 and L2) to an increased flexibility and reactivity of its active site with potential substrates. The aim of this work is to study the structural, dynamical, and functional effects of the most flexible regions close to the active site and to determine the impact of mutations on the protein. For both models, wild-type (PON1wild) and PON1 mutant (PON1mut) models, the L1 loop and Q/R and L/M mutations were constructed using MODELLER software. Molecular dynamics simulations of 20 ns at 300 K on fully modeled PON1wild and PON1mut apoenzyme have been done. Detailed analyses of the root-mean-square deviation and fluctuations, H-bonding pattern, and torsion angles have been performed. The PON1wild results were then compared with those obtained for the PON1mut. Our results show that the active site in the wild-type structure is characterized by two distinct movements of opened and closed conformations of the L1 and L2 loops. The alternating and repetitive movement of loops at specific times is consistent with the presence of 11 defined hydrogen bonds. In the PON1mut, these open-closed movements are therefore totally influenced and repressed by the Q/R and L/M mutations. In fact, these mutations seem to impact the PON1mut active site by directly reducing the catalytic core flexibility, while maintaining a significant mobility of the switch regions delineated by the loops surrounding the active site. The impact of the studied mutations on structure and dynamics proprieties of the protein may subsequently contribute to the loss of both flexibility and activity of the PON1 enzyme. PMID:26417201

  20. A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

    PubMed

    Xiao, Xunjun; Jones, Gabrielle; Sevilla, Wednesday A; Stolz, Donna B; Magee, Kelsey E; Haughney, Margaret; Mukherjee, Amitava; Wang, Yan; Lowe, Mark E

    2016-10-28

    Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

  1. Preliminary crystallographic analysis of a double mutant of the acetyl xylo-oligosaccharide esterase Axe2 in its dimeric form.

    PubMed

    Lansky, Shifra; Alalouf, Onit; Salama, Rachel; Dvir, Hay; Shoham, Yuval; Shoham, Gil

    2014-04-01

    Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacterium Geobacillus stearothermophilus T6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space group P212121, with unit-cell parameters a = 71.1, b = 106.0, c = 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

  2. Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants

    NASA Astrophysics Data System (ADS)

    Tosatto, Laura; Horrocks, Mathew H.; Dear, Alexander J.; Knowles, Tuomas P. J.; Dalla Serra, Mauro; Cremades, Nunilo; Dobson, Christopher M.; Klenerman, David

    2015-11-01

    Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson’s disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.

  3. A Pharmacogenetic Approach to Identify Mutant Forms of α-Galactosidase A that Respond to a Pharmacological Chaperone for Fabry Disease

    PubMed Central

    Wu, Xiaoyang; Katz, Evan; Valle, Maria Cecilia Della; Mascioli, Kirsten; Flanagan, John J; Castelli, Jeffrey P; Schiffmann, Raphael; Boudes, Pol; Lockhart, David J; Valenzano, Kenneth J; Benjamin, Elfrida R

    2011-01-01

    Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc. PMID:21598360

  4. A weakly pathogenic Rauscher spleen focus-forming virus mutant that lacks the carboxyl-terminal membrane anchor of its envelope glycoprotein.

    PubMed Central

    Machida, C A; Bestwick, R K; Kabat, D

    1985-01-01

    A mutant Rauscher spleen focus-forming virus (mutant 4-3) that causes mild splenic erythroblastosis in mice has a 44-base-pair deletion in the 3' region of its envelope glycoprotein (env) gene. The encoded glycoprotein terminates prematurely, lacks a hydrophobic membrane anchor, and has a shortened intracellular lifespan. An active site for causing erythroblast proliferation may occur in the undamaged amino-terminal domain of the env glycoprotein. Images PMID:3973973

  5. Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

    PubMed

    Kodaira, K I; Taketo, A

    1977-05-17

    Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

  6. Preliminary crystallographic analysis of two oligomerization-deficient mutants of the aerolysin toxin, H132D and H132N, in their proteolyzed forms

    PubMed Central

    Pernot, Lucile; Schiltz, Marc; van der Goot, F. Gisou

    2010-01-01

    Aerolysin is a major virulence factor produced by the Gram-negative bacterium Aeromonas hydrophila and is a member of the β-pore-forming toxin family. Two oligomerization-deficient aerolysin mutants, H132D and H132N, have been overproduced, proteolyzed by trypsin digestion and purified. Crystals were grown from the proteolyzed forms and diffraction data were collected for the two mutants to 2.1 and 2.3 Å resolution, respectively. The prism-shaped crystals belonged to space group C2. The crystal structure of the mutants in the mature, but not heptameric, aerolysin form will provide insight into the intermediate states in the oligomerization process of a pore-forming toxin. PMID:21139211

  7. Preliminary crystallographic analysis of two oligomerization-deficient mutants of the aerolysin toxin, H132D and H132N, in their proteolyzed forms.

    PubMed

    Pernot, Lucile; Schiltz, Marc; van der Goot, F Gisou

    2010-12-01

    Aerolysin is a major virulence factor produced by the Gram-negative bacterium Aeromonas hydrophila and is a member of the β-pore-forming toxin family. Two oligomerization-deficient aerolysin mutants, H132D and H132N, have been overproduced, proteolyzed by trypsin digestion and purified. Crystals were grown from the proteolyzed forms and diffraction data were collected for the two mutants to 2.1 and 2.3 Å resolution, respectively. The prism-shaped crystals belonged to space group C2. The crystal structure of the mutants in the mature, but not heptameric, aerolysin form will provide insight into the intermediate states in the oligomerization process of a pore-forming toxin.

  8. Internal Dynamics and Ionization States of the Macrophage Migration Inhibitory Factor: Comparison Between Wild-Type and Mutant Forms

    SciTech Connect

    Soares, Thereza A.; Lins, Roberto D.; Straatsma, TP; Briggs, J. M.

    2002-11-15

    The macrophage migration inhibitory factor (MIF) is a cytokine which shares a common structural architecture and catalytic strategy with three isomerases: 4-oxalocrotonate tautomerase, 5-carboxymethyl-2-hydroxymuconate isomerase and D-dopachrome tautomerase. A highly conserved N-terminal proline acts as a base\\acid during the proton transfer reaction catalyzed by these enzymes. Such unusual catalytic strategy appears to be possible only due to the N-terminal proline pKa be shifted to 5.0-6.0 units. Mutations of this residue result in a significant decrease of the catalytic activity of MIF. Two hypotheses have been proposed to explain the catalytic inefficiency of MIF: the lower basicity of primary amines with regard to secondary ones and the increased flexibility resulting from the replacement of a proline by residues like glycine. To investigate that, we have performed molecular dynamics simulations of MIF-wt and its mutant P1G as well as calculated the protonation properties of several mutant forms. It has been found that the N-terminal glycine does not show larger fluctuations compared to proline, but the former residue is more exposed to the solvent throughout the simulations. The apparent pKa of these residues displays very little change (as expected from the structural rigidity of MIF) and is not significantly affected by the surrounding ionizable residues. Instead, the hydrophobic character of the active site seems to be the main factor in determining the pKa of the N-terminal residue and the catalytic efficiency of MIF.

  9. Amelioration of the cardiovascular effects of cocaine in rhesus monkeys by a long-acting mutant form of cocaine esterase.

    PubMed

    Collins, Gregory T; Carey, Kathy A; Narasimhan, Diwahar; Nichols, Joseph; Berlin, Aaron A; Lukacs, Nicholas W; Sunahara, Roger K; Woods, James H; Ko, Mei-Chuan

    2011-04-01

    A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5-10 min, and saline-like HR measures restored within 20-40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.

  10. Structure-function analysis of dynein light chain 1 identifies viable motility mutants in bloodstream-form Trypanosoma brucei.

    PubMed

    Ralston, Katherine S; Kisalu, Neville K; Hill, Kent L

    2011-07-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

  11. The Peripheral Neuropathy-Linked Trembler and Trembler-J Mutant Forms of Peripheral Myelin Protein 22 are Folding-Destabilized†

    PubMed Central

    Myers, Jeffrey K.; Mobley, Charles K.; Sanders, Charles R.

    2008-01-01

    Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth Type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and folding stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D (Trembler phenotype), and L16P (Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the 3-D structures of these disease-linked mutants are similar to the folded wild type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant. PMID:18795802

  12. De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

    PubMed Central

    Sebastian, Jees; Swaminath, Sharmada; Nair, Rashmi Ravindran; Jakkala, Kishor; Pradhan, Atul

    2016-01-01

    ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli. PMID:27895008

  13. De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro.

    PubMed

    Sebastian, Jees; Swaminath, Sharmada; Nair, Rashmi Ravindran; Jakkala, Kishor; Pradhan, Atul; Ajitkumar, Parthasarathi

    2017-02-01

    Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli. Copyright © 2017 American Society for Microbiology.

  14. Congenital Cataract Causing Mutants of αA-Crystallin/sHSP Form Aggregates and Aggresomes Degraded through Ubiquitin-Proteasome Pathway

    PubMed Central

    Raju, Ilangovan; Abraham, Edathara C.

    2011-01-01

    Background Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy. Methodology/Principal Findings YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control. Conclusions/Significance Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells

  15. Cytomegalovirus UL128 homolog mutants that form a pentameric complex produce virus with impaired epithelial and trophoblast cell tropism and altered pathogenicity in the guinea pig.

    PubMed

    Coleman, Stewart; Choi, K Yeon; McGregor, Alistair

    2017-09-01

    Guinea pig cytomegalovirus (GPCMV) encodes a homolog pentameric complex (PC) for specific cell tropism and congenital infection. In human cytomegalovirus, the PC is an important antibody neutralizing target and GPCMV studies will aid in the development of intervention strategies. Deletion mutants of the C-terminal domains of unique PC proteins (UL128, UL130 and UL131 homologs) were unable to form a PC in separate transient expression assays. Minor modifications to the UL128 homolog (GP129) C-terminal domain enabled PC formation but viruses encoding these mutants had altered tropism to renal and placental trophoblast cells. Mutation of the presumptive CC chemokine motif encoded by GP129 was investigated by alanine substitution of the CC motif (codons 26-27) and cysteines (codons 47 and 62). GP129 chemokine mutants formed PC but GP129 chemokine mutant viruses had reduced epitropism. A GP129 chemokine mutant virus pathogenicity study demonstrated reduced viral load to target organs but highly extended viremia. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli

    PubMed Central

    Vetouli, E. E.; Bozavoutoglou, E.; Lebessi, E.; Tzelepi, E.; Tzouvelekis, L. S.

    2015-01-01

    The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did. PMID:26438499

  17. Purification, crystallization and preliminary X-ray analysis of truncated and mutant forms of VP4 protease from infectious pancreatic necrosis virus

    SciTech Connect

    Lee, Jaeyong; Feldman, Anat R.; Chiu, Elaine; Chan, Charlena; Kim, You-Na; Delmas, Bernard; Paetzel, Mark

    2006-12-01

    Various truncated and mutant forms of the protease VP4 from infectious pancreatic necrosis virus were used to generate two different crystal forms of VP4 which diffracted to beyond 2.4 Å resolution. In viruses belonging to the Birnaviridae family, virus protein 4 (VP4) is the viral protease responsible for the proteolytic maturation of the polyprotein encoding the major capsid proteins (VP2 and VP3). Infectious pancreatic necrosis virus (IPNV), the prototype of the aquabirnavirus genus, is the causative agent of a contagious disease in fish which has a large economic impact on aquaculture. IPNV VP4 is a 226-residue (24.0 kDa) serine protease that utilizes a Ser/Lys catalytic dyad mechanism (Ser633 and Lys674). Several truncated and mutant forms of VP4 were expressed in a recombinant expression system, purified and screened for crystallization. Two different crystal forms diffract beyond 2.4 Å resolution. A triclinic crystal derived from one mutant construct has unit-cell parameters a = 41.7, b = 69.6, c = 191.6 Å, α = 93.0, β = 95.1, γ = 97.7°. A hexagonal crystal with space group P6{sub 1}22/P6{sub 5}22 derived from another mutant construct has unit-cell parameters a = 77.4, b = 77.4, c = 136.9 Å.

  18. [Physical-chemical properties of the mutant (protein) form of D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN].

    PubMed

    Fonin, A V; Stepanenko, O V; Povarova, O I; Volova, E A; Filippova, E M; Bublikov, G S; Kuznetsova, I M; Demchenko, A P; Turoverov, K K

    2013-01-01

    The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system.

  19. Restoration of DNA-Binding and Growth-Suppressive Activity of Mutant Forms of p53 Via a PCAF-Mediated Acetylation Pathway

    PubMed Central

    PEREZ, RICARDO E.; KNIGHTS, CHAD D.; SAHU, GEETARAM; CATANIA, JASON; KOLUKULA, VAMSI K.; STOLER, DANIEL; GRAESSMANN, ADOLF; OGRYZKO, VASILY; PISHVAIAN, MICHAEL; ALBANESE, CHRISTOPHER; AVANTAGGIATI, MARIA LAURA

    2013-01-01

    Tumor-derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell-type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re-induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild-type p53 is modulated by various acetyl-transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl-transferase is down-regulated in tumors harboring p53 mutants, where its re-expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA-binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53-regulated promoters and transcriptional activity in vivo. These data suggest that PCAF-mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis-regulation of PCAF activity is a pre-requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53. PMID:20589832

  20. Herbicide-resistant forms of Arabidopsis thaliana acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to inhibitors of four defined mutants.

    PubMed Central

    Chang, A K; Duggleby, R G

    1998-01-01

    Acetohydroxyacid synthase (AHAS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. Four mutants (A122V, W574S, W574L and S653N) of the AHAS gene from Arabidopsis thaliana were constructed, expressed in Escherichia coli, and the enzymes were purified. Each mutant form and wild-type was characterized with respect to its catalytic properties and sensitivity to nine herbicides. Each enzyme had a pH optimum near 7.5. The specific activity varied from 13% (A122V) to 131% (W574L) of the wild-type and the Km for pyruvate of the mutants was similar to the wild-type, except for W574L where it was five-fold higher. The activation by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined. A122V showed reduced affinity for all three cofactors, whereas S653N bound FAD more strongly than wild-type AHAS. Six sulphonylurea herbicides inhibited A122V to a similar degree as the wild-type but S653N showed a somewhat greater reduction in sensitivity to these compounds. In contrast, the W574 mutants were insensitive to these sulphonylureas, with increases in the Kiapp (apparent inhibition constant) of several hundred fold. All four mutants were resistant to three imidazolinone herbicides with decreases in sensitivity ranging from 100-fold to more than 1000-fold. PMID:9677339

  1. Process of consecutive cell divisions and separations in a regular tetrads-forming mutant of Micrococcus lysodeikticus (luteus).

    PubMed

    Monodane, T; Matsushima, Y; Kotani, S

    1978-01-01

    A mutant MT of Micrococcus lysodeikticus (luteus) IFO 3333, whose minimum growing unit is not a single cell, but a tetrad unlike the wild-type divides by binary fission of each monococcus, and then separates first into two daughter tetrads, second into four tetrads and third into eight tetrads. The three planes of either the cell division or the cell separation are equivalent to one another and oriented at right angles in three dimensions, respectively. The process of consecutive cell divisions and separations of the mutant tetrads was schematically illustrated.

  2. Cl- -dependent photovoltage responses of bacteriorhodopsin: comparison of the D85T and D85S mutants and wild-type acid purple form.

    PubMed

    Kalaidzidis, I V; Kaulen, A D

    1997-12-01

    Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl- -induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl- translocation in the cytoplasmic direction. Cl- translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild-type acid purple form, respectively. Cl- induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild-type acid purple form. This phase is supposed to be associated with the reversible Cl- movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.

  3. A polymorphic form of steroidogenic factor 1 associated with ACTH receptor deficiency in mouse adrenal cell mutants.

    PubMed

    Schimmer, Bernard P; Cordova, Martha; Tsao, Jennivine; Frigeri, Claudia

    2003-06-01

    We have described a family of adrenocortical tumor cell mutants (including clones OS3, Y6, and 10r9) that are resistant to ACTH because they fail to express the gene encoding the ACTH receptor (MC2R). The MC2R deficiency results from a mutation that impairs the activity of the nuclear receptor steroidogenic factor 1 (SF1) at the MC2R promoter. In this report, we show that ACTH resistance in the mutant clones is associated with a Sf1 gene that has Ser at codon 172 instead of Ala. In two of the three mutant clones, this Sf1 allele is amplified together with flanking DNA from chromosome 2 that includes the genes encoding germ cell nuclear factor and the beta-type proteosome subunit Psmb7. SF1(A172) and SF1(S172) exhibit little or no difference in transcriptional activity in SF1-dependent reporter gene assays, suggesting that SF1(S172) per se is not directly responsible for the loss of MC2R expression. Instead, the Sf1(S172) allele appears to be a marker of ACTH resistance in this family of adrenocortical tumor cell mutants, possibly reflecting the activity of a neighboring gene.

  4. The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

    PubMed Central

    Peres, Lázaro Eustáquio Pereira

    2012-01-01

    Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively. PMID:22915742

  5. An over-oxidized form of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with bulbar onset shares a toxic mechanism with mutant SOD1

    PubMed Central

    Guareschi, Stefania; Cova, Emanuela; Cereda, Cristina; Ceroni, Mauro; Donetti, Elena; Bosco, Daryl A.; Trotti, Davide; Pasinelli, Piera

    2012-01-01

    Recent studies suggest that Cu/Zn superoxide dismutase (SOD1) could be pathogenic in both familial and sporadic amyotrophic lateral sclerosis (ALS) through either inheritable or nonheritable modifications. The presence of a misfolded WT SOD1 in patients with sporadic ALS, along with the recently reported evidence that reducing SOD1 levels in astrocytes derived from sporadic patients inhibits astrocyte-mediated toxicity on motor neurons, suggest that WT SOD1 may acquire toxic properties similar to familial ALS-linked mutant SOD1, perhaps through posttranslational modifications. Using patients’ lymphoblasts, we show here that indeed WT SOD1 is modified posttranslationally in sporadic ALS and is iper-oxidized (i.e., above baseline oxidation levels) in a subset of patients with bulbar onset. Derivatization analysis of oxidized carbonyl compounds performed on immunoprecipitated SOD1 identified an iper-oxidized SOD1 that recapitulates mutant SOD1-like properties and damages mitochondria by forming a toxic complex with mitochondrial Bcl-2. This study conclusively demonstrates the existence of an iper-oxidized SOD1 with toxic properties in patient-derived cells and identifies a common SOD1-dependent toxicity between mutant SOD1-linked familial ALS and a subset of sporadic ALS, providing an opportunity to develop biomarkers to subclassify ALS and devise SOD1-based therapies that go beyond the small group of patients with mutant SOD1. PMID:22416121

  6. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    PubMed

    Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  7. Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin.

    PubMed

    Adachi, H; Ohta, T; Matsuzawa, H

    1991-02-15

    Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme.

  8. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

    PubMed Central

    Reed, Matthew D.; Wilder, Julie A.; Mega, William M.; Hutt, Julie A.; Kuehl, Philip J.; Valderas, Michelle W.; Chew, Lawrence L.; Liang, Bertrand C.; Squires, Charles H.

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg. PMID:26207820

  9. Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease.

    PubMed

    Lelan, Faustine; Boyer, Cécile; Thinard, Reynald; Rémy, Séverine; Usal, Claire; Tesson, Laurent; Anegon, Ignacio; Neveu, Isabelle; Damier, Philippe; Naveilhan, Philippe; Lescaudron, Laurent

    2011-01-01

    A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn) human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinson's disease (PD). This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ) and the olfactory bulbs (OB) as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.

  10. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.

    PubMed

    Vidal, O; Longin, R; Prigent-Combaret, C; Dorel, C; Hooreman, M; Lejeune, P

    1998-05-01

    Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized E. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652-655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the ompR allele described in this work. Transduction of knockout mutations in either csgA or ompR caused the loss of the adherence properties of several biofilm-forming E. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

  11. Characterization of a stable spheroplast type L-form of Proteus mirabilis D 52 as cell envelope mutant. I. Isolation, growth characteristics, biochemical activities, and sensitivity to bacteriophages.

    PubMed

    Gumpert, J; Taubeneck, U

    1975-01-01

    A stable spheroplast type L-form could be isolated by transferring 627 single colonies and 195 agar blocks with several colonies of unstable L-forms of Proteus mirabilis D 52 on agar media without supplements of penicillin. The L-form grows well on complex and synthetic agar media, however, it failed to grow in any of the liquid media which have been proved. With one exception (formation of acid from maltose) the L-form shows the same bioche mical activities like the parent rod-shaped bacterium. However, the insensitivity for various phages and the failure of DAP in the envelopes demonstrate that there are profound alterations in the biosynthesis and structure of the murein and of the outer wall layers. The results of these investigations and an ultrastructural analysis (Gumpert and Taubeneck 1975) show that the stable spheroplast type L-form LD 52 B of Proteus mirabilis must be considered as a true cell envelope mutant.

  12. Mutant alpha-synuclein causes age-dependent neuropathology in monkey brain.

    PubMed

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua; Li, Xiao-Jiang

    2015-05-27

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. Copyright © 2015 the authors 0270-6474/15/358345-14$15.00/0.

  13. Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

    PubMed Central

    Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua

    2015-01-01

    Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. PMID:26019347

  14. Structure of recombinant capsids formed by the beta-annulus deletion mutant -- rCP (Delta48-59) of Sesbania mosaic virus.

    PubMed

    Pappachan, Anju; Subashchandrabose, Chinnathambi; Satheshkumar, P S; Savithri, H S; Murthy, M R N

    2008-05-25

    A unique feature of several T=3 icosahedral viruses is the presence of a structure called the beta-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T=3 capsid assembly. In order to examine the importance of the beta-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48-59 involved in the formation of the beta-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Delta48-59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 A resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the beta-annulus. Therefore, the beta-annulus is not essential for T=3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the beta-annulus could be closely similar.

  15. Crystallization and preliminary crystallographic analysis of decameric and monomeric forms of C49S mutant thioredoxin-dependent AhpC from Helicobacter pylori

    SciTech Connect

    Supangat; Seo, Kyung Hye; Furqoni, Ahmad; Kwon, Young-Chul; Cho, Myung-Je; Rhee, Kwang-Ho; Lee, Sang Yeol; Lee, Kon Ho

    2008-05-01

    Decameric and monomeric forms of recombinant C49S mutant AhpC from H. pylori have been crystallized. Diffraction data were collected to 2.8 and 2.25 Å, respectively. Cys49Ser mutant Helicobacter pylori alkyl hydroperoxide reductase (C49S HpAhpC) was purified under reducing conditions in monomeric and decameric forms. The monomeric form was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.25 Å resolution and belonged to space group C2, with unit-cell parameters a = 245.8, b = 140.7, c = 189.5 Å, β = 127°, and contained 20 molecules in the asymmetric unit. A crystal of the decameric form was obtained by the microbatch crystallization method and diffracted to 2.8 Å resolution. It belonged to space group C222, with unit-cell parameters a = 257.5, b = 417.5, c = 95.6 Å. The structure of the monomeric form of C49S HpAhpC has been solved by the molecular-replacement method.

  16. A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts.

    PubMed

    Mayer, T; Meyer, M; Janning, A; Schiedel, A C; Barnekow, A

    1999-03-25

    The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

  17. Expression and properties of wild-type and mutant forms of the Drosophila sex comb on midleg (SCM) repressor protein.

    PubMed

    Bornemann, D; Miller, E; Simon, J

    1998-10-01

    The Sex comb on midleg (Scm) gene encodes a transcriptional repressor of the Polycomb group (PcG). Here we show that SCM protein is nuclear and that its expression is widespread during fly development. SCM protein contains a C-terminal domain, termed the SPM domain, which mediates protein-protein interactions. The biochemical function of another domain consisting of two 100-amino-acid-long repeats, termed "mbt" repeats, is unknown. We have determined the molecular lesions of nine Scm mutant alleles, which identify functional requirements for specific domains. The Scm alleles were tested for genetic interactions with mutations in other PcG genes. Intriguingly, three hypomorphic Scm mutations, which map within an mbt repeat, interact with PcG mutations more strongly than do Scm null alleles. The strongest interactions produce partial synthetic lethality that affects doubly heterozygous females more severely than males. We show that mbt repeat alleles produce stable SCM proteins that associate with normal sites in polytene chromosomes. We also analyzed progeny from Scm mutant germline clones to compare the effects of an mbt repeat mutation during embryonic vs. pupal development. We suggest that the mbt repeat alleles produce altered SCM proteins that incorporate into and impair function of PcG protein complexes.

  18. FTIR spectroscopic study of biofilms formed by the rhizobacterium Azospirillum brasilense Sp245 and its mutant Azospirillum brasilense Sp245.1610

    NASA Astrophysics Data System (ADS)

    Tugarova, Anna V.; Scheludko, Andrei V.; Dyatlova, Yulia A.; Filip'echeva, Yulia A.; Kamnev, Alexander A.

    2017-07-01

    Biofilms are spatially and metabolically structured communities of microorganisms, representing a mode of their existence which is ubiquitous in nature, with cells localised within an extracellular biopolymeric matrix, attached to each other, at an interface. For plant-growth-promoting rhizobacteria (PGPR), the formation of biofilms is of special importance due to their primary localisation at the surface of plant root systems. In this work, FTIR spectroscopy was used, for the first time for bacteria of the genus Azospirillum, to comparatively study 6-day-mature biofilms formed on the surface of ZnSe discs by the rhizobacterium Azospirillum brasilense Sp245 and its mutant A. brasilense Sp245.1610. The mutant strain, having an Omegon Km insertion in the gene of lipid metabolism fabG1 on the plasmid AZOBR_p1, as compared to the wild-type strain Sp245 (see http://dx.doi.org/10.1134/S1022795413110112)

  19. Cell surface of a tetrads-forming mutant of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface.

    PubMed

    Monodane, T; Tokunaga, M; Torii, M

    1990-01-01

    Tetrads-forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four "unit monococci" constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild-type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165-174, 1989) and the mutant MT cells is discussed.

  20. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

    PubMed

    Conway de Macario, E; Ellis, J; Guzman, R; Rotman, B

    1978-02-01

    Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,

  1. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

    PubMed Central

    de Macario, E C; Ellis, J; Guzman, R; Rotman, B

    1978-01-01

    Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody, PMID:416439

  2. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

    SciTech Connect

    Sukumar, Narayanasami; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F. Scott

    2009-06-01

    The crystal structure of the G81A mutant form of the chimera of (S)-mandelate dehydrogenase and of its complexes with two of its substrates reveal productive and non-productive modes of binding for the catalytic reaction. The structure also indicates the role of G81A in lowering the redox potential of the flavin co-factor leading to an ∼200-fold slower catalytic rate of substrate oxidation. (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed ∼100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 Å resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by

  3. Stoichiometric selection of tight-binding inhibitors by wild-type and mutant forms of malarial (Plasmodium falciparum) dihydrofolate reductase.

    PubMed

    Kamchonwongpaisan, Sumalee; Vanichtanankul, Jarunee; Tarnchompoo, Bongkoch; Yuvaniyama, Jirundon; Taweechai, Supannee; Yuthavong, Yongyuth

    2005-03-01

    A simple method for screening combinatorial and other libraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has been developed, based on the affinities of the inhibitors with the enzyme. In the presence of limiting amounts of the enzyme, a number of inhibitors in the library were bound to extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatment to release bound inhibitors, the amounts of free and bound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The differences in the patterns reflected the binding of high-affinity components compared with the other members in the library. A good correlation was found between the inhibition constants (Ki values) and the extent of binding of inhibitors to wild-type, double (C59R+S108N) and quadruple mutant (N51I+C59R+S108N+I164L) of PfDHFR, as well as human DHFR. In addition to identifying lead components of the libraries with high affinities (low Ki values) and stabilities (low k(off) rates), this simple method also provides an alternative way for quickly and accurately calculating enzyme binding affinities of inhibitors in combinatorial chemical libraries.

