Impact of monomeric and aggregated wild‐type and A30P/A53T double‐mutant α‐synuclein on antioxidant mechanism and glutamate metabolic profile of cultured astrocytes

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

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Aspergillus nidulans Protein O-Mannosyltransferases Play Roles in Cell Wall Integrity and Developmental Patterning

Eukaryotic Cell ◽

10.1128/ec.00040-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1475-1485 ◽

Cited By ~ 33

Author(s):

Thanyanuch Kriangkripipat ◽

Michelle Momany

Keyword(s):

Cell Wall ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Elevated Temperatures ◽

Cell Wall Integrity ◽

Wild Type ◽

Spatial Cues ◽

Developmental Patterning ◽

In The Wild ◽

Increased Sensitivity

ABSTRACT Protein O-mannosyltransferases (Pmts) initiate O-mannosyl glycan biosynthesis from Ser and Thr residues of target proteins. Fungal Pmts are divided into three subfamilies, Pmt1, -2, and -4. Aspergillus nidulans possesses a single representative of each Pmt subfamily, pmtA (subfamily 2), pmtB (subfamily 1), and pmtC (subfamily 4). In this work, we show that single Δpmt mutants are viable and have unique phenotypes and that the ΔpmtA ΔpmtB double mutant is the only viable double mutant. This makes A. nidulans the first fungus in which all members of individual Pmt subfamilies can be deleted without loss of viability. At elevated temperatures, all A. nidulans Δpmt mutants show cell wall-associated defects and increased sensitivity to cell wall-perturbing agents. The Δpmt mutants also show defects in developmental patterning. Germ tube emergence is early in ΔpmtA and more frequent in ΔpmtC mutants than in the wild type. In ΔpmtB mutants, intrahyphal hyphae develop. All Δpmt mutants show distinct conidiophore defects. The ΔpmtA strain has swollen vesicles and conidiogenous cells, the ΔpmtB strain has swollen conidiophore stalks, and the ΔpmtC strain has dramatically elongated conidiophore stalks. We also show that AN5660, an ortholog of Saccharomyces cerevisiae Wsc1p, is modified by PmtA and PmtC. The Δpmt phenotypes at elevated temperatures, increased sensitivity to cell wall-perturbing agents and restoration to wild-type growth with osmoticum suggest that A. nidulans Pmts modify proteins in the cell wall integrity pathway. The altered developmental patterns in Δpmt mutants suggest that A. nidulans Pmts modify proteins that serve as spatial cues.

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Thiotetrazole alkynylacetanilides as potent and bioavailable non-nucleoside inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2007.06.012 ◽

2007 ◽

Vol 17 (16) ◽

pp. 4437-4441 ◽

Cited By ~ 38

Author(s):

Alexandre Gagnon ◽

Ma’an H. Amad ◽

Pierre R. Bonneau ◽

René Coulombe ◽

Patrick L. DeRoy ◽

...

Keyword(s):

Double Mutant ◽

Wild Type ◽

Reverse Transcriptases ◽

Nucleoside Inhibitors ◽

Hiv 1

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Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

10.21203/rs.3.rs-243105/v1 ◽

2021 ◽

Author(s):

Satoshi Yawata ◽

Kenichi Noda ◽

Ai Shimomura ◽

Akio Kuroda

Keyword(s):

Sodium Chloride ◽

Double Mutant ◽

Luciferase Activity ◽

Firefly Luciferase ◽

Activity Level ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Original Activity ◽

Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

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STY1 and STY2 promote the formation of apical tissues during Arabidopsis gynoecium development

Development ◽

10.1242/dev.129.20.4707 ◽

2002 ◽

Vol 129 (20) ◽

pp. 4707-4717 ◽

Cited By ~ 1

Author(s):

Sandra Kuusk ◽

Joel J. Sohlberg ◽

Jeff A. Long ◽

Ingela Fridborg ◽

Eva Sundberg

Keyword(s):

Double Mutant ◽

Ectopic Expression ◽

Ring Finger ◽

Regional Differentiation ◽

35S Promoter ◽

Insertion Mutation ◽

Wild Type ◽

Transposon Insertion ◽

Visible Effect ◽

Gynoecium Development

Gynoecium ontogenesis in Arabidopsis is accomplished by the co-ordinated activity of genes that control patterning and the regional differentiation of tissues, and ultimately results in the formation of a basal ovary, a short style and an apical stigma. A transposon insertion in the STYLISH1 (STY1) gene results in gynoecia with aberrant style morphology, while an insertion mutation in the closely related STYLISH2 (STY2) gene has no visible effect on gynoecium development. However, sty1-1 sty2-1 double mutant plants exhibit an enhanced sty1-1 mutant phenotype and are characterized by a further reduction in the amount of stylar and stigmatic tissues and decreased proliferation of stylar xylem. These data imply that STY1 and STY2 are partially redundant and that both genes promote style and stigma formation and influence vascular development during Arabidopsis gynoecium development. Consistently, STY1 and STY2 are expressed in the apical parts of the developing gynoecium and ectopic expression of either STY1 or STY2 driven by the CaMV 35S promoter is sufficient to transform valve cells into style cells. STY1::GUS and STY2::GUS activity is detected in many other organs as well as the gynoecium, suggesting that STY1 and STY2 may have additional functions. This is supported by the sty1-1 sty2-1 double mutants producing rosette and cauline leaves with a higher degree of serration than wild-type leaves. STY1 and STY2 are members of a small gene family, and encode proteins with a RING finger-like motif. Double mutant analyses indicate that STY1 genetically interacts with SPATULA and possibly also with CRABS CLAW.

