Kinetic Characterization of Wild-Type and S229A Mutant MurB:  Evidence for the Role of Ser 229 as a General Acid†

Biochemistry ◽  
1997 ◽  
Vol 36 (4) ◽  
pp. 796-805 ◽  
Author(s):  
Timothy E. Benson ◽  
Christopher T. Walsh ◽  
Vincent Massey
Keyword(s):  
Kinetic Characterization ◽  
Wild Type ◽  
General Acid

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

scholarly journals Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael J Prigge ◽  
Matthieu Platre ◽  
Nikita Kadakia ◽  
Yi Zhang ◽  
Kathleen Greenham ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Early Embryo ◽  
Wild Type ◽  
The Family ◽  
Dependent Inhibition ◽  
Root Gravitropism ◽  
Specialized Function ◽  
Mutant Lines

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

1984 ◽  
Vol 26 (3) ◽  
pp. 386-389 ◽  
Author(s):  
Linda J. Reha-Krantz ◽  
Sükran Parmaksizoglu
Keyword(s):  
Dna Polymerase ◽  
Low Temperatures ◽  
Bacteriophage T4 ◽  
Effect Of Temperature ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

scholarly journals Kinetic characterization of the Escherichia coli oligopeptidase A (OpdA) and the role of the Tyr607 residue

2010 ◽  
Vol 500 (2) ◽  
pp. 131-136 ◽  
Author(s):  
Ricardo Z. Lorenzon ◽  
Carlos E.L. Cunha ◽  
Marcelo F. Marcondes ◽  
Maurício F.M. Machado ◽  
Maria A. Juliano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Characterization

Role of Na/K/Cl cotransport in astrocytes

1992 ◽  
Vol 70 (S1) ◽  
pp. S260-S262 ◽  
Author(s):  
Wolfgang Walz
Keyword(s):  
Volume Regulation ◽  
Ion Homeostasis ◽  
Combined Cycle ◽  
Kinetic Characterization ◽  
Cultured Astrocytes ◽  
Net Uptake ◽  
Swelling Volume ◽  
External K

The kinetic characterization of the Na/K/Cl cotransport of cultured astrocytes and evidence for its involvement in volume regulation and K+ net uptake during K+ clearance are reviewed. Emphasis is put on experimental evidence for a proposed sodium cycle in astrocytes; this cycle involves a Na+–K+ ATPase that is stimulated by both a high external K+ and intracellular Na+. Elevated external K+ also stimulates the Na/K/Cl carrier, transporting these ions inward. As a result Na+ is cycled across the membrane, carried inward by the Na/K/Cl carrier, and returned by the Na+–K+ ATPase. Both functionally coupled mechanisms lead to intracellular KCl accumulation and inward movements of water to compensate for increased osmolarity. The combined cycle is expected to play a major role in the regulation of physiological K+ levels in the brain.Key words: furosemide, ion homeostasis, Na+–K+ ATPase, swelling, volume regulation.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

scholarly journals Characterization of the Opp Peptide Transporter ofCorynebacterium pseudotuberculosisand Its Role in Virulence and Pathogenicity

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Pablo M. R. O. Moraes ◽  
Nubia Seyffert ◽  
Wanderson M. Silva ◽  
Thiago L. P. Castro ◽  
Renata F. Silva ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Etiologic Agent ◽  
Peptide Transporter ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Cell Nutrition ◽  
Oligopeptide Permease ◽  
Extracellular Environment

Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused byCorynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp inC. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of theoppDgene sequence. As occurred to the wild-type, the ΔoppDstrain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppDmutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.

scholarly journals Characterization of Integrin αIIbβ3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets

2020 ◽  
Vol 21 (18) ◽  
pp. 6563
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Soochong Kim
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Tyrosine Kinase 2 ◽  
Platelet Spreading ◽  
Protein Kinase Cδ

Engagement of integrin αIIbβ3 promotes platelet–platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbβ3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbβ3 activation caused PKCδ activation. αIIbβ3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbβ3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbβ3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

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