  4. Crystal structure of the 70S ribosome bound with the Q253P mutant form of release factor RF2.

    PubMed

    Santos, Natalia; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Noller, Harry F

    2013-07-02

    Bacterial translation termination is mediated by release factors RF1 and RF2, which recognize stop codons and catalyze hydrolysis of the peptidyl-tRNA ester bond. The catalytic mechanism has been debated. We proposed that the backbone amide NH group, rather than the side chain, of the glutamine of the universally conserved GGQ motif participates in catalysis by H-bonding to the tetrahedral transition-state intermediate and by product stabilization. This was supported by complete loss of RF1 catalytic activity when glutamine is replaced by proline, the only residue that lacks a backbone NH group. Here, we present the 3.4 Å crystal structure of the ribosome complex containing the RF2 Q253P mutant and find that its fold, including the GGP sequence, is virtually identical to that of wild-type RF2. This rules out proline-induced misfolding and further supports the proposal that catalytic activity requires interaction of the Gln-253 backbone amide with the 3' end of peptidyl-tRNA.

  5. Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

    PubMed Central

    Barbeito, Ana G.; Garringer, Holly J.; Baraibar, Martin A.; Gao, Xiaoying; Arredondo, Miguel; Núñez, Marco T.; Smith, Mark A.; Ghetti, Bernardino; Vidal, Ruben

    2009-01-01

    Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain. PMID:19519778

  6. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1

    PubMed Central

    Ghadge, Ghanashyam D.; Pavlovic, John; Koduvayur, Sujatha P.; Kay, Brian K.; Roos, Raymond P.

    2013-01-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as ‘intrabodies’ within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. PMID:23607939

  7. Complementation in trans of altered thymocyte development in knock-in mice expressing mutant forms of SLP76

    PubMed Central

    Jordan, Martha S.; Smith, Jennifer E.; Burns, Jeremy C.; Austin, Jessica-Elise T.; Nichols, Kim E.; Aschenbrenner, Anna C.; Koretzky, Gary A.

    2008-01-01

    Summary The adaptor protein SLP76 directs signaling downstream of the TCR and is essential for thymocyte development. SLP76 contains three tyrosines in its N-terminus that are critical for its function. To define the role of these residues in thymocyte development, we generated two lines of KI mice, one expressing a mutation in tyrosine 145 (Y145F) and a second harboring two point mutations at tyrosines 112 and 128 (Y112/128F). We show here that while thymocyte development requires both Y145 and Y112/128-generated signals, selection is more dependent upon Y145. While several proximal TCR signaling events were defective in both KI mice, phosphorylation of Vav1 and activation of Itk-dependent pathways were differentially affected by mutations at Y112/128 or Y145, respectively. Analysis of mice expressing one Y145F and one Y112/128F allele revealed that these mutants could complement one another in trans, demonstrating cooperativity between two or more SLP76 molecules. PMID:18342008

  8. Structural studies of mutant forms of the PQQ-forming enzyme PqqC in the presence of product and substrate.

    PubMed

    Puehringer, Sandra; RoseFigura, Jordan; Metlitzky, Moritz; Toyama, Hirohide; Klinman, Judith P; Schwarzenbacher, Robert

    2010-08-15

    Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial cofactor in numerous alcohol dehydrogenases including methanol dehydrogenase and glucose dehydrogenase. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF and proceeds by an unknown pathway. PqqC is one of two metal free oxidases of known structure and catalyzes the last step of PQQ biogenesis which involves a ring closure and an eight-electron oxidation of the substrate [3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ)]. PqqC has 14 conserved active site residues, which have previously been shown to be in close contact with bound PQQ. Herein, we describe the structures of three PqqC active site variants, H154S, Y175F, and the double mutant R179S/Y175S. The H154S crystal structure shows that, even with PQQ bound, the enzyme is still in the "open" conformation with helices alpha5b and alpha6 unfolded and the active site solvent accessible. The Y175F PQQ complex crystal structure reveals the closed conformation indicating that Y175 is not required for the conformational change. The R179S/Y175S AHQQ complex crystal structure is the most mechanistically informative, indicating an open conformation with a reaction intermediate trapped in the active site. The intermediate seen in R179S/Y175S is tricyclic but nonplanar, implying that it has not undergone oxidation. These studies implicate a stepwise process in which substrate binding leads to the generation of the closed protein conformation, with the latter playing a critical role in O(2) binding and catalysis. 2010 Wiley-Liss, Inc.

  9. Spectral characteristics of the mutant form GGBP/H152C of D-glucose/D-galactose-binding protein labeled with fluorescent dye BADAN: influence of external factors

    PubMed Central

    Fonin, Alexander V.; Stepanenko, Olga V.; Povarova, Olga I.; Volova, Catherine A.; Philippova, Elizaveta M.; Bublikov, Grigory S.; Kuznetsova, Irina M.; Demchenko, Alexander P.

    2014-01-01

    The mutant form GGBP/H152C of the D-glucose/D-galactose-binding protein with the solvatochromic dye BADAN linked to cysteine residue Cys 152 can be used as a potential base for a sensitive element of glucose biosensor system. We investigated the influence of various external factors on the physical-chemical properties of GGBP/H152C-BADAN and its complex with glucose. The high affinity (Kd = 8.5 µM) and high binding rate of glucose make GGBP/H152C-BADAN a good candidate to determine the sugar content in biological fluids extracted using transdermal techniques. It was shown that changes in the ionic strength and pH of solution within the physiological range did not have a significant influence on the fluorescent characteristics of GGBP/H152C-BADAN. The mutant form GGBP/H152C has relatively low resistance to denaturation action of GdnHCl and urea. This result emphasizes the need to find more stable proteins for the creation of a sensitive element for a glucose biosensor system. PMID:24711960

  10. X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

    PubMed

    Yoshida, Hiromi; Nishi, Nozomu; Wada, Kenji; Nakamura, Takanori; Hirashima, Mitsuomi; Kuwabara, Naoyuki; Kato, Ryuichi; Kamitori, Shigehiro

    2017-09-02

    Galectin-9 (G9) is a tandem-repeat type β-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers.

    PubMed

    Hansson, A; Willows, R D; Roberts, T H; Hansson, M

    2002-10-15

    Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg(2+) into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA(+)) family of proteins. AAA(+) proteins are Mg(2+)-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.

  12. Differential interaction between iron and mutant alpha-synuclein causes distinctive Parkinsonian phenotypes in Drosophila.

    PubMed

    Zhu, Zhou-Jing; Wu, Ka-Chun; Yung, Wing-Ho; Qian, Zhong-Ming; Ke, Ya

    2016-04-01

    Alpha-synuclein aggregation is the central hallmark of both sporadic and familial Parkinson's disease (PD). Patients with different PD-causing genetic defects of alpha-synuclein usually show distinctive clinical features that are atypical to sporadic PD. Iron accumulation is invariably found in PD. Recent studies showed that mutant and wild-type alpha-synuclein may have differential interaction with iron and mutant alpha-synuclein toxicity could be preferentially exacerbated by iron. We hence hypothesized that iron overload could selectively influence mutant alpha-synuclein toxicity and disease phenotypes. To test the hypothesis, we investigated if Drosophila melanogaster over-expressing A53T, A30P, and wild-type (WT) alpha-synuclein have different responses to iron treatment. We showed that iron treatment induced similar reduction of survival rate in all flies but induced a more severe motor decline in A53T and A30P mutant alpha-synuclein expressing flies, suggesting interaction between mutant alpha-synuclein and iron. Although no significant difference in total head iron content was found among these flies, we demonstrated that iron treatment induced selective DA neuron loss in motor-related PPM3 cluster only in the flies that express A53T and A30P mutant alpha-synuclein. We provided the first in vivo evidence that iron overload could induce distinctive neuropathology and disease phenotypes in mutant but not WT alpha-synuclein expressing flies, providing insights to the cause of clinical features selectively exhibited by mutant alpha-synuclein carriers.

  13. Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Phage T4 Vertex Protein Gp24 and its Mutant Forms

    SciTech Connect

    Boeshans,K.; Liu, F.; Peng, G.; Idler, W.; Jang, S.; Marekov, L.; Black, L.; Ahvazi, B.

    2006-01-01

    The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6 {angstrom} resolution compared to wild-type gp24 at 3.80 {angstrom} resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

  14. Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo- and apo-forms.

    PubMed Central

    Sau, A K; Chen, C A; Cowan, J A; Mazumdar, S; Mitra, S

    2001-01-01

    Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe(4)S(4)](3+)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the [Fe(4)S(4)](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent. PMID:11566801

  15. Use of resistant ACCase mutants to screen for novel inhibitors against resistant and susceptible forms of ACCase from grass weeds.

    PubMed

    Shukla, Amit; Nycholat, Corwin; Subramanian, Mani V; Anderson, Richard J; Devine, Malcolm D

    2004-08-11

    The aryloxyphenoxypropionic acid (AOPP) and cyclohexanedione (CHD) herbicides inhibit the first committed enzyme in fatty acid biosynthesis, acetyl CoA carboxylase (ACCase). The frequent use of AOPP and CHD herbicides has resulted in the development of resistance to these herbicides in many grass weed species. New herbicides that inhibit both the susceptible and resistant forms of ACCase in grass weeds would have obvious commercial appeal. In the present study, an attempt was made to identify molecules that target both the herbicide-sensitive and -resistant forms of ACCase. Seven experimental compounds, either CHD-like or AOPP-CHD hybrids, were synthesized and assayed against previously characterized susceptible and resistant forms of ACCase. All seven compounds inhibited ACCase from sensitive biotypes of Setaria viridis and Eleusine indica (I50 values from 6.4 to >100 microM) but were not particularly potent compared to some commercialized herbicides (I50 values of 0.08-5.6 microM). In almost all cases, the I50 values for each compound assayed against the resistant ACCases were higher than those against the corresponding sensitive ACCase, indicating reduced binding to the resistant ACCases. One compound, a CHD analogue, was almost equally effective against the resistant and susceptible ACCases, although it was not a very potent ACCase inhibitor per se (I50 of 51 and 76 microM against susceptible ACCase from S. viridis and E. indica, respectively). The AOPP-CHD hybrid molecules also inhibited some of the resistant ACCases, with I50 values ranging from 6.4 to 50 microM. These compounds may be good leads for developing ACCase inhibitors that target a wider range of ACCase isoforms, including those found in AOPP- and CHD-resistant weed biotypes.

  16. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence

    PubMed Central

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E.; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R.; Ross, Ian; Parton, Robert G.; Böcking, Till; Gambin, Yann

    2016-01-01

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer’s and Parkinson’s disease. Parkinson’s disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson’s disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils. PMID:27892477

  17. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence.

    PubMed

    Sierecki, Emma; Giles, Nichole; Bowden, Quill; Polinkovsky, Mark E; Steinbeck, Janina; Arrioti, Nicholas; Rahman, Diya; Bhumkar, Akshay; Nicovich, Philip R; Ross, Ian; Parton, Robert G; Böcking, Till; Gambin, Yann

    2016-11-28

    Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.

  18. Probing the folding and unfolding of wild-type and mutant forms of bacteriorhodopsin in micellar solutions: evaluation of reversible unfolding conditions.

    PubMed

    Chen, G Q; Gouaux, E

    1999-11-16

    Wild-type and mutant forms of bacteriorhodopsin (sbR) from Halobacterium salinarium, produced by Escherichia coli overexpression of a synthetic gene, were reversibly unfolded in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxyl-1-propane (CHAPSO), and sodium dodecyl sulfate (SDS) mixed micelles. To study the effect on protein stability by substitutions on the hydrophobic surface with polar residues, the unfolding behavior of a G113Q, G116Q mutant [sbR(Q2)] was compared to the wild-type sbR [sbR(WT)]. sbR(Q2) was more sensitive to SDS-induced unfolding than sbR(WT) under equilibrium conditions, and kinetic experiments showed that sbR(Q2) was more sensitive to acid-induced denaturation and thermal unfolding than sbR(WT). Since the mutations in sbR(Q2) were on the detergent-embedded hydrophobic surface of sbR, protein destabilization by these mutations supports the concept that the membrane-embedded segments are important for the stability of sbR. Our experiments provide the basis for studying the thermodynamic stability of sbR by evaluating reversible folding and unfolding conditions in DMPC/CHAPSO/SDS mixed micelles.

  19. Dimerization, oligomerization, and aggregation of human amyotrophic lateral sclerosis copper/zinc superoxide dismutase 1 protein mutant forms in live cells.

    PubMed

    Kim, Jiho; Lee, Honggun; Lee, Joo Hyun; Kwon, Do-yoon; Genovesio, Auguste; Fenistein, Denis; Ogier, Arnaud; Brondani, Vincent; Grailhe, Regis

    2014-05-23

    More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.

  20. Protein structure refinement based on paramagnetic NMR shifts: applications to wild-type and mutant forms of cytochrome c.

    PubMed Central

    Gochin, M.; Roder, H.

    1995-01-01

    A new approach to NMR solution structure refinement is introduced that uses paramagnetic effects on nuclear chemical shifts as constraints in energy minimization or molecular dynamics calculations. Chemical shift differences between oxidized and reduced forms of horse cytochrome c for more than 300 protons were used as constraints to refine the structure of the wild-type protein in solution and to define the structural changes induced by a Leu 94 to Val mutation. A single round of constrained minimization, using the crystal structure as the starting point, converged to a low-energy structure with an RMS deviation between calculated and observed pseudo-contact shifts of 0.045 ppm, 7.5-fold lower than the starting structure. At the same time, the procedure provided stereospecific assignments for more than 45 pairs of methylene protons and methyl groups. Structural changes caused by the mutation were determined to a precision of better than 0.3 A. Structure determination based on dipolar paramagnetic (pseudocontact) shifts is applicable to molecules containing anisotropic paramagnetic centers with short electronic relaxation times, including numerous naturally occurring metalloproteins, as well as proteins or nucleic acids to which a paramagnetic metal ion or ligand may be attached. The long range of paramagnetic shift effects (up to 20 A from the iron in the case of cytochrome c) provides global structural constraints, which, in conjunction with conventional NMR distance and dihedral angle constraints, will enhance the precision of NMR solution structure determination. PMID:7757018

  1. Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

    PubMed Central

    Sun, Xiankui; Fontaine, Jean-Marc; Hoppe, Adam D.; Carra, Serena; DeGuzman, Cheryl; Martin, Jody L.; Simon, Stephanie; Vicart, Patrick; Welsh, Michael J.; Landry, Jacques

    2010-01-01

    A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing. PMID:20157854

  2. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    PubMed

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  3. X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

    PubMed

    Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro

    2016-12-01

    Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

  4. Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: The blue form is inactive in proton translocation

    SciTech Connect

    Subramaniam, S.; Marti, T.; Khorana, H.G. )

    1990-02-01

    Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85----Glu and Arg-82----Ala mutants exist in a pH-dependent equilibrium between purple (lambda max approximately 550/540 nm) and blue (lambda max approximately 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85----Asn mutant displays a blue chromophore (lambda max approximately 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212----Asn mutant is purple (lambda max approximately 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.

  5. Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

    PubMed

    Subramaniam, S; Marti, T; Khorana, H G

    1990-02-01

    Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85----Glu and Arg-82----Ala mutants exist in a pH-dependent equilibrium between purple (lambda max approximately 550/540 nm) and blue (lambda max approximately 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85----Asn mutant displays a blue chromophore (lambda max approximately 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212----Asn mutant is purple (lambda max approximately 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.

  6. Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei ▿ †

    PubMed Central

    Ralston, Katherine S.; Kisalu, Neville K.; Hill, Kent L.

    2011-01-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness. PMID:21378260

  7. Pro-oxidant copper-binding mode of the Apo form of ALS-linked SOD1 mutant H43R denatured at physiological temperature.

    PubMed

    Fujimaki, Nobuhiro; Kitamura, Furi; Takeuchi, Hideo

    2013-08-06

    The mutation of Cu,Zn-superoxide dismutase (SOD1), a major antioxidant enzyme, is associated with amyotrophic lateral sclerosis (ALS). In a previous study, we showed that the metal-depleted apo form of an ALS-linked mutant, H43R, undergoes denaturation at physiological temperature (37 °C) in 90 min and acquires pro-oxidant activity in the presence of Cu(2+) and H2O2. In this study, we have examined the Cu(2+)-binding mode of denatured apo-H43R by circular dichroism (CD), fluorescent oxidation, UV Raman spectroscopy, and photooxidation. CD spectroscopy indicates that denatured apo-H43R loses native β-barrel structure and the binding of Cu(2+) to the denatured apo form induces local refolding. Fluorescent-oxidation assays in the absence and presence of Cu(2+) chelators show that denatured apo-H43R contains two Cu(2+)-binding sites with higher and lower Cu(2+) affinities and with pro-oxidant activities in the reverse order. UV Raman spectroscopy gives evidence that His residues are bound to Cu(2+) mainly through the imidazole Nτ atom at the higher-affinity site and through the Nπ atom at the lower-affinity site, sharing one His residue with each other. The Cu(2+)-binding mode of denatured apo-H43R is analogous to but different from the Cu,Zn-binding mode of the native holo form. Photooxidation experiments confirm the involvement of His residues in the pro-oxidant activity. Taken together, it is suggested that the binding of Cu(2+) induces the local refolding of denatured apo-H43R to create toxic catalytic centers that convert the enzyme from antioxidant to pro-oxidant, leading to the pathogenesis of ALS. His residues are essential for both Cu(2+)-binding and pro-oxidant activities.

  8. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  9. Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease.

    PubMed

    de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; Garrido-Maraver, Juan; Cordero, Mario D; Villanueva Paz, Marina; Delgado Pavón, Ana; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Ybot-González, Patricia; Paula Zaderenko, Ana; Ortiz Mellet, Carmen; García Fernández, José M; Sánchez-Alcázar, José A

    2015-06-05

    Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N'-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.

  10. Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

    PubMed Central

    de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; Garrido-Maraver, Juan; Cordero, Mario D.; Villanueva Paz, Marina; Delgado Pavón, Ana; Alcocer-Gómez, Elizabet; de Lavera, Isabel; Ybot-González, Patricia; Paula Zaderenko, Ana; Ortiz Mellet, Carmen; Fernández, José M. García; Sánchez-Alcázar, José A.

    2015-01-01

    Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD. PMID:26045184

  11. Dioxygen activation at non-heme diiron centers: characterization of intermediates in a mutant form of toluene/o-xylene monooxygenase hydroxylase.

    PubMed

    Murray, Leslie J; García-Serres, Ricardo; Naik, Sunil; Huynh, Boi Hanh; Lippard, Stephen J

    2006-06-14

    We report the generation and characterization of an intermediate in a mutant form of the toluene/o-xylene monooxygenase hydroxylase component from Pseudomonas stutzeri OX1. The reaction of chemically reduced I100W variant in the presence of the coupling protein, ToMOD, with dioxygen was monitored by stopped-flow UV/visible spectroscopy. Rapid-freeze quench (RFQ) samples were also generated for EPR and Mössbauer spectroscopy. A transient species is observed in the UV/visible spectrum with an absorption maximum at 500 nm. EPR and Mössbauer spectra of RFQ samples identified this species as a diiron(III,IV) cluster spin-coupled to a neutral W radical. A diamagnetic precursor to the mixed-valent diiron(III,IV) was also observed at an earlier time point, with Mössbauer parameters typical of high-spin FeIII. We have tentatively assigned this antiferromagnetically coupled diiron(III) intermediate as a peroxo-bridged cluster, and this complex has also been observed in preliminary studies of the wild-type hydroxylase.

  12. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.

    PubMed

    Chishti, M A; Yang, D S; Janus, C; Phinney, A L; Horne, P; Pearson, J; Strome, R; Zuker, N; Loukides, J; French, J; Turner, S; Lozza, G; Grilli, M; Kunicki, S; Morissette, C; Paquette, J; Gervais, F; Bergeron, C; Fraser, P E; Carlson, G A; George-Hyslop, P S; Westaway, D

    2001-06-15

    We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.

  13. Increased body temperature accelerates aggregation of the Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy.

    PubMed Central

    Abrahamson, M; Grubb, A

    1994-01-01

    Hereditary cystatin C amyloid angiopathy is a dominantly inherited disorder, characterized by dementia, paralysis, and death from cerebral hemorrhage in early adult life. A variant of the cysteine proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the patients and their spinal-fluid level of cystatin C is abnormally low. The disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in an Escherichia coli expression system and isolated by use of denaturing buffers, immunosorption, and gel filtration. Parallel physicochemical and functional investigations of L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, respectively) but differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological activity immediately after it is transferred to a nondenaturing buffer. The dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. The aggregation at physiological concentrations is likewise increased at 40 compared to 37 degrees C, by approximately 60%. These properties of L68Q-cystatin C have bearing upon our understanding of the pathophysiological process of hereditary cystatin C amyloid angiopathy. They might also be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. Images PMID:8108423

  14. Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

    PubMed

    Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

    2017-03-01

    In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases.

  15. Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

    PubMed Central

    Thompson, L M; Raffioni, S; Wasmuth, J J; Bradshaw, R A

    1997-01-01

    Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to

  16. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates

    SciTech Connect

    Sukumar, Narayanasami; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F. Scott

    2009-06-12

    (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed {approx}100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 {angstrom} resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

  17. Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

    PubMed

    Sukumar, Narayanasami; Dewanti, Asteriani; Merli, Angelo; Rossi, Gian Luigi; Mitra, Bharati; Mathews, F Scott

    2009-06-01

    (S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

  18. PDGFRA-mutant syndrome.

    PubMed

    Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

    2015-07-01

    Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

  19. Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

    PubMed

    Deng, Jiahui; Lv, E; Yang, Jian; Gong, Xiaoli; Zhang, Wenzhong; Liang, Xibin; Wang, Jiazeng; Jia, Jun; Wang, Xiaomin

    2015-05-28

    The acupuncture or electroacupuncture (EA) shows the therapeutic effect on various neurodegenerative diseases. This effect was thought to be partially achieved by its ability to alleviate existing neuroinflammation and glial dysfunction. In this study, we systematically investigated the effect of EA on abnormal neurochemical changes and motor symptoms in a mouse neurodegenerative disease model. The transgenic mouse which expresses a mutant α-synuclein (α-syn) protein, A53T α-syn, in brain astrocytic cells was used. These mice exhibit extensive neuroinflammatory and motor phenotypes of neurodegenerative disorders. In this study, the effects of EA on these phenotypic changes were examined in these mice. EA improved the movement detected in multiple motor tests in A53T mutant mice. At the cellular level, EA significantly reduced the activation of microglia and prevented the loss of dopaminergic neurons in the midbrain and motor neurons in the spinal cord. At the molecular level, EA suppressed the abnormal elevation of proinflammatory factors (tumor necrosis factor-α and interleukin-1β) in the striatum and midbrain of A53T mice. In contrast, EA increased striatal and midbrain expression of a transcription factor, nuclear factor E2-related factor 2, and its downstream antioxidants (heme oxygenase-1 and glutamate-cysteine ligase modifier subunits). These results suggest that EA possesses the ability to ameliorate mutant α-syn-induced motor abnormalities. This ability may be due to that EA enhances both anti-inflammatory and antioxidant activities and suppresses aberrant glial activation in the diseased sites of brains.

  20. Ferritin H subunit gene is specifically expressed in melanophore precursor-derived white pigment cells in which reflecting platelets are formed from stage II melanosomes in the periodic albino mutant of Xenopus laevis.

    PubMed

    Fukuzawa, Toshihiko

    2015-09-01

    "White pigment cells" are derived from melanophore precursors and contain both melanophore-specific and iridophore-specific pigment organelles. Whereas melanophores differentiate in the wild type regenerating tail, white pigment cells appear in the regenerating tail in the periodic albino mutant (a(p)/a(p)) of Xenopus laevis. The localization and density of white pigment cells in the mutant regenerating tail are similar to those of melanophores in the wild type regenerating tail. Here, white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea (PTU), which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells. Ultrastructural analysis shows that reflecting platelet formation in white pigment cells is different from that in iridophores. Reflecting platelets in iridophores are formed from spherical vesicles with electron-dense material, whereas they are formed from stage II melanosomes characteristic of melanophore precursors in white pigment cells. Ultrastructural features of pigment organelles, except reflecting platelets, are similar between mutant melanophores and white pigment cells. In an attempt to identify specific genes in white pigment cells, a subtracted cDNA library enriched for mutant cDNAs has been prepared. Subtracted cDNA fragments have been cloned and selected by whole mount in situ hybridization. Among cDNA fragments examined so far, the ferritin H subunit gene is specifically expressed in white pigment cells, but not in melanophores. Pigment organellogenesis and specific gene expression in white pigment cells are also discussed.

  1. Structural and preliminary molecular dynamics studies of the Rhodobacter sphaeroides reaction center and its mutant form L(M196)H + H(M202)L

    NASA Astrophysics Data System (ADS)

    Klyashtorny, V. G.; Fufina, T. Yu.; Vasilieva, L. G.; Shuvalov, V. A.; Gabdulkhakov, A. G.

    2014-07-01

    Pigment-protein interactions are responsible for the high efficiency of the light-energy transfer and conversion in photosynthesis. The reaction center (RC) from the purple bacterium Rhodobacter sphaeroides is the most convenient model for studying the mechanisms of primary processes of photosynthesis. Site-directed mutagenesis can be used to study the effect of the protein environment of electron-transfer cofactors on the optical properties, stability, pigment composition, and functional activity of RC. The preliminary analysis of RC was performed by computer simulation of the amino acid substitutions L(M196)H + H(M202)L at the pigment-protein interface and by estimating the stability of the threedimensional structure of the mutant RC by the molecular dynamics method. The doubly mutated reaction center was overexpressed, purified, and crystallized. The three-dimensional structure of this mutant was determined by X-ray crystallography and compared with the molecular dynamics model.

  2. Molecular dynamics and molecular docking studies on E166A point mutant, R274N/R276N double mutant, and E166A/R274N/R276N triple mutant forms of class A β-lactamases.

    PubMed

    Kumar, K M; Lavanya, P; Anbarasu, Anand; Ramaiah, Sudha

    2014-12-01

    Bacterial resistance to β-lactams antibiotics is a serious threat to human health. The most common cause of resistance to the β-lactams is the production of β-lactamase that inactivates β-lactams. Specifically, class A extended-spectrum β-lactamase produced by antibiotic resistant bacteria is capable of hydrolyzing extended-spectrum Cephalosporins and Monobactams. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. In this present study, the E166A point mutant, R274N/R276N double mutant, and E166A/R274N/R276N triple mutant class A β-lactamases are analyzed. Molecular dynamics (MD) simulations are done to understand the consequences of mutations in class A β-lactamases. Root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessibility surface area, hydrogen bond, and essential dynamics analysis results indicate notable loss in stability for mutant class A β-lactamases. MD simulations of native and mutant structures clearly confirm that the substitution of alanine at the position of 166, Asparagine at 274 and 276 causes more flexibility in 3D space. Molecular docking results indicate the mutation in class A β-lactamases which decrease the binding affinity of Cefpirome and Ceftobiprole which are third and fifth generation Cephalosporins, respectively. MD simulation of Ceftobiprole-native and mutant type Class A β-lactamases complexes reveal that E166A/R274N/R276N mutations alter the structure and notable loss in the stability for Ceftobirole-mutant type Class A β-lactamases complexes. Ceftobiprole is currently prescribed for patients with serious bacterial infections; this phenomenon is the probable cause for the effectiveness of Ceftobiprole in controlling bacterial infections.

  3. Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26).

    PubMed Central

    Dilsiz, N; Crabbe, M J

    1995-01-01

    The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane. Images Figure 5 Figure 6 Figure 7 PMID:7848273

  4. Isolation of a herpes simplex virus type 1 mutant with a deletion in the virion host shutoff gene and identification of multiple forms of the vhs (UL41) polypeptide.

    PubMed Central

    Read, G S; Karr, B M; Knight, K

    1993-01-01

    The virion host shutoff (vhs) gene (UL41) of herpes simplex virus type 1 (HSV-1) encodes a virion component that induces degradation of host mRNAs and the shutoff of most host protein synthesis. Subsequently, the vhs protein accelerates the turnover of all kinetic classes of viral mRNA. To identify the vhs (UL41) polypeptide within infected cells and virions, antisera raised against a UL41-lacZ fusion protein were used to characterize the polypeptides encoded by wild-type HSV-1 and two mutants: vhs1, a previously characterized mutant that lacks detectable virion host shutoff activity, and vhs-delta Sma, a newly constructed mutant containing a deletion of 196 codons from UL41. Two forms of the vhs (UL41) polypeptide were identified in cells infected with the wild-type virus or vhs1. Wild-type HSV-1 produced a major 58-kDa polypeptide, as well as a less abundant 59.5-kDa form of the protein, while vhs1 produced 57- and 59-kDa polypeptides that were approximately equally abundant. Although for either virus, both forms of the protein were phosphorylated, they differed in the extent of phosphorylation. While both vhs polypeptides were found in infected cells, only the faster migrating, less phosphorylated form was incorporated into virions. vhs-delta Sma encoded a smaller, 31-kDa polypeptide which, although present in infected cells, was not incorporated into virions. The results identify multiple forms of the vhs (UL41) polypeptide and suggest that posttranslational processing affects its packaging into virions, as well as its ability to induce mRNA degradation. Images PMID:8230437

  5. Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme.

    PubMed

    Morningstar, Marshall L; Roth, Thomas; Farnsworth, David W; Smith, Marilyn Kroeger; Watson, Karen; Buckheit, Robert W; Das, Kalyan; Zhang, Wanyi; Arnold, Eddy; Julias, John G; Hughes, Stephen H; Michejda, Christopher J

    2007-08-23

    In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.44 microM, and TC50 >/= 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

  6. Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae

    SciTech Connect

    Oliveira, Marcos Antonio de Genu, Victor; Discola, Karen Fulan; Alves, Simone Vidigal; Netto, Luis Eduardo Soares; Guimarães, Beatriz Gomes

    2007-08-01

    A recombinant mutant (C47S) of cytosolic thioredoxin peroxidase 1 from S. cerevisiae was expressed, purified and crystallized by the hanging-drop vapour-diffusion method from protein previously treated with 1,4-dithiothreitol. The crystals belong to the monoclinic space group C2 and diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His{sub 6}-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress.

  7. Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

    PubMed Central

    Dauvillée, David; Colleoni, Christophe; Shaw, Eudean; Mouille, Gregory; D'Hulst, Christophe; Morell, Matthew; Samuel, Michael S.; Bouchet, Brigitte; Gallant, Daniel J.; Sinskey, Anthony; Ball, Steven

    1999-01-01

    In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii. PMID:9880375

  8. Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

    PubMed

    Pratter, S M; Eixelsberger, T; Nidetzky, B

    2015-12-01

    A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production.

  9. Synthesis, Biological Activity, and Crystal Structure of Potent Nonnucleoside Inhibitors of HIV-1 Reverse Transcriptase That Retain Activity against Mutant Forms of the Enzyme†

    PubMed Central

    Morningstar, Marshall L.; Roth, Thomas; Farnsworth, David W.; Smith, Marilyn Kroeger; Watson, Karen; Buckheit, Robert W.; Das, Kalyan; Zhang, Wanyi; Arnold, Eddy; Julias, John G.; Hughes, Stephen H.; Michejda, Christopher J.

    2010-01-01

    In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 μM, EC50 = 0.44 μM, and TC50 ≥ 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants. PMID:17663538

  10. Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type, Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer's Disease Pathology.

    PubMed

    Pahrudin Arrozi, Aslina; Shukri, Siti Nur Syazwani; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum; Ahmad Damanhuri, Mohd Hanafi; Makpol, Suzana

    2017-04-17

    Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer's disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer's disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.

  11. Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line.

    PubMed

    El-Moghazy, Samir M; George, Riham F; Osman, Essam Eldin A; Elbatrawy, Ahmed A; Kissova, Miroslava; Colombo, Ambra; Crespan, Emmanuele; Maga, Giovanni

    2016-11-10

    Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  12. Cell wall-bridge maintaining three dimensional structure of cell packets formed by the localized suppression of cell separation of a Micrococcus lysodeikticus (luteus) mutant.

    PubMed

    Monodane, T; Matsushima, Y; Kotani, S

    1979-01-01

    Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells. The mechanism of cell wall-bridge formation is discussed in connection with cell separation.

  13. Synthesis and biological evaluation of azole-diphenylpyrimidine derivatives (AzDPPYs) as potent T790M mutant form of epidermal growth factor receptor inhibitors.

    PubMed

    Song, Zhendong; Jin, Yue; Ge, Yang; Wang, Changyuan; Zhang, Jianbin; Tang, Zeyao; Peng, Jinyong; Liu, Kexin; Li, Yanxia; Ma, Xiaodong

    2016-11-01

    A series of novel azole-diphenylpyrimidine derivatives (AzDPPYs) were synthesized and biologically evaluated as potent EGFR(T790M) inhibitors. Among these analogues, the most active inhibitor 6e not only displayed high activity against EGFR(T790M/L858R) kinase (IC50=3.3nM), but also was able to repress the replication of H1975 cells harboring EGFR(T790M) mutation at a concentration of 0.118μmol/L. In contrast to the lead compound rociletinib, 6e slightly reduces the key EGFRT790M-minduced drug resistance. Significantly, inhibitor 6e demonstrates high selectivity (SI=299.3) for T790M-containing EGFR mutants over wild type EGFR, hinting that it will cause less side effects.

  14. Aberrant trafficking of human melanocortin 1 receptor variants associated with red hair and skin cancer: Steady-state retention of mutant forms in the proximal golgi.

    PubMed

    Sánchez-Laorden, Berta L; Herraiz, Cecilia; Valencia, Julio C; Hearing, Vincent J; Jiménez-Cervantes, Celia; García-Borrón, José C

    2009-09-01

    The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to reddish, poorly photoprotective pheomelanins. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation (the RHC phenotype) and increased skin cancer risk. Three highly penetrant RHC variants, R151C, R160W, and D294H are loss-of-function MC1R mutants with altered cell surface expression. In this study, we show that forward trafficking was normal for D294H. Conversely, export traffic was impaired for R151C, which accumulated in the endoplasmic reticulum (ER), and for R160W, which was enriched in the cis-Golgi. This is the first report of steady-state retention in a post-ER secretory compartment of a GPCR mutant found in the human population. Residues R151 and R160 are located in the MC1R second intracellular loop (il2). Two other mutations in il2, T157A preventing T157 phosphorylation and R162P disrupting a (160)RARR(163) motif, also caused intracellular retention. Moreover, T157 was phosphorylated in wild-type MC1R and a T157D mutation mimicking constitutive phosphorylation allowed normal traffic, and rescued the retention phenotype of R160W and R162P. Therefore, MC1R export is likely regulated by T157 phosphorylation and the (160)RARR(163) arginine-based motif functions as an ER retrieval signal. These elements are conserved in mammalian MC1Rs and in all five types of human melanocortin receptors. Thus, members of this GPCR subfamily might share common mechanisms for regulation of plasma membrane expression.

  15. Glial Innate Immunity Generated by Non-Aggregated Alpha-Synuclein in Mouse: Differences between Wild-type and Parkinson's Disease-Linked Mutants

    PubMed Central

    Roodveldt, Cintia; Labrador-Garrido, Adahir; Gonzalez-Rey, Elena; Fernandez-Montesinos, Rafael; Caro, Marta; Lachaud, Christian C.; Waudby, Christopher A.; Delgado, Mario; Dobson, Christopher M.; Pozo, David

    2010-01-01

    Background Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn). Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown. Methods and Findings In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T). Conclusions These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD. PMID:21048992

  16. Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4.

    PubMed

    Bjørnstad, Linn G; Zoppellaro, Giorgio; Tomter, Ane B; Falnes, Pål Ø; Andersson, K Kristoffer

    2011-03-15

    The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV-visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a g(max) of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.

  17. Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid.

    PubMed

    Ichi, Shunsuke; Nakazaki, Hiromichi; Boshnjaku, Vanda; Singh, Ravneet Monny; Mania-Farnell, Barbara; Xi, Guifa; McLone, David G; Tomita, Tadanori; Mayanil, Chandra Shekhar K

    2012-01-20

    Although maternal intake of folic acid (FA) prevents neural tube defects in 70% of the population, the exact mechanism of prevention has not been elucidated. We hypothesized that FA affects neural stem cell (NSC) proliferation and differentiation. This hypothesis was examined in a folate-responsive spina bifida mouse model, Splotch (Sp(-/-)), which has a homozygous loss-of-function mutation in the Pax3 gene. Neurospheres were generated with NSCs from the lower lumbar neural tube of E10.5 wild-type (WT) and Sp(-/-) embryos, in the presence and absence of FA. In the absence of FA, the number of neurospheres generated from Sp(-/-) embryos compared with WT was minimal (P<0.05). Addition of FA to Sp(-/-) cultures increased the expression of a Pax3 downstream target, fgfr4, and rescued NSC proliferative potential, as demonstrated by a significant increase in neurosphere formation (P<0.01). To ascertain if FA affected cell differentiation, FA-stimulated Sp(-/-) neurospheres were allowed to differentiate in the continued presence or absence of FA. Neurospheres from both conditions expressed multi-potent stem cell characteristics and the same differentiation potential as WT. Further, multiple neurospheres from both WT and FA-stimulated Sp(-/-) cell cultures formed extensive synaptic connections. On the whole, FA-mediated rescue of neural tube defects in Sp(-/-) embryos promotes NSC proliferation at an early embryonic stage. FA-stimulated Sp(-/-) neurospheres differentiate and form synaptic connections, comparable to WT.

  18. Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility*

    PubMed Central

    Ingold, Irina; Aichler, Michaela; Yefremova, Elena; Roveri, Antonella; Buday, Katalin; Doll, Sebastian; Tasdemir, Adrianne; Hoffard, Nils; Wurst, Wolfgang; Walch, Axel; Ursini, Fulvio; Friedmann Angeli, José Pedro; Conrad, Marcus

    2015-01-01

    The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4−/− embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility. PMID:25922076

  19. Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility.

    PubMed

    Ingold, Irina; Aichler, Michaela; Yefremova, Elena; Roveri, Antonella; Buday, Katalin; Doll, Sebastian; Tasdemir, Adrianne; Hoffard, Nils; Wurst, Wolfgang; Walch, Axel; Ursini, Fulvio; Friedmann Angeli, José Pedro; Conrad, Marcus

    2015-06-05

    The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4(-/-) embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility.

  20. Nif- Hup- mutants of Rhizobium japonicum.

    PubMed Central

    Moshiri, F; Stults, L; Novak, P; Maier, R J

    1983-01-01

    Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity. Images PMID:6874648

  1. The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin.

    PubMed

    McClintock, Dayle; Ratner, Desiree; Lokuge, Meepa; Owens, David M; Gordon, Leslie B; Collins, Francis S; Djabali, Karima

    2007-12-05

    Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals.

  2. Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.).

    PubMed Central

    Andersen, M D; Jensen, A; Robertus, J D; Leah, R; Skriver, K

    1997-01-01

    To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role. PMID:9148754

  3. Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity

    PubMed Central

    McAlary, Luke; Aquilina, J. Andrew; Yerbury, Justin J.

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (<1 year to ~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of

  4. [Synergism between aggregation mutants of Dictyostelium discoideum].

    PubMed

    Barra, J

    1977-02-21

    The cells of an aggregateless mutant of Dictyostelium discoïdeum, agip 235, can cooperate with other aggregateless or wild strains to form differentiated aggregates. A soluble mediator liberated by the coaggregating cells seems responsible for the development of agip 235. In most cases, the development of mutant agip 235 stops at the aggregation stage; however, its coaggregation with the mutant 518 results in cosporulation, with the production of viable spores of each genotype, effecting a phenotypic suppression of both mutations.

  5. Functional expression in yeast of an N-deleted form of At-ACA8, a plasma membrane Ca(2+)-ATPase of Arabidopsis thaliana, and characterization of a hyperactive mutant.

    PubMed

    Bonza, Maria Cristina; Luoni, Laura; De Michelis, Maria Ida

    2004-03-01

    A constitutively active form of At-ACA8, a plasma membrane Ca(2+)-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (delta74- At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca(2+) transport systems. Delta74- At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. Delta74- At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca(2+) uptake and Ca(2+)-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca(2+)-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the (794)HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of delta74- At-ACA8. The (794)HE-->KK mutant was also about 6-fold more sensitive than delta74- At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E(2) state during the catalytic cycle.

  6. Studies on the defect underlying the lysosomal storage of sialic acid in Salla disease. Lysosomal accumulation of sialic acid formed from N-acetyl-mannosamine or derived from low density lipoprotein in cultured mutant fibroblasts.

    PubMed Central

    Renlund, M; Kovanen, P T; Raivio, K O; Aula, P; Gahmberg, C G; Ehnholm, C

    1986-01-01

    Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane. PMID:3944269

  7. Temperature-sensitive mutants of the slime mould Physarum polycephalum. I. Mutants of the amoebal phase.

    PubMed

    Wheals, A E; Grant, W D; Jockusch, B M

    1976-11-24

    A replica plating method for isolating it amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10(-3) per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.

  8. O-Antigen-Deficient Francisella tularensis Live Vaccine Strain Mutants Are Ingested via an Aberrant Form of Looping Phagocytosis and Show Altered Kinetics of Intracellular Trafficking in Human Macrophages

    PubMed Central

    Lee, Bai-Yu; Horwitz, Marcus A.

    2012-01-01

    We examined the uptake and intracellular trafficking of F. tularensis Live Vaccine Strain (LVS) and LVS with disruptions of wbtDEF and wbtI genes essential for synthesis of the O antigen of lipopolysaccharide. Unlike parental bacteria, O-antigen-deficient LVS is efficiently killed by serum with intact complement but not by serum lacking terminal complement components. Opsonization of O-antigen-deficient LVS in serum lacking terminal complement components allows efficient uptake of these live bacteria by macrophages. In the presence of complement, whereas parental F. tularensis LVS is internalized within spacious pseudopod loops, mutant LVS is internalized within tightly juxtaposed multiple onion-like layers of pseudopodia. Without complement, both parental and mutant LVSs are internalized within spacious pseudopod loops. Thus, molecules other than O antigen are important in triggering dramatic pseudopod extensions and uptake by spacious pseudopod loops. Following uptake, both parental and mutant LVSs enter compartments that show limited staining for the lysosomal membrane glycoprotein CD63 and little fusion with secondary lysosomes. Subsequently, both parental and mutant LVSs lose their CD63 staining. Whereas the majority of parental LVS escapes into the cytosol by 6 h after uptake, mutant LVS shows a marked lag but does escape by 1 day after uptake. Despite the altered kinetics of phagosome escape, both mutant and parental strains grow to high levels within human macrophages. Thus, the O antigen plays a role in the morphology of uptake in the presence of complement and the kinetics of intracellular growth but is not essential for escape, survival, altered membrane trafficking, or intramacrophage growth. PMID:22202123

  9. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  10. A Subset of Tumor-Derived Mutant Forms of p53 Down-Regulate p63 and p73 through a Direct Interaction with the p53 Core Domain

    PubMed Central

    Gaiddon, C.; Lokshin, M.; Ahn, J.; Zhang, T.; Prives, C.

    2001-01-01

    The p53 protein is related by sequence homology and function to the products of two other genes, p63 and p73, that each encode several isoforms. We and others have discovered previously that certain tumor-derived mutants of p53 can associate and inhibit transcriptional activation by the α and β isoforms of p73. In this study we have extended these observations to show that in transfected cells a number of mutant p53 proteins could bind and down-regulate several isoforms not only of p73 (p73α, -β, -γ, and -δ) but also of p63 (p63α and -γ; ΔNp63α and -γ). Moreover, a correlation existed between the efficiency of p53 binding and the inhibition of p63 or p73 function. We also found that wild-type p63 and p73 interact efficiently with each other when coexpressed in mammalian cells. The interaction between p53 mutants and p63 or p73 was confirmed in a physiological setting by examining tumor cell lines that endogenously express these proteins. We also demonstrated that purified p53 and p73 proteins interact directly and that the p53 core domain, but not the tetramerization domain, mediates this interaction. Using a monoclonal antibody (PAb240) that recognizes an epitope within the core domain of a subset of p53 mutants, we found a correlation between the ability of p53 proteins to be immunoprecipitated by this antibody and their ability to interact with p73 or p63 in vitro and in transfected cells. Based on these results and those of others, we propose that interactions between the members of the p53 family are likely to be widespread and may account in some cases for the ability of tumor-derived p53 mutants to promote tumorigenesis. PMID:11238924

  11. Acyl-CoA synthetase activity links wild-type but not mutant α-synuclein to brain arachidonate metabolism

    PubMed Central

    Golovko, Mikhail Y.; Rosenberger, Thad A.; Faergeman, Nils J.; Feddersen, Søren; Cole, Nelson B.; Pribill, Ingrid; Berger, Johannes; Nussbaum, Robert L.; Murphy, Eric J.

    2008-01-01

    Because α-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of α-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-14C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetases (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wt mouse or human α-synuclein, but not by A30P, E46K, and A53T forms of α-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools was markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that α-synuclein bound free 20:4n-6 (Kd of 3.7 μM), but did not bind 20:4n-6-CoA. These data suggest α-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that α-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum localized acyl-CoA synthetase activity, although mutants forms of α-synuclein fail to restore this activity. PMID:16734431

  12. Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3′-terminal psoralen on a third strand directed to the mutant base pair

    PubMed Central

    Broitman, Steven L.; Amosova, Olga; Fresco, Jacques R.

    2003-01-01

    Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell β-globin gene mutation and the comparable wild-type β-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3′-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established. PMID:12907707

  13. Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3'-terminal psoralen on a third strand directed to the mutant base pair.

    PubMed

    Broitman, Steven L; Amosova, Olga; Fresco, Jacques R

    2003-08-15

    Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell beta-globin gene mutation and the comparable wild-type beta-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3'-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established.

  14. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  15. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity.

    PubMed

    Lippincott, B B; Lippincott, J A

    1966-10-01

    Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

  16. The mitochondrial permeability transition pore regulates Parkinson’s disease development in mutant α-synuclein transgenic mice

    PubMed Central

    Martin, Lee J.; Semenkow, Samantha; Hanaford, Allison; Wong, Margaret

    2013-01-01

    Parkinson’s disease (PD) is a movement disorder caused by neurodegeneration in neocortex, substantia nigra (SN) and brainstem and synucleinopathy. Some inherited PD is caused by mutations in α-synuclein (αSyn), and inherited and idiopathic PD are associated with mitochondrial perturbations. However, the mechanisms of pathogenesis are unresolved. We characterized a human αSyn transgenic mouse model and tested the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the disease mechanisms. C57BL/6 mice expressing human A53T-mutant αSyn driven by a Thy1 promoter develop a severe, age-related, fatal movement disorder involving ataxia, rigidity, and postural instability. These mice develop synucleinopathy and neocortical, SN, and cerebello-rubro-thalamic degeneration involving mitochondriopathy and apoptotic and non-apoptotic neurodegeneration. Interneurons undergo apoptotic degeneration in young mice. Mutant αSyn associated with dysmorphic neuronal mitochondria and bound voltage-dependent anion channels. Genetic ablation of cyclophilin D, an mPTP modulator, delayed disease onset and extended lifespans of mutant αSyn mice. Thus, mutant αSyn transgenic mice on a C57BL/6 background develop PD-like phenotypes, and the mPTP is involved in their disease mechanisms. PMID:24325796

  17. Brassinosteroid Mutants of Crops.

    PubMed

    Bishop, Gerard J.

    2003-12-01

    Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs' essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.

  18. Human tau protein forms complex with PrP and some GSS- and fCJD-related PrP mutants possess stronger binding activities with tau in vitro.

    PubMed

    Wang, Xiao-Fan; Dong, Chen-Fang; Zhang, Jin; Wan, Yan-Zhen; Li, Feng; Huang, Yin-Xia; Han, Lu; Shan, Bing; Gao, Chen; Han, Jun; Dong, Xiao-Ping

    2008-03-01

    Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1-91) and tandem repeats region (amino acids 186-283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.

  19. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

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

  20. A mutant with aberrant extracellular LcrV-YscF interactions fails to form pores and translocate Yop effector proteins but retains the ability to trigger Yop secretion in response to host cell contact.

    PubMed

    Harmon, Dana E; Murphy, Julia L; Davis, Alison J; Mecsas, Joan

    2013-05-01

    The plasmid-encoded type three secretion system (TTSS) of Yersinia spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires Yersinia to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but Yersinia still retains the ability to sense cell contact.

  1. A Mutant with Aberrant Extracellular LcrV-YscF Interactions Fails To Form Pores and Translocate Yop Effector Proteins but Retains the Ability To Trigger Yop Secretion in Response to Host Cell Contact

    PubMed Central

    Harmon, Dana E.; Murphy, Julia L.; Davis, Alison J.

    2013-01-01

    The plasmid-encoded type three secretion system (TTSS) of Yersinia spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires Yersinia to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but Yersinia still retains the ability to sense cell contact. PMID:23475976

  2. Misfolded opsin mutants display elevated β -sheet structure

    DOE PAGES

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; ...

    2015-09-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate themore » aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.« less

  3. Misfolded Opsin Mutants Display Elevated β-Sheet Structure

    PubMed Central

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; Park, Paul S.–H.

    2015-01-01

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. PMID:26358292

  4. Misfolded opsin mutants display elevated β-sheet structure.

    PubMed

    Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun; Park, Paul S-H

    2015-10-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.

  5. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, O.E.; Pan, D.

    1994-07-19

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

  6. Mutant maize variety containing the glt1-1 allele

    DOEpatents

    Nelson, Oliver E.; Pan, David

    1994-01-01

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

  7. Iron-molybdenum cofactor synthesis in Azotobacter vinelandii Nif- mutants.

    PubMed Central

    Imperial, J; Shah, V K; Ugalde, R A; Ludden, P W; Brill, W J

    1987-01-01

    Nif- mutants of Azotobacter vinelandii defective in dinitrogenase activity synthesized iron-molybdenum cofactor (FeMo-co) and accumulated it in two protein-bound forms: inactive dinitrogenase and a possible intermediate involved in the FeMo-co biosynthetic pathway. FeMo-co from both these proteins could activate apo-dinitrogenase from FeMo-co-deficient mutants. PMID:3470286

  8. High Persister Mutants in Mycobacterium tuberculosis

    PubMed Central

    Torrey, Heather L.; Keren, Iris; Via, Laura E.; Lee, Jong Seok; Lewis, Kim

    2016-01-01

    Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection. PMID:27176494

  9. The zebrafish early arrest mutants.

    PubMed

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

    1996-12-01

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

  10. Auxin transport inhibitor induced low complexity petiolated leaves and sessile leaf-like stipules and architectures of heritable leaf and stipule mutants in Pisum sativum suggest that its simple lobed stipules and compound leaf represent ancestral forms in angiosperms.

    PubMed

    Kumar, Arvind; Sharma, Vishakha; Khan, Moinuddin; Hindala, Mali Ram; Kumar, Sushil

    2013-04-01

    In angiosperms, leaf and stipule architectures are inherited species-specific traits. Variation in leaf and stipule sizes, and forms result from the interaction between abiotic and biotic stimuli, and gene regulatory network(s) that underlie the leaf and stipule developmental programme(s). Here, correspondence between variation in leaf and stipule architectures described for extant angiosperms and that induced mutationally and by imposition of stress in model angiosperm species, especially in Pisum sativum, was detected. Following inferences were drawn from the observations. (i) Several leaf forms in P. sativum have origin in fusion of stipule and leaf primordia. Perfoliate (and amplexicaul and connate) simple sessile leaves and sessile adnate leaves are the result of such primordial fusions. Reversal of changes in the gene regulatory network responsible for fusion products are thought to restore original stipule and leaf conditions. (ii) Compound leaf formation in several different model plants, is a result of promotion of pathways for such condition by gene regulatory networks directed by KNOx1 and LEAFY transcription factors or intercalation of the gene networks directed by them. (iii) Gene regulatory network for compound leaves in P. sativum when mutated generates highly complex compound leaves on one hand and simple leaves on other hand. These altered conditions are mutationally reversible. (vi) Simple leaves in model plants such as Arabidopsis thaliana despite overexpression of KNOx1 orthologues do not become compound. (v) All forms of leaves, including simple leaf, probably have origins in a gene regulatory network of the kind present in P. sativum.

  11. Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

    PubMed Central

    Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

    2013-01-01

    Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

  12. Native Mutant Huntingtin in Human Brain

    PubMed Central

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  13. ECB deacylase mutants

    DOEpatents

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

    2002-01-01

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

  14. [Pigment composition and photosynthetic activity of pea chlorophyll mutants].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthine; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO2 assimilation and oxygen production in the mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29-30% lower than in the control plants; in their hybrids, however, it is 1.5-2 higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that the mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.

  15. [Eremothecium ashbyii mutants resistant to 2,6-diaminopurine].

    PubMed

    Stepanov, A I; Beburov, M Iu; Zhdanov, V G

    1975-01-01

    3 groups of Eremothecium ashbyii mutants resistant to 5-10(-3) M 2,6-diaminopurine (DAP) ahve been obtained. The mutants of the 1st group (Dap-r) are selected from the initial susceptible strain by the ability to grow in the presence of 5-10(-3) M DAP. The mutants of the 2nd group (Azg-Dap-r) are selected in the selective background of two analogues of 5-10(-3) M DAP and 10(-4) M 8-azaguanine (AG). The mutants of the 3rd group (Azg-r - DAP-r) are isolated from the mutant Azg-r 34 resistant to 10(-4) M AG. The results of studying cross-resistance of mutants to DAP, AG and 8-azaadenine (AA) show that Dap-r and Azg-Dap-r mutants in contrast to Azg-r - Dap-r, have common phenotypic properties and can grow only on the analogues of adenine. DAP, but not AA, eliminates the inhibitory effect of AG on the growth of these mutants. This effect is probably due to deaminating DAP to guanine. Mutants Azg-r - Dap-r retain the initial resistance to 10(-4) M AG, but are susceptible to higher concentrations of AG and in this case DAP does not eliminate the inhibitory effect of AG. In all mutants obtained the effectiveness of the incorporation of 14C-adenine (but not 14C-guanine) is sharply reduced, thus indicating the absence of adenosine-monophosphate pyrophosphorylase activity. The mutants do not excrete purine-like compounds into the medium. In the course of the continuous growth of mutants in the presence of DAP but not of guanine the red intracellular pigment is formed which seems to be a complex of riboflavin with DAP. A disturbance in the synthesis of adenosine monophosphate pyrophosphorylase does not influence practically the level of the synthesis of riboflavin in E. ashbyii.

  16. Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

    PubMed Central

    Bryan, L E; Kwan, S

    1981-01-01

    Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells. PMID:6791588

  17. Silibinin meglumine, a water-soluble form of milk thistle silymarin, is an orally active anti-cancer agent that impedes the epithelial-to-mesenchymal transition (EMT) in EGFR-mutant non-small-cell lung carcinoma cells.

    PubMed

    Cufí, Sílvia; Bonavia, Rosa; Vazquez-Martin, Alejandro; Corominas-Faja, Bruna; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; Martin-Castillo, Begoña; Barrajón-Catalán, Enrique; Visa, Joana; Segura-Carretero, Antonio; Bosch-Barrera, Joaquim; Joven, Jorge; Micol, Vicente; Menendez, Javier A

    2013-10-01

    Silibinin is the primary active constituent of a crude extract (silymarin) from milk thistle plant (Silybum marianum) seeds. We explored the ability of an oral milk thistle extract formulation that was enriched with a water-soluble form of silibinin complexed with the amino-sugar meglumine to inhibit the growth of non-small-cell lung carcinoma (NSCLC) mouse xenografts. As a single agent, oral silibinin meglumine notably decreased the overall volumes of NSCLC tumors as efficiently as did the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Concurrent treatment with silibinin meglumine impeded the regrowth of gefitinib-unresponsive tumors, resulting in drastic tumor growth prevention. Because the epithelial-to-mesenchymal transition (EMT) is required by a multiplicity of mechanisms of resistance to EGFR TKIs, we evaluated the ability of silibinin meglumine to impede the EMT in vitro and in vivo. Silibinin-meglumine efficiently prevented the loss of markers associated with a polarized epithelial phenotype as well as the de novo synthesis of proteins associated with the mesenchymal morphology of transitioning cells. Our current findings with this non-toxic, orally active, and water-soluble silibinin formulation might facilitate the design of clinical trials to test the administration of silibinin meglumine-containing injections, granules, or beverages in combination with EGFR TKIs in patients with EGFR-mutated NSCLC. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin

    PubMed Central

    Tan, Z; Dai, W; van Erp, T G M; Overman, J; Demuro, A; Digman, M A; Hatami, A; Albay, R; Sontag, E M; Potkin, K T; Ling, S; Macciardi, F; Bunney, W E; Long, J D; Paulsen, J S; Ringman, J M; Parker, I; Glabe, C; Thompson, L M; Chiu, W; Potkin, S G

    2015-01-01

    Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT. PMID:26100538

  19. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  20. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, James P.

    1997-01-01

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

  1. Method for rapid isolation of sensitive mutants

    DOEpatents

    Freyer, J.P.

    1997-07-29

    Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

  2. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    SciTech Connect

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  3. Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

    SciTech Connect

    Chandra, P. Manish; Brannigan, James A.; Prabhune, Asmita; Pundle, Archana; Turkenburg, Johan P.; Dodson, G. Guy; Suresh, C. G.

    2005-01-01

    The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants. The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.

  4. Tetrahymena mutants with short telomeres.

    PubMed Central

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

    1998-01-01

    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

  5. Mutant chaperonin proteins: new tools for nanotechnology

    NASA Astrophysics Data System (ADS)

    Li, Y.; Paavola, C. D.; Kagawa, H.; Chan, S. L.; Trent, J. D.

    2007-11-01

    Much effort has gone into finding peptides that bind potentially useful nanoparticles, but relatively little effort has focused on the scaffolds that organize these peptides into useful nanostructures. Chaperonins are protein complexes with 14-18 protein subunits that self-assemble into double-ring complexes and function as scaffolds for peptides or amino acids that bind metallic and semiconductor quantum dots. The utility of chaperonins as scaffolds depends on their structure and their ability to self-assemble into double-rings and higher-order structures, such as filaments and two-dimensional arrays. To better understand the structure of chaperonins, we constructed a model of a group II chaperonin and, based on this model, genetically constructed five mutant subunits with significant deletions. We expressed these mutants as recombinant proteins and observed by native polyacrylamide gel electrophoresis (PAGE) and transmission electron microscopy (TEM) that they all self-assembled into double rings. Our model predicted and TEM confirmed that these deletions did not significantly change the 17 nm diameter of the wild-type double rings, but decreased their height and opened their central cavities. Four of the five mutants formed higher-order structures: chains of rings, bundles of chains or filaments, and two-dimensional arrays, which we suggest can be useful nanostructures.

  6. A computational study of λ-lac mutants

    NASA Astrophysics Data System (ADS)

    Werner, Maria; Aurell, Erik

    2009-12-01

    We present a comprehensive, computational study of the properties of bacteriophage λ mutants designed by Atsumi and Little (2006 Proc. Natl. Acad. Sci. 103 4558-63). These phages underwent a genetic reconstruction where Cro was replaced by a dimeric form of the Lac repressor. To clarify the theoretical characteristics of these mutants, we built a detailed thermodynamic model. The mutants all have a different genetic wiring than the wild-type λ. One group lacks regulation of PRM by the lytic protein. These mutants only exhibit the lysogenic equilibrium, with no transiently active PR. The other group lacks the negative feedback from CI. In this group, we identify a handful of bi-stable mutants, although the majority only exhibit the lysogenic equilibrium. The experimental identification of functional phages differs from our predictions. From a theoretical perspective, there is no reason why only 4 out of 900 mutants should be functional. The differences between theory and experiment can be explained in two ways. Either, the view of the λ phage as a bi-stable system needs to be revised, or the mutants have in fact not undergone a modular replacement, as intended by Atsumi and Little, but constitute instead a wider systemic change.

  7. Mouse infection and pathogenesis by Trypanosoma brucei motility mutants.

    PubMed

    Kisalu, Neville K; Langousis, Gerasimos; Bentolila, Laurent A; Ralston, Katherine S; Hill, Kent L

    2014-06-01

    The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream-form motility mutant in 427-derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time-course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.

  8. Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

    PubMed Central

    Noel, K D; Stacey, G; Tandon, S R; Silver, L E; Brill, W J

    1982-01-01

    Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads. Images PMID:6956566

  9. Characterization of Helicobacter pylori urease mutants.

    PubMed Central

    Segal, E D; Shon, J; Tompkins, L S

    1992-01-01

    The association between Helicobacter pylori, gastritis, and peptic ulcer is well established, and the association of infection with gastric cancer has been noted in several developing countries. However, the pathogenic mechanism(s) leading to disease states has not been elucidated. The H. pylori urease is thought to be a determinant of pathogenicity, since the enzyme is produced by all H. pylori clinical isolates. Evidence indicates that some H. pylori strains are more cytotoxic than others, with a correlation between the activity of the urease and the presence of a vacuolating cytotoxin having been made. However, the number of cytotoxins remains unknown at this time. The relationship between the urease and cytotoxicity has previously been examined with chemical inhibitors. To examine the role of the urease and its relationship to cytotoxicity, urease-deficient mutants were produced following ethyl methanesulfonate mutagenesis of H. pylori 87A300. Two mutants (the ure1 and ure5 mutants) which were entirely deficient in urease activity (Ure-) were selected. Characterization of the isolates at the protein level showed that the urease subunits lacked the ability to complex and form the active urease enzyme. The ure1 mutant was shown to be sensitive to the effects of low pH in vitro and exhibited no cytotoxicity to eucaryotic cells, whereas the parental strain (Ure+) produced a cytotoxic effect in the presence of urea. Interaction between the H. pylori Ure+ and Ure- strains and Caco-2 cells appeared to be similar in that both bacterial types elicited pedestal formation and actin condensation. These results indicate that the H. pylori urease may have many functions, among them (i) protecting H. pylori against the acidic environment of the stomach, (ii) acting as a cytotoxin, with human gastric cells especially susceptible to its activity, and (iii) disrupting cell tight junctions in such a manner that the cells remain viable but an ionic flow between the cells occurs

  10. Mutants of Myxococcus xanthus dsp defective in fibril binding.

    PubMed Central

    Chang, B Y; Dworkin, M

    1996-01-01

    The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils

  11. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction

    PubMed Central

    Sot, Begoña; Rubio-Muñoz, Alejandra; Leal-Quintero, Ahudrey; Martínez-Sabando, Javier; Marcilla, Miguel; Roodveldt, Cintia; Valpuesta, José M.

    2017-01-01

    The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington’s disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson’s disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism. PMID:28102321

  12. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction.

    PubMed

    Sot, Begoña; Rubio-Muñoz, Alejandra; Leal-Quintero, Ahudrey; Martínez-Sabando, Javier; Marcilla, Miguel; Roodveldt, Cintia; Valpuesta, José M

    2017-01-19

    The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson's disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism.

  13. Endospore degradation in an oligosporogenic, crystalliferous mutant of Bacillus thuringiensis.

    PubMed

    Sierra-Martínez, Pável; Ibarra, Jorge E; de la Torre, Mayra; Olmedo, Gabriela

    2004-02-01

    We isolated a new oligosporogenic mutant from Bacillus thuringiensis var. kurstaki HD73 that retains the ability to produce insecticidal crystal inclusions. Sporulation in this mutant initiates in a manner similar to the wild-type strain, and under the electron microscope endospores are seen, but these do not reach maturity (except for 0.2% of them). At a late stage, the coat surrounding the forespore seems to lack shape and to be empty. Most mutant cells exhibit a well-formed bipyramidal crystal but are completely devoid of the forespore. The mutant has a functional SigK holoenzyme, which is required for the expression of genes involved in the formation of spore coat and cortex and for cry1A transcription from the BtII promoter. Defective maturation of spores could be due to an inadequate forespore coat or cortex structure resulting in the arrest of sporulation at late stage III or early stage IV.

  14. Stability of Osaka Mutant and Wild-Type Fibril Models.

    PubMed

    Berhanu, Workalemahu M; Alred, Erik J; Hansmann, Ulrich H E

    2015-10-15

    Single amino acid mutations in amyloid-beta (Aβ) peptides can lead to early onset and increased severity of Alzheimer's disease. An example is the Osaka mutation (Aβ1-40E22D), which is more toxic than wild-type Aβ1-40. This mutant quickly forms early stage fibrils, one of the hallmarks of the disease, and these fibrils can even seed fibrilization of wild-type monomers. Using molecular dynamic simulations, we show that because of formation of various intra- and intermolecular salt bridges the Osaka mutant fibrils are more stable than wild-type fibrils. The mutant fibril also has a wider water channel with increased water flow than the wild type. These two observations can explain the higher toxicity and aggregation rate of the Osaka mutant over the wild type.

  15. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

    PubMed Central

    Noel, K D; Sanchez, A; Fernandez, L; Leemans, J; Cevallos, M A

    1984-01-01

    Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained. Images PMID:6325385

  16. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form.

    PubMed

    Baum, Bernhard; Lecker, Laura S M; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N; Brenk, Ruth

    2015-08-01

    Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  17. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  18. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.

    PubMed

    Krebs, M P; Mollaaghababa, R; Khorana, H G

    1993-03-01

    A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli.

  19. Gene replacement in Halobacterium halobium and expression of bacteriorhodopsin mutants.

    PubMed Central

    Krebs, M P; Mollaaghababa, R; Khorana, H G

    1993-01-01

    A gene replacement method has been developed to express bacteriorhodopsin mutants in the archaeon Halobacterium halobium. Selectable plasmids carrying the bacterioopsin gene (bop) were integrated at the chromosomal bop locus of H. halobium. Under nonselective conditions, recombinants were isolated that had lost the integrated plasmid and retained a single chromosomal copy of the bop gene. This approach was used to construct a bop deletion strain. By using this strain, recombinants were obtained that express wild-type bacteriorhodopsin and mutants known to be defective in proton translocation. The expressed proteins were purified in a membrane fraction similar to purple membrane and were characterized in this form. UV/visible spectra of dark- and light-adapted bacteriorhodopsin from wild-type and Asp-96 mutants were identical to those of purple membrane. Arg-82, Asp-85, and Asp-212 mutants had 10- to 50-nm red shifts in their absorption maxima and showed altered light adaptation. The proton translocation activity of the wild-type samples and purple membrane was comparable, whereas the mutants had 0-60% of wild-type activity. These results support earlier studies of proton translocation mutants expressed in Escherichia coli. Images Fig. 2 Fig. 3 Fig. 6 PMID:8446619

  20. Bacteriorhodopsin mutants of Halobacterium sp. GRB. II. Characterization of mutants.

    PubMed

    Soppa, J; Otomo, J; Straub, J; Tittor, J; Meessen, S; Oesterhelt, D

    1989-08-05

    The bacterioopsin genes of Halobacterium sp. GRB (Ebert, K., Goebel, W., and Pfeifer, F. (1984) Mol. & Gen. Genet. 194, 91-97) wild type and 10 independent mutants of different phenotypes have been cloned and sequenced. The wild type gene has two conservative changes compared to the gene of Halobacterium halobium, so that the proteins of the two species are identical. Six different mutations at five different codons have been found, leading to the following amino acid changes compared to the wild type: Trp10----Cys (three cases), Tyr57----Asn, Asp85----Glu, Asp06----Asn (three cases), Asp96----Gly, Trp138----Arg. A first characterization of the mutant proteins is given, and their implications for models of bacteriorhodopsin structure and function are discussed.

  1. Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants.

    PubMed

    D'Argenio, David A; Calfee, M Worth; Rainey, Paul B; Pesci, Everett C

    2002-12-01

    Two distinctive colony morphologies were noted in a collection of Pseudomonas aeruginosa transposon insertion mutants. One set of mutants formed wrinkled colonies of autoaggregating cells. Suppressor analysis of a subset of these mutants showed that this was due to the action of the regulator WspR and linked this regulator (and the chemosensory pathway to which it belongs) to genes that encode a putative fimbrial adhesin required for biofilm formation. WspR homologs, related in part by a shared GGDEF domain, regulate cell surface factors, including aggregative fimbriae and exopolysaccharides, in diverse bacteria. The second set of distinctive insertion mutants formed colonies that lysed at their center. Strains with the most pronounced lysis overproduced the Pseudomonas quinolone signal (PQS), an extracellular signal that interacts with quorum sensing. Autolysis was suppressed by mutation of genes required for PQS biosynthesis, and in one suppressed mutant, autolysis was restored by addition of synthetic PQS. The mechanism of autolysis may involve activation of the endogenous prophage and phage-related pyocins in the genome of strain PAO1. The fact that PQS levels correlated with autolysis suggests a fine balance in natural populations of P. aeruginosa between survival of the many and persistence of the few.

  2. Mutant power: using mutant allele collections for yeast functional genomics

    PubMed Central

    Norman, Kaitlyn L.

    2016-01-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. PMID:26453908

  3. Mutant power: using mutant allele collections for yeast functional genomics.

    PubMed

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Revertants and Secondary arom-2 Mutants Induced in Non-Complementing Mutants in the arom Gene Cluster of Neurospora Crassa

    PubMed Central

    Case, Mary E.; Giles, Norman H.

    1974-01-01

    Extensive genetical and biochemical studies have been performed with revertants and secondary arom-2 mutants induced in two different primary non-complementing mutants which map within the arom gene cluster of Neurospora crassa. These studies indicate that mutant M54 but not M25 can revert by super-suppressor mutations in unlinked genes, thus confirming previous evidence that M54 contains a nonsense codon. At least three new super suppressors of M54 have been detected. All four super suppressors (including one previously detected) when combined with M54 result in high levels of all five of the arom enzymic activities in the form of arom multienzyme complexes very similar to (but not necessarily identical with) that in wild type (WT).—Evidence has also been obtained that the two non-complementing mutants can yield revertants which appear to result from true back mutations and produce arom aggregates essentially indistinguishable from that of WT. In addition, M25, but not M54, when plated on quinic acid yields revertants (secondary mutants) some of which are phenotypically indistinguishable from arom-2 primary mutants and others of which, although also mapping within the arom-2 gene, exhibit unusual properties. Genetic evidence indicates that the M25 secondary mutants are localized within the arom-2 gene, but that they arise from mutational events more complex than ones resulting in single base pair changes in the M25 codon.—The recovery of secondary arom-2 mutants as revertants of non-complementing arom mutants provides strong evidence, independent of earlier recombination data, that non-complementing arom mutants are located within the arom-2 structural gene of the arom gene cluster. In addition, the occurrence and characteristics of these secondary arom-2 mutants provide strong evidence, independent of the results with nonsense suppressors, that the arom gene cluster is transcribed, beginning with the arom-2 gene, as a single polycistronic messenger

  5. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    PubMed

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  6. Effects of mutant rat dynamin on endocytosis

    PubMed Central

    1993-01-01

    Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non- coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2- terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over- expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis. PMID:8335685

  7. Misfolded opsin mutants display elevated β -sheet structure

    SciTech Connect

    Miller, Lisa M.; Gragg, Megan; Kim, Tae Gyun; Park, Paul S. -H.

    2015-09-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Also, both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. In conclusion, the effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.

  8. Analysis of spontaneous suppressor mutants from the photomixotrophically grown pmgA-disrupted mutant in the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Nishijima, Yoshiki; Kanesaki, Yu; Yoshikawa, Hirofumi; Ogawa, Takako; Sonoike, Kintake; Nishiyama, Yoshitaka; Hihara, Yukako

    2015-12-01

    The pmgA-disrupted (ΔpmgA) mutant in the cyanobacterium Synechocystis sp. PCC 6803 suffers severe growth inhibition under photomixotrophic conditions. In order to elucidate the key factors enabling the cells to grow under photomixotrophic conditions, we isolated spontaneous suppressor mutants from the ΔpmgA mutant derived from a single colony. When the ΔpmgA mutant was spread on a BG11 agar plate supplemented with glucose, colonies of suppressor mutants appeared after the bleaching of the background cells. We identified the mutation site of these suppressor mutants and found that 11 mutants out of 13 had a mutation in genes related to the type 1 NAD(P)H dehydrogenase (NDH-1) complex. Among them, eight mutants had mutations within the ndhF3 (sll1732) gene: R32stop, W62stop, V147I, G266V, G354W, G586C, and deletion of 7 bp within the coding region. One mutant had one base insertion in the putative -10 box of the ndhC (slr1279) gene, leading to the decrease in the transcripts of the ndhCKJ operon. Two mutants had one base insertion and deletion in the coding region of cupA (sll1734), which is co-transcribed with ndhF3 and ndhD3 and comprises together a form of NDH-1 complex (NDH-1MS complex) involved in inducible high-affinity CO2 uptake. The results indicate that the loss of the activity of this complex effectively rescues the ΔpmgA mutant under photomixotrophic condition with 1 % CO2. However, little difference among WT and mutants was observed in the activities ascribed to the NDH-1MS complex, i.e., CO2 uptake and cyclic electron transport. This may suggest that the NDH-1MS complex has the third, currently unknown function under photomixotrophic conditions.

  9. Proposed Nomenclature for Mutants of Adenoviruses

    PubMed Central

    Ginsberg, Harold S.; Williams, James F.; Doerfler, Walter H.; Shimojo, Hiroto

    1973-01-01

    In accord with the nomenclature proposed for mutants of simian virus 40 the same rules, with minor modifications, are recommended for naming mutants of adenoviruses. It is further suggested that these rules, which pertain to a system of classification based primarily upon complementation analysis, also be applied to mutants of other DNA-containing animal viruses. PMID:4355864

  10. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  11. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  12. Isolation and characterization of yeast monomorphic mutants of Candida albicans.

    PubMed Central

    Elorza, M V; Sentandreu, R; Ruiz-Herrera, J

    1994-01-01

    A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the levels of chitin and glucans, and in specific mannoproteins, some of them recognizable by specific polyclonal and monoclonal antibodies. It is suggested that these structural alterations hinder the construction of a normal hyphal wall. Images PMID:8157600

  13. Identification and Characterization of Aspergillus Nidulans Mutants Defective in Cytokinesis

    PubMed Central

    Harris, S. D.; Morrell, J. L.; Hamer, J. E.

    1994-01-01

    Filamentous fungi undergo cytokinesis by forming crosswalls termed septa. Here, we describe the genetic and physiological controls governing septation in Aspergillus nidulans. Germinating conidia do not form septa until the completion of their third nuclear division. The first septum is invariantly positioned at the basal end of the germ tube. Block-and-release experiments of nuclear division with benomyl or hydroxyurea, and analysis of various nuclear division mutants demonstrated that septum formation is dependent upon the third mitotic division. Block-and-release experiments with cytochalasin A and the localization of actin in germlings by indirect immunofluorescence showed that actin participated in septum formation. In addition to being concentrated at the growing hyphal tips, a band of actin was also apparent at the site of septum formation. Previous genetic analysis in A. nidulans identified four genes involved in septation (sepA-D). We have screened a new collection of temperature sensitive (ts) mutants of A. nidulans for strains that failed to form septa at the restrictive temperature but were able to complete early nuclear divisions. We identified five new genes designated sepE, G, H, I and J, along with one additional allele of a previously identified septation gene. On the basis of temperature shift experiments, nuclear counts and cell morphology, we sorted these cytokinesis mutants into three phenotypic classes. Interestingly, one class of mutants fails to form septa and fails to progress past the third nuclear division. This class of mutants suggests the existence of a regulatory mechanism in A. nidulans that ensures the continuation of nuclear division following the initiation of cytokinesis. PMID:8150280

  14. Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    PubMed Central

    Abdul-Hussein, Saba; Rahl, Karin; Moslemi, Ali-Reza; Tajsharghi, Homa

    2013-01-01

    Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein. PMID:24039757

  15. [LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

    PubMed

    Semenova, G A; Chekunova, E M; Ladygin, V G

    2015-01-01

    The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed.

  16. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    PubMed

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

  17. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

    PubMed Central

    Howden, R; Goldsbrough, P B; Andersen, C R; Cobbett, C S

    1995-01-01

    An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants. PMID:7770517

  18. Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi)

    SciTech Connect

    Klittich, C.J.R.; Leslie, J.F.

    1988-03-01

    Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactors that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. The authors concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.

  19. A metal-accumulator mutant of Arabidopsis thaliana.

    PubMed Central

    Delhaize, E

    1996-01-01

    A mutation designated man1 (for manganese accumulator) was found to cause Arabidopsis thaliana seedlings to accumulate a range of metals. The man1 mutation segregated as a single recessive locus located on chromosome 3. When grown on soil, mutant seedlings accumulated Mn (7.5 times greater than wild type), Cu (4.6 times greater than wild type), Zn (2.8 times greater than wild type), and Mg (1.8 times greater than wild type) in leaves. In addition to these metals, the man1 mutant accumulated 2.7-fold more S in leaves, primarily in the oxidized form, than wild-type seedlings. Analysis of seedlings grown by hydroponic culture showed a similar accumulation of metals in leaves of man1 mutants. Roots of man1 mutants also accumulated metals, but unlike leaves they accumulated 10-fold more total Fe (symplasmic and apoplasmic combined) than wild-type roots. Roots of man1 mutants possessed greater (from 1.8- to 20-fold) ferric-chelate reductase activity than wild-type seedings, and this activity was not responsive to changes of Mn nutrition in either genotype. Taken together, these results suggest that the man1 mutation disrupts the regulation of metal-ion uptake or homeostasis in Arabidopsis. PMID:8754685

  20. Nanoformulated cell-penetrating survivin mutant and its dual actions

    PubMed Central

    Sriramoju, Bhasker; Kanwar, Rupinder K; Kanwar, Jagat R

    2014-01-01

    In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. PMID:25045261

  1. Interaction of metronidazole with DNA repair mutants of Escherichia coli.

    PubMed Central

    Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

    1984-01-01

    It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light. PMID:6367636

  2. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes

    PubMed Central

    1987-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve

  3. Biochemical characterization and molecular genetics of nine mutants of Penicillium chrysogenum impaired in penicillin biosynthesis.

    PubMed

    Cantoral, J M; Gutiérrez, S; Fierro, F; Gil-Espinosa, S; van Liempt, H; Martín, J F

    1993-01-05

    Nine mutants of Penicillium chrysogenum (npe1 to npe8 and npe10) impaired in penicillin biosynthesis were screened after nitrosoguanidine mutation. Mutants npe1, npe4, npe5, npe6, npe7, npe8, and npe10 failed to synthesize significant levels of penicillin, whereas strains npe2 and npe3 synthesized about 20% of the penicillin level produced by the parental strain. Mutants npe5 and npe10 did not show alpha-aminoadipylcysteinyl-valine (ACV) synthetase activity in vitro and did not form ACV in vivo. Immunoblotting analysis of the different mutants using antibodies raised against Aspergillus nidulans ACV-synthetase showed that mutants npe5 and npe10 lacked this multienzyme protein, which in the parental strain had a molecular mass of about 420 kDa, and mutants npe2 and npe3 formed reduced level of this protein. All mutants showed normal levels of isopenicillin N synthase, as shown by Western blot analysis and enzyme assays (except npe10 that lacked this enzyme and npe2 and npe3 that formed reduced levels); npe1, npe4, npe6, npe7, npe8, and npe10 lacked isopenicillin N acyltransferase. Southern hybridizations of total DNA of the parental strain and mutants npe5, npe6, npe8, and npe10 with probes internal to the pcbAB, pcbC, and penDE genes showed that mutants npe5, npe6, and npe8 had the same arrangement of the penicillin gene cluster carrying probably point mutations, but mutant npe10 lacked the three penicillin biosynthetic genes, suggesting that it had suffered a deletion of the entire penicillin cluster. Southern hybridization with a pyrG probe as control and fingerprinting analysis of total DNA of npe10 as compared to several P.chrysogenum strains and other Penicillium and Aspergillus species, confirmed that npe10 is a deletion mutant of P. chrysogenum that had lost the penicillin biosynthetic genes.

  4. Oral Immunization Against Experimental Salmonellosis I. Development of Temperature-Sensitive Mutant Vaccines

    PubMed Central

    Fahey, K. J.; Cooper, G. N.

    1970-01-01

    Mutant strains of Salmonella enteritidis were selected for their inability to proliferate at 37 C; when exposed to this temperature, these organisms formed tangled masses of long filaments in liquid media, presumably as a result of their inability to form cross septa. The mutants were also incapable of synthesizing flagella protein. A study of the biological charateristics of the mutants indicated that in most respects they resembled the parent strain of S. enteritidis; however, they were avirulent for mice, presumably because of the restriction of growth imposed by the body temperature of the animal. Preliminary studies have suggested that these mutants are highly effective in inducing protection against severe challenge infections of S. enteritidis; of especial interest is the fact that, when given orally, the mutants conferred a substantial degree of protection against oral infection with the virulent strain. PMID:16557726

  5. Oral immunization against experimental salmonellosis I. Development of temperature-sensitive mutant vaccines.

    PubMed

    Fahey, K J; Cooper, G N

    1970-03-01

    Mutant strains of Salmonella enteritidis were selected for their inability to proliferate at 37 C; when exposed to this temperature, these organisms formed tangled masses of long filaments in liquid media, presumably as a result of their inability to form cross septa. The mutants were also incapable of synthesizing flagella protein. A study of the biological charateristics of the mutants indicated that in most respects they resembled the parent strain of S. enteritidis; however, they were avirulent for mice, presumably because of the restriction of growth imposed by the body temperature of the animal. Preliminary studies have suggested that these mutants are highly effective in inducing protection against severe challenge infections of S. enteritidis; of especial interest is the fact that, when given orally, the mutants conferred a substantial degree of protection against oral infection with the virulent strain.

  6. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  7. Genetic interactions among homologous recombination mutants in Candida albicans.

    PubMed

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Purification of recombinant C-reactive protein mutants

    PubMed Central

    Thirumalai, Avinash; Singh, Sanjay K.; Hammond, David J.; Gang, Toh B.; Ngwa, Donald N.; Pathak, Asmita; Agrawal, Alok

    2017-01-01

    C-reactive protein (CRP) is an evolutionarily conserved protein, a component of the innate immune system, and an acute phase protein in humans. In addition to its raised level in blood in inflammatory states, CRP is also localized at sites of inflammation including atherosclerotic lesions, arthritic joints and amyloid plaque deposits. Results of in vivo experiments in animal models of inflammatory diseases indicate that CRP is an anti-pneumococcal, anti-atherosclerotic, anti-arthritic and an anti-amyloidogenic molecule. The mechanisms through which CRP functions in inflammatory diseases are not fully defined; however, the ligand recognition function of CRP in its native and non-native pentameric structural conformations and the complement-activating ability of ligand-complexed CRP have been suggested to play a role. One tool to understand the structure-function relationships of CRP and determine the contributions of the recognition and effector functions of CRP in host defense is to employ site-directed mutagenesis to create mutants for experimentation. For example, CRP mutants incapable of binding to phosphocholine are generated to investigate the importance of the phosphocholine-binding property of CRP in mediating host defense. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. While the methods to purify wild-type CRP are well established, different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either calcium or phosphocholine. In this article, we report the methods used to purify pentameric recombinant wild-type and mutant CRP expressed in and secreted by mammalian cells. PMID:1460031

  9. Purification and in vitro complementation of mutant histidinol dehydrogenases. [Salmonella typhimurium

    SciTech Connect

    Lee, S.Y.; Grubmeyer, C.T.

    1987-09-01

    The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the his01242 (constitutive overproducer) attenuator mutations and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl/sub 2/, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound /sup 54/Mn/sup 2 +/ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound /sup 54/Mn/sup 2 +/ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 20-mercaptoethanol was prevented by low levels of MnCl/sub 2/.

  10. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents.

    PubMed

    Rogers, P; Palosaari, N

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid, and the Spo mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  11. Cytochrome abnormalities and cyanide-resistant respiration in extranuclear mutants of Aspergillus nidulans.

    PubMed Central

    Turner, G; Rowlands, R T

    1976-01-01

    The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature and at the temperature of liquid N2 and compared with those of the wild-type strain. The oligomycin-resistant, slow growing mutant contained an increased amount of cytochrome c without any loss of cytochromes b and a,a3. The cold-sensitive mutant, apparently normal when grown at 37 C, showed an increased amount of cytochrome c and a partial loss of cytochromes b and a,a3 when grown at 20 C. A combination of these effects was observed in the double-mutant recombinant. Cyanide-resistant respiration was present in both mutant strains and in the recombinant at much higher levels than in the wild-type strain. In the oligomycin-resistant mutant, this was usually present together with cyanide-sensitive respiration, whereas in the cold-sensitive mutant and recombinant grown at 20 C cyanide-resistant approached 100%. Inhibitor and growth yield studies indicated that the cyanide-resistant pathway was not used by the cold-sensitive mutant during growth at 20 C. PMID:1107321

  12. Altered desferrioxamine-mediated iron utilization is a common trait of bald mutants of Streptomyces coelicolor.

    PubMed

    Lambert, Stéphany; Traxler, Matthew F; Craig, Matthias; Maciejewska, Marta; Ongena, Marc; van Wezel, Gilles P; Kolter, Roberto; Rigali, Sébastien

    2014-08-01

    Streptomyces coelicolor is an important model organism for developmental studies of filamentous GC-rich actinobacteria. The genetic characterization of mutants of S. coelicolor blocked at the vegetative mycelium stage, the so-called bald (bld) mutants that are unable to erect spore-forming aerial hyphae, has opened the way to discovering the molecular basis of development in actinomycetes. Desferrioxamine (DFO) production and import of ferrioxamines (FO; iron-complexed DFO) are key to triggering morphogenesis of S. coelicolor and we show here that growth of S. coelicolor on the reference medium for Streptomyces developmental studies is fully dependent on DFO biosynthesis. UPLC-ESI-MS analysis revealed that all bld mutants tested are affected in DFO biosynthesis, with bldA, bldJ, and ptsH mutants severely impaired in DFO production, while bldF, bldK, crr and ptsI mutants overproduce DFO. Morphogenesis of bldK and bldJ mutants was recovered by supplying exogenous iron. Transcript analysis showed that the bldJ mutant is impaired in expression of genes involved in the uptake of FO, whereas transcription of genes involved in both DFO biosynthesis and FO uptake is increased in bldK mutants. Our study allows proposing altered DFO production and/or FO uptake as a novel phenotypic marker of many S. coelicolor bld mutants, and strengthens the role of siderophores and iron acquisition in morphological development of actinomycetes.

  13. Arabidopsis Mutant bik1 Exhibits Strong Resistance to Plasmodiophora brassicae

    PubMed Central

    Chen, Tao; Bi, Kai; He, Zhangchao; Gao, Zhixiao; Zhao, Ying; Fu, Yanping; Cheng, Jiasen; Xie, Jiatao; Jiang, Daohong

    2016-01-01

    Botrytis-induced kinase1 (BIK1), a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1, and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2, and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40–50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS) at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA) treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2) and npr1-1 [non-expresser of PR genes that regulate systemic acquired resistance (SAR)] mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms. PMID:27679580

  14. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  15. Electrophysiological study of Drosophila rhodopsin mutants

    PubMed Central

    1986-01-01

    Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1-6 photoreceptors, and a mutant of the ninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodopsin content in R1-6 photoreceptors is reduced by 10(2)-10(6)-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologically normal. Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model. Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps. Mutant bumps are strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletion of rhodopsin content, rather than to any specific alterations in the opsin protein. Lowering or buffering the intracellular calcium concentration by EGTA injection mimics the effects of the mutations on the bump amplitude, but, unlike the mutations, it also affects the latency and kinetics of light responses. PMID:3097245

  16. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    SciTech Connect

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  17. Rhizobium meliloti mutants unable to synthesize anthranilate display a novel symbiotic phenotype.

    PubMed Central

    Barsomian, G D; Urzainqui, A; Lohman, K; Walker, G C

    1992-01-01

    Analyses of Rhizobium meliloti trp auxotrophs suggest that anthranilate biosynthesis by the R. meliloti trpE(G) gene product is necessary during nodule development for establishment of an effective symbiosis. trpE(G) mutants, as well as mutants blocked earlier along this pathway in aromatic amino acid biosynthesis, form nodules on alfalfa that have novel defects. In contrast, R. meliloti trp mutants blocked later in the tryptophan-biosynthetic pathway form normal, pink, nitrogen-fixing nodules. trpE(G) mutants form two types of elongated, defective nodules containing unusually extended invasion zones on alfalfa. One type contains bacteroids in its base and is capable of nitrogen fixation, while the other lacks bacteroids and cannot fix nitrogen. The trpE(G) gene is expressed in normal nodules. Models are discussed to account for these observations, including one in which anthranilate is postulated to act as an in planta siderophore. Images PMID:1320610

  18. Mutant TP53 Posttranslational Modifications: Challenges and Opportunities

    PubMed Central

    Nguyen, Thuy-Ai; Menendez, Daniel; Resnick, Michael A.; Anderson, Carl W.

    2014-01-01

    The wild-type human p53 (TP53) tumor suppressor can be posttranslationally modified at over 60 of its 393 residues. These modifications contribute to changes in TP53 stability and in its activity as a transcription factor in response to a wide variety of intrinsic and extrinsic stresses in part through regulation of protein-protein and protein-DNA interactions. The TP53 gene frequently is mutated in cancers, and in contrast to most other tumor suppressors the mutations are mostly missense often resulting in the accumulation of mutant protein, which may have novel or altered functions. Most mutant TP53s can be posttranslationally modified at the same residues as in wild-type TP53. Strikingly, however, codons for modified residues are rarely mutated in human tumors, suggesting that TP53 modifications are not essential for tumor suppression activity. Nevertheless, these modifications might alter mutant TP53 activity and contribute to a gain-of-function leading to increased metastasis and tumor progression. Furthermore, many of the signal transduction pathways that result in TP53 modifications are altered or disrupted in cancers. Understanding the signaling pathways that result in TP53 modification and the functions of these modifications in both wild-type TP53 and its many mutant forms may contribute to more effective cancer therapies. PMID:24395704

  19. Mutant calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation

    PubMed Central

    Elf, Shannon; Abdelfattah, Nouran S.; Chen, Edwin; Perales-Patón, Javier; Rosen, Emily A.; Ko, Amy; Peisker, Fabian; Florescu, Natalie; Giannini, Silvia; Wolach, Ofir; Morgan, Elizabeth A.; Tothova, Zuzana; Losman, Julie-Aurore; Schneider, Rebekka K.; Al-Shahrour, Fatima; Mullally, Ann

    2016-01-01

    Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. PMID:26951227

  20. Electron microscope studies of temperature-sensitive mutants of herpes simplex virus type 2.

    PubMed Central

    Cabral, G A; Schaffer, P A

    1976-01-01

    Nine temperature-sensitive mutants of herpes simplex virus type 2 representing eight complementation groups were assigned to two classes as a consequence of the virion forms and virus-specific cellular alterations observed in thin sections of mutant-infected human embryonic lung cells grown at the nonpermissive temperature. Mutants in class A, one DNA- and one DNA +, failed to synthesize detectable virus particles. Mutants in class B, 4DNA- and 3DNA+, produced moderate to large numbers of empty nucleocapsids. Dense-cored nucleocapsids were not observed in thin sections of cells infected with any of the nine mutants at this temperature. Virus-specific cellular alterations consisted primarily of margination of chromating and nulcear membrane thickening and duplication. Images PMID:178905

  1. Electron microscope studies of temperature-sensitive mutants of herpes simplex virus type 2.

    PubMed

    Cabral, G A; Schaffer, P A

    1976-05-01

    Nine temperature-sensitive mutants of herpes simplex virus type 2 representing eight complementation groups were assigned to two classes as a consequence of the virion forms and virus-specific cellular alterations observed in thin sections of mutant-infected human embryonic lung cells grown at the nonpermissive temperature. Mutants in class A, one DNA- and one DNA +, failed to synthesize detectable virus particles. Mutants in class B, 4DNA- and 3DNA+, produced moderate to large numbers of empty nucleocapsids. Dense-cored nucleocapsids were not observed in thin sections of cells infected with any of the nine mutants at this temperature. Virus-specific cellular alterations consisted primarily of margination of chromating and nulcear membrane thickening and duplication.

  2. Enhancers of Conidiation Mutants in Aspergillus Nidulans

    PubMed Central

    Gems, D. H.; Clutterbuck, A. J.

    1994-01-01

    Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.'' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies. PMID:8056325

  3. Regulation of Mutant p53 Protein Expression.

    PubMed

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

    2015-01-01

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

  4. Eyespot-assembly mutants in Chlamydomonas reinhardtii.

    PubMed Central

    Lamb, M R; Dutcher, S K; Worley, C K; Dieckmann, C L

    1999-01-01

    Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell. PMID:10511552

  5. A halotolerant mutant of Saccharomyces cerevisiae.

    PubMed Central

    Gaxiola, R; Corona, M; Zinker, S

    1996-01-01

    FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

  6. Saint Louis Encephalitis Temperature-Sensitive Mutants.

    DTIC Science & Technology

    1979-09-01

    Several mutants have been used in pairwise crosses to determine complementation Accessionl For VTIS G!RA& DTIC T %B 3 fl!, -r,. AD) Av ’Di [ Annual...Ghendon (1973) indicate that a large number of polio virus ts mutants producing a pathologic change in infected monkeys were assayed for virus produccion

  7. Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation.

    PubMed

    Kojima, Tomoko; Saito, Katsuharu; Oba, Hirosuke; Yoshida, Yuma; Terasawa, Junya; Umehara, Yosuke; Suganuma, Norio; Kawaguchi, Masayoshi; Ohtomo, Ryo

    2014-05-01

    Several symbiotic mutants of legume plants defective in nodulation have also been shown to be mutants related to arbuscular mycorrhizal (AM) symbiosis. The origin of the AM symbiosis can be traced back to the early land plants. It has therefore been postulated that the older system of AM symbiosis was partially incorporated into the newer system of legume-rhizobium symbiosis. To unravel the genetic basis of the establishment of AM symbiosis, we screened about 34,000 plants derived from ethyl methanesulfonate (EMS)-mutagenized Lotus japonicus seeds by microscopic observation. As a result, three lines (ME778, ME966 and ME2329) were isolated as AM-specific mutants that exhibit clear AM-defective phenotypes but form normal effective root nodules with rhizobial infection. In the ME2329 mutant, AM fungi spread their hyphae into the intercellular space of the cortex and formed trunk hyphae in the cortical cells, but the development of fine branches in the arbuscules was arrested. The ME2329 mutant carried a nonsense mutation in the STR-homolog gene, implying that the line may be an str mutant in L. japonicus. On the ME778 and ME966 mutant roots, the entry of AM fungal hyphae was blocked between two adjacent epidermal cells. Occasionally, hyphal colonization accompanied by arbuscules was observed in the two mutants. The genes responsible for the ME778 and ME966 mutants were independently located on chromosome 2. These results suggest that the ME778 and ME966 lines are symbiotic mutants involved in the early stage of AM formation in L. japonicus.

  8. Radiation-sensitive mutants of Arabidopsis thaliana

    SciTech Connect

    Jenkins, M.E.; Harlow, G.R.; Liu, Z.

    1995-06-01

    Five Arabidopsis mutants have been isolated on the basis of hypersensitivity of leaf tissue to UV light. For each mutant, the UV-hypersensitive phenotype (uvh) was inherited as a single recessive Mendelian trait. In addition, each uvh mutant represented a separate complementation group. Three of the mutations producing the UV hypersensitive phenotype have been mapped relative to either genetic markers or physical microsatellite polymorphisms. Locus UVH1 is linked to nga76 on chromosome 5, UVH3 to GL1 on chromosome three, and UVH6 to nga59 on chromosome 1. Each uvh mutant has a characteristic pattern of sensitivity based on UV sensitivity of leaf tissue, UV sensitivity of root tissue, and ionizing radiation sensitivity of seeds. On the basis of these patterns, possible molecular defects in these mutants are discussed. 30 refs., 3 figs., 5 tabs.

  9. The TOC159 mutant of Arabidopsis thaliana accumulates altered levels of saturated and polyunsaturated fatty acids.

    PubMed

    Afitlhile, Meshack; Fry, Morgan; Workman, Samantha

    2015-02-01

    We evaluated whether the TOC159 mutant of Arabidopsis called plastid protein import 2-2 (ppi2-2) accumulates normal levels of fatty acids, and transcripts of fatty acid desaturases and galactolipid synthesis enzymes. The ppi2-2 mutant accumulates decreased pigments and total fatty acid content. The MGD1 gene was downregulated and the mutant accumulates decreased levels of monogalactosyldiacylglycerol (MGDG) and 16:3, which suggests that the prokaryotic pathway was impaired in the mutant. The HY5 gene, which encodes long hypocotyl5 transcription factor, was upregulated in the mutant. The DGD1 gene, an HY5 target was marginally increased and the mutant accumulates digalactosyldiacylglycerol at the control level. The mutant had increased expression of 3-ketoacyl-ACP synthase II gene, which encodes a plastid enzyme that elongates 16:0 to 18:0. Interestingly, glycerolipids in the mutant accumulate increased levels of 18:0. A gene that encodes stearoyl-ACP desaturase (SAD) was expressed at the control level and 18:1 was increased, which suggest that SAD may be strongly regulated at the posttranscriptional level. The molar ratio of MGDG to bilayer forming plastid lipids was decreased in the cold-acclimated wild type but not in the ppi2-2 mutant. This indicates that the mutant was unresponsive to cold-stress, and is consistent with increased levels of 18:0, and decreased 16:3 and 18:3 in the ppi2-2 mutant. Overall, these data indicate that a defective Toc159 receptor impaired the synthesis of MGDG, and affected desaturation of 16 and 18-carbon fatty acids. We conclude that expression of the MGD1 gene and synthesis of MGDG are tightly linked to plastid biogenesis.

  10. crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

    PubMed

    McCusker, J H; Haber, J E

    1988-06-01

    Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

  11. Phenotypic and Transcriptomic Characterization of Bacillus subtilis Mutants with Grossly Altered Membrane Composition▿ †

    PubMed Central

    Salzberg, Letal I.; Helmann, John D.

    2008-01-01

    The Bacillus subtilis membrane contains diacylglycerol-based lipids with at least five distinct headgroups that together help to define the physical and chemical properties of the lipid bilayer. Here, we describe the phenotypic characterization of mutant strains lacking one or more of the following lipids: glycolipids (ugtP mutants), phosphatidylethanolamine (pssA and psd mutants), lysylphosphatidylglycerol (mprF), and cardiolipin (ywnE and ywjE). Alterations of membrane lipid headgroup composition are generally well-tolerated by the cell, and even severe alterations lead to only modest effects on growth proficiency. Mutants with decreased levels of positively charged lipids display an increased sensitivity to cationic antimicrobial compounds, and cells lacking glycolipids are more sensitive to the peptide antibiotic sublancin and are defective in swarming motility. A quadruple mutant strain (ugtP pssA mprF ywnE), with a membrane comprised predominantly of phosphatidylglycerol, is viable and grows at near-wild-type rates, although it forms long, coiled filaments. Transcriptome comparisons identified numerous regulons with altered expression in cells of the ugtP mutant, the pssA mprF ywnE triple mutant, and the ugtP pssA mprF ywnE quadruple mutant. These effects included a general decrease in expression of the SigD and FapR regulons and increased expression of cell envelope stress responses mediated by σM and the YvrGHb two-component system. PMID:18820022

  12. Conditional Expression of Parkinson disease-related Mutant α-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1

    PubMed Central

    Lin, Xian; Parisiadou, Loukia; Sgobio, Carmelo; Liu, Guoxiang; Yu, Jia; Sun, Lixin; Shim, Hoon; Gu, Xing-Long; Luo, Jing; Long, Cai-Xia; Ding, Jinhui; Mateo, Yolanda; Sullivan, Patricia H.; Wu, Ling-Gang; Goldstein, David S.; Lovinger, David; Cai, Huaibin

    2012-01-01

    α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD. PMID:22764233

  13. Dedifferentiation of neurons precedes tumor formation in Lola mutants.

    PubMed

    Southall, Tony D; Davidson, Catherine M; Miller, Claire; Carr, Adrian; Brand, Andrea H

    2014-03-31

    The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. Here, we identify Lola as a transcription factor that is required to maintain neurons in a differentiated state. We show that Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Dedifferentiation of Neurons Precedes Tumor Formation in lola Mutants

    PubMed Central

    Southall, Tony D.; Davidson, Catherine M.; Miller, Claire; Carr, Adrian; Brand, Andrea H.

    2014-01-01

    Summary The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. Here, we identify Lola as a transcription factor that is required to maintain neurons in a differentiated state. We show that Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants. PMID:24631403

  15. Improved solubility of replication factor C (RFC) Walker A mutants.

    PubMed

    Marzahn, Melissa R; Bloom, Linda B

    2012-06-01

    Protein insolubility often poses a significant problem during purification protocols and in enzyme assays, especially for eukaryotic proteins expressed in a recombinant bacterial system. The limited solubility of replication factor C (RFC), the clamp loader complex from Saccharomyces cerevisiae, has been previously documented. We found that mutant forms of RFC harboring a single point mutation in the Walker A motif were even less soluble than the wild-type complex. The addition of maltose at 0.75 M to the storage and assay buffers greatly increases protein solubility and prevents the complex from falling apart. Our analysis of the clamp loading reaction is dependent on fluorescence-based assays, which are environmentally sensitive. Using wt RFC as a control, we show that the addition of maltose to the reaction buffers does not affect fluorophore responses in the assays or the enzyme activity, indicating that maltose can be used as a buffer additive for further downstream analysis of these mutants.

  16. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  17. Biochemical characterization of the chlB mutant of E. coli

    SciTech Connect

    Johnson, J.L.; Indermauer, L.W.; Rajagopalan, K.V. )

    1991-03-11

    The chlorate resistant mutants of E. coli exhibit a pleiotropic loss of the activities of several molybdoenzymes suggestive of defective molybdenum cofactor synthesis. Indeed, mutants at the chlA and chlE loci have been shown to be deficient in molybdenum cofactor. ChlB mutants, on the other hand, contain high levels of molybdenum cofactor as measured by conversion to the Form A derivative and by reconstitution of the nitrate reductase in the high molecular weight fraction of extracts of the Neurospora crassa nit-1 mutant. The recent discovery that the molybdenum cofactors of E. coli nitrate reductase and formate dehydrogenase contain molybdopterin guanine dinucleotide (MGD) rather than the simpler molybdopterin (MPT) raised the possibility that the chlB locus could be essential for the biosynthesis of MGD from MPT. To test this, conditions were devised for conversion of MGD to a fluorescent, stable derivative, Form A-GMP, and the absorption, fluorescence and chromatographic properties of Form A-GMP were established. Both Form A, arising from MPT, and Form A-GMP arising from MGD, were quantitated in extracts of wild type and chlB cells. Wild type cells were found to contain both Form A and Form A-GMP. In contrast, chlB cells contained elevated levels of Form A but no Form A-GMP. These results suggest that the chlB gene product is essential for the conversion of MPT to MGD.

  18. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  19. HIV-1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

    Cancer.gov

    Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor.

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

    PubMed Central

    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

    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

  1. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    PubMed

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  2. Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

    PubMed Central

    Ramaswamy, S; Dworkin, M; Downard, J

    1997-01-01

    Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates

  3. Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

    PubMed

    Ramaswamy, S; Dworkin, M; Downard, J

    1997-05-01

    Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.

  4. TEMPERATURE-SENSITIVE MUTANTS OF BACILLUS SUBTILIS BACTERIOPHAGE SP3 II.

    PubMed Central

    Nishihara, Mutsuko; Romig, W. R.

    1964-01-01

    Nishihara, Mutsuko (University of California, Los Angeles), and W. R. Romig. Temperature-sensitive mutants of Bacillus subtilis bacteriophage SP3. II. In vivo complementation studies. J. Bacteriol. 88:1230–1239. 1964.—A plate-spotting procedure was used in initial attempts to group the temperature-sensitive Bacillus subtilis phage SP3 mutants by complementation. The results obtained did not show any clear patterns of reactions among the mutants. Crosses were, therefore, repeated in broth at a temperature of 49 C, which greatly reduced the extent of replication of each mutant type alone. The data on mixed infections indicated that there was a minimum of six complementation groups. Of the 12 isolates, 7 did not seem to complement with each other; the rest complemented with each other and with the seven noncomplementing mutants. There was a positive correlation between the complementation reaction of a pair and the recovery of wild-phenotype phages from a 49 C broth lysate. The relative proportion of phages capable of forming wild-phenotype plaques on plates incubated at 46 C to the total number of plaque-forming units was higher in a lysate of a mixed infection with two mutants than in lysates of each mutant alone. Moreover, this frequency was higher for a mixed lysate made at 49 C than for a lysate of the same two mutants made at 37 C. These observations suggested that genetic recombination might occur at 49 C, and that the increased recovery of wild-phenotype phages in lysates made at this temperature might be due to a selective advantage for these phages. Recombination experiments at 37 C with some complementing pairs gave frequencies of 2.0 to 4.8%. The ratio of wild-phenotype revertants to total phages in the stock lysates used for these crosses at 37 C was less than 10−6. The noncomplementing mutants were not conclusively shown to be nonidentical. PMID:14234775

  5. Morphological mutants of Gibberella fujikuroi for enhanced production of gibberellic acid.

    PubMed

    Lale, G; Jogdand, V V; Gadre, R V

    2006-01-01

    To examine the production of gibberellic acid by selected morphological mutants of Gibberella fujikuroi in liquid cultures. Mutants of G. fujikuroi having different morphological characteristics were selected after UV irradiation. The production of gibberellic acid by mutants that had different hyphal lengths was examined in shake flasks in media with different concentrations of nutrients as well as different volumes of the medium. Fed-batch fermenter study was performed to evaluate the mutant Mor-25 for growth and production of gibberellic acid. The broth was analysed by high performance liquid chromatography for fusaric acid, the common mycotoxin produced by strains of Fusarium. A variety of morphological mutants having different mycelial and soluble pigmentation as well as colony morphologies were generated from G. fujikuroi upon exposure to UV radiation. A nonpigmented mutant (Car-1) was selected as intermediate parent and later, mutants Mor-1 and Mor-25 were selected based on their distinct morphology. The colonies on regeneration agar plates were small, compact and dry. In liquid medium, mutant Mor-25 grew in a micro-pelleted form and the mycelium had short, highly branched hyphae, curly at tips with thick, swollen cells. Mutant Mor-25 grew rapidly in a low-cost medium containing defatted groundnut flour, sucrose and salts. In media with higher nutrient concentrations as well as larger volumes, it produced twofold more gibberellic acid than the parent. Fusaric acid, the common mycotoxin, was absent in the fermentation broth of mutant Mor-25. The mutants have been deposited in National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India under following culture collection numbers (Car-1, NCIM 1323; Mor-1, NCIM 1322; and Mor-25, NCIM 1321). Growth of unpigmented, morphological mutants of G. fujikuroi that led to lower viscosity in fermentation broth resulted in increased production of gibberellic acid. The use of

  6. Yeast mutants auxotrophic for choline or ethanolamine.

    PubMed Central

    Atkinson, K D; Jensen, B; Kolat, A I; Storm, E M; Henry, S A; Fogel, S

    1980-01-01

    Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally. However, mitochondrial abnormalities were evident even when ethanolamine and choline were supplied. Diploids homozygous for the cho1 mutation were defective in sporulation. Growth on nonfermentable carbon sources was slow, and a high proportion of respiratory-deficient (petite) cells were generated in cho1 cultures. PMID:6988386

  7. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  8. Muscle development in mdx mutant mice.

    PubMed

    Dangain, J; Vrbova, G

    1984-01-01

    Mechanical and contractile properties of tibialis anterior (TA) muscles from X-linked muscular dystrophic (mdx) mutant mice at different stages of development are compared to those of muscles from normal control animals. There is no difference between the tension output, speeds of contraction and relaxation, and weight of TA muscles from mutant adults and normal control animals. However, it is found that in 3-4-week-old mutant animals, tension output and muscle weight are very much reduced, and half relaxation time is prolonged. Thus, during this stage of development, muscles from mdx mice do not function properly. Histological examination of these muscles provides further evidence that, in these animals, rapid muscle destruction occurs at a particular time of development and that it is followed by complete recovery. This new mutant therefore presents an interesting case of muscle destruction and rapid regeneration. However, it is not an adequate model for Duchenne muscular dystrophy.

  9. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...mutations found in breast cancer using both structural and cell based assays. We have now have evidence for the effects of the most recurrent

  10. Mutant IDH1 and thrombosis in gliomas.

    PubMed

    Unruh, Dusten; Schwarze, Steven R; Khoury, Laith; Thomas, Cheddhi; Wu, Meijing; Chen, Li; Chen, Rui; Liu, Yinxing; Schwartz, Margaret A; Amidei, Christina; Kumthekar, Priya; Benjamin, Carolina G; Song, Kristine; Dawson, Caleb; Rispoli, Joanne M; Fatterpekar, Girish; Golfinos, John G; Kondziolka, Douglas; Karajannis, Matthias; Pacione, Donato; Zagzag, David; McIntyre, Thomas; Snuderl, Matija; Horbinski, Craig

    2016-12-01

    Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

  11. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1-Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...ORGANIZATION: Sloan Kettering Institute for Cancer Research New York, NY 10065 REPORT DATE: September 2015 TYPE OF REPORT: Annual Technical Report...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1-Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT

  12. Permission Forms

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    The prevailing practice in public schools is to routinely require permission or release forms for field trips and other activities that pose potential for liability. The legal status of such forms varies, but they are generally considered to be neither rock-solid protection nor legally valueless in terms of immunity. The following case and the…

  13. Permission Forms

    ERIC Educational Resources Information Center

    Zirkel, Perry A.

    2005-01-01

    The prevailing practice in public schools is to routinely require permission or release forms for field trips and other activities that pose potential for liability. The legal status of such forms varies, but they are generally considered to be neither rock-solid protection nor legally valueless in terms of immunity. The following case and the…

  14. Quantitative Analysis of Triple Mutant Genetic Interactions

    PubMed Central

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E.; Wu, Qiuqin; Haber, James E.; Krogan, Nevan J.

    2014-01-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven effective for characterizing cellular functions but can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed Triple Mutant Analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, that is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principle actors are deleted. TMA has also uncovered double mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete, and measures interactions for up to 30 double mutants against a library of 1536 single mutants. PMID:25010907

  15. Isolation and characterization of unusual gin mutants.

    PubMed Central

    Klippel, A; Cloppenborg, K; Kahmann, R

    1988-01-01

    Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation. Images PMID:2974801

  16. Crystal Structure of a Thermally Stable Rhodopsin Mutant

    PubMed Central

    Standfuss, Jörg; Xie, Guifu; Edwards, Patricia C.; Burghammer, Manfred; Oprian, Daniel D.; Schertler, Gebhard F. X.

    2007-01-01

    We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N-terminus and loop E3 which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10°C. It furthermore allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5μm x-ray beam were used to collect data along a single needle measuring 5x5x90μm3. The disulfide introduces only minor changes but fixes the N-terminal cap over the β-sheet lid covering the ligand binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins. PMID:17825322

  17. Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

    PubMed

    McConnell, S J; Stewart, L C; Talin, A; Yaffe, M P

    1990-09-01

    The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

  18. Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants.

    PubMed

    Schultz, J R; Clarke, C F

    1999-01-01

    Ubiquinol (QH2) is a lipid-soluble molecule that participates in cellular redox reactions. Previous studies have shown that yeast mutants lacking QH2 are hypersensitive to treatment with polyunsaturated fatty acids (PUFAs) indicating that QH2 can function as an antioxidant in vivo. In this study the effect of 1 mM linolenic acid on levels of Q6 and Q6H2 is assessed in both wild-type and respiration-deficient (atp2 delta) strains. The response of Q-deficient mutants to other forms of oxidative stress is further characterized to define those conditions where QH2 acts as an antioxidant. Endogenous antioxidant defense systems were also assessed in wild-type, Q-deficient, and atp2 delta strains. Superoxide dismutase (SOD) activity decreased and catalase activity increased in both Q-deficient and atp2 delta mutants compared to wild-type cells, suggesting that such changes result from the loss of respiration rather than the lack of Q.

  19. A naturally occurring deletion mutant of figwort mosaic virus (caulimovirus) is generated by RNA splicing.

    PubMed

    Scholthof, H B; Wu, F C; Richins, R D; Shepherd, R J

    1991-09-01

    A naturally occurring deletion mutant is observed in plants infected with figwort mosaic virus (FMV), a caulimovirus. The encapsidated mutant genome is formed spontaneously in association with two different strains of FMV in four host plant species. The mutant also appears when cloned wild-type viral DNA is used as the inoculum. The deletion mutant alone is not infectious and it appears unable to replicate after its formation, even in the presence of wild-type virus. The gene for chloramphenicol acetyltransferase was inserted at different positions in the deletion mutant genome, and subsequent transient assays showed that gene expression of the mutant occurs despite the deletion. Sequence analyses of the mutant genome revealed a deletion of 1237-bp segment encompassing a major portion of the coat protein gene and the 5' end of the downstream reverse transcriptase gene. This deletion is associated with consensus signals for RNA splicing including the conserved 5' and 3' splice sites plus surrounding sequences, putative branch point(s) for lariat formation, and an extremely high adenosine content (41%) of the removed fragment. This suggests that splicing of the FMV full-length transcript has occurred prior to reverse transcription and this accounts for the presence and accumulation of encapsidated DNAs with the same deletion.

  20. New nodulation mutants responsible for infection thread development in Lotus japonicus.

    PubMed

    Yano, Koji; Tansengco, Myra L; Hio, Taihei; Higashi, Kuniko; Murooka, Yoshikatsu; Imaizumi-Anraku, Haruko; Kawaguchi, Masayoshi; Hayashi, Makoto

    2006-07-01

    Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

  1. Interlaboratory comparison: liver spontaneous mutant frequency from lambda/lacI transgenic mice (Big Blue) (II).

    PubMed

    Young, R R; Rogers, B J; Provost, G S; Short, J M; Putman, D L

    1995-03-01

    Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

  2. Proton movement and photointermediate kinetics in rhodopsin mutants.

    PubMed

    Lewis, James W; Szundi, Istvan; Kazmi, Manija A; Sakmar, Thomas P; Kliger, David S

    2006-05-02

    The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.

  3. GREEN FLUORESCENT PIGMENT ACCUMULATED BY A MUTANT OF CELLVIBRIO GILVUS.

    PubMed

    LOVE, S H; HULCHER, F H

    1964-01-01

    Love, Samuel H. (Bowman Gray School of Medicine, Winston-Salem, N.C.), and Frank H. Hulcher. Green fluorescent pigment accumulated by a mutant of Cellvibrio gilvus. J. Bacteriol. 87:39-45. 1964.-A mutant of Cellvibrio gilvus, designated strain 139A, liberated a green, fluorescent pigment into the surrounding culture medium. A study of the factors which affected the accumulation of this pigment led to the development of a chemically defined medium which supported maximal pigment accumulation in aerated, liquid cultures. d-Glucose, glycine or l-serine, l-phenylalanine, l-proline, and l-lysine comprised the organic components of this medium. The visible absorption spectrum of the pigment showed a maximal band at 400 mmu (pH 7.0). A difference spectrum between reduced and oxidized pigment showed loss of the band at 400 mmu upon oxidation. However, a methanol-extractable, flavinelike compound occurred in the wild strain but not in the mutant. Ferric ions added to the defined medium stimulated growth, with a concomitant reduction of pigment accumulation. Pigment was formed at a maximal rate during the stationary growth phase, and the highest yield was obtained by 18 hr. Organic solvents did not extract the pigment from water solutions. One and sometimes two, compounds absorbing at 400 mmu could be eluted by ion-exchange chromatography on Cellex-P (H(+)), which was used to separate the pigment from other components in the culture supernatants so that the radioactivity of the pigment could be measured. The mutant synthesized C(14)-labeled pigment from d-glucose-U-C(14) and from each of four amino acids (glycine-1-C(14), l-phenylalanine-U-C(14), l-proline-U-C(14), and l-lysine-U-C(14). Delta-Amino-levulenic acid-4-C(14) did not contribute C(14) to the pigment.

  4. Prion propagation in cells expressing PrP glycosylation mutants.

    PubMed

    Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

    2011-04-01

    Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

  5. Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development.

    PubMed

    Groth, Martin; Kosuta, Sonja; Gutjahr, Caroline; Haage, Kristina; Hardel, Simone Liesel; Schaub, Miriam; Brachmann, Andreas; Sato, Shusei; Tabata, Satoshi; Findlay, Kim; Wang, Trevor L; Parniske, Martin

    2013-07-01

    Arbuscular mycorrhiza (AM) fungi form nutrient-acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM-defective mutants via a microscopic screen of an ethyl methanesulfonate-mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild-type-like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  6. Outer membranes of gram-negative bacteria. XV. Transmembrane diffusion rates in lipoprotein-deficient mutants of Escherichia coli.

    PubMed Central

    Nikaido, H; Bavoil, P; Hirota, Y

    1977-01-01

    Permeability of the outer membrane to 6-aminopenicillanic acid was unaltered in an lpo mutant, lacking the Braun lipoprotein, a result suggesting that the lipoproteins by themselves form no or few diffusion pores. PMID:200601

  7. Quantum chemical modeling of rhodopsin mutants displaying switchable colors.

    PubMed

    Melaccio, Federico; Ferré, Nicolas; Olivucci, Massimo

    2012-09-28

    We look at the possibility to compute and understand the color change occurring upon mutation of a photochromic protein. Accordingly, ab initio multiconfigurational quantum chemical methods are used to construct basic quantum-mechanics/molecular-mechanics (QM/MM) models for a small mutant library of the sensory rhodopsin of Anabaena (Nostoc) sp. PCC7120 cyanobacterium. Together with the wild-type forms, a set of 26 absorption maxima spanning a ca. 80 nm range is obtained. We show that these models can be used to capture the electrostatic change controlling the computed color variation and the change in the ionization of specific side chains.

  8. Mutant TDP-43 does not impair mitochondrial bioenergetics in vitro and in vivo.

    PubMed

    Kawamata, Hibiki; Peixoto, Pablo; Konrad, Csaba; Palomo, Gloria; Bredvik, Kirsten; Gerges, Meri; Valsecchi, Federica; Petrucelli, Leonard; Ravits, John M; Starkov, Anatoly; Manfredi, Giovanni

    2017-05-08

    Mitochondrial dysfunction has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Functional studies of mitochondrial bioenergetics have focused mostly on superoxide dismutase 1 (SOD1) mutants, and showed that mutant human SOD1 impairs mitochondrial oxidative phosphorylation, calcium homeostasis, and dynamics. However, recent reports have indicated that alterations in transactivation response element DNA-binding protein 43 (TDP-43) can also lead to defects of mitochondrial morphology and dynamics. Furthermore, it was proposed that TDP-43 mutations cause oxidative phosphorylation impairment associated with respiratory chain defects and that these effects were caused by mitochondrial localization of the mutant protein. Here, we investigated the presence of bioenergetic defects in the brain of transgenic mice expressing human mutant TDP-43 (TDP-43(A315T) mice), patient derived fibroblasts, and human cells expressing mutant forms of TDP-43. In the brain of TDP-43(A315T) mice, TDP-43 mutant fibroblasts, and cells expressing mutant TDP-43, we tested several bioenergetics parameters, including mitochondrial respiration, ATP synthesis, and calcium handling. Differences between mutant and control samples were evaluated by student t-test or by ANOVA, followed by Bonferroni correction, when more than two groups were compared. Mitochondrial localization of TDP-43 was investigated by immunocytochemistry in fibroblasts and by subcellular fractionation and western blot of mitochondrial fractions in mouse brain. We did not observe defects in any of the mitochondrial bioenergetic functions that were tested in TDP-43 mutants. We detected a small amount of TDP-43(A315T) peripherally associated with brain mitochondria. However, there was no correlation between TDP-43 associated with mitochondria and respiratory chain dysfunction. In addition, we observed increased calcium uptake in mitochondria from TDP-43(A315T) mouse

  9. Isolation of Rhodospirillum centenum Mutants Defective in Phototactic Colony Motility by Transposon Mutagenesis

    PubMed Central

    Jiang, Ze-Yu; Rushing, Brenda G.; Bai, Yong; Gest, Howard; Bauer, Carl E.

    1998-01-01

    The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria. PMID:9495765

  10. Erroneously elevated glucose values due to maltose interference in mutant glucose dehydrogenase pyrroloquinolinequinone (mutant GDH-PQQ) based glucometer.

    PubMed

    Chakraborty, Partha Pratim; Patra, Shinjan; Bhattacharjee, Rana; Chowdhury, Subhankar

    2017-05-12

    Currently available glucose test strip enzymes include glucose oxidase (GOD) and glucose dehydrogenase (GDH). In GDH-based glucometers, glucose oxidation can be catalysed by different cofactors: nicotinamide adenine dinucleotide (GDH-NAD), flavin adenine dinucleotide (GDH-FAD), pyrroloquinolinequinone (GDH-PQQ) and mutant GDH-PQQ. GOD-based and GDH-NAD-based glucometers are substrate-specific and do not react with sugars other than glucose. GDH-FAD reacts with xylose only in addition to glucose. GDH-PQQ is not glucose-specific; in addition to glucose, it reacts with different other sugars and produces falsely high values of capillary glucose in the presence of such substances. There are reports of several deaths associated with usage of GDH-PQQ-based test strips. A modified form of GDH-PQQ, the so-called mutant GDH-PQQ, is supposedly free from such interferences. In this article spuriously high glucose values due to maltose interference in a glucometer using the mutant GDH-PQQ chemistry are being reported. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  11. Site-directed mutagenesis in bacteriorhodopsin mutants and their characterization for bioelectrical and biotechnological equipment.

    PubMed

    Saeedi, Pardis; Moosaabadi, Jafar Mohammadian; Sebtahmadi, Seyed Sina; Behmanesh, Mehrdad; Mehrabadi, Jalil Fallah

    2012-03-01

    Bacteriorhodopsin (BR) mutagenesis plays an important role in the development of BR-based materials and tools with enhanced optical and electrical properties. Previously reported protocols for generating BR mutations are inefficient for the preparation and purification of mutant proteins. Therefore, a series of BR mutations were generated by using improved methods, which are described in further detail. The functional activity of the recombinant proteins was confirmed by spectroscopic and electrochemical assays. Modified proteins with different wavelengths and activities form a foundation for color-sensitive sensors and can be utilized to produce unique bioelectrical and biotechnological tools and materials. The proton-pumping activity of the generated mutant D85E was normal, indicating that the mutant could be used in light batteries. However, mutants D85Q and D85N were almost inactive; and D85N had a prolonged M state, suggesting that it could be utilized in light memories.

  12. Generation and analysis of bacteriorhodopsin mutants with the potential for biotechnological applications.

    PubMed

    Saeedi, P; Moosaabadi, J Mohammadian; Sebtahmadi, S Sina; Mehrabadi, J Fallah; Behmanesh, M; Nejad, H Rouhani; Nazaktabar, A

    2012-01-01

    The properties of bacteriorhodopsin (BR) can be manipulated by genetic engineering. Therefore, by the methods of gene engineering, Asp85 was replaced individually by two other amino acids (D85V, D85S). The resulting recombinant proteins were assembled into soybean vesicles retinylated to form functional BR-like nano-particles. Proton translocation was almost completely abrogated by the mutant D85S, while the D85V mutant was partially active in pumping protons. Compared with wild type, maximum absorption of the mutants, D85V and D85S, were 563 and 609 nm, which illustrated 5 nm reductions (blue shift) and 41 nm increases (red shift), respectively. Since proton transport activity and spectroscopic activities of the mutants are different, a wide variety of membrane bioreactors (MBr) have been developed. Modified proteins can be utilized to produce unique photo/Electro-chromic materials and tools.

  13. Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

    PubMed Central

    Shin, Seung-Min; Choi, Dong-Ki; Jung, Keunok; Bae, Jeomil; Kim, Ji-sun; Park, Seong-wook; Song, Ki-Hoon; Kim, Yong-Sung

    2017-01-01

    Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology. PMID:28489072

  14. ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function.

    PubMed

    Sharma, Aarti; Lyashchenko, Alexander K; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z; Shneider, Neil A

    2016-02-04

    Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations.

  15. Characterization of shrunken endosperm mutants in barley.

    PubMed

    Ma, Jian; Jiang, Qian-Tao; Wei, Long; Wang, Ji-Rui; Chen, Guo-Yue; Liu, Ya-Xi; Li, Wei; Wei, Yu-Ming; Liu, Chunji; Zheng, You-Liang

    2014-04-10

    Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1-seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

  16. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    SciTech Connect

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

    2014-11-07

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  17. Stability of Iowa mutant and wild type Aβ-peptide aggregates

    NASA Astrophysics Data System (ADS)

    Alred, Erik J.; Scheele, Emily G.; Berhanu, Workalemahu M.; Hansmann, Ulrich H. E.

    2014-11-01

    Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid β-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two Aβ-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.

  18. Characterization of a Lignified Secondary Phloem Fibre‐deficient Mutant of Jute (Corchorus capsularis)

    PubMed Central

    SENGUPTA, GARGI; PALIT, P.

    2004-01-01

    • Background and Aims High lignin content of lignocellulose jute fibre does not favour its utilization in making finer fabrics and other value‐added products. To aid the development of low‐lignin jute fibre, this study aimed to identify a phloem fibre mutant with reduced lignin. • Methods An x‐ray‐induced mutant line (CMU) of jute (Corchorus capsularis) was morphologically evaluated and the accession (CMU 013) with the most undulated phenotype was compared with its normal parent (JRC 212) for its growth, secondary fibre development and lignification of the fibre cell wall. • Key Results The normal and mutant plants showed similar leaf photosynthetic rates. The mutant grew more slowly, had shorter internodes and yielded much less fibre after retting. The fibre of the mutant contained 50 % less lignin but comparatively more cellulose than that of the normal type. Differentiation of primary and secondary vascular tissues throughout the CMU 013 stem was regular but it did not have secondary phloem fibre bundles as in JRC 212. Instead, a few thin‐walled, less lignified fibre cells formed uni‐ or biseriate radial rows within the phloem wedges of the middle stem. The lower and earliest developed part of the mutant stem had no lignified fibre cells. This developmental deficiency in lignification of fibre cells was correlated to a similar deficiency in phenylalanine ammonia lyase activity, but not peroxidase activity, in the bark tissue along the stem axis. In spite of severe reduction in lignin synthesis in the phloem cells this mutant functioned normally and bred true. • Conclusions In view of the observations made, the mutant is designated as deficient lignified phloem fibre (dlpf). This mutant may be utilized to engineer low‐lignin jute fibre strains and may also serve as a model to study the positional information that coordinates secondary wall thickening of fibre cells. PMID:14707004

  19. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

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

    1993-06-01

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

  20. Computing border bases using mutant strategies

    NASA Astrophysics Data System (ADS)

    Ullah, E.; Abbas Khan, S.

    2014-01-01

    Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

  1. Isolation and characterization of transcription fidelity mutants.

    PubMed

    Strathern, Jeffrey N; Jin, Ding Jun; Court, Donald L; Kashlev, Mikhail

    2012-07-01

    Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

  2. Sleep restores behavioral plasticity to Drosophila mutants.

    PubMed

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J

    2015-05-18

    Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

  3. The Truncated C-terminal Fragment of Mutant ATXN3 Disrupts Mitochondria Dynamics in Spinocerebellar Ataxia Type 3 Models

    PubMed Central

    Hsu, Jung-Yu; Jhang, Yu-Ling; Cheng, Pei-Hsun; Chang, Yu-Fan; Mao, Su-Han; Yang, Han-In; Lin, Chia-Wei; Chen, Chuan-Mu; Yang, Shang-Hsun

    2017-01-01

    Spinocerebellar ataxia type 3 (SCA3), known as Machado-Joseph disease, is an autosomal dominant disease caused by an abnormal expansion of polyglutamine in ATXN3 gene, leading to neurodegeneration in SCA3 patients. Similar to other neurodegenerative diseases, the dysfunction of mitochondria is observed to cause neuronal death in SCA3 patients. Based on previous studies, proteolytic cleavage of mutant ATXN3 is found to produce truncated C-terminal fragments in SCA3 models. However, whether these truncated mutant fragments disturb mitochondrial functions and result in pathological death is still unclear. Here, we used neuroblastoma cell and transgenic mouse models to examine the effects of truncated mutant ATXN3 on mitochondria functions. In different models, we observed truncated mutant ATXN3 accelerated the formation of aggregates, which translocated into the nucleus to form intranuclear aggregates. In addition, truncated mutant ATXN3 caused more mitochondrial fission, and decreased the expression of mitochondrial fusion markers, including Mfn-1 and Mfn-2. Furthermore, truncated mutant ATXN3 decreased the mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rate. In transgenic mouse models, truncated mutant ATXN3 also led to more mitochondrial dysfunction, neurodegeneration and cell death in the cerebellums. This study supports the toxic fragment hypothesis in SCA3, and also provides evidence that truncated mutant ATXN3 is severer than full-length mutant one in vitro and in vivo. PMID:28676741

  4. The Truncated C-terminal Fragment of Mutant ATXN3 Disrupts Mitochondria Dynamics in Spinocerebellar Ataxia Type 3 Models.

    PubMed

    Hsu, Jung-Yu; Jhang, Yu-Ling; Cheng, Pei-Hsun; Chang, Yu-Fan; Mao, Su-Han; Yang, Han-In; Lin, Chia-Wei; Chen, Chuan-Mu; Yang, Shang-Hsun

    2017-01-01

    Spinocerebellar ataxia type 3 (SCA3), known as Machado-Joseph disease, is an autosomal dominant disease caused by an abnormal expansion of polyglutamine in ATXN3 gene, leading to neurodegeneration in SCA3 patients. Similar to other neurodegenerative diseases, the dysfunction of mitochondria is observed to cause neuronal death in SCA3 patients. Based on previous studies, proteolytic cleavage of mutant ATXN3 is found to produce truncated C-terminal fragments in SCA3 models. However, whether these truncated mutant fragments disturb mitochondrial functions and result in pathological death is still unclear. Here, we used neuroblastoma cell and transgenic mouse models to examine the effects of truncated mutant ATXN3 on mitochondria functions. In different models, we observed truncated mutant ATXN3 accelerated the formation of aggregates, which translocated into the nucleus to form intranuclear aggregates. In addition, truncated mutant ATXN3 caused more mitochondrial fission, and decreased the expression of mitochondrial fusion markers, including Mfn-1 and Mfn-2. Furthermore, truncated mutant ATXN3 decreased the mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rate. In transgenic mouse models, truncated mutant ATXN3 also led to more mitochondrial dysfunction, neurodegeneration and cell death in the cerebellums. This study supports the toxic fragment hypothesis in SCA3, and also provides evidence that truncated mutant ATXN3 is severer than full-length mutant one in vitro and in vivo.

  5. Polyomavirus middle T-antigen NPTY mutants.

    PubMed Central

    Druker, B J; Sibert, L; Roberts, T M

    1992-01-01

    A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg. Images PMID:1326642

  6. Cold-adapted poliovirus mutants bypass a postentry replication block.

    PubMed

    Dove, A W; Racaniello, V R

    1997-06-01

    In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.

  7. IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation.

    PubMed

    Bayraktar, Oznur; Oral, Ozlem; Kocaturk, Nur Mehpare; Akkoc, Yunus; Eberhart, Karin; Kosar, Ali; Gozuacik, Devrim

    2016-01-01

    The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies.

  8. IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation

    PubMed Central

    Bayraktar, Oznur; Akkoc, Yunus; Eberhart, Karin; Kosar, Ali

    2016-01-01

    The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget’s Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies. PMID:27768726

  9. Genetics of a dwarf mutant in groundnut.

    PubMed

    Patil, S H; Mouli, C

    1975-01-01

    A spontaneous dwarf mutant of groundnut variety, Kopergaon-3, showed differential expression for plant height and secondary branching characters in the reciprocal F1 populations. These differences were assumed to be due to the interaction of nuclear and cytoplasmic factors which mutated with dwarfness.Segregation for dwarfness in the F2 and F3 generations confirmed the monogenic inheritance. The mutant expression was, therefore, controlled by a pair of recessive factors designated d(v)d(v), indicating dwarfism in the Valencia group.

  10. Fluoroquinolone-resistant mutants of Burkholderia cepacia.

    PubMed

    Pope, C F; Gillespie, S H; Pratten, J R; McHugh, T D

    2008-03-01

    Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

  11. Ovarian abnormalities in the staggerer mutant mouse.

    PubMed

    Guastavino, Jean-Marie; Boufares, Salima; Crusio, Wim E

    2005-08-24

    Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

  12. Nonphotic phase shifting in hamster clock mutants.

    PubMed

    Mrosovsky, N; Salmon, P A; Menaker, M; Ralph, M R

    1992-01-01

    Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.

  13. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  14. Mutants with Enhanced Nitrogenase Activity in Hydroponic Azospirillum brasilense-Wheat Associations

    PubMed Central

    Pereg Gerk, Lily; Gilchrist, Kate; Kennedy, Ivan R.

    2000-01-01

    The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism. PMID:10788397

  15. Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Dube, D K; Loeb, L A

    1994-01-01

    We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site [K.M. Munir, D.C. French, D.K. Dube and L.A. Loeb (1992) J. Biol. Chem., 167, 6584-6589]. In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine. Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type. It was able to phosphorylate thymidine more rapidly than the wild type both in vivo and in vitro. The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of [methyl-3H]thymidine into E. coli, (ii) a 2.4 times higher rate of [methyl-3H]thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type. Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat. The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.

  16. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals

    PubMed Central

    Hisa, Tomoyuki; Spence, Sally E; Rachel, Rivka A; Fujita, Masami; Nakamura, Takuro; Ward, Jerrold M; Devor-Henneman, Deborah E; Saiki, Yuriko; Kutsuna, Haruo; Tessarollo, Lino; Jenkins, Nancy A; Copeland, Neal G

    2004-01-01

    Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC. PMID:14713950

  17. Reduced chlorophyll biosynthesis in heterozygous barley magnesium chelatase mutants.

    PubMed

    Braumann, Ilka; Stein, Nils; Hansson, Mats

    2014-05-01

    Chlorophyll biosynthesis is initiated by magnesium chelatase, an enzyme composed of three proteins, which catalyzes the insertion of Mg2+ into protoporphyrin IX to produce Mg-protoporphyrin IX. In barley (Hordeum vulgare L.) the three proteins are encoded by Xantha-f, Xantha-g and Xantha-h. Two of the gene products, XanH and XanG, belong to the structurally conserved family of AAA+ proteins (ATPases associated with various cellular activities) and form a complex involving six subunits of each protein. The complex functions as an ATP-fueled motor of the magnesium chelatase that uses XanF as substrate, which is the catalytic subunit responsible for the insertion of Mg2+ into protoporphyrin IX. Previous studies have shown that semi-dominant Xantha-h mutations result in non-functional XanH subunits that participate in the formation of inactive AAA complexes. In the present study, we identify severe mutations in the barley mutants xantha-h.38, -h.56 and -h.57. A truncated form of the protein is seen in xantha-h.38, whereas no XanH is detected in xantha-h.56 and -h.57. Heterozygous mutants show a reduction in chlorophyll content by 14-18% suggesting a slight semi-dominance of xantha-h.38, -h.56 and -h.57, which otherwise have been regarded as recessive mutations.

  18. Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

    PubMed Central

    Mehta, K D; Gupta, R S

    1983-01-01

    Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are

  19. Activation of the thrombopoietin receptor by mutant calreticulin in CALR-mutant myeloproliferative neoplasms.

    PubMed

    Araki, Marito; Yang, Yinjie; Masubuchi, Nami; Hironaka, Yumi; Takei, Hiraku; Morishita, Soji; Mizukami, Yoshihisa; Kan, Shin; Shirane, Shuichi; Edahiro, Yoko; Sunami, Yoshitaka; Ohsaka, Akimichi; Komatsu, Norio

    2016-03-10

    Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell-derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.

  20. Agravitropic mutants of the moss Ceratodon purpureus do not complement mutants having a reversed gravitropic response.

    PubMed

    Cove, David J; Quatrano, Ralph S

    2006-07-01

    New mutants of the moss Ceratodon purpureus have been isolated, which showed abnormal gravitropic responses. The apical cells of protonemal filaments of wild-type strains respond to gravity by growing upwards and are well aligned to the gravity vector. This response only occurs in darkness. Mutants show a range of phenotypes. Some are insensitive to gravity, showing symmetrical growth, while others align to the gravity vector but orient growth downwards. A further class grows in darkness as though it were in light, showing insensitivity to gravity and continued chlorophyll synthesis. Somatic hybrids between mutants and wild-type strains and between pairs of mutants have been selected using transgenic antibiotic resistance as selective markers. Hybrids between wild-type strains and all of the mutants have a wild-type phenotype, and so all mutants therefore have recessive phenotypes. Mutants comprise three complementation groups. One group has a single member, while another has three members. The third has at least 16 members and shows a complex pattern of complementation consistent with a single gene product functioning in both orientation and alignment to gravity, as well as contributing more than one subunit to the mature product.

  1. Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting

    SciTech Connect

    Hollocher, H.; Place, A.R.

    1987-06-01

    Using protein blotting and an immuno-overlay procedure, the authors have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutations have retained the ability form dimers under native conditions. None of the inactive mutant proteins has the ability to form the adduct-bound isozyme. The authors have found no correlation between protein pI and i vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

  2. Genotyping-by-sequencing of glossy mutants

    USDA-ARS?s Scientific Manuscript database

    Glossy mutants are a common occurrence in Brassica oleracea L. and they have been documented in most crop varieties of the species including cabbage, kale, broccoli, and collard. Glossy phenotypes have been of particular interest to researchers due to observations that they influence insect behavior...

  3. Comprehensive transposon mutant library of Pseudomonas aeruginosa

    PubMed Central

    Jacobs, Michael A.; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V.; Manoil, Colin

    2003-01-01

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. PMID:14617778

  4. Comprehensive transposon mutant library of Pseudomonas aeruginosa.

    PubMed

    Jacobs, Michael A; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V; Manoil, Colin

    2003-11-25

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

  5. Conformational stability of adrenodoxin mutant proteins.

    PubMed Central

    Burova, T. V.; Beckert, V.; Uhlmann, H.; Ristau, O.; Bernhardt, R.; Pfeil, W.

    1996-01-01

    Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found. PMID:8880913

  6. Yeast mutants overproducing iso-cytochromes c

    SciTech Connect

    Sherman, F.; Cardillo, T.S.; Errede, B.; Friedman, L.; McKnight, G.; Stiles, J.I.

    1980-01-01

    For over 15 years, the iso-cytochrome c system in the yeast Saccharomyces cerevisiae has been used to investigate a multitude of problems in genetics and molecular biology. More recently, attention has been focused on using mutants for examining translation and transcriptional processes and for probing regulatory regions governing gene expression. In an effort to explore regulatory mechanisms and to investigate mutational alterations that lead to increased levels of gene products, we have isolated and characterized mutants that overproduce cytochrome c. In this paper we have briefly summarized background information of some essential features of the iso-cytochrome c system and we have described the types of mutants that overproduce iso-1-cytochrome c or iso-2-cytochrome c. Genetic procedures and recombinant DNA procedures were used to demonstrate that abnormally high amounts of gene products occur in mutants as result of duplications of gene copies or of extended alteration of regulatory regions. The results summarized in this paper point out the requirements of gross mutational changes or rearrangements of chromosomal segments for augmenting gene products.

