Different Sensitivities of Mutants and Chimeric Forms of Human Muscle and Liver Fructose- 1,6-Bisphosphatases towards AMP

2003 ◽  
Vol 384 (1) ◽  
pp. 51-58 ◽  
Author(s):  
D. Rakus ◽  
H. Tillmann ◽  
R. Wysocki ◽  
S. Ulaszewski ◽  
K. Eschrich ◽  
...  
Keyword(s):  
Liver Enzyme ◽  
Human Liver ◽  
Human Muscle ◽  
Wild Type ◽  
Muscle Enzyme ◽  
Double Mutation ◽  
Binding Domains ◽  
Fructose 1,6 Bisphosphatase ◽  
Amp Inhibition ◽  
Acid Exchange

Abstract AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 uM and 4.8 uM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the Nterminal region of the muscle enzyme towards liver FBPase (Lys20→Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20→Lys and double mutation Glu19→Asp/Glu20→Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 uM). The decrease of sensitivity for AMP of the muscle mutant Lys20→Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20→Lys and Glu19→Asp/Glu20→Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

scholarly journals Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method.

1989 ◽  
Vol 37 (5) ◽  
pp. 611-615 ◽  
Author(s):  
S Ito ◽  
A Iwasaki ◽  
J Syundo ◽  
Y Tamura ◽  
S Kishi ◽  
...  
Keyword(s):  
Liver Enzyme ◽  
Specific Antibody ◽  
Immunohistochemical Staining ◽  
Human Liver ◽  
Liver Extract ◽  
Precipitin Line ◽  
Tissue Sections ◽  
Immunohistochemical Reaction ◽  
Enzyme Method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).

scholarly journals CRISPR knockouts of pmela and pmelb engineered a golden tilapia by regulating relative pigment cell abundance

2021 ◽  
Author(s):  
Chenxu Wang ◽  
Jia Xu ◽  
Thomas D. Kocher ◽  
Minghui Li ◽  
Deshou Wang
Keyword(s):  
Pigment Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Homozygous Mutation ◽  
Body Color ◽  
Wild Type ◽  
Double Mutation ◽  
Vertical Bars ◽  
White Plumage ◽  
New Strategies

Premelanosome protein (pmel) is a key gene for melanogenesis in vertebrates. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study we found that most fishes have two pmel genes arising from the teleost-specific whole genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9 gene editing. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypo-pigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela -/- mutants, without effects on RPE pigmentation. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela -/- mutants, and also an increase in the number and size of xanthophores, producing a strong golden body color without bars in the trunk. The RPE pigmentation of pmela -/ - ;pmelb -/- was similar to pmela -/- mutants, with much less pigmentation than pmelb -/- mutants and wild-type fish. Taken together, our results indicate that, while both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.

scholarly journals A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC

2021 ◽  
Author(s):  
M. Veronica Lopez ◽  
Sabrina E Vinzon ◽  
Eduardo G. A. Cafferata ◽  
Felipe J Nunez ◽  
Ariadna Soto ◽  
...  
Keyword(s):  
Immune Response ◽  
Clinical Trials ◽  
Dendritic Cells ◽  
Neutralizing Antibodies ◽  
Human Muscle ◽  
T Cell Immunity ◽  
Single Shot ◽  
Wild Type ◽  
Cell Immunity ◽  
Human Muscle Cells

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials.

scholarly journals Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

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.

scholarly journals A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1106
Author(s):  
M. Verónica López ◽  
Sabrina E. Vinzón ◽  
Eduardo G. A. Cafferata ◽  
Felipe J. Núñez ◽  
Ariadna Soto ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Single Dose ◽  
Neutralizing Antibodies ◽  
Human Adenovirus ◽  
Human Muscle ◽  
T Cell Immunity ◽  
Wild Type ◽  
Cell Immunity ◽  
Human Muscle Cells ◽  
Adenovirus 5

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.

Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes

2007 ◽  
Vol 12 (3) ◽  
pp. 313-323 ◽  
Author(s):  
Giovanna Di Nardo ◽  
Andrea Fantuzzi ◽  
Anastasia Sideri ◽  
Paola Panicco ◽  
Carlo Sassone ◽  
...  
Keyword(s):  
Human Liver ◽  
Liver Enzymes ◽  
Wild Type

Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

Entropic effect and residue specific entropic contribution to the cooperativity in streptavidin–biotin binding

Nanoscale ◽  
2020 ◽  
Vol 12 (13) ◽  
pp. 7134-7145 ◽  
Author(s):  
Yalong Cong ◽  
Kaifang Huang ◽  
Yuchen Li ◽  
Susu Zhong ◽  
John Z. H. Zhang ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Md Simulations ◽  
Specific Charge ◽  
Wild Type ◽  
Double Mutation ◽  
Biotin Binding ◽  
Entropic Effect ◽  
Entropic Contribution

Molecular dynamics (MD) simulations were performed employing the polarized protein-specific charge (PPC) to explore the origin of the cooperativity in streptavidin–biotin systems (wild type, two single mutations and one double-mutation).

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis-Mediated Gene Deletion Indicates a Requirement for Chlamydia trachomatis Tarp during In Vivo Infectivity and Reveals a Specific Role for the C Terminus during Cellular Invasion

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Susmita Ghosh ◽  
Elizabeth A. Ruelke ◽  
Joshua C. Ferrell ◽  
Maria D. Bodero ◽  
Kenneth A. Fields ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Host Cell ◽  
Host Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Content Type ◽  
Allelic Exchange ◽  
Binding Domains

ABSTRACT The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

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