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Abscisic acid resistant mutants in the fern Ceratopteris: characterization and genetic analysis

Canadian Journal of Botany ◽

10.1139/b85-220 ◽

1985 ◽

Vol 63 (9) ◽

pp. 1582-1585 ◽

Cited By ~ 23

Author(s):

Leslie G. Hickok

Keyword(s):

Abscisic Acid ◽

Genetic Analysis ◽

Sexual Differentiation ◽

Double Mutant ◽

Acid Resistance ◽

The Other ◽

Additive Effects ◽

Wild Type ◽

Resistant Mutants ◽

Moderate Resistance

Abscisic acid normally inhibits growth and male sexual differentiation (antheridia formation) in gametophytes of the fern Ceratopteris. Abscisic acid resistant mutants show increased growth and sexual differentiation in comparison with the wild type when cultured in the presence of abscisic acid. Two different mutants that confer resistance to the effects of abscisic acid have been fully characterized. One shows moderate resistance and the other strong resistance. The mutations involve separate but linked loci. Recombination between the loci yields double mutant (cis) recombinants that exhibit additive effects and show exceptional levels of abscisic acid resistance.

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Evolutionary expansion of poly-aspartate motif in the activation peptide of mouse cationic trypsinogen limits autoactivation and protects against pancreatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00265.2021 ◽

2021 ◽

Author(s):

Anna Orekhova ◽

Balazs Csaba Nemeth ◽

Zsanett Jancso ◽

Andrea Geisz ◽

Dora Mosztbacher ◽

...

Keyword(s):

Double Mutant ◽

Early Age ◽

Hereditary Pancreatitis ◽

Wild Type ◽

Trypsinogen Activation ◽

Histological Damage ◽

Cationic Trypsinogen ◽

Activation Peptide ◽

Aspartate Residue

The activation peptide of mammalian trypsinogens typically contains a tetra-aspartate motif (positions P2-P5 in Schechter-Berger numbering) that inhibits autoactivation and facilitates activation by enteropeptidase. This evolutionary mechanism protects the pancreas from premature trypsinogen activation while allowing physiological activation in the gut lumen. Inborn mutations that disrupt the tetra-aspartate motif cause hereditary pancreatitis in humans. A subset of trypsinogen orthologs, including the mouse cationic trypsinogen (isoform T7), harbor an extended penta-aspartate motif (P2-P6) in their activation peptide. Here, we demonstrate that deletion of the extra P6 aspartate residue (D23del) increased autoactivation of T7 trypsinogen 3-fold. Mutagenesis of the P6 position in wild-type T7 trypsinogen revealed that bulky hydrophobic side-chains are preferred for maximal autoactivation and deletion-induced shift of the P7 Leu to P6 explains the autoactivation increase in the D23del mutant. Accordingly, removal of the P6 Leu by N-terminal truncation with chymotrypsin C reduced autoactivation of the D23del mutant. Homozygous T7D23del mice carrying the D23del mutation did not develop spontaneous pancreatitis and severity of cerulein-induced acute pancreatitis was comparable to that of C57BL/6N controls. However, sustained stimulation with cerulein resulted in markedly increased histological damage in T7D23del mice relative to C57BL/6N mice. Furthermore, when the T7D23del allele was crossed to a chymotrypsin-deficient background, the double-mutant mice developed spontaneous pancreatitis at an early age. Taken together, the observations argue that evolutionary expansion of the poly-aspartate motif in mouse cationic trypsinogen contributes to the natural defenses against pancreatitis and validate the role of the P6 position in autoactivation control of mammalian trypsinogens.

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Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas

Journal of Cell Science ◽

10.1242/jcs.109.7.1899 ◽

1996 ◽

Vol 109 (7) ◽

pp. 1899-1907 ◽

Cited By ~ 1

Author(s):

G. Habermacher ◽

W.S. Sale

Keyword(s):

Okadaic Acid ◽

Protein Phosphatase ◽

Double Mutant ◽

Sliding Velocity ◽

Wild Type ◽

Phosphatase Inhibitors ◽

Radial Spokes ◽

Gamma S ◽

Type 1 Phosphatase

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

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An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽

10.1534/genetics.120.303476 ◽

2020 ◽

Vol 216 (2) ◽

pp. 381-393

Author(s):

Anastasiia N. Klimova ◽

Steven J. Sandler

Keyword(s):

Escherichia Coli ◽

Double Mutant ◽

Strand Break ◽

Wild Type ◽

Epistasis Analysis ◽

Chromatid Cohesion ◽

K 12 ◽

Structural Maintenance Of Chromosomes ◽

Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

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Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster

Anesthesiology ◽

10.1097/00000542-200310000-00019 ◽

2003 ◽

Vol 99 (4) ◽

pp. 867-875 ◽

Cited By ~ 16

Author(s):

Sumiko Gamo ◽

Junya Tomida ◽

Katsuyuki Dodo ◽

Dai Keyakidani ◽

Hitoshi Matakatsu ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Double Mutant ◽

Developmental Stages ◽

Northern Blotting ◽

General Anesthetics ◽

Wild Type ◽

Mutant Strains ◽

Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

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