  7. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    PubMed

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  8. Ethanol production using engineered mutant E. coli

    DOEpatents

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  9. Phenotypic mutant library: potential for gene discovery

    USDA-ARS?s Scientific Manuscript database

    The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

  12. The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

    PubMed

    Ahne, F; Merlos-Lange, A M; Lang, B F; Wolf, K

    1984-09-01

    The three mutator strains ana (r)-8, ana (r)-14, and diu (r)-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

  13. Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

    PubMed Central

    Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A.; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K.; Altenbach, Christian; Hong, Yuan; Hanson, Susan M.; Palazzo, Maria C.; Chen, Jeannie; Hubbell, Wayne L.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

    2013-01-01

    Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. PMID:24012956

  14. Extended region of nodulation genes in Rhizobium meliloti 1021. I. Phenotypes of Tn5 insertion mutants

    SciTech Connect

    Swanson, J.A.; Tu, J.K.; Ogawa, J.; Sanga, R.; Fisher, R.F.; Long, S.R.

    1987-10-01

    Rhizobium meliloti Nod/sup -/ mutant WL131, a derivative of wild-type strain 102F51, was complemented by a clone bank of wild-type R. meliloti 1021 DNA, and clone pRmJT5 was recovered. Transfer of pRmJT5 conferred alfalfa nodulation on other Rhizobium species, indicating a role in host range determination for pRmJT5. Mutagenesis of pRmJT5 revealed several segments in which transposon insertion causes delay in nodulation, and/or marked reduction of the number of nodules formed on host alfalfa plants. The set of mutants indicated five regions in which nod genes are located; one mutant, nod-216, is located in a region not previously reported to encode a nodulation gene. Other mutant phenotypes correlated with the positions of open reading frames for nodH, nodF and nodE, and with a 2.2-kb EcoRI fragment. A mutant in nodG had no altered phenotype in this strain. One nodulation mutant was shown to be a large deletion of the common nod gene region. The authors present a discussion comparing the various studies made on this extended nod gene region.

  15. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics.

    PubMed

    Howe, Douglas G; Bradford, Yvonne M; Conlin, Tom; Eagle, Anne E; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E; Westerfield, Monte

    2013-01-01

    ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos.

  16. DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

    PubMed Central

    Torreblanca, J.; Casadesus, J.

    1996-01-01

    Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic'') plasmid. PMID:8878670

  17. Characterization of a novel gravitropic mutant of morning glory, weeping2

    NASA Astrophysics Data System (ADS)

    Kitazawa, Daisuke; Miyazawa, Yutaka; Fujii, Nobuharu; Nitasaka, Eiji; Takahashi, Hideyuki

    2008-09-01

    In higher plants, gravity is a major environmental cue that governs growth orientation, a phenomenon termed gravitropism. It has been suggested that gravity also affects other aspects of morphogenesis, such as circumnutation and winding movements. Previously, we showed that these aspects of plant growth morphology require amyloplast sedimentation inside gravisensing endodermal cells. However, the molecular mechanism of the graviresponse and its relationship to circumnutation and winding remains obscure. Here, we have characterized a novel shoot gravitropic mutant of morning glory, weeping2 ( we2). In the we2 mutant, the gravitropic response of the stem was absent, and hypocotyls exhibited a severely reduced gravitropic response, whereas roots showed normal gravitropism. In agreement with our previous studies, we found that we2 mutant has defects in shoot circumnutation and winding. Histological analysis showed that we2 mutant forms abnormal endodermal cells. We identified a mutation in the morning glory homolog of SHORT-ROOT ( PnSHR1) that was genetically linked to the agravitropic phenotype of we2 mutant, and which may underlie the abnormal differentiation of endodermal cells in this plant. These results suggest that the phenotype of we2 mutant is due to a mutation of PnSHR1, and that PnSHR1 regulates gravimorphogenesis, including circumnutation and winding movements, in morning glory.

  18. d14, a strigolactone-insensitive mutant of rice, shows an accelerated outgrowth of tillers.

    PubMed

    Arite, Tomotsugu; Umehara, Mikihisa; Ishikawa, Shinji; Hanada, Atsushi; Maekawa, Masahiko; Yamaguchi, Shinjiro; Kyozuka, Junko

    2009-08-01

    Recent studies using highly branched mutants of pea, Arabidopsis and rice have demonstrated that strigolactones, a group of terpenoid lactones, act as a new hormone class, or its biosynthetic precursors, in inhibiting shoot branching. Here, we provide evidence that DWARF14 (D14) inhibits rice tillering and may act as a new compo-nent of the strigolactone-dependent branching inhibition pathway. The d14 mutant exhibits increased shoot branch-ing with reduced plant height like the previously characterized strigolactone-deficient and -insensitive mutants d10 and d3, respectively. The d10-1 d14-1 double mutant is phenotypically indistinguishable from the d10-1 and d14-1 single mutants, consistent with the idea that D10 and D14 function in the same pathway. However, unlike with d10, the d14 branching phenotype could not be rescued by exogenous strigolactones. In addition, the d14 mutant contained a higher level of 2'-epi-5-deoxystrigol than the wild type. Positional cloning revealed that D14 encodes a protein of the alpha/beta-fold hydrolase superfamily, some members of which play a role in metabolism or signaling of plant hormones. We propose that D14 functions downstream of strigolactone synthesis, as a component of hormone signaling or as an enzyme that participates in the conversion of strigolactones to the bioactive form.

  19. Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

    PubMed

    Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

    2017-04-04

    Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10(5) colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

  20. ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics

    PubMed Central

    Howe, Douglas G.; Bradford, Yvonne M.; Conlin, Tom; Eagle, Anne E.; Fashena, David; Frazer, Ken; Knight, Jonathan; Mani, Prita; Martin, Ryan; Moxon, Sierra A. Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruef, Barbara J.; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Sprunger, Brock; Van Slyke, Ceri E.; Westerfield, Monte

    2013-01-01

    ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos. PMID:23074187

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

    PubMed

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

    2010-02-01

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

  2. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    SciTech Connect

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  3. GAMPMS: Genetic algorithm managed peptide mutant screening.

    PubMed

    Long, Thomas; McDougal, Owen M; Andersen, Tim

    2015-06-30

    The prominence of endogenous peptide ligands targeted to receptors makes peptides with the desired binding activity good molecular scaffolds for drug development. Minor modifications to a peptide's primary sequence can significantly alter its binding properties with a receptor, and screening collections of peptide mutants is a useful technique for probing the receptor-ligand binding domain. Unfortunately, the combinatorial growth of such collections can limit the number of mutations which can be explored using structure-based molecular docking techniques. Genetic algorithm managed peptide mutant screening (GAMPMS) uses a genetic algorithm to conduct a heuristic search of the peptide's mutation space for peptides with optimal binding activity, significantly reducing the computational requirements of the virtual screening. The GAMPMS procedure was implemented and used to explore the binding domain of the nicotinic acetylcholine receptor (nAChR) α3β2-isoform with a library of 64,000 α-conotoxin (α-CTx) MII peptide mutants. To assess GAMPMS's performance, it was compared with a virtual screening procedure that used AutoDock to predict the binding affinity of each of the α-CTx MII peptide mutants with the α3β2-nAChR. The GAMPMS implementation performed AutoDock simulations for as few as 1140 of the 64,000 α-CTx MII peptide mutants and could consistently identify a set of 10 peptides with an aggregated binding energy that was at least 98% of the aggregated binding energy of the 10 top peptides from the exhaustive AutoDock screening.

  4. Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model

    PubMed Central

    Kikuchi, Hitoshi; Almer, Gabriele; Yamashita, Satoshi; Guégan, Christelle; Nagai, Makiko; Xu, Zuoshang; Sosunov, Alexander A.; McKhann, Guy M.; Przedborski, Serge

    2006-01-01

    Mutation in superoxide dismutase-1 (SOD1), which is a cause of ALS, alters the folding patterns of this protein. Accumulation of misfolded mutant SOD1 might activate endoplasmic reticulum (ER) stress pathways. Here we show that transgenic mice expressing ALS-linked SOD1 mutants exhibit molecular alterations indicative of a recruitment of ER's signaling machinery. We demonstrate by biochemical and morphological methods that mutant SOD1 accumulates inside the ER, where it forms insoluble high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein. These alterations are age- and region-specific, because they develop over the course of the disease and occur in the affected spinal cord but not in the nonaffected cerebellum in transgenic mutant SOD1 mice. Our results suggest a toxic mechanism for mutant SOD1 by which this ubiquitously expressed pathogenic protein could affect motor neuron survival and contribute to the selective motor neuronal degeneration in ALS. PMID:16595634

  5. Cell-free ring expansion of penicillin N to deacetoxycephalosporin C by Cephalosporium acremonium CW-19 and its mutants.

    PubMed Central

    Yoshida, M; Konomi, T; Kohsaka, M; Baldwin, J E; Herchen, S; Singh, P; Hunt, N A; Demain, A L

    1978-01-01

    To examine microbiological ring expansion of penicillin N to a cephalosporin, we obtained five mutants of Cephalosporium acremonium blocked in beta-lactam antibiotic biosynthesis from 2500 survivors of mutagenesis. In submerged fermentation, mutants M-0198, M-0199, and M-2351 produced no beta-lactam antibiotic (type A), whereas mutants M-1443 and M-1836 formed penicillin N but not cephalosporin C (type B). Cell-free extracts of type A mutants converted penicillin N to a cephalosporin; those of type B mutants did not. The product of the cell-free reaction was identified as deacetoxycephalosporin C by thin-layer chromatography, paper chromatography, paper electrophoresis, and enzyme tests. These data strongly support our hypothesis that penicillin N is an intermediate of cephalosporin biosynthesis. PMID:282643

  6. Sirtuin 3 rescues neurons through the stabilisation of mitochondrial biogenetics in the virally-expressing mutant α-synuclein rat model of parkinsonism.

    PubMed

    Gleave, Jacqueline A; Arathoon, Lindsay R; Trinh, Dennison; Lizal, Kristin E; Giguère, Nicolas; Barber, James H M; Najarali, Zainab; Khan, M Hassan; Thiele, Sherri L; Semmen, Mahin S; Koprich, James B; Brotchie, Jonathan M; Eubanks, James H; Trudeau, Louis-Eric; Nash, Joanne E

    2017-10-01

    Parkinson's disease (PD) is a neurodegenerative movement disorder, which affects approximately 1-2% of the population over 60years of age. Current treatments for PD are symptomatic, and the pathology of the disease continues to progresses over time until palliative care is required. Mitochondria are key players in the pathology of PD. Genetic and post mortem studies have shown a large number of mitochondrial abnormalities in the substantia nigra pars compacta (SNc) of the parkinsonian brain. Furthermore, physiologically, mitochondria of nigral neurons are constantly under unusually high levels of metabolic stress because of the excitatory properties and architecture of these neurons. The protein deacetylase, Sirtuin 3 (SIRT3) reduces the impact subcellular stresses on mitochondria, by stabilising the electron transport chain (ETC), and reducing oxidative stress. We hypothesised that viral overexpression of myc-tagged SIRT3 (SIRT3-myc) would slow the progression of PD pathology, by enhancing the functional capacity of mitochondria. For this study, SIRT3-myc was administered both before and after viral induction of parkinsonism with the AAV-expressing mutant (A53T) α-synuclein. SIRT3-myc corrected behavioural abnormalities, as well as changes in striatal dopamine turnover. SIRT3-myc also prevented degeneration of dopaminergic neurons in the SNc. These effects were apparent, even when SIRT3-myc was transduced after the induction of parkinsonism, at a time point when cell stress and behavioural abnormalities are already observed. Furthermore, in an isolated mitochondria nigral homogenate prepared from parkinsonian SIRT3-myc infected animals, SIRT3 targeted the mitochondria, to reduce protein acetylation levels. Our results demonstrate that transduction of SIRT3 has the potential to be an effective disease-modifying strategy for patients with PD. This study also provides potential mechanisms for the protective effects of SIRT3-myc. Crown Copyright © 2017. Published by

  7. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae

    PubMed Central

    Hathroubi, S.; Hancock, M. A.; Langford, P. R.; Tremblay, Y. D. N.; Labrie, J.

    2015-01-01

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. PMID:26483403

  8. Surface Polysaccharide Mutants Reveal that Absence of O Antigen Reduces Biofilm Formation of Actinobacillus pleuropneumoniae.

    PubMed

    Hathroubi, S; Hancock, M A; Bossé, J T; Langford, P R; Tremblay, Y D N; Labrie, J; Jacques, M

    2015-10-19

    Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides, including lipopolysaccharides (LPS) and capsular polysaccharides (CPS), are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement for these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well-characterized mutants were used: an O-antigen LPS mutant, a truncated core LPS mutant with an intact O antigen, a capsule mutant, and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the findings for the biofilm of the parental or other strains, the biofilm of the O antigen and the PGA mutants was dramatically reduced, and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR, and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to that in the biofilm cells of the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance, but the lack of O antigen did not abolish these interactions. In conclusion, the absence of the O antigen reduces the ability of A. pleuropneumoniae to form a biofilm, and this is associated with the reduced expression and production of PGA. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

    PubMed

    Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji

    2016-10-28

    Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

  10. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers.

  11. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants.

    PubMed

    Wu, Xi; Singh, Atul K; Wu, Xiaoyu; Lyu, Yuan; Bhunia, Arun K; Narsimhan, Ganesan

    2016-07-01

    Antimicrobial peptides (AMPs) are relatively short peptides that have the ability to penetrate the cell membrane, form pores leading to cell death. This study compares both antimicrobial activity and cytotoxicity of native melittin and its two mutants, namely, melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity. The antimicrobial activity against different strains of Listeria was investigated by bioassay, viability studies, fluorescence and transmission electron microscopy. Cytotoxicity was examined by lactate dehydrogenase (LDH) assay on mammalian Caco-2 cells. The minimum inhibitory concentration of native, mutant I17K, mutant G1I against Listeria monocytogenes F4244 was 0.315±0.008, 0.814±0.006 and 0.494±0.037μg/ml respectively, whereas the minimum bactericidal concentration values were 3.263±0.0034, 7.412±0.017 and 5.366±0.019μg/ml respectively. Lag time for inactivation of L. monocytogenes F4244 was observed at concentrations below 0.20 and 0.78μg/ml for native and mutant melittin I17K respectively. The antimicrobial activity against L. monocytogenes F4244 was in the order native>G1I>I17K. Native melittin was cytotoxic to mammalian Caco-2 cells above concentration of 2μg/ml, whereas the two mutants exhibited negligible cytotoxicity up to a concentration of 8μg/ml. Pore formation in cell wall/membrane was observed by transmission electron microscopy. Molecular dynamics (MD) simulation of native and its mutants indicated that (i) surface native melittin and G1I exhibited higher tendency to penetrate a mimic of bacterial cell membrane and (ii) transmembrane native and I17K formed water channel in mimics of bacterial and mammalian cell membranes.

  12. Mutants deleted in the agnogene of simian virus 40 define a new complementation group.

    PubMed Central

    Mertz, J E; Murphy, A; Barkan, A

    1983-01-01

    Analysis of the DNA sequence of the late leader region of simian virus 40 indicates that it might encode a 61-amino acid, highly basic protein, LP-1. Mutants deleted in this region are viable, but they produce infectious progeny more slowly than wild-type virus in established monkey cells. On the basis of the rates of appearance and the sizes of mixed plaques formed after cotransfections with pairs of mutants, we found that mutants defective in the synthesis of LP-1 complementation was also observed in infections with virions and was bidirectional. Therefore, these mutants define a new complementation group, group G. In addition, a protein of the appropriate molecular weight for LP-1 (approximately 8 X 10(3) ) was synthesized by wild-type virus-infected cells but not by mock-infected or group G gene mutant-infected cells. This protein, whose identity has been established definitively by Jay et al. (Nature (London) 291:346-349, 1981), was synthesized at a high rate at late times after infection, was present predominantly in the cytoplasmic fraction of cells, possessed a fairly short half-life, and was absent from mature virions. Once formed, virions of group G gene mutants behaved biologically and physically like virions of wild-type virus. On the basis of these findings and other known properties of LP-1 and mutants defective in LP-1 synthesis, we hypothesize that LP-1 functions to facilitate virion assembly, possibly by serving as a nonreusable scaffolding protein. Images PMID:6296443

  13. Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

    PubMed

    Gordo-Gilart, Raquel; Andueza, Sara; Hierro, Loreto; Jara, Paloma; Alvarez, Luis

    2016-01-01

    Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.

  14. Defective Kernel Mutants of Maize II. Morphological and Embryo Culture Studies.

    PubMed

    Sheridan, W F; Neuffer, M G

    1980-08-01

    This report presents the initial results of our study of the immature kernel stage of 150 defective kernel maize mutants. They are single gene, recessive mutants that map throughout the genome, defective in both endosperm and embryo development and, for the most part, lethal (Neuffer and Sheridan 1980). All can be distinguished on immature ears, and 85% of them reveal a mutant phenotype within 11 to 17 days post-pollination. Most have immature kernels that are smaller and lighter in color than their normal counterparts. Forty of the mutants suffer from their defects early in kernel development and are blocked in embryogenesis before their primordia differentiate, or, if primordia are formed, they are unable to germinate when cultured as immature embryos or tested at maturity; a few begin embryo degeneration prior to the time that mutant kernels became visually distinguishable. The others express the associated lesion later in kernel development and form at least one leaf primordium by the time kernels are distinguishable and will germinate when cultured or tested at maturity. In most cases, on a fresh weight basis, the mutants have embryos that are more severely defective than the endosperm; their embryos usually are no more than one-half to two-thirds the size, and lag behind by one or two developmental stages. in comparison with embryos in normal kernels from the same ear. One hundred and two mutants were examined by culturing embryos on basal and enriched media; 21 simply enlarged or completely failed to grow on any of the media tested; and 81 produced shoots and roots on at least one medium. Many grew equally well on basal and enriched media; 16 grew at a faster rate on basal medium and 23 displayed a superior growth on enriched medium. Among the latter group, 10 may be auxotrophs. One of these mutants and another mutant isolated by E. H. Coe are proline-requiring mutants, allelic to pro-1. Considering their diversity of expression as evidenced by their

  15. Defective Kernel Mutants of Maize II. Morphological and Embryo Culture Studies

    PubMed Central

    Sheridan, William F.; Neuffer, M. G.

    1980-01-01

    This report presents the initial results of our study of the immature kernel stage of 150 defective kernel maize mutants. They are single gene, recessive mutants that map throughout the genome, defective in both endosperm and embryo development and, for the most part, lethal (Neuffer and Sheridan 1980). All can be distinguished on immature ears, and 85% of them reveal a mutant phenotype within 11 to 17 days post-pollination. Most have immature kernels that are smaller and lighter in color than their normal counterparts. Forty of the mutants suffer from their defects early in kernel development and are blocked in embryogenesis before their primordia differentiate, or, if primordia are formed, they are unable to germinate when cultured as immature embryos or tested at maturity; a few begin embryo degeneration prior to the time that mutant kernels became visually distinguishable. The others express the associated lesion later in kernel development and form at least one leaf primordium by the time kernels are distinguishable and will germinate when cultured or tested at maturity. In most cases, on a fresh weight basis, the mutants have embryos that are more severely defective than the endosperm; their embryos usually are no more than one-half to two-thirds the size, and lag behind by one or two developmental stages. in comparison with embryos in normal kernels from the same ear. One hundred and two mutants were examined by culturing embryos on basal and enriched media; 21 simply enlarged or completely failed to grow on any of the media tested; and 81 produced shoots and roots on at least one medium. Many grew equally well on basal and enriched media; 16 grew at a faster rate on basal medium and 23 displayed a superior growth on enriched medium. Among the latter group, 10 may be auxotrophs. One of these mutants and another mutant isolated by E. H. Coe are proline-requiring mutants, allelic to pro-1. Considering their diversity of expression as evidenced by their

  16. Mutant p53 regulates ovarian cancer transformed phenotypes through autocrine matrix deposition

    PubMed Central

    Iwanicki, Marcin P.; Chen, Hsing-Yu; Iavarone, Claudia; Zervantonakis, Ioannis K.; Muranen, Taru; Novak, Marián; Ince, Tan A.; Brugge, Joan S.

    2016-01-01

    High-grade serous ovarian carcinoma (HGS-OvCa) harbors p53 mutations and can originate from the epithelial cell compartment of the fallopian tube fimbriae. From this site, neoplastic cells detach, survive in the peritoneal cavity, and form cellular clusters that intercalate into the mesothelium to form ovarian and peritoneal masses. To examine the contribution of mutant p53 to phenotypic alterations associated with HGS-OvCA, we developed live-cell microscopy assays that recapitulate these early events in cultured fallopian tube nonciliated epithelial (FNE) cells. Expression of stabilizing mutant variants of p53, but not depletion of endogenous wild-type p53, in FNE cells promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of cell clusters with mesothelium-intercalation capacity. Mutant p53R175H-induced phenotypes were dependent on fibronectin production, α5β1 fibronectin receptor engagement, and TWIST1 expression. These results indicate that FNE cells expressing stabilizing p53 mutants acquire anchorage independence and subsequent mesothelial intercalation capacity through a mechanism involving mesenchymal transition and matrix production. These findings provide important new insights into activities of mutant p53 in the cells of origin of HGS-OvCa. PMID:27482544

  17. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid.

    PubMed

    Cheng, Man Hei; Tam, Chung Nga; Choy, Kwong Wai; Tsang, Wai Hung; Tsang, Sze Lan; Pang, Chi Pui; Song, You Qiang; Sham, Mai Har

    2016-01-01

    Secc proteins with reduced solubility and prone to form aggregates within lens cells. Accumulation of mutant proteins in the lens fibers would lead to cataract formation in the Secc mutant.

  18. A γA-Crystallin Mouse Mutant Secc with Small Eye, Cataract and Closed Eyelid

    PubMed Central

    Cheng, Man Hei; Tam, Chung Nga; Choy, Kwong Wai; Tsang, Wai Hung; Tsang, Sze Lan; Pang, Chi Pui; Song, You Qiang; Sham, Mai Har

    2016-01-01

    Secc proteins with reduced solubility and prone to form aggregates within lens cells. Accumulation of mutant proteins in the lens fibers would lead to cataract formation in the Secc mutant. PMID:27513760

  19. Genome sequence of an Adoxophyes orana granulovirus (AdorGV) occlusion body morphology mutant

    USDA-ARS?s Scientific Manuscript database

    - Genome sequence of an Adoxophyes orana granulovirus (AdorGV) occlusion body morphology mutant Baculoviruses package virus particles in a crystalline matrix of viral protein to form occlusion bodies. Occlusion bodies are used in biological control because the crystalline matrix protects the viral ...

  20. An Integrated Structural and Computational Study of the Thermostability of Two Thioredoxin Mutants from Alicyclobacillus acidocaldarius

    PubMed Central

    Bartolucci, Simonetta; De Simone, Giuseppina; Galdiero, Stefania; Improta, Roberto; Menchise, Valeria; Pedone, Carlo; Pedone, Emilia; Saviano, Michele

    2003-01-01

    We report a crystallographic and computational analysis of two mutant forms of the Alicyclobacillus acidocaldarius thioredoxin (BacTrx) done in order to evaluate the contribution of two specific amino acids to the thermostability of BacTrx. Our results suggest that the thermostability of BacTrx may be modulated by mutations affecting the overall electrostatic energy of the protein. PMID:12837806

  1. Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.

    PubMed

    Khan, A R; Williams, K A; Boggs, J M; Deber, C M

    1995-09-26

    The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and

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

    PubMed Central

    2014-01-01

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

  3. Substantially elevating the levels of αB-crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation.

    PubMed

    Xu, Guilian; Fromholt, Susan; Ayers, Jacob I; Brown, Hilda; Siemienski, Zoe; Crosby, Keith W; Mayer, Christopher A; Janus, Christopher; Borchelt, David R

    2015-05-01

    There has been great interest in enhancing endogenous protein maintenance pathways such as the heat-shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in amyotrophic lateral sclerosis and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co-expression of αB-crystallin (αB-crys) has been shown to inhibit the formation of detergent-insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB-crys at > 6-fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB-crys alone contained 20% more motor neurons per section than littermate controls. Raising αB-crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent-insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB-crys in spinal motor neurons by 6-fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation. Enhancing the protein chaperone function may present a therapeutic approach to amyotrophic lateral sclerosis caused by mutations in SOD1, and other neurodegenerative disorders characterized by cytosolic protein aggregation. Previous studies in cell models suggested that the chaperone known as αB-crystallin (αB-crys) can prevent mutant SOD1 aggregation. We report that transgenic expression of αB-crys at > 6-fold the normal level in spinal

  4. [Pigment accumulation and functional activity of chloroplasts in common Pisum sativum L. mutants with low chlorophyll level (chlorotica)].

    PubMed

    Ladygin, V G

    2003-01-01

    Pea mutants chlorotica 2004 and 2014 with a low content of chlorophyll were studied. The mutant 2004 has light green leaves and stem, and the mutant 2014 has yellow green leaves and stem. They accumulate approximately 80 and 50% chlorophylls of the parent form of pea Torsdag cv. The content of carotene in carotenoids of the mutant 2004 was much lower, and the accumulation of lutein and violaxanthine was increased. The accumulation of all carotenoids in the mutant 2014 decreased almost proportionally to a decrease in the chlorophyll content. The rate of CO2 evolution in mutant chlorotica 2004 and 2014 was established to be lower. The quantum efficiency of photosynthesis in the mutants was 29-30% lower as compared to the control, and in hybrid plants it was 1.5-2-fold higher. It is assumed that the increase in the activity of the night-time respiration in gas exchange of chlorotica mutants and the drop of photosynthesis lead to a decrease in biomass increment. The results obtained allow us to conclude that the mutation of chlorotica 2004 and 2014 affects the genes controlling the formation and functioning of different components of the photosynthetic apparatus.

  5. Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

    PubMed Central

    Fortnagel, Peter; Freese, Ernst

    1968-01-01

    Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from 14C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in α-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation. Images PMID:4967197

  6. Amuvatinib has cytotoxic effects against NRAS-mutant melanoma but not BRAF-mutant melanoma.

    PubMed

    Fedorenko, Inna V; Fang, Bin; Koomen, John M; Gibney, Geoffrey T; Smalley, Keiran S M

    2014-10-01

    Effective targeted therapy strategies are still lacking for the 15-20% of melanoma patients whose melanomas are driven by oncogenic NRAS. Here, we report on the NRAS-specific behavior of amuvatinib, a kinase inhibitor with activity against c-KIT, Axl, PDGFRα, and Rad51. An analysis of BRAF-mutant and NRAS-mutant melanoma cell lines showed the NRAS-mutant cohort to be enriched for targets of amuvatinib, including Axl, c-KIT, and the Axl ligand Gas6. Increasing concentrations of amuvatinib selectively inhibited the growth of NRAS-mutant, but not BRAF-mutant melanoma cell lines, an effect associated with induction of S-phase and G2/M-phase cell cycle arrest and induction of apoptosis. Mechanistically, amuvatinib was noted to either inhibit Axl, AKT, and MAPK signaling or Axl and AKT signaling and to induce a DNA damage response. In three-dimensional cell culture experiments, amuvatinib was cytotoxic against NRAS-mutant melanoma cell lines. Thus, we show for the first time that amuvatinib has proapoptotic activity against melanoma cell lines, with selectivity observed for those harboring oncogenic NRAS.

  7. alpha Pix enhances mutant huntingtin aggregation.

    PubMed

    Eriguchi, Makoto; Mizuta, Haruo; Luo, Shouqing; Kuroda, Yasuo; Hara, Hideo; Rubinsztein, David C

    2010-03-15

    Huntington's disease is caused by polyglutamine-expanded mutant huntingtin (muhtt), an aggregation-prone protein. We identified the Pak-interacting exchange factor (alpha Pix/Cool2) as a novel huntingtin (htt) interacting protein, after screening actin-cytoskeleton organization-related factors. Using immunoprecipitation experiments, we show that alpha Pix binds to both the N-terminal of wild-type htt (wthtt) and mutant htt (muthtt). Colocalization studies revealed that alpha Pix accumulates in muthtt aggregates. Deletion analysis suggested that the dbl homology (DH) and pleckstrin homology (PH) domains of alpha Pix are required for its interaction with htt. Overexpression of alpha Pix enhanced muthtt aggregation by inducing SDS-soluble muthtt-muthtt interactions. Conversely, knocking down alpha Pix attenuated muhtt aggregation. These findings suggest that alpha Pix plays an important role in muthtt aggregation.

  8. Oxygen sensitivity of an Escherichia coli mutant.

    PubMed Central

    Adler, H; Mural, R; Suttle, B

    1992-01-01

    Genetic evidence indicates that Oxys-6, an oxygen-sensitive mutant of Escherichia coli AB1157, is defective in the region of the hemB locus. Oxys-6 is capable of growth under aerobic conditions only if cultures are initiated at low-inoculum levels. Aerobic liquid cultures are limited to a cell density of 10(7) cells per ml by the accumulation of a metabolically produced, low-molecular-weight, heat-stable material in complex organic media. Both Oxys-6 and AB1157 cells produce the material, but only aerobic cultures of the mutant are inhibited by it. The material is produced by both intact cells and cell extracts in complex media. This reaction also occurs when the amino acid L-lysine is substituted for complex media. Images PMID:1551829

  9. Acriflavine-Resistant Mutant of Streptococcus cremoris†

    PubMed Central

    Sinha, R.P.

    1977-01-01

    Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures. PMID:907329

  10. Intact Interval Timing in Circadian CLOCK Mutants

    PubMed Central

    Cordes, Sara; Gallistel, C. R.

    2008-01-01

    While progress has been made in determining the molecular basis for the circadian clock, the mechanism by which mammalian brains time intervals measured in seconds to minutes remains a mystery. An obvious question is whether the interval timing mechanism shares molecular machinery with the circadian timing mechanism. In the current study, we trained circadian CLOCK +/− and −/− mutant male mice in a peak-interval procedure with 10 and 20-s criteria. The mutant mice were more active than their wild-type littermates, but there were no reliable deficits in the accuracy or precision of their timing as compared with wild-type littermates. This suggests that expression of the CLOCK protein is not necessary for normal interval timing. PMID:18602902

  11. Mutant KRAS promotes malignant pleural effusion formation.

    PubMed

    Agalioti, Theodora; Giannou, Anastasios D; Krontira, Anthi C; Kanellakis, Nikolaos I; Kati, Danai; Vreka, Malamati; Pepe, Mario; Spella, Magda; Lilis, Ioannis; Zazara, Dimitra E; Nikolouli, Eirini; Spiropoulou, Nikolitsa; Papadakis, Andreas; Papadia, Konstantina; Voulgaridis, Apostolos; Harokopos, Vaggelis; Stamou, Panagiota; Meiners, Silke; Eickelberg, Oliver; Snyder, Linda A; Antimisiaris, Sophia G; Kardamakis, Dimitrios; Psallidas, Ioannis; Marazioti, Antonia; Stathopoulos, Georgios T

    2017-05-16

    Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

  12. EMMA--the European mouse mutant archive.

    PubMed

    Hagn, Michael; Marschall, Susan; Hrabè de Angelis, Martin

    2007-09-01

    The European Mouse Mutant Archive (EMMA) offers the worldwide scientific community a free archiving service for its mutant mouse lines and access to a wide range of disease models and other research tools. EMMA is currently comprised of seven partners who operate as the primary mouse repository in Europe. EMMA' s primary objectives are to establish and manage a unified repository for maintaining mouse mutations and to make them available to the scientific community. In addition to these core services, the consortium can generate germ-free (axenic) mice for its customers and also hosts courses in cryopreservation. EMMA is a founder member of the Federation of International Mouse Resources (FIMRe). The EMMA network is funded by the participating institutes, national research programmes and the European Commission Research Infrastructures Programme.

  13. Computational and Experimental Study of Neuroglobin and Mutants

    NASA Astrophysics Data System (ADS)

    Nelson, Lauren; Cho, Samuel; Kim-Shaprio, Daniel

    Neuroglobin (Ngb) is a hexacoordinated heme protein that is closely related to hemoglobin and myoglobin and normally found in the brain and nervous systems. It is involved in cellular oxygen homeostasis and reversibly binds to oxygen with a higher binding affinity than hemoglobin. To protect the brain tissue from hypoxic or ischemic conditions, Ngb increases oxygen availability. We have previously shown that a mutant form of Ngb reduces nitrite to nitric oxide 50x faster than myoglobin and 500x faster than hemoglobin. It also tightly binds to carbon monoxide (CO) with an association rate that is 500x faster than hemoglobin. To analyze the structure of neuroglobin and the characteristics causing these phenomena, we performed 3 sets of 1 microsecond molecular dynamic (MD) simulations of wild-type oxidized and reduced human Ngb and their C46A, C55A, H64L, and H64Q mutants. We also directly compare our MD simulations with time-resolved absorption spectroscopy. These studies will help identify treatments for diseases involving low nitric oxide availability and carbon monoxide poisoning. This research was supported by an NIH NSRA predoctoral fellowship in the Structural and Computational Biophysics Program training Grant (T32GM095440-05).

  14. Microfilament Distribution in Maize Meiotic Mutants Correlates with Microtubule Organization.

    PubMed Central

    Staiger, CJ; Cande, WZ

    1991-01-01

    Microtubules and microfilaments often codistribute in plants; their presumed interaction can be tested with drugs although it is not always clear that these are without side effects. In this study, we exploited mutants defective in meiotic cell division to investigate in a noninvasive way the relationship between the two cytoskeletal elements. By staining unfixed, permeabilized cells with rhodamine-phalloidin, spatial and temporal changes in microfilament distribution during maize meiosis were examined. In wild-type microsporocytes, a microtubule array that radiates from the nucleus disappeared during spindle formation and returned at late telophase. This result differed from the complex cytoplasmic microfilament array that is present at all stages, including karyokinesis and cytokinesis. During division, a second class of microfilaments also was observed in the spindle and phragmoplast. To analyze this apparent association of microtubules and microfilaments, we examined several meiotic mutants known to have stage-specific disruptions in their microtubule arrays. Two mutations that altered the number or form of meiotic spindles also led to a dramatic reorganization of F-actin. In contrast, rearrangement of nonspindle, cytoplasmic microtubules did not lead to concomitant changes in F-actin distribution. These results suggested that microtubules and microfilaments interact in a cell cycle-specific and site-specific fashion during higher plant meiosis. PMID:12324607

  15. Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

    PubMed

    Lambright, D G; Balasubramanian, S; Boxer, S G

    1989-05-05

    Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

  16. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  17. Isolation of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Chidambaram, M; Sharma, B; Petras, S F; Reese, C P; Froshauer, S; Weinstock, G M

    1995-01-01

    Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

  18. Sleep restores behavioral plasticity to Drosophila mutants

    PubMed Central

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J.

    2015-01-01

    SUMMARY Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular-lesion. Sleep was increased using three independent strategies: activating the dorsal Fan Shaped Body (FB), increasing the expression of Fatty acid binding protein (dFabp) or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or Long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using Aversive Phototaxic Suppression (APS) and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer’s disease. Together these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggests that increasing sleep may benefit patients with certain neurological disorders. PMID:25913403

  19. Mutant Sodium Channel for Tumor Therapy

    PubMed Central

    Tannous, Bakhos A; Christensen, Adam P; Pike, Lisa; Wurdinger, Thomas; Perry, Katherine F; Saydam, Okay; Jacobs, Andreas H; García-Añoveros, Jaime; Weissleder, Ralph; Sena-Esteves, Miguel; Corey, David P; Breakefield, Xandra O

    2009-01-01

    Viral vectors have been used to deliver a wide range of therapeutic genes to tumors. In this study, a novel tumor therapy was achieved by the delivery of a mammalian brain sodium channel, ASIC2a, carrying a mutation that renders it constitutively open. This channel was delivered to tumor cells using a herpes simplex virus-1/Epstein–Barr virus (HSV/EBV) hybrid amplicon vector in which gene expression was controlled by a tetracycline regulatory system (tet-on) with silencer elements. Upon infection and doxycycline induction of mutant channel expression in tumor cells, the open channel led to amiloride-sensitive sodium influx as assessed by patch clamp recording and sodium imaging in culture. Within hours, tumor cells swelled and died. In addition to cells expressing the mutant channel, adjacent, noninfected cells connected by gap junctions also died. Intratumoral injection of HSV/EBV amplicon vector encoding the mutant sodium channel and systemic administration of doxycycline led to regression of subcutaneous tumors in nude mice as assessed by in vivo bioluminescence imaging. The advantage of this direct mode of tumor therapy is that all types of tumor cells become susceptible and death is rapid with no time for the tumor cells to become resistant. PMID:19259066

  20. Amphimeric mitochondrial genomes of petite mutants of yeast. I. Flip-flop amphimers make up the mitochondrial genomes of "palindromic" petite mutants of yeast.

    PubMed

    Rayko, E; Goursot, R

    1996-07-31

    The mitochondrial (mt) genomes of three spontaneous cytoplasmic "palindromic" petite mutants of yeast were studied by restriction-enzyme analysis. These mt genomes were shown to be made up of an amplified "master basic unit" consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them. The basic unit was called "amphimeric", this term having been first proposed for certain lambda-phage mutants. We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two - "flip" and "flop" - tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively. These repeat units make two types of "amphimeric" mt genomes which exist in equal proportions in the cell. In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a...A...a...A...), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d...d...D...D...d...d...andt...t...T...T...t...t...). The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers. In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d...t and D...T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D...t and d...T). Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way. Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called "palindromic" or "rearranged".

  1. Mutant p53: One, No One, and One Hundred Thousand.

    PubMed

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer.

  2. Isolation of a novel mutant from Bacillus subtilis natto.

    PubMed

    Yoshida, Kazuo

    2006-01-01

    For the construction of strains with full probiotics function in intestines, deoxycholate resistant mutants were isolated from Bacillus subtilis natto. The partial characterization of the mutants was carried out and described.

  3. Testing Current and Developing Novel Therapies for NF1-Mutant Sarcomas in a Genetically Engineered Mouse Model

    DTIC Science & Technology

    2015-04-01

    valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE April 2015 2. REPORT TYPE Annual 3. DATES COVERED 15 ...NF1-mutant sarcomas.    These studies may identify more efficacious treatments for patients with NF1-mutant sarcomas. 15 . SUBJECT TERMS sarcoma...training for two high school students. Mr. Matt Mosca, a high-school sophomore , worked in our lab over the summer and received scientific training

  4. HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

    Cancer.gov

    On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry by the inhibitor. Cover design by Joseph Myer

  5. A Mutant of Mycobacterium smegmatis Defective in Dipeptide Transport

    PubMed Central

    Bhatt, Achal; Green, Renee; Coles, Roswell; Condon, Michael; Connell, Nancy D.

    1998-01-01

    A mutant of Mycobacterium smegmatis unable to use the dipeptide carnosine (β-alanyl-l-histidine) as a sole carbon or nitrogen source was isolated. Carnosinase activity and the ability to grow on β-Ala and/or l-His were similar in the mutant and the wild type. However, the mutant showed significant impairment in the uptake of carnosine. This study is the first description of a peptide utilization mutant of a mycobacterium. PMID:9852030

  6. Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

    PubMed Central

    Leighton, T. J.; Stock, J. J.

    1970-01-01

    Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed. PMID:4992372

  7. Targeting Oncogenic Mutant p53 for Cancer Therapy.

    PubMed

    Parrales, Alejandro; Iwakuma, Tomoo

    2015-01-01

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

  8. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    DOE PAGES

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; ...

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes ofmore » mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.« less

  9. An annotated database of Arabidopsis mutants of acyl lipid metabolism

    SciTech Connect

    McGlew, Kathleen; Shaw, Vincent; Zhang, Meng; Kim, Ryeo Jin; Yang, Weili; Shorrosh, Basil; Suh, Mi Chung; Ohlrogge, John

    2014-12-10

    Mutants have played a fundamental role in gene discovery and in understanding the function of genes involved in plant acyl lipid metabolism. The first mutant in Arabidopsis lipid metabolism (fad4) was described in 1985. Since that time, characterization of mutants in more than 280 genes associated with acyl lipid metabolism has been reported. This review provides a brief background and history on identification of mutants in acyl lipid metabolism, an analysis of the distribution of mutants in different areas of acyl lipid metabolism and presents an annotated database (ARALIPmutantDB) of these mutants. The database provides information on the phenotypes of mutants, pathways and enzymes/proteins associated with the mutants, and allows rapid access via hyperlinks to summaries of information about each mutant and to literature that provides information on the lipid composition of the mutants. Mutants for at least 30 % of the genes in the database have multiple names, which have been compiled here to reduce ambiguities in searches for information. Furthermore, the database should also provide a tool for exploring the relationships between mutants in acyl lipid-related genes and their lipid phenotypes and point to opportunities for further research.

  10. Registration of two allelic erect leaf mutants of sorghum

    USDA-ARS?s Scientific Manuscript database

    Two allelic sorghum [Sorghum bicolor (L.) Moench] erect leaf (erl) mutants were isolated from an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) mutant library developed at the Plant Stress and Germplasm Development Unit, at Lubbock, Texas. The two mutants, erl1-1 and erl1-2, were isol...

  11. A human brain tumor-derived PDGFR-alpha deletion mutant is transforming.

    PubMed

    Clarke, I D; Dirks, P B

    2003-02-06

    Aberrant receptor tyrosine kinase signaling plays an important role in the molecular pathogenesis of brain tumors. We have been studying a previously identified human glioblastoma-derived PDGFR-alpha mutant that has an in-frame deletion in the extracellular domain, causing loss of exons 8 and 9 (PDGFR-alpha(delta8,9)). In the primary tumor, this deletion mutant receptor was shown to be amplified and overexpressed. The purpose of this study was to determine the expression, activity, localization, and transformation properties of this deletion mutant. In the absence of serum, or PDGF-AA, PDGFR-alpha(delta8,9) was phosphorylated on tyrosine residues, indicating ligand-independent autoactivation. Localization by staining and cell surface biotinylation studies revealed expression of the deletion mutant predominantly in the cytoplasm, with very little present on the cell surface. To determine if PDGFR-alpha(delta8,9) was oncogenic, we transfected wild-type and mutant receptors into Rat1 cells and performed analyses of cell growth, in vitro transformation, and subcutaneous growth in the nude mouse. PDGFR-alpha(delta8,9)-expressing cells displayed enhanced cell growth and survival in low serum, and formed foci in monolayer cultures. PDGFR-alpha(delta8,9)-expressing Rat1 cells were also tumorigenic when injected subcutaneously into nude mice. Expression of PDGFR-alpha(delta8,9) was also associated with increased c-Jun phosphorylation in the absence of PDGF ligand, demonstrating also that the mutant receptor is associated with altered intracellular signaling. These data demonstrate that PDGFR-alpha(delta8,9) is transforming, and it is the first demonstration of a naturally occurring tumor-derived mutant PDGFR-alpha with oncogenic properties.

  12. Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

    PubMed

    Gemel, Joanna; Simon, Adria R; Patel, Dakshesh; Xu, Qin; Matiukas, Arvydas; Veenstra, Richard D; Beyer, Eric C

    2014-09-01

    Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20-31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition). Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Bacteriochlorophyll homolog compositions in the bchU mutants of green sulfur bacteria.

    PubMed

    Tsukatani, Yusuke; Harada, Jiro; Mizoguchi, Tadashi; Tamiaki, Hitoshi

    2013-12-01

    Chlorosomes of the green sulfur bacterium Chlorobaculum limnaeum contain a large number of self-aggregated bacteriochlorophyll (BChl) e molecules. The ΔbchU mutant of this organism lacks BchU, a C20-methyltransferase, and therefore produces BChl f, which is the C20-unsubstituted form of BChl e. The BChl e homolog compositions, in terms of degrees of C8(2)-methylation, were not changed in the wild type during growth, while the BChl f homolog patterns in the mutant were significantly altered at various time periods of growth. BChl f with an isobutyl group at the C8 position was dominant at the early stage of growth, whereas the proportion of BChl f with the C8-ethyl group increased in the late exponential phase. We also constructed the ΔbchU mutant of C. tepidum which originally produces BChl c: the mutant therefore produces BChl d. BChl d homologs highly methylated at the C8(2) position also increased in the ΔbchU mutant of C. tedium compared to those in the wild type. These phenomena suggest that BchU interferes with the methylation ability of BchQ, a C8(2)-methyltransferase, and that the enzymes might compete in terms of obtaining S-adenosyl-methionine, the source of a methyl group. As a result, when grown to the late log phase, the ΔbchU mutant of C. limnaeum had similar heterogeneities of pigment homolog compositions compared to those in the wild type. Chlorosomes with a high proportion of C8-ethylated BChl homologs might be important for fine-tuning the light-harvesting or energy-transfer efficiency. Chlorosomes of the ΔbchU mutants at the various growth stages will be good materials for investigating effects of C8(2)-methylations on supramolecular structures of self-aggregated pigments.

  14. Photophysics and optical switching in green fluorescent protein mutants

    PubMed Central

    Creemers, T. M. H.; Lock, A. J.; Subramaniam, V.; Jovin, T. M.; Völker, S.

    2000-01-01

    We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable “one-color” systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in “color” on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species. PMID:10716703

  15. Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules.

    PubMed

    Walls, Zachary F; Goodell, Stefanie; Andrews, Chasity D; Mathis, Jonathan; Lee, Kyung-Dall

    2013-04-15

    Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. The enl mutants enhance the lrx1 root hair mutant phenotype of Arabidopsis thaliana.

    PubMed

    Diet, Anouck; Brunner, Susanne; Ringli, Christoph

    2004-06-01

    The development of root hairs serves as an excellent model to study cell growth using both cytological and genetic approaches. In the past, we have characterized LRX1, an extracellular protein of Arabidopsis consisting of an LRR-domain and a structural extensin domain. LRX1 is specifically expressed in root hairs and lrx1 mutants show severe deficiencies in root hair development. In this work, we describe the characterization of enl (enhancer of lrx1) mutants that were isolated in a visual screen of an ethylmethanesulfonate -mutagenized lrx1 line for plants exhibiting an enhanced lrx1 phenotype. Four recessive enl mutants were analyzed, three of which define new genetic loci involved in root hair development. The mutations at the enl loci and lrx1 result in additive phenotypes in enl/lrx1 double mutants. One enl mutant is affected in the ACTIN2 gene and encodes a protein with a 22 amino acid deletion at the C-terminus. The comparison of molecular and phenotypic data of different actin2 alleles suggests that the truncated ACTIN2 protein is still partially functional.

  17. Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment

    PubMed Central

    Kimmel, Robin A.; Dobler, Stefan; Schmitner, Nicole; Walsen, Tanja; Freudenblum, Julia; Meyer, Dirk

    2015-01-01

    Diabetes mellitus is characterized by disrupted glucose homeostasis due to loss or dysfunction of insulin-producing beta cells. In this work, we characterize pancreatic islet development and function in zebrafish mutant for pdx1, a gene which in humans is linked to genetic forms of diabetes and is associated with increased susceptibility to Type 2 diabetes. Pdx1 mutant zebrafish have the key diabetic features of reduced beta cells, decreased insulin and elevated glucose. The hyperglycemia responds to pharmacologic anti-diabetic treatment and, as often seen in mammalian diabetes models, beta cells of pdx1 mutants show sensitivity to nutrient overload. This unique genetic model of diabetes provides a new tool for elucidating the mechanisms behind hyperglycemic pathologies and will allow the testing of novel therapeutic interventions in a model organism that is amenable to high-throughput approaches. PMID:26384018

  18. An improved reversibly dimerizing mutant of the FK506-binding protein FKBP

    PubMed Central

    Barrero, Juan J.; Papanikou, Effrosyni; Casler, Jason C.; Day, Kasey J.; Glick, Benjamin S.

    2016-01-01

    ABSTRACT FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP. PMID:27738551

  19. Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging

    PubMed Central

    Kennedy, Julie; Brear, Andrea G.; Prahlad, Veena; Blacque, Oliver E.; Sengupta, Piali

    2016-01-01

    The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies. PMID:27906968

  20. SUMO3 Modification Accelerates the Aggregation of ALS-Linked SOD1 Mutants

    PubMed Central

    Niikura, Takako; Kita, Yoshiko; Abe, Yoichiro

    2014-01-01

    Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS. PMID:24971881

  1. [Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

    PubMed

    Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I

    2015-01-01

    Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

  2. The Chlamydomonas zygospore: mutant strains of Chlamydomonas monoica blocked in zygospore morphogenesis comprise 46 complementation groups.

    PubMed Central

    VanWinkle-Swift, K; Baron, K; McNamara, A; Minke, P; Burrascano, C; Maddock, J

    1998-01-01

    Chlamydomonas monoica undergoes homothallic sexual reproduction in response to nitrogen starvation. Mating pairs are established in clonal culture via flagellar agglutination and fuse by way of activated mating structures to form the quadriflagellate zygote. The zygote further matures into a dormant diploid zygospore through a series of events that we collectively refer to as zygosporulation. Mutants that arrest development prior to the completion of zygosporulation have been obtained through the use of a variety of mutagens, including ultraviolet irradiation, 5-fluorodeoxyuridine, ethyl methanesulfonate, and methyl methanesulfonate. Complementation analysis indicates that the present mutant collection includes alleles affecting 46 distinct zygote-specific functions. The frequency with which alleles at previously defined loci have been recovered in the most recent mutant searches suggests that as many as 30 additional zygote-specific loci may still remain to be identified. Nevertheless, the present collection should provide a powerful base for ultrastructural, biochemical, and molecular analysis of zygospore morphogenesis and dormancy in Chlamydomonas. PMID:9475727

  3. Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins.

    PubMed

    Canto, Tomas; Palukaitis, Peter

    2005-04-01

    The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.

  4. Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

    PubMed Central

    Urzainqui, A; Walker, G C

    1992-01-01

    The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each. Images PMID:1577707

  5. Isolation and characterization of high-temperature-induced Dauer formation mutants in Caenorhabditis elegans.

    PubMed Central

    Ailion, Michael; Thomas, James H

    2003-01-01

    Dauer formation in Caenorhabditis elegans is regulated by at least three signaling pathways, including an insulin receptor-signaling pathway. These pathways were defined by mutants that form dauers constitutively (Daf-c) at 25 degrees. Screens for Daf-c mutants at 25 degrees have probably been saturated, but failed to identify all the components involved in regulating dauer formation. Here we screen for Daf-c mutants at 27 degrees, a more strongly dauer-inducing condition. Mutations identified include novel classes of alleles for three known genes and alleles defining at least seven new genes, hid-1-hid-7. Many of the genes appear to act in the insulin branch of the dauer pathway, including pdk-1, akt-1, aex-6, and hid-1. We also molecularly identify hid-1 and show that it encodes a novel highly conserved putative transmembrane protein expressed in neurons. PMID:14504222

  6. Structural and Functional Recovery of Sensory Cilia in C. elegans IFT Mutants upon Aging.

    PubMed

    Cornils, Astrid; Maurya, Ashish K; Tereshko, Lauren; Kennedy, Julie; Brear, Andrea G; Prahlad, Veena; Blacque, Oliver E; Sengupta, Piali

    2016-12-01

    The majority of cilia are formed and maintained by the highly conserved process of intraflagellar transport (IFT). Mutations in IFT genes lead to ciliary structural defects and systemic disorders termed ciliopathies. Here we show that the severely truncated sensory cilia of hypomorphic IFT mutants in C. elegans transiently elongate during a discrete period of adult aging leading to markedly improved sensory behaviors. Age-dependent restoration of cilia morphology occurs in structurally diverse cilia types and requires IFT. We demonstrate that while DAF-16/FOXO is dispensable, the age-dependent suppression of cilia phenotypes in IFT mutants requires cell-autonomous functions of the HSF1 heat shock factor and the Hsp90 chaperone. Our results describe an unexpected role of early aging and protein quality control mechanisms in suppressing ciliary phenotypes of IFT mutants, and suggest possible strategies for targeting subsets of ciliopathies.

  7. Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

    PubMed Central

    Ernst, A; Black, T; Cai, Y; Panoff, J M; Tiwari, D N; Wolk, C P

    1992-01-01

    Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane. Images PMID:1328150

  8. Rescue of αB Crystallin (HSPB5) Mutants Associated Protein Aggregation by Co-Expression of HSPB5 Partners.

    PubMed

    Hussein, Rasha M; Benjamin, Ivor J; Kampinga, Harm H

    2015-01-01

    HSPB5 (also called αB-crystallin) is a ubiquitously expressed small heat shock protein. Mutations in HSPB5 have been found to cause cataract, but are also associated with a subgroup of myofibrillar myopathies. Cells expressing each of these HSPB5 mutants are characterized by the appearance of protein aggregates of primarily the mutant HSPB5. Like several members of the HSPB family, HSPB5 can form both homo-oligomeric and hetero-oligomeric complexes. Previous studies showed that co-expression of HSPB1 and HSPB8 can prevent the aggregation associated with the HSPB5 (R120G) mutant in cardiomyocytes and in transgenic mice. In this study, we systematically compared the effect of co-expression of each of the members of the human HSPB family (HSPB1-10) on the aggregation of three different HSPB5 mutants (R120G, 450 Δ A, 464 Δ CT). Of all members, co-expression of HSPB1, HSPB4 and HSPB5 itself, most effectively prevent the aggregation of these 3 HSPB5 mutants. HSPB6 and HSPB8 were also active but less, whilst the other 5 HSPB members were ineffective. Co-expression of Hsp70 did not reduce the aggregation of the HSPB5 mutants, suggesting that aggregate formation is most likely not related to a toxic gain of function of the mutants per se, but rather related to a loss of chaperone function of the oligomeric complexes containing the HSPB5 mutants (dominant negative effects). Our data suggest that the rescue of aggregation associated with the HSPB5 mutants is due to competitive incorporation of its partners into hetero-oligomers hereby negating the dominant negative effects of the mutant on the functioning of the hetero-oligomer.

  9. Ultrastructural Characterization of Infection and Colonization of Maize Leaves by Colletotrichum graminicola, and by a C. graminicola Pathogenicity Mutant.

    PubMed

    Mims, C W; Vaillancourt, L J

    2002-07-01

    ABSTRACT Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 36 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable

  10. An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes

    PubMed Central

    Lyell, Noreen L.; Septer, Alecia N.; Dunn, Anne K.; Duckett, Drew; Stoudenmire, Julie L.

    2016-01-01

    ABSTRACT Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ. Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg2+ to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment. IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most

  11. An Expanded Transposon Mutant Library Reveals that Vibrio fischeri δ-Aminolevulinate Auxotrophs Can Colonize Euprymna scolopes.

    PubMed

    Lyell, Noreen L; Septer, Alecia N; Dunn, Anne K; Duckett, Drew; Stoudenmire, Julie L; Stabb, Eric V

    2017-03-01

    Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a Vibrio fischeri mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in alr, glr (murI), glmS, several heme biosynthesis genes, and ftsA, as well as a mutant disrupted 14 bp upstream of ftsQ Mutants with insertions in ftsA or upstream of ftsQ were recovered by addition of Mg(2+) to LBS, but their cell morphology and motility were affected. The ftsA mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in glmS, alr, or glr was recovered with N-acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to V. fischeri in the Euprymna scolopes light organ; however, none of these mutants colonized the host effectively. In contrast, hemA and hemL mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide Bradyrhizobium symbionts with ALA, but they contrast with virulence phenotypes of hemA mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment.IMPORTANCE By supplementing a rich yeast-based medium, we were able to recover V. fischeri mutants with insertions in conditionally essential genes